Saturday, 17 November 2012

Charlotte Brontë on being involved and observing

Charlotte Brontë was famed, even back in her day, for writing in a very personal manner. The emotional involvement she put into her books was so real that it convinced the young George Eliot, who admired both Jane Eyre and Villette. It has also been the subject of criticism, because she could not write heroines who did not share some affinity to herself. But if you look closer her heroines come in two classes, those who are involved in the story, and those who are observers.

Jane Eyre is most certainly personally involved. The sufferings at Lowood are felt by her, the scenes that take place at Thornfield especially affect her life. Bertha Mason's existence affects her because she is Jane's impediment to marrying Mr Rochester. Mr Rochester's gloom is meant to be reformed by Jane. Even when she doesn't take part in the scenes where the high society people visit Rochester, she feels strongly because her rival in affections, Blanche Ingram, is present. Her escape to Moor End shows her personal development to renounce fleshly instincts, only to renounce sacrificing herself to religion. 

Then she observes St John's unfulfilled passion for Rosamond Oliver. But she does try to urge them to marry, only for St John to refuse it and propose to Jane instead as a substitute, because she is stronger and more determined than shallow Rosamond. 

Caroline Helstone is a quiet person and therefore doesn't really converse with many people whom she dislikes in her uncle's congregation. She is also painfully shy. But her thoughts are always with us, and her emotions often over-wrought. It is also the ordinary people of her parish that she doesn't really deal with. She is not, however, as alone as Charlotte Brontë would have us think. Apart from Shirley she doesn't seem to have a female friend her age in the district, but she does have the affections of Cyril and Margaret Hall, who have fatherly and motherly feelings towards her. William Farren is also fond of her, and so are Robert and Hortense Moore. With Caroline you are looking at a childlike girl, unlike Jane Eyre's more solitary surroundings. But like Jane, the world of the novel does revolve around her, even with the Luddite riots and the numerous subplots.  She is involved in Miss Ainley's charitable projects, Shirley's whimsical conversation and the so-called love triangle involving her, Robert and Shirley. But Caroline is essentially loveable to the reader (unless you hate weak weeping girls who pine for lost love) as those whom the author is sympathetic to are fond of her. Early readers loved her and thought her the best-drawn character in the book. Jessy and Rose Yorke speak to the older girl as equals with intelligence and affection, Martin Yorke has a schoolboy crush on her.  Even Mr Yorke has a slight tenderness for her as she reminds him of his old dead love. Louis Moore is perhaps one of the few sympathetic characters who seek to avoid her. Though we must consider he has her wellbeing in mind when he asks Shirley on purpose to inform Caroline when Robert is shot. One suspects that Louis Moore is aware of Caroline's feelings for Robert. 

Lucy Snowe on the other hand is an enigma. She observes, feels but is often distant from the subplots. Dr John's and Ginevra's courtship are not part of her life, neither is that of Dr John and Paulina. Though Mrs Bretton may invite her out, she is never fully part of their circle, and they do not understand the nature of her melancholy. She sees Madame Beck's peeping, spying and manoeuvres but until the end, they are observations of a character rather than part of a plot. Villette is a series of character sketches - to show us the sort of people Lucy sees and not so much how she is involved, but how she is NOT involved, because she is deficient in charisma, humour and liveliness. No wonder some people think she is manic-depressive or bipolar or something.  Mrs Bretton is fond of her, Paulina likes her as a friend, but there is still that distance, because both are in higher circles - not so much money-wise, but charisma-wise. They have connections whereas Lucy does not. She enjoys some solitude but she also hates being alone with no close friend in the world. Part of her affinity with Paulina is due to the fact Paulina can sit still and keep quiet instead of harrassing her with confidences. She enjoys the easy familiarity with her friends but their souls cannot come closer, because they do not say much to each other. There cannot be too much silence in a friendship. 

There are scenes where she is involved - her teaching of the horrible pupils of the Pensionnat - but there is a distance between them, and it shows you Lucy's drudgery and development rather than form the plot. Villette is definitely a novel of character. The mysterious nun and the notes that drop from nowhere do not involve her - they are merely incidents that she happens to notice. Why put all these useless incidents in, Charlotte? Only to illustrate how detached Lucy is from everyone around her. And it is a clever device. We do see her response, her snarkiness and bitterness with everyone else's doings that she is not part of. Which shows her character - detached, lonely and rational.

M. Paul does try to make her a part of his life, by getting her a new school and loving her romantically. But in the end he dies which brings us back to square one. Ironically Lucy's happiness when he is away reflects Charlotte's later need for time alone when she had married Nicholls who insisted that she accompanied him on his duties. Poor Charlotte. Perhaps she anticipated this side of her character - wanting time alone, and yet languishing away due to loneliness. If Lucy had married Paul how would it be? He would want her to be involved in his life, and she could not fit in with society there. She may succeed professionally, but emotionally and socially she has not really progressed.

Putting in the King and Queen is curious - she is definitely not part of them and the observations are short. Perhaps Charlotte was impressed by the King of Belgium, but I suspect she put him in to show how upper-class society is rather than merely an individual she chanced to notice. She wanted to show how hypochondria affects even the upper classes - that the King is so, could mean he is a symbol for the country - and therefore hypochondria could be a widespread problem. There was much concern and preoccupation in the Victorian era compared to the previous era, with industrialisation, lack of jobs, and material success as opposed to the spiritual and natural things that Charlotte associated with the Romantics, her idols. While the Prince Regent was known for excess and partying, the King of Belgium could reflect the more sober Victorian era - the qualities Prince Albert was known for. But this is mere guesswork. We do know that she wrote to her sister Emily about Queen Victoria's landing in Brussels to see King Leopold, and that Queen Victoria was a jovial, unaffected woman. Nothing too uppish about her - while the Regency upper classes were certainly uppish. The King's melancholy is one similarity between him and Lucy, and may perhaps align both middle-class teacher and royal King into a more equal position. Which is one thing she doesn't seem to share with her other middle-class acquaintances (according to the novel, that is). Could it be Charlotte's way of saying that melancholy is not so much a class issue, but a matter of the individual? Certainly Lucy is upset when Dr John tells her to cultivate happiness, because it doesn't come to her the way it comes to him. He is naturally jovial and charismatic: she is not. They are two opposite individuals despite being of the same class.

Thursday, 15 November 2012

Eukaryotic DNA packaging mechanisms

The Nucleosome Family: Dynamic and growing by Zlatanova
When digested by nucleases, chromatin gives protecting limiting DNA siexe of 146+/- 2bp. This DNA and octamer of histone molecules (2 each of H2A, H2B, H3 and H4) around which DNA wraps, is the nucleosome core particle.  Repeating unit is composed of core particle and a linker region that connects core particles.

Nucleosome is not a unique unit but a dynamic family of particles. They differ in extent of wrapping of DNA around histone proteins or in composition of internal histone core or both.






Table 1. Summary of Nucleosome Particles with Suggested and Traditional Names
Suggested NameTraditional NameHistone Composition/Stoichiometry
Nucleosome (alternative equivalent names: L-octasome, L-nucleosome)NucleosomeH2A/H2B (H3/H4)2 H2A/H2B
R-nucleosome (R-octasome)ReversomeH2A/H2B (H3/H4)2 H2A/H2B
HexasomeHexasomeH2A/H2B (H3/H4)2 or (H3/H4)2H2A/H2B
L-tetrasomeTetrasome(H3/H4)2
R-tetrasomeRight-handed tetrasome(H3/H4)2
Split nucleosomeSplit nucleosomeH2A/H2B/H3/H4 + H3/H4/H2A/H2B
HemisomeHalf-nucleosomeH2A/H2B/H3/H4 (could be histone variant specific)
Particles containing nonhistone proteins (NHP) in lieu of histones should have specific names, depending on the NHP they contain and the protein stoichiometry. For example, the proposed Scm3 (CenH3/H4)2 Scm3 particle might be called Scm3 hexasome.


Octasome: a particle containing one H3/H4 tetramer and 2 H2A/H2B dimers
Steady-state variability of DNA wrapped around histone octamer. 
In S. pombe, repeat length of chromatin is only 156+/- 2bp.  It is suggested that chromatin fibre must be right-handed solenoid due to very short 10bp linker. It is possible that nucleosomal DNA in these particles is permanently unwound to produce longer linkers. On linker cleavage, DNA may revert to 146bp wrapping. This creates the false impression that S. pombe chromatin has highly short linkers. It has been directly observed that linkers in short repeat-length fibres are extended at expense of wrapped DNA.  Oligonucleosomes reconstituted to saturation on DNA consisting of 172bp repeats of a nucleosome positioning sequence showed a narrow distribution of linker lengths of 73bp, leaving only 100bp on histone core.

Spontaneous conformational transitions in nucleosome
Widom showed the existence of partial uncoiling or breathing motions of nucleosomal DNA. This allows protein access to binding sites hidden inside nucleosomes. Site exposure model was substantiated by FRET studies. Nucleosomal DNA stays fully wrapped for 250ms. Unwrapped state lasts 10-50ms.

Zlanova used single-pair FRET with donor and acceptor dyes placed 80bp apart on 2 gyres on nucleosomal DNA on opposite side of dyad axis. Frequent transitions between a high and low FRET state was interpreted as a long-range unwrapping of nucleosomal DNA.  Transitions representing long-range opening was observed.  Breathing (short range) and opening (longer range) excursions of nucleosomal DNA occur. They contribute to accessibility of nucleosomal DNA to protein binding.


Figure 2. Nucleosome Breathing, Opening, and Gaping(A) Breathing and opening involve breaking of histone/DNA contacts from the end of the octasomes: short-range in breathing and long-range in opening (see text).(B) The gaping transition involves breaking of histone/histone contacts. Inset: The two docking domains that are broken are depicted as magenta clouds. The orange arrow stands for the axis around which the hinge opening takes place.

Gaping transition was predicted by Mozziconacci and Victor.  Occurs through inge opening of 30°rees; round an axis at H3C110/H3C110 interface. Both docking domains break: H2A contacts with H3'/H4' along with H2A' contacts with H3/H4. This transition may affect chromatin fibre compaction at different levels.  Gaping enables stacking between adj nucleosomes. This may provide a functional 2-state structure to fibre: a locked-gaped and stacked-structure in constitutive heterochromatin and an unlocked structure in euchromatin.

Prunell suggested that nucleosome particles fluctuate among 3 DNA conformational states: a conformation with a negative crossing of incoming and outgoing linkers, an open conformation with uncrossed DNAs and a conformation which cross positively.

Dynamic regulation of nucleosome positiong in human genome by Schones
Positioning of nucleosomes with respect to DNA regulates transcription. Schones et al generate genome-wide maps of nucleosome positions in resting and activated human AD4+ T cells by sequencing nucleosome ends using Solexa.

Nucleosomes are highly phased relative to transcription start sites (TSS) of expressed genes. Phasing disappears for unexpressed genes. Expressed and unexpressed genes show differential positioning for +1 nucleosome.

Promoters with stalled PolII show nucleosome phasing similar to promoters of transcriptionally active genes. Gene activation by T cell receptor signalling accompanies nucleosome reorganisation in promoters and enhancers. H2A.Z eviction or H3K4me3 modification may facilitate nucleosome eviction or repositionng in promoter regions of human genome.


Figure 1. The Solexa Sequencing Tags Define Nucleosome Boundaries in the Human Genome(A) Nucleosome profile at the FZD2 promoter. The mononucleosome-sized DNA was isolated from MNase-digested chromatin of human T cells and sequenced to read 25 bp from the end using the Solexa sequencing technique. The sequence reads mapped to a given region were used to generate the nucleosome profiles using a scoring function (see Experimental Procedures) as shown by the black track. Blue ovals indicate inferred nucleosome positions in this study and red ovals indicate nucleosome positions identified previously (Ozsolak et al., 2007).(B) Nucleosome profile at the BRCA1 and NBR2promoters.(C) Nucleosomes identified in the gene body of NBPF10 (region shown is chr1:16763501-16764673).(D) Confirmation of nucleosome boundaries using LM-PCR. The mononucleosome DNA was ligated to a pair of Solexa adaptors, followed by amplification using one Solexa primer and one sequence-specific primer recognizing one of the nucleosomes indicated in Figure 1C. The product was labeled using a 32P-labeled nested primer, resolved by polyacrylamide gel electrophoresis, and visualized by exposing to X-ray films. An arrowhead indicates the major nucleosome boundaries.

To analse nucleosome positioning across genome, mononucleosome-sized DNA was isolated from MNase-digested chromatin. DNA ends were sequenced using Solexa.

Number of sequence reads (tags) from sense and antisense strands of DNA in 5 bp windows surrounding TSSs were counted for expressed and unexpressed genes. 8 phased nucleosomes were detected (filled ovals from -3 to +5), 3 upstream and 5 downstream of TSSs in promoter regions of expressed genes. Only 1 well-positioned nucleosome, the +1 nucleosome was detected in promoter regions of unexpressed genes.

To validate nucleosome distribution, they examined distribution of histone H3. They sequenced ChIP DNA using an H3 antibody and chromatin fragmented by sonication of formaldehyde-cross-linked CD4+ T cells. Generally similar nucleosome levels in most regions.




Figure 2. Nucleosomes near the TSS of Actively Transcribed Genes Are Strongly Phased(A) The nucleosomes near the TSS of expressed genes are phased with respect to the TSS. The y axis shows the normalized number of sequence tags from the sense strand (red) and antisense strand (green) of DNA at each position. The inferred nucleosomes are shown by the filled ovals that are numbered as indicated.(B) Only one well-positioned nucleosome exists near the TSS of unexpressed genes (see panel A for details).(C) Histone distribution surrounding the TSS of expressed genes analyzed by ChIP-Seq using an H3 antibody and crosslinked and sonicated chromatin. The y axis shows the normalized number of sequence tags from the sense strand (red) and antisense strand (green) of DNA at each position.(D) The +1 nucleosomes are differentially positioned in expressed and unexpressed genes. The nucleosome tags from the sense strand of DNA of expressed (indicated as 5′ Exp Nuc, red) and unexpressed (indicated as 5′ Non Nuc, blue) genes are shown. The Pol II tags obtained from the ChIP-Seq analysis (Barski et al., 2007) are also shown for the expressed and unexpressed genes.

Nucleosome positioning surrounding TSSs
To examine relationship between positioning of nucleosome and transcriptional activity they plotted nucleosome and Pol II tags from sense strand for expressed and unexpressed promoters.  5'end of +1 nucleosome in actibe promoters peaked at +40bp. 5'end of +1 nucleosome in inactibe promoters peaked at +10bp. Examination of Pol II binding in promoter region of active genes indicates it peaks at +10bp, overlapping with nucleosome peak in inactive promoters.

Nucleosome phasing near TSSs is correlated with Pol II binding
Better phasing with higher levels of Pol II. Genes without any Pol II binding had low levels of expression. Genes with elongating Poll I showed broad range of expression.

igure 3. Promoters with Stalled Pol II Exhibit Similar Patterns of Nucleosome Phasing to the Promoters with Elongating Pol II(A) Expression patterns of the genes with elongating, stalled, or no Pol II (see Experimental Procedures for details). They axis indicates the number of genes exhibiting the expression level shown by the x axis.(B) The nucleosome pattern near the TSS of the genes with elongating Pol II. The y axis shows the normalized number of sequence tags from the sense strand (red) and antisense strand (green) of DNA at each position.(C) The nucleosome pattern near the TSS of the genes with stalled Pol II in the promoter region.(D) The nucleosome pattern near the TSS of the genes without any Pol II binding in the promoter region.
T cell activation induces nucleosome reorganisation
TCR signalling activates human CD$+ T cells. This activated 417 and repressed 580 genes.  Increase in +1 and +2 nucleosome levels downstream of TSS.   TCR signalling may have swticed Pol II from stalled to elongateding form. More hypophosphorylated Pol II than ser5-phosphorylated Pol I was detected at inducible promoters before TCR signalling. Reverse after TCR signalling.

Nucleosome level at -1 nucleosome position increased. This may repress genes.

Nucleosome reorganisation at functional enhancers
After T cell activation, nucleosomes became more localised.  Nucleosomes were removed or shifted so that CNS (conserved noncoding senquence  b, c, d, e amd f were now in linker regions. They may have become accessible to regulatory factors. Localisation of regulatory sequences in linker regions may help regulate transcription. Many CNSs are in linker regions.

Modification of Promoter-proximal nucleosomes
Promoter regions are associated with H3K4 methylation. Nucleosomesnear TSS are modified with H3K4me3 and nucleosomes further being modified with H3K4me2 and H3K4me1. H3K4 modification tags and nucleosomes tags were examined near TSSs of actibe gemes.

H3K4me3 modification was associated with -2, +1, +2 and +3 nucleosomes. H3K4m32 peaks with +3 and +4 nucleosomes. H3K4me1 peaks with +5 and +6 nucleosomes.

Histone variant H2A.Z is indicated to be associated with -3, -2, +1, +2 amd +3 nucleosomes of actively transcribed genes in human genome. H2A.Z are preferentially lost in -1 nucleosome region.  Loss of H2A.Z-containing nucleosome from -1 nucleosome position in TCR-inducible genes in resting T cells was similar to loss in expressed genes.

Preferential loss of H3K4me3-modified nucleosomes from -1 nucleosomes.  Deposition of H2A.Z or modifification by H3K4me3 may facilitate nucleosome evictgion or repositioning in -1 nucleosome region.

Molecular modelling of chromatosome particle by Bharath
Structure of rat histone H1d showed a 3-helical bundle fold which is a HTH variant. It could bind to DNA at major groove. Primary binding site of globular domain inetracts with extra 20bp of DNA of entering duplex at major groove. Secondary binding site interacts with minor groove of central gyre of DNA superhelix of the nucleosomal core. Structure was stemlike.

Nucleosome arrays reveal two-start organisation of chromatin fibre by Dorigo
DNA in eukaryotic cell nuclei assembles with istones into chromatin.  Stricture of 30nm fibre or nucleosoms higher order structurehas been contentious.  Models are in 2 classes

1), the one-start helix, with bent linker DNA connecting each pair of nucleosome cores. They follow each other immediately along same helical path.

2) two-start helix, based onstraight linker DNA connecting between 2 adjacent stacks of helically arranged nuclrosomes cores.

In one-start model, most prominent is solenoid. Nucleosomes coil around a central cavity with 6-8 nucleosomes per turn.

2-start class is divided between helical ribbon model and crossed-linker mode.
Fig. 1.
Models for the DNA path in the chromatin fiber. Higher order structure models: (A) one-start solenoidal (6), (B) two-start supercoiled (7), and (C) two-start twisted (12). Upper views have the fiber axis running vertically; lower views are down the fiber axis. DNA associated with the nucleosome core is red/blue, and linker DNA running between cores is yellow. These models are idealized, with nucleosome cores in each start contacting each other. The open three-dimensional zigzag seen in conditions not fully compacting may be a precursor

Mutants were constructed of hstones H2A, H2B and H4 by replacing one aa with cysteine in each version. This stabilises higher order structure by forming disulfides.   Nucleosome arrays were assembled.

Chromatin fibre comprises 2 tacks of nucleosomes in accord with two-start model.
Fig. 4.
Electron micrographs showing the two-start organization of nucleosome arrays. (A) 48-mer nucleosome arrays were cross-linked via H4-V21C/H2A-E64C–mediated disulfide formation under compacting conditions and prepared for EM with the use of negative stain. (B) Arrays were treated with 100 mM DTT to relieve the disulfide cross-link and then prepared as for (A). (C) Arrays (three separate examples are shown) prepared as for (A) were cleaved at the Sca I site in the linker DNA. Scale bars indicate 50 nm. EM magnification was 13,000× for (A) and (B) and 26,000× for (C).
Formation of higher order secondary and tertiary chromatin structures by genomic mouse mammary tumour virus promoters by Georgel
Agarose multigel electrophoresis charactersised structural features of isolated genomic mouse mammary tumour virus (MMTV) promoters. Mouse 3134 cells contain 200 stably integrated tandem repeats of a 2.4kb MMTV promoter fragment.

Inactive, basally active and hormonally actibated genomic promoters were liberated by restriction digestion of isolated nuclei, recovered in low=sal nuclear extracts, and electrophoresed in multigels. Spec bands were detected and characterised by Southern and Wester blotting.

Transcriptionally inactive promtoers contain TBP and high levels of histone H1. They are present to varying extent in untreated and dexamethasone-treated 3134 cells.

Basally active promoter, present in untreated cells, is bound to RNAP II, TBP and Oct1. It contains acetylated H3 tail domains and is depleted of histone H1.
All forms of MMTV promoter condense into higher order 2dary or 3ary chromatin structures in vitro with Mg2+.  Genomic MMTV can still form classical higher order structures under physiological salt conditions even after dissocation of H1 and binding of TFs and multiprotein complexes.


Chromatin fragments from basally active and hormonally induced genomic MMTV promoters completely lost H1. Consist of nucleosomal arrays stably bound to various TFs (Oct1) and RNAPII. However genomic promoter fragments can still form salt-dependent 2dary chromatin structure in same manner as model nucleosomal arrays.
Revised model for eukaryotic promoter activation. (A) Present model of transcriptional activation. Inactive and active promoters are depicted in their primary chromatin structure. (B) Proposed model of transcriptional activation. Inactive and active promoters are depicted in folded secondary chromatin structures. “X” and “Y” refer to hypothetical protein–protein interactions involving the upstream regulatory element and the proximal promoter.

TBP is thought of as being a component of eukaryotic PIC. However, genomic chromatin from transcriptionally inactive MMTV promoters contained bound TBP and histone H1. TBP did not interfere with Mg2+ dependent 2dary and 3ary chromatin structure in vitro. TBP was tightly associated with highly condensed chromatin in metaphase chromosomes. It is possible that TBP can bind transcriptionally repressed, H1-stabilised higher order chromatin structures  Possibly to preset or mark promoters for activation before structural reorganisation of promoter chromatin associated with transcription initiation.


Roles of transition nuclear proteins in spermiogenesis by Meistrich
Nuclear changes during spermiogenesis
Spermiogenesis in vertebrates is divided into 2 phases. In first phase, nucleus is round, contains histones as major basic nuclear proteins, and is transcriptionally active.  2nd phase, dramatic changes in chromatin structure  nuclear shaping and condensation.

After nuclear elongation starts, transcription stops. In many species histones are removed and replaced by other proteins. This causes sperm to have one of more protamines as major nuclear protein. In mammals the transition protein (TP) are called transition nuclear proteins. They are intermediate proteins in histone to protamine transition.

Protamines are all highly basic, arginine-rich proteins.  Mouse contains 2 protamines, P1 and P2.  They are arginine- and cysteine-rich.

Replacement of histones and deposition of protamines involve chromatin remodelling.  May cause genome-wide cessation of RNA transcription at this stage.  Nucleosomal DNA containing histones is highly supercoiled. This characteristic is lost during transformation into protamine-containing sperm chromatin.

A high level of DNA strand breaks are formed at time of histone removal during spermiogenesis. Most are reparied by ligation. Sperm still seems to have higher levels of DNA strand breaks than somatic cells.

Histone to protamine transition seems to occur directly in fish and birds. In mammals, TPs are deposited on chromatin as histones are removed and chromatin condensation is initiated. Later these are replaced by protamines.

Transition proteins
TP1 and TP2 are prominent in rodent spermatids. TP1 is 6200 Da. It has 20% arginine and 20% lysine and no cysteine. TP2 is 13000 Da. It has 10% arginine, 10% lysine, and 5% cysteine. TP1 is abundantly expressed and its sequence is highly conserved in various mammals. TP2 sequence is poorly conserved. Its expression levels vary between species.

Fig. 1.  Sequence of appearance of histones, transition proteins ( TPs), and protamines (P1 and P2) during spermiogenesis in the mouse (Mayer and Zirkin 1979; Courtens and Loir 1981; Balhorn et al. 1984; Biggiogera et al.1992; Alfonso and Kistler 1993; Lee et al. 1995; Wu and Means 2000; Yu et al. 2000). The steps of spermatid development and the nuclear shape and condensation state are given at the bottom. The sequence of proteins whose presence has been identified biochemically is indicated by solid bars, the width of which represents the relative levels of the protein. In some instances information on the rat has been extrapolated to the mouse (Meistrich et al. 1994; Kistler et al. 1996). The dashed lines indicate that the presence of the protein has only been identified immunohistochemically

Some studies show that TP1 decreases melting temperature of DNA, relaxes DNA in nucleosomal core particles and stimulates DNA-relaxing activity of topoisomerase 1. One function may be to facilitate histone removal. However other studies indicate that neither TP1 nor TP2 produce any topological changes in supercoiling of DNA.

TP2 increases melting temperature of DNA and compacts DNA in nucleosomal cores. It has been proposed to be a DNA-condensing protein.

TP1 can stimulate single-strand break repair in vitro and in transfected somatic cells. TPs may have important role in repair of DNA strand breaks during chromatin remodelling associated with histone loss.

Meistrich shows that either TPs are not essential for prodcution of fertile sperm. Better sperm function when there is one copy of each Tnp gene than when there are two copies of either gene.

A walk through vertebrate and invertebrate protamines by Lewis
In eutheria (placental mammals) cysteine is important part of protamine sequence. Cysteine is uncommon in other chromosomal proteins.

Protamines show random coil conformation in solution. They adopt  a degree of 2dary structure on neutralisation of arginine positive charge due to electrostatic interactions with phosphate backbone of DNA. Charge neutralisation causes DNA bending. This causes highly compact toroidal nucleoprotamine sturctures in mammalian sperm.

Protamine has a high rate of evolution, like other reproductive proteins. This allows protamines to be used as evolutionary markers to distinguish between closely related species.

Packaging paternal chromosomes with protamine by Braun
Replacing histones with protamines may generate more hydrodynamic pserm head to speed transit through female reproductive tract and across zona pellucida urrounding egg. Nucleoprotamine struvture may protect genetic material in sperm head from physical and chemical damage. Packing sperm chromatin may reprogram paternal genome so appropriate genes from father's chromosomes are expressed in early embryo.

Cho used gene targeting in mouse embryos. They showed that mutation of either haplod-expressed gene causes defective sperm. Removing one copy of Prm1 or Prm2 is detrimental to postmeiotic spermatids.

Incomplete cytokinesis during mitosis and meiosis generates clustrs of haploid spermatids that remina connected through cytoplasmic bridges. Intercellular bridges connecting spermatids equilibrate 1x dosage produced by wt sptermatide and zero dosage of mutant spermatid to 0.5x.  Protamine reduction causes defects in DNA compaction.

Prelude to protamines
When Tnp1 is deleted in experiments, transition proteins have redundant functions  Mutants homozygous for Tnp1 or Tnp2 are fertile but litter sizes are smaller and sperm have minor head abnormalities.

Cho et al showed that in Prm2+/- chimeras, there is also less Prm1 protein in sperm. This indicates that Prm1 deposition requirs normal amounts of Prm2. In transgenic mice that overexpress Prm1 at normal time during spermatid differentiation, ratio of protamine 1 to protamine 2 in mature sperm is similar to that in wt sperm.  Posttranslational modification may ensure precise ratio.

Nuclear condensation
Histone displacement by transition proteins and protamines is accompanied by post-translational modifications. Histone acetylation esp histone H4 acetylation, ubiquination and phosphorylation facilitate displacement of histones.

Chromatin remodelling may require chaperones that actively displace posttranslationally modified histones.
Phosphorylation of transition proteins and protamines, which neutralises highly basic proteins may be important for chromatin compaction.

Targeted mutations in HR6B ubiquitin-conjugating enzyme, Ube2b and Camk4 disrupt terminal stages of spermatid ddifferentiation and cause sterility. Camk4 encodes CaMKIV and phosphorylates Prm2.

Packaging chromatin. A model of chromatin packaging in somatic cells (left) and mammalian sperm (right). In somatic cells, the DNA is wound twice around histone octamers to form nucleosomes, which are then coiled into solenoids. The solenoids are attached at intervals to the nuclear matrix at their bases and form DNA loop domains. In the sperm nucleus, protamines replace the histones and the protamine-DNA complex is coiled into a doughnut shape. Inset shows the tight compacting of protamine-DNA strands. Displacement of the histones is facilitated by post-translational modifications of the proteins, in the form of histone H4 acetylation, ubiquitination and phosphorylation. Phosphorylation and dephosphorylation of the transition proteins facilitate their displacement before protamines bind.
DNA Condensation by protamine and arginine-rich peptides: analysis of toroid stability using single DNA molecules by Balhorn
Scanning probe microscopy studies of sperm chromatin and synthetic DNA-protamine complexes indicate that DNA coiling into toroidal subunits can be micmicked by adding protamine to DNA in vitro. This coiling is initiated in maturing spermatid to prepare genome for delivery into egg.

DNA-protamine has increased resistance to nuclease digestion. It is also structurally similar to native sperm chromatin. This suggests that DNA packaging by protamine may offer new approach for improving efficiency of DNA uptake by sperm.

Number of arginine residues in DNA binding domain of protamine affects stability of DNA-protamine.

Binding of protamine and complex condensation seem to protect DNA during its transport to egg and ensure all genetic activity is suppressed until sperm's genome is reactivated after fertilisation.

Microspheres with attached DNA molecules were introduced into one port of flow cell and trapped. It is checked to ensure only 1 DNA molecule was attached to bead. Portamine binding was inititated by moving bead and its attached DNA across interface into buffer stream containing protamine. Fluorescnce of DNA was imagaed using CCD camera. As protein dissociated from molecule, coiled DNA reextended until it reached its original length.

Similar experiments done using synthetic peptides. Study confirms that number of arginine residues in molecule affects its rate of dissociation from DNA.  More arginine residues, lower off rate (molecules/sec) and higher dissociation time.

This information can be used to design protamine-like molecules to package genes to facilitate uptake and integration by somatic cells and sperm.  Mammalian eggs have a mechanism to remove protamine form DNA once sperm head enters cytoplasm. Somatic cells do not encounter protamine. Use a synthetic protein containing a DNA binding domain with fewer Arg residues linked to a nuclear localisation signal sequence. Protein would dissociate from DNA more rapidly and might direct sequence to nucleus before it completely dissociates.

Protamine-induced condensation and decondensation of the same DNA molecule by Brewer
Condensation and decondensation experiments with λ-phage DNA show that toroid formation and stability are influenced by the number of arginine-rich anchoring domains in protamine. The results explain why protamines contain so much arginine and suggest that these proteins must be actively removed from sperm chromatin after fertilization

Protamine and other polycations have been shown to coil DNA into toroidal structures containing up to 60 kb of DNA (1–3). Individual bacteriophage appear to contain a single toroid folded inside the protein capsid (3), whereas a sperm cell contains as many as 50,000 toroids packed inside its nucleus (1). The protamines responsible for inducing torus formation and packaging DNA in maturing spermatids contain a series of arginine-rich anchoring domains (4) that bind to the phosphodiester backbone of DNA in a base sequence–independent fashion (5). One protamine molecule is bound to each turn [∼11 base pairs (bp)] of DNA (56), and adjacent arginines in the anchoring domains interlock both strands of the helix. Arginine-rich sequences are also present in the proteins that package DNA in several viruses (7), but the viral proteins contain fewer anchoring domains per molecule.

To examine toroid formation under conditions that preclude aggregation and precipitation and allow a detailed analysis of kinetics, we used an optical trap to isolate individual DNA molecules and fluorescence microscopy to monitor the formation of toroids in real time as they are induced by protamine (or Arg6) binding.
λ-phage DNA concatemers (20 to 80 μm long) were tagged at one end with a biotinylated oligonucleotide attached to a 1-μm streptavidin-coated polystyrene bead and stained with the intercalating dye YOYO-1 (14). These molecules were introduced through one port of a “bifurcated flow cell” (Fig. 1A) and the condensing agent protamine (or Arg6) through another port so that the two solutions flowed side by side with minimal mixing. An infrared optical trap (15) (Fig. 1B) was used to move an individual DNA molecule, via its attached bead, from the sample (DNA) side to the condensing agent (protein) side of the flow cell. The molecule was extended by the force of the flowing buffer, and its entire length became visible because of the fluorescence of the intercalated dye. Toroid formation (condensation) (Fig. 1C) was monitored in real time by measuring the change in length of the molecule as a function of time after moving it into the buffer stream containing protein (16).


Figure 1
(A) Top view of the flow cell (25) showing how the DNA molecules (attached to beads) and protamine enter the cell and form an interface (−−−) with little or no mixing. (B) Side view of the system showing the optical trap (orange) holding a bead attached to a single DNA molecule. The cell is illuminated from beneath by a 1-mW argon-ion laser, λ = 488 nm, to excite the YOYO-1 dye bound to the DNA. (C) Model of a DNA molecule condensing in protamine (protamine molecules not shown). The upper molecule shows the initiation of coiling and the lower molecule depicts the progression of coiling to form the torus.
Activation mechanism of nuclear chaperone nucleoplasm: role of core domain by Banuelos
Nucleoplasmin (NP) is a nuclear protein. It mediate nucleosome assembly by removing basic proteins from sperm chromatin and exchanging them with histones. This is modulated by NP phosphorylation at multiple sites.  After fertilisation NP may remodel highly condensed paternal chromatin by replacing sperm basic proteins with histones.

NP is pentameric. Each monomer is 200 aa long. It has 2 domains: a core, which forms a stable ringlike pentamer, and a tail that holds a polyQ tract and a NLS.

Core domain is responsible for NP oligomerisation. It gives the protein extreme stability. X-ray structures show that the monomers fold in an 8-stranded β-barrel.  Conserved apolar residues form a continuous ring. They confer thermostability.

Tail domain has a region rich in negatively charged residues (Asp and Glu), called a polyQ tract an a nuclear localisation signal.

Activity of NP is modulated by phosphorylation at multiple residues.

Higher phosphorylation of NP from eggs correlates with higher sperm decondensation activity. On hyperphosphorylation  NP increases its affinity for sperm-specific basic proteins and maintians its ability to bind histones. It was shown that core domain of hyperphosphorylated NP binds basic proteins and decondenses chromatin using fluorescence microscopy.

Recombinant core can be activated through mutations that mimic phosphorylation.

The Crystal structure of nucleoplasmin-core: implications for histone binding and nucleosome assembly by Dutta
Np binds H2A-H2B dimer. N1 chaperones H3-H4 dimer. Xenopus oocyte nuclei contains enough histones to assmble chromatin in 10, 000 cells. Np and N 1 are most abundant nonhistone proteins in these nuclei. Np and N1 may form histone storage complexes that are mobilised during fertilisation and early embryogenesis to assmble nucleosomes.

Np core monomer forms an 8-stranded β-barrel with jellyroll topology.
Figure 2. Structure of the Np Monomer and Pentamer(A) A face view of the Np-core pentamer is shown as a ribbon diagram, viewed from the pentamer-pentamer interface. A single monomer is highlighted in cranberry. Note the prominent β hairpin that extends radially from the subunit-subunit interface.(B) An Np-core monomer is shown at higher magnification, and each β strand is labeled.(C) The Np-core monomer in (B) was rotated ∼90° away from the reader to present a side view, as seen from outside the pentamer. Positions of the conserved A1 tract (red dots), β hairpin, AKDE, and GSGP loops are indicated.(D) The Np-core monomer is shown in a similar orientation as in (C), except that it is viewed from the central 5-fold axis

Acidic tracts mobility may facilitate interaction with basic histones.
Figure 4. Structure of the Np Decamer within Crystals(A) The Np decamer is shown, as viewed along the 5-fold axis. The two pentamers are offset by ∼15°, and individual monomers within the top and bottom pentamers are labeled A′–E′ and A–E, respectively.(B) The Np decamer has an hourglass shape when viewed from the side, along a 2-fold axis. Localized negative charges are present near the pentamer-pentamer interface. In addition, a pair of opposing Lys57 residues are marked with an asterisk (see [D] and [F]).(C) A surface view of the Np pentamer is shown from within the pentamer-pentamer interface. Alternating positive (blue) and negative (red) charges that arise from Lys82 and Asp58 form an inner ring (black dots with white circles). At higher radius, Lys57 and Glu59 from the AKDE motif are revealed (see labeling key). The β hairpins form a distinctive projection.(D) A side view is shown of two Np monomers that face each other across the pentamer-pentamer interface. Lys82 and Asp58 form a pair of water-mediated, charge-based interactions that span the interface. In addition, Lys57 and Glu59 form an intramonomer salt bridge (see [F]).(E) A close-up is shown of a pair of charge-based interactions formed by Lys82, Asp58, and intervening waters.(F) A close-up is shown of an intramonomer salt bridge formed by Lys57, Glu59, and a bound water molecule. This salt bridge may neutralize potentially destabilizing interactions between opposing Lys57 residues (see [B] and [D])
At pentamer-pentamer interface, the conserved GSGP motif is stabilised by a water molecule.

Histone binding by Np has a 1:1 stoichiometry, according to Superose column. rNp binds specficially H2a/H2B dimers.

It is noted that no octamers were formed in control experiments without Np.






Figure 6. A Model of Np Histone Complexes(A) A model is shown of the proposed Np histone complexes. The model is viewed from the top along the 5-fold axis. The inner ring (R1) corresponds to a decamer-dimer complex, while the outer ring (R2) corresponds to a decamer-octamer complex. Pentamers are colored blue and cranberry. The histone dimers are colored in yellow and the tetramers in purple.(B) Visual docking reveals a surface complimentarity between the histone octamer and the Np decamer. The concave lateral surface of the Np decamer is created by two opposing β hairpins. Hypothetical positions of the A1 and A2 tracts are indicated by dotted lines. The flexible N termini of H2A/H2B have been omitted for clarity. The Np decamer-octamer complex is viewed from the side

A Np-core decamer formed in crystal. A network of H bonded waters stitched together opposing pentamers. Stability is provided by ten charge-based interactions between Asp58 from AKDE motif and Lys82 mediated by water molecules. An intramonomer salt bridge between Lys57, Glu59 and a bound water helps to neutralise destabilising interactions between Lys 57 residues in opposing pentamers.

Decamer assembly with an offset of 15° may help to define binding site for a pair of H2A-H2B dimers.

Decamer may be stabilised by histone binding, as pairs of H2A/H2B dimers would form a bridge between opposing pentamers. Waters in interface may be expelled when histones are bound, bringing Np pentamers closer together.

Dutta suggests that Dp decamers are formed in presence of 4 core histones and that H2A/H2B dimers bind Np directly. Decamer formation means that a small concentration of negative charge is on lateral surface of decamer near pentamer-pentamer interface.

A1 tracts each contain 5 residues that are disordered in model. These will generate an extensive, negatively charged region on distal and lateral faces of decmaer.

A2 and A3 tracts in decamer will contribute more negative residues to decamer.

These flexible tracts may form an acidic cloud that envelops molecule. With A1 may affect histone binding and release.

Np-core is sufficient to mediate assembly of histone octamers. A2 and A3 tracts are not needed for assembly. They may stabilise Np histone complexes in vivo.

The proposed model for Np histone complex formation. Np decamer forms a central hub provides docking sites for 5 pairs of histone dimers on its lateral surface. Addition of 5 histone tetramers completes octamer assembly. Chaperone may preassemble dimers and octamers in a geometry conducive to sperm chromatin decondensation or nucleosome assembly.

Docking suggests why Np cannot form a direct complex with H3/H4 tetramer. Model suggests that flexible, positively-charged N termini of H2A/H2B dimers will become reorganised on binding to Np decamer.   This minimises steric clashes and maximises charge complimentarity. Large A2 acidic tracts may bind positively charged histone residues that form ramp on which DNA binds.

DNA binding surface on histone dimer may be blocked in putative chaperone complex. DNA may play active role in displacing histones from Np.



Figure 7. Histone Storage and Nucleosome AssemblyNucleoplasmin pentamers (1) may form decamers (2) in nuclei that are stabilized by the lateral binding of H2A/H2B dimers, to form decamer-dimer complexes (3). In the presence of N1-tetramer complex (4), the Np decamer-dimer may participate in the assembly of nucleosomes. Alternatively, H3/H4 tetramers (either free or complexed with N1, shown here in green) may reversibly bind to Np decamer-dimer complexes to form Np-HOC assemblies (5) and free N1 (6). The Np decamer-dimer complexes may function in nucleosome assembly and in the exchange reactions that promote decondensation of sperm chromatin. The decamer-octamer complexes may function in histone storage or may participate in nucleosome assembly


Sequential model of nucleosome assembly suggets that N1 delivers a H3/H4 tetramer to DNA. followed by Np-mediated addition of H2A/H2B dimers.

In first step, a tetrasome is assembled by N1-mediated addition of a tetramer that loosely organises 2 DNA loops. A pair of H2A/H2B dimers is delivered to tetrasome by Np.

Significance of Np phosporylation
During nucleosome assembly, chaperons eg Np must transfer bound histones onto DNA. Hyperphosphorylation of Np correlates with enhanced H2A/H2B dimer exchange during decondensation of sperm chromatin.

Tetrasomes or DNA may bind exposd histone surfaces in chaperone complexes.  Release of DNA-histone complexes may be facilitated by local charge repulsion between DNA and hyperphophorylated chaperone.




Promoter recognition

The RNA polymerase II core promoter - the gateway to transcription by Juven-Gorshen, Hsu, Theisen, and Kadonaga and RNA polymerase II core promoter by Smale and Kadonaga
Focused versus dispersed core promoters
In focused core promoters there is either single transcription start site or a distinct cluster of short sites in a short region of several nucleotides. Most uk core promoters appear to be focused core promoters. In vertebrates only 1/3 core promoters are focused; the majority of genes contain dispersed core promoters.  A number of transcription start sites are distributed over a broad region that might range from 50-100 nts.

TATA box, BRE, Inr, MTE, DPE, and DCE are found in focused core promoters. These core promoter elements are not universal. Each is present in only a subset of core promoters.  TATA box and BRE are most ancient of core promoter motifs.

TATA box, BRE and cognate protein factors, TBP, TFIIB are conserved from archaea to humans. TATA  box is in plant promoters.

Dispersed core promoters are found in CpG islands in vertebrates and lack TATA, DEP and MTE.

Focused core promotesr are more ancient and used in a broader range of organisms than dispersed promoters.

Initiator (Inr)
Inr encompases the transcription start site.  Inr consensus is YYANWYY in humans and TCAKTY ni Drosophila. A in the middle is often the +1 start site in focused core promoters.  It is the most common sequence motif in focused core promoters. Inr is a recognition site for TFIID binding.  Sequence specificity of TFIID binding to Inr region of core promoter is identifcal to Inrs consensus sequence.

It can function independently of TATA box in an analysis of lymphocyte-specific terminal transferase promoter. Transcription from this promoter initiates at a single start site. But region between -25 and -30 is G/C-rich and is unimportant for promoter activity. Mutant analysis showed that sequence between -3 and +5 is necessary and sufficient for accurate transcription.

Tnr functions synergestically with TATA box when separated by 25 bp. When an Inr is inserted into a synthetic promoter downstream of 6 binding sites for transcription factor Sp1 (absence of TATA box), Inr supports high levels of transcription that initiate at a spec start site within Inr. When Inr is inserted at a diff location relative to Sp1 sites, RNA synthesis begins at nt dictated by Inr. When Inr is absent, transcription begins at heterogenous start sites at much lower frequencies.

Mammalian Inr consensus is suggested to be YR (R is +1 start site). Drosophila core promoters have a stricter consensus sequence of TCAGTY. Interestingly, transcription does not need to being at +1 nutcleotide for Inr to function.

TATA and Inr act independently when separated by more than 30 bp. When separated by 15 or 20 bp synergy is retained, but ocation of start site is dictated by location of TATA rather than that of Inr.

TFIID complex is essential for Inr activity. TBP is insufficient.  DNase I footprinting studies with purified human TFIID and simple synthetic promoters containing only TATA and Inr elements showed weak TFIID interactions at Inr.  When Inr was disrupted by point mutation, a much weaker footprint was detected that was confined to TATA box. Disruptin TATA box eliminated the entire footprint except an enhanced DNse I cleavage site at Inr. Cooperative binding of TFIID was disrupted by increasing or decrasing TATA- Inr spacing by 5 bp.

Inr is essential for stable TFIID binding.

Mammalian TFs evolved to function with a broader range of Inr sequences.

TATA box
TATA box has a consensus of TATAWAAR, where upstream T is most commonly at -31 or -30 relative to A+1 (pr G+1) in Inr. Mutations in TATA box reduced activity of cellular and viral promoters.  TATA box is uncommon in vertebrates, only 1/3 or less of vertebrate core promoters are focused and only a fraction of focused core promoters contain a TATA box.

However, in S. cerevisiae, TATA boxes was 40-120 bp from start site.

TATA box is recognised and bound by TBP, a subunit of TFIID complex in eukaryotes. TBF-associated factors, TAF, are components of TFIID complex.  TAFs contribute to core promoter recognition, kinase activity  ubiquitin activating and conjugating activity, coactivator functions.

DNA-binding region of TBP folds into a saddle structure. Saddle consists of 2 quasi-symmetrical domains. N-terminal domain contacts 3' half of a consensus TATA box. C-terminal domain contacts 5' half. The concave surface of the saddle consists of 10 β-sheets. 8 of 10 sheets contact minor groove of duplex DNA. TBP bindings to minor groove relies on hydrophobic interactions. TBP induces kinks in DNA at 5' and 3' ends of TATA box. It partially unwinds duplex due to insertion of phenylalanine residues. Distorted DNA structure is restricted to the region that is directly contacted by TBP.

Start site selection in TATA-containing promoters.  Mechanism that determines distance from TATA box to transcription start site has been studied.  By swapping basal factors purified from S. cerevisiae and S. pombe, TFIIB and RNAPII were found to dictate this distance. When S. cerevisiae TFIIB and RNAPII were combined with other S. pombe basal factors, transcription initiated 40-120 bp downstream of TATA box. When S. pombe TFIIB and RNAPII were combined with other S. cerevisiae basal factors, transcription initiated 25-30bp downstream of TATA.  TFIIB was implicated as mutations in its N-terminal charged cluster domain shifts the location of start sites by a few nts in yeast and mammalian promoters.

In TBP-DNA cocrystals, TBP binds in a polar manner to asymmetrical TATA sequences, TATAAAAG and TATAAA.

BRE 
BRE (TFIIB recognition element) is a TFIIB-binding sequence. TFIIB can bind upstream or downstream of TATA box at the BREu (upstream BRE, immediately upstream) or BREd (downstream BRE).  BREu consensus is SSRGCC. GREd is immediately downstream of TATA box. It has a consensus of RTDKKKK. BREu and BREd can act in either  a positive of negative manner.

TFIIB interacts with DNA in a sequence specific manner.  Archaeal TBP and TFB (archaeal homolog of TFIIB) bound cooperatively to T6 promoter when TATA box and upstream element were present. In presence of TFB, strong preference for purines 3 and 6 bp upstream of TATA, with weaker nt preferences at other positions. Recognition is BRE is mediated by a HTH motif at C-terminus of TFIIB. This motif is missing in yeast and plants. BRE may not contribute to gene regulation in these.

BRE represses transcription in vitro with crude nuclear extracts and in vivo in transfection assays. Repression of TFIIb-BRE was relieved when transcriptional activators bound distal sites. Tramscriptional activation increased.

CpG islands
CpG dinucleotide, a DNA methyltransferase substrate is under-represented in genomes of many vertebrates, 5-methylcytosine deaminates to form thymine, which is not repaired by DNA repair enzymes. 0.5-2kbp stretches of DNA possess a high density of CpG islands.  Human genome has 29 000 islands.

In mammals CpG islands are associated with half of promoters for protein-coding genes.

They lack consensus or near-consensus TATa boxes, DPE elements or Inbr. Have multiple transcription start sites that span 100 bp or more.  Mutations in vicinity of start site can cause use of alternative start sites but promoter strength is often unaffected.

Has multiple binding sites of Sp1. Transcription start sites are 40-80bp downstream of Sp1 sites. Sp1 may direct basal machinery to form a preinitiation complex.


DPE
DPE (downstream core promoter elements) is a downstream TFIID recognition sequence important for basal transcription activity. IT is required for binding of purified TFIID to a subset of TATA-less promoters. TFIID binds cooperatively to DPE and Inr.

DPE is conserved from Drosophila to humans. It is +28 to +33 relative to A+1 in Inr. DPE consensus is RGWYVT in Drosophila.

DPE functions cooperatively with Inr. Spacing between Inr and DPE is critical for optimal transcription. Single nt increase of decrease in spacing reduces TFIID binding and transcriptional activity.

Photocrosslinking studies show DPE is proximity to TFIID subunits TAF6 (TAFII60) and TAF9 (TAFII40) which contain histone fold motifs and are related to histones Hr and H3. It is possible that TAF6-TAF9 subunits of TFIID interfact with DPE similar to binding of histones H3-H4 to DNA in nucleosomes.

Similarities and differences between DPE and TATA box. Both are recognition sites for binding of TFIID. On the other hand, TATA box but not DPE can function independently of Inr. If TATA-dependent promoter is inactivated by mutation of TATA motif, core promoter activity can be restored by adding DEP at its downstream position.

An activity stimulates DPE-dependent transcription and repressed TATA-dependent transcription  This acivity is mediated by NC2/Dr1-Drap1, a repressor of TATA-dependent transcription.

MTE
MTE(motif ten element) has a consensus of CSARCSSAAC from +18 to +27 relative to A+1 in Inr in Drosophila.  Mutations of nt from +18 to +22 can abolish MTE actibity in vitro and in  cultured cells.

MTE functions cooperatively with Inr with strict Inr-MTE spacing requirement.  MTE addition compesnates for loss of basal transcription activity that occurs on TATA box or DPE mutation.

MTE shows synergy with TATA and DPE.  Synergy between MTE and other core promoter motifs inspired design of a Super Core Promoter.

DCE and XCPE1 motifs
DCE (downstream core element) has been found in human beta-globin promoter and characterised in adenovirus major late promoter. Occurs often with TATA box.  Appears to be distrinct from DPE.

DCE consists of 3 subelements: S1, CTTC from +6 to +11; SII, CTGT from +16 to +21; and SIII, AGC from +30 to +34.

XCPE1 (X core promoter element 1) motif is from -8 to +2 relative to +1 start site. It is in 1% of human core promoters, most of which are TATA-less.  Shows little activity by itself.  Acts with sequence-specific activators.

TFIIB
Binding of TBP to TATA box nucleates formation of pre-initiation complex (PIC) consisting of RNAPII, TFIIA, B, D, E, F amd H.  Tsai and Sigler (2000) crystallised a human TFIIBc-TBPc complex bound to an idealised and extended adenovirus major late promoter (MLP).

TFIIB makes spec base contacts in major groove of BRE and minor groove immediately downstream of TATA box. Upstream interactions are mediated by HTH motif. There are 5 ternary complexes linked.

Side chains of Lys 189 and Arg 193 of TFIIBc H bond with phosphate backbone at upstream end of TATA box and immediately downstream if ut, These bridging polar interactions between TFIIBc and promoter require deformation of TATa box. May stabilise ternary complex and reinforce TBP-induced deformation of DNA.

Human TFIIBc interacts with major groove upstream of TATA box through HTH motif. In this structure, Val283 makes vdW contacts with C5-C6 edge of C(-34'), 3 bp upstream of TATA box, in all 5 copies of ternary complex. A G:C bp 3 bp upstream of TATA box is conserved.  Mutagenesis indicates Cal 283 confers specificity for a G:C bp at position -34 of BRE. (Langrange 1998).

Side chain of mutational sensitive ARg 286 makes water-mediated contacts with G(-38) in 4 copies, G (-37) in 2 copies, and vdW with phosphoribose backbone of G(-38) in all 5 copies of complex.

A Role for TBP dimerisation in preventing unregulated gene expression by Jackson-Fisher
TBP dimerisation
TBP negatively autoregulates accessibility to promoter DNA in yeast through dimerisation. In absence of DNA, carboxy-terminal domain of TBP crystallised as a dimer, in which DNA-binding and dimerisation surfaces overlapped.

Jackson-Fisher et al (1999) applied site-directed mutagenesis at interfacial residues N69, V71 and V161. 2 alleles targeted at each site: Bulky charged substitution (Arg, expected to be severe) and a smaller potentially less severe mutation (Glu or Ser). A pulldown assay quantitated the binding of mutant or wild-type TBPs to immobilised GST-TBPs, which contains a wt yeast TBP C-terminal domain. Wt TBP retained on resin. Mutants was retained over a range of efficiencies. H bonds and hydrophobic interactions stabilised dimer structure.



Figure 1. Mutations in the Crystallographic TBP Dimer Interface Affect Dimerization(A and B) Space-filling model of the core yeast TBP/TATA complex and TBP dimers, respectively ( [7] and [24]).(C) Ribbon diagram of TBP showing the location of V161, N69, and V71 in a space-filling representation.(D) A ball and stick representation of the crystallographic dimer interface between one monomer and amino acids V161, N69, and V71 on the other monomer. A dashed line indicates an intermolecular hydrogen bond.(E) GST-TBP(181C) (10 nM) was immobilized on glutathione agarose and incubated with the indicated yeast TBP mutants (100 nM). The resins were washed several times, and the amount of bound TBP was determined by Western blotting. Assuming a 1:1 stoichiometry in the “heterodimer,” a maximum of 10 nM TBP is expected to be retained. For wild-type TBP, retention of 10%–20% of this level was observed, which is expected given the competition from homodimerization and the extensive washing steps involved.
TBP dimer instability correlates with activator-dependent transcription
If dimer association is rate limiting for gene expression, then dimerisation-defective mutants should stimulate expression of a basal promoter.  "Basal" is level of trasncription in the absence of activator function. Residual levels of monomer TBP would cause low level of basal transcription with wt TBP. This model predicts that TBP over-expression would largely increase dimer pool and marginally increase monomer pool.

If dimer dissociation is rate limiting without activator function, and activators accelerate rate-limiting steps, then activated transcription may not be positively affected by dimer instability.  Activators might discoate dimers. An activator might facilitate dimer dissociation through interactions with TBP, TAFs, chromatin remodelling complexes or components of Pol II holoenzyme.

At repressed promoters, dimer dissocation may not be rate limiting. Disscoation of a repressor might be primary step in initiating transcription complex assembly.

None of mutants could support cell viability at 22, 30 or 37C. Shuffling in wt TBP supported cell growth.

In vivo assay for basal transcription was adopted. Uses truncated ADH1 promoter fused to a lacZ promoter gene. Gal4-binding sites are upstream of this promoter. To maintain basal level expression, where Gal4 is repressed, cells were grown in glucose.

For activated trasncription, a fusion of entire PGK promoter to lacZ was used.  Reporter constructs were on high copy plasmids.  Test TBP was terminally HA-tagged and expressed from a low-copy plasmid using TBP promoter (low expression) or PGK promoter (high expression). TBP expressed under control of promoters is targeted to nucleus.

Correlation between decreased dimer stability in vitro and elevated basal levels of β-galactosidase activity in vivo at low and high levels of test TBP.  This trend was not apparent with activated transcription.








Table 1. In Vivo Transcription and TBP Levels in the Presence of TBP Mutants
Promoter of lacZ Reporter
DimerTBPBasal (low)Basal (high)Activated (high)Chromosomal Basal (high)Relative TBP levels (low)(high)
Null/endogenous1115000.0020/10/1
100WT1115000.002.3714
110V71E35310000.013.405
39V161E269000.007.101.9
12N69S1020*ND0.014.131.3*
8V71R17819000.023.10.68
1V161R2515010000.026.05.35
0.5N69R541306000.048.04.25
Dimerization data (column 1) represent relative binding to GST-TBP(181C) as determined in Figure 1E and are an average of six sets of data. See Figure 5 for standard errors. β-galactosidase activity (columns 3–6) is relative to wild-type test TBP in column 3 (1.0 = 3 Miller units using the high-sensitivity substrate CPRG) and represents an average of at least three independent determinations. All data were obtained in the linear range of the assay. The null allele expresses only the first 81 amino acids of TBP. “Test” HA-tagged TBP levels were determined by quantitative Western blotting with TBP antibodies as described in Figure 5B. TBP levels are relative to the endogenous (untagged) TBP (1 = 17,000 ± 2,000 molecules per cell). The standard error for all data is presented graphically in Figure 5E. Expression of the TBPEBmutants did not cause a decrease in the expression of TAFII145 (data not shown). “Low” in columns 3 and 7 indicates that the test TBP was driven by the TBP promoter; “high” in columns 4–6 and 8 indicates that the test TBP was driven by the PGK promoter.ND, not determined.
*
Strains harboring TBP(N69S) expressed at high levels grew very slowly.

Dimer dissociation is not rate limiting under these conditions.




Figure 2. A Model for How TBP Dimers Might Prevent Activator-Independent Transcription(A) Dimerization prevents TBP from binding to DNA. The little that does escape might give rise to low levels of basal transcription, occurring in the absence of a functional activator.(B) TBP dimer dissociation and DNA binding are facilitated either directly or indirectly by a functional promoter-bound activator, giving rise to high levels of transcription.(C) Dimer dissociation might not be rate limiting at repressed promoters

TBP dimer stability correlates with steady-state levels of TBP in vivo
To investigate if increase in basal transcription is caused by greatly increased expression of TBP mutants, the steady-state level of TBP was examined under the control of either TBP (low) or PGK (high) promoter. N-terminal tags on test TBPs cause slower mobilities in SDS PAGE, distinguishing it from endogenous TBP.

Under TBP promoter, expression level of mutants were below that of uniformly expressed endogenous wt TBP.  Highest expression levels were wt TBP and V71E, expressed at 40% level of endogenous TBP. Mutants (V161R and N69R) with highest β-galactosidase activity were present at lowesr levels. Elevated basal lacZ expression cannot be explained by increased expression of TBPEB mutants.

When test TBPs were expressed at high levels under PGK promoter, greater fluctuation in their steady-state levels whereas endogenous TBP levels remained uniform.  Wt TBP was expressed 14-fold higher levels than endogenous wt TBP. V161R and N69R were expressed at less than half level of endogenous TBP.  Strong correlation between dimer stability and in vivo levels of TBP. This suggests that TBP dimerisation stabilises TBP against degradation.

Strong correlation between dimer instability and specific activity of TBP. Higher dimer stability, lower specific activity of TBP. TBP dimerisation may be barrier to expression of uninduced genes.

Competition by TATA DNA
When yeast TBP was preincubated with increasing concentrations of TATA DNA, mutant TATA, or GC-rich DNA, only TATA efficiently inhibited crosslinking of TBP into dimers. When pure TATA and TBP are equimolar and at equilibrium, TBP prefers binding TATA to binding to itself 

General role for TBP dimerisation in preventing unregulated gene expression

TBP dimers might be general TBP status before promoter binding. Dimers block promoter binding and expression of uninduced genes. TBP dimersiation occludes its TFIIB-binding surface and may prevent TFIIB from associating with TBP until TBP is bound to DNA.

TBP Antagonists
Crystal structure of Negative Cofactor 2 recognising the TBP-DNA transcription complex by Kamada (2001)
Positive and negative cofactors interact with general transcription machinery. NC2 is the best characterised negative cofactor.  It inhibited transcription initiation by RNAPII by direct interactions with TBP-DNA complex in human tumour cells.

NC2 consists of 2 subunits, α: 22kDa and β: 20kDa.  They are conserved among eukaryotes.  AA sequences of α and β are related to histones H2A and H2B. They heterodimerise through N-terminal core histone-like regions.  In vitro, NC2 recognises TBP-DNA complex and inhibits incorporation of TFIIB and positive cofactor TFIIA, blocking PIC assembly.

NC2 was thought to be general negative regulator of transcription initiation.  NC2 is suggested to establish appropriate balance between positive and negative cofactors.  Inactivation of NC2 α in yeast shows limited positive effects. It has a positive role in transcription initiation controlled by DPE in certain D. melanogaster gene promoters.  It has been associated with PICs assembled on actively transcribing promoters in yeast.

Kamada determined X-ray structure of human NC2 recognising a preformed binary complex of human TBP bound to a TATA.  N-terminal portions of NC2α and β resemble H2A and H2B.

NC2α and β  form a heterodimer that binds underside of TBP-DNA. This allows C-terminus of NC2β to spec contact upper surface of saddle and block TFIIB entry. NC2 heterodimer acts as a molecular clamp, gripping upper and lower surfaces of TBP-DNA complex. NC2 and TFIIB cannot bind at the same time.

NC2 heterodimer binds major groove face of TATA element beneath molecular saddle. It makes protein-protein contacts with N-terminal stirrup of TBP.  Remainder of NCβ traverses up and over the saddle.

α helix H4 interacts with DNA backbone  α helix H5 interacts with upper surface of TBP C-terminal domain.  N-terminal half of F4 is stabilised by interactions with NC2α and within NC2β.  It is likely that H5 and remainded of H4 are random coil in absence of TBP-DNA.

NC2 and H2A/B show similar DNA binding properties.  The calculated electrostatic potential of upper surface of histonelike portion of NC2 heterodimre is highly basic. Positive electrostatic potential allows favourable polar interactions with negatively charged nucleic acid backbones bound beneath molecular saddle. NC2α makes salt bridges and water-mediated H bonds with backbone of top DNA strand upstream of TATA and with backbone of bottom DNA strand.  NC2β interacts with backbone of top DNA strand downstream of TATA element.  In TATA box, Lys 29 of NC2α projects into minor groove.

2 C-terminal α helices of NC2β support TBP and TFIID recognition.  Remained of NC2 molecular clamp consists of C-terminal α helices H4 and H5. Polar penultimate helix, H4 makes backbone contacts with both DNA strands on 3' side of TATA.  C-terminal helix, H5 lies on upper surface of TBP.

Residues 115-133 of H5 of NC2β participate in salt bridges, H bonds, and vdW contacts with TBP.

Structure of ternary complex makes it likely that C-terminal half of H4 and all of H5 are random coils in solution. It is suggested that NC2 binding occurs in 2 stages.Core histonelike heterodimer interacts with DNA on underside of TBP-DNA via electrostatic interactions. Structural consequences of target induced disorder-to-order transitions in C terminus of NC2β dictates directional NC2 binding.

It is presumed that Lys95, Arg 101 and Lys 102 contribute to this process by contacting backbones of both DNA strands downstream of TATA,. facilitating random coil to helix transition, giving rise to H4.

Transcriptional regulation by NC2. TBP-induced bend in core promoter is unaffected by NC2 binding. It seems to be recognised by core histone-like portion of NC2 heterodimer.  NC2 recognises TBP-TaTA complex, making protein-protien contacts with both TBP domains.  Steric clash between H4 of NC2 β and TFIIB.  NC2 blocks TFIA binding by steric hindrance. NC2 represses transcription initiation by acting as a molecular clamp that recognises TBP-DNA and physically blocks binding of TFIIB and TFIIA.

H5 supports molecular recognition of upper surface of TBP. H4 steric interference inhibits TFIIB binding and transcription initiation.

Transcriptional activators could bind certain promoters and target H4 and/or H5 of NC2 β. Disrupting interactions between NC2 and TBP-TATA could allow TFIIB to replace NC2 heterodimer.  This selectively relives repression of only those genes  Transcriptional activators and/or positive cofactors could bind to and alter conformations of  H4 and/or H5 of NC2 β. This allows productive TFIIB binding to an NC2-TBP-promoter complex. Histone-like portion of NC2 may remain bound to core promoter.

Kadonaga showed that NC2 can inhibit assembly of functional PIC at TATA and activate DPE-dependent transcription on separate DNA templates.  Activating transcription from a DPE-dependent promoter requires only H2A/H2B-like heterodimeric portion of NC2.

Albert 2010 Genome Biology
Albert et al (2010) did a genome-wide analysis on promoter association of human TFIIB and NC2 and correlated with gene expression and core promoter architecture.  TFIIB/NC2 ratio increased towards most highly expressed genes.  In 81% of TFIIB-dominated genes but in only 38% of NC2-dominated genes, at least one core promoter motif was present. TATA was most strongly enriched motif.  TFIIB strongly selects for TATA.

NC2 is more frequent on genes with multiple start sites lacking defined core promoter elements.

TFIIB/NC2 ratios are influenced by activators and core promoter elements.
Transcription complexes were assembled on a Gal4-responsive heterologous promoter template with wt and mutant TATA box in presence and absence of activator Gal4-VP!6. Activator enhanced TFIIB binding. NC2 did not respond to activator. Positive activator effect on TFIIB was stronger for mutant TATA than wt.

PIC (or TBP-TFIIB) association correlates with TATA or is independent of core elements altogether. NC2 association is largely independent of underlying core promoter structure.

NC2 occupancy shows a positive correlation of binding with TATA presence. It becomes negative relative to competing TFIIB.  TFIIB/NC2 ratios increase in strongly expressed 5% of B cell genes. Indicates negative role for NC2 at strongly expressed genes carrying intact core promoters.

TATA is positively correlated with TFIIB binding.  Genes with high TFIIB/NC2 ratio often carry G/C-rich regions.

High TFIIB/NC2 ratios select for promoters with focused start sites and conserved core elements. High NC2/TFIIB ratios correlate to multiple start sites promoters lacking defined core elements.

The TATA box regulates TBP dynamics in vivo by Tora
Tora proposes that TATA box role in RNAPII transcription assists rapid TBP dissociation from a subset of highly regulated PolII promoters. DNA bending on TBP binding coul release TBP rapidly from TATA by regulators. Eg NC2 and ATPases BTAF1 and Mot1p.

Regulation of TBP activity
NC2 binding to TBP-TATA blocks incorporation of basal trascription factors TFIIA and TFIIB intoPIC.  NC2 association induces structural changes in TBP-TATA. This allows DNA sliding and recognition of nonTATA sequences by TBP.

Mot1p in yeast cells is an ATP-dependent inhibitor of TBP-TATA complex formation.  MOT1 regulates pol II transcription posiitvely and negatively.  Mot1p-TBP has a high affinity for DNA but relaxed specificity for TATA.  NC2-TBP lacks prference for TATA sequences.  Mot1p and Nc2 collaborate to regulate TBP function in pol II transcription.  (See 30 and 39).

TATA plays a role in rapid TBP dissociation in vivo
DNA bending is suggested to play a role in functional distinctio of TBP between TATA-containing and TATA-less promoters. It is assumed that in vivo binding of TATA box by TBP or TFIID creates a bent DNA conformation.  Mot1p and Nc2 action would release strained conformation. This assists in rapid dissociation of TBP from TATA.

TATA would contribute to rapid dissociation of TBP and possibly PIC.  Single TATA box mutations reduce TBP-induced bending.  These promoters may not release TBP as rapidly as TATA=containing promoters on action of Mot1p and NC2.


Figure 2. Bending of the TATA-box acts as a ‘spring’ for rapid release of TBP. (a) Binding and rapid release of TBP from TATA-box containing promoters (i). Upon binding to TATA the DNA adopts a bent conformation (ii). NC2 approaches the TBP–TATA complex from the underside and BTAF1 (Mot1p) from the top (iii). BTAF1 (Mot1p) and NC2 are directly involved in dissociation of the protein–DNA complex (indicated by dashed arrows). The bent conformation of the TATA box contributes to the rapid release of TBP (iv). (b) TATA-less promoters carrying broad and dispersed initiation sites. TBP can bind to different positions on these promoters, but for clarity, only a single TBP is indicated. TBP binding does not result in bending of promoter DNA (i, ii). Therefore, release of TBP by the combined action of BTAF1 (Mot1p) and NC2 is less rapid (iii, iv). It is important to stress that although the bent arrows emanating from the DNA indicate transcription start sites, they do not reflect transcriptional activity.

Non-optimal TATA elements exhibit diverse mechanistic consequences
Certain off-consensus TATA elements form poor binding sites for TBP. This interferes with complex formation with TFIIA and /or TFIIB. Recruitment step of TBP affected.

Other motifs (CATAAA and TATAAG) do not affect initial formation of TBP, TFIIA-TBP or TFIIB-TBP complexes. But unable to form stable TFIIA-TBP-DNA complex in vitro.

Single Cytosine of Guanine Replacements in TATAAA element diminish gene expression in vivo
Less induction of β-galactosidase for mutants when induced by Gal4.

Internal base substitutions in TATA sequence can disrupt TBP binding.
Each of the C and G series elements was tested for ability to form TBP-DNA complex.  Abilities to form TBP-DNA complexes varied for each.  Some were disrupted.

Higher Order complex formation also depends on sequence of core element.
TFIIA-TBP-DNA complex formation was decreased.  Similar trend for TFIIB-TBP-DNA complex formation.

TFIIA-TBP-DNA complex is destabilised on TATAAG element
Electrophoretic mobility shift assays measured reltaive binding and stability of TFIIA-TBP complex on TATAAA and ATAAG. TFII-TBP was stable on TATAAA. TFIIA-TBP on TATAAG showed great loss of complex.

TFIIB-TBP-DNA behaved similarly on both TATAAA and TATAAG. TBP binds TATAAA amd TATAAG in a manner that allows stable association of TFIIB-TBP higher order complex and difference in stability of TFIIA-TBP-DNA is specific to complex containing TFIIA.

Stability of TFIIB-TBP-DNA complex depends strongly on sequence of TATA element
Fusion of TBP and TFIIA results in an increase in expression from CATAAA and TATAAG in vivo.
Used yeast strains expressing TFIIa-TBP and TFIIB-TBP as fusion molecules. TFIIA-TBP fusion yeast strains containing CATAAA and TATAAG showed dramatic increase in activity to 30% of TATAAA activity. This increase is specific to TFIIA. No such increase in TFIIB-TBP fusion strain.

Unexpected roles for core promoter recognition factors in cell-type-specific transcription and gene regulation by Goodrich (2010)
TFIID subunits function in most RNAPII promoters in higher eukaryotes.

RFIID consists of TBP and 13 or 14 TAF subunits. It binds core promoter DNA through multiple subunits (TBP, TAF1, 2, 6, 9).

TBP subunit binds TATA bozes. Several TAFs bind promoter elements downstream of TATA box. TAF1 and TAF2 bind initiator element, which spans TSS.  TAF6 and TAF9 bind the downstream promoter element. TAF1 is in close proximity to downstream core leement when TFIID is bound to promoters.

Some TAF subunits are also targets of transcriptional activators. This allows TFIID to integrate signals from activators to core promoter.

Multiple subunits of the transcription factor IID (TFIID) complex bind core promoter elements12. TATA-box-binding protein (TBP) binds TATA boxes. TBP-associated factor 1 (TAF1) and TAF2 bind the initiator element (Inr). TAF6 and TAF9 bind the downstream promoter element (DPE).
Pathology of TBP mutations
Spinocerebellar atazia 17 (SCA17) and Huntington's disease-like 4 (HDL-4) by Stevanin
Autosomal dominant cerebellar ataxis are characterised by progressive cerebellar ataxia. Uncoordinated movements, unsteady gait, dysarthia.

15 genes and mutations have been identified. Translated (CAG)n/polyglutamine repeat expansions are responsible for disease caused by 7 of these genes, SCA 1-3, 6, 7, 17 and DRPLA.

SCA17/HDLA
A rare neurodegenerative disorder belonging to the polygluatminopathy group.  Can lead to death. Number of glutamines observed in pathological proteins varies from 21 to more than 400. Phenotype usually manifests above a repeat number between 35 and 40.

SCA17 was first reported in a complex neurological disorder with cerebellar ataxia. Patient carried 63 trinucleotide repeats in gene encoding TBP on chromosome 6q27.

Size and structure of normal repeat in TBP gene
N-terminus of TBP contains a long stretch of glutamines. Repeat is impure nd is encoded by 3 CAG stretches, interrupted by 1 to 3 CAA codons. Qstretch is polymorphic in normal populations. Normal range is between 25-42 residues.  Most alleles contain 32-39 repeats.

SCA17, a novel autosomal dominant cerebellar ataxia caused by an expanded polyQ in TBP by Nakamura
IC2 antibody screening was used to identify polyQ tract in lymphoblastoid cell lines.  Antibody recognises proteins of largely expanded polyQ tracts. A polyQ protein of 49 kDa was found in one proband.  Its MW is similar to TBP. Might be mutant TBP, as the monoclonal IC2 Ab was originally raised against TBP.

Using primer pairs flanking CAG/CAA repeat of TBP gene it was found that proband and his affected sibling had same repeat expansions. Western blot confirmed identity of 49kDa band using monoclonal anti-TBP antibody.

The CAA/CAG repeat numbers of TBP genes were determined in two affected siblings and their mother.  CAA/CAG repeat in healthy mother was 37/39. In affected siblings was heterozygous, 37/55 and 39/55.

Number of CAG/CAA repeats in TBP gene ranged from 29 to 42 in healthy control chromosomes. A TBP gene having a CAG/CAA number in excess of 43 is pathological.

Immunocytochemical study of post mortem brain tissues
Immunocytochemical examination of post mortem brain tissues from a patient who had expanded polQ repeats in his TBP. Shrinkage and moderate loss of small neurons with gliosis in caudate nucleus and putamen.

Immunocytochemical analysis was performed with anti-ubiquitin and anti-TBP antibodies. Showed neuronal intranuclear inclusion bodies. Most neuronal nuclei were diffusely stained with 1C2-Ab. None were stained in healthy control brains. Neuronal inclusion bodies may provide marker of disease process in many polyQ diseases.

Polyglutamine domain modulates the TBP-TFIIB interaction: implications for its normal function and neurodegeneration by Friedman
PolyQ regions are present in many eukaryotic TFs inc vertebrate TBPs. Length of polyQ varies among vertebrates species. It is higher in mammals. Friedman (2007) showed that expanded polyQ tract in TBP influences its dimerisation and interaction with TFIIB.

They generated transgenic SCA17 mice expressing mutant TBP. cDNAs encoded human TBP with polyQ tracts of different length. PolyQ-expanded TBPs were preferentially detected with polyQ-spec antibody 1C2.

Immunofluorescent staining showed that transfected TBPs localised mainly to nucleous.  TBPs with pathologic polyQ tracts formed nuclear aggregates.

Neurological phenotype and neuropathology of SCA17 mice
Abnormal posture in TBP-105Q mice. All SCA17 mice had reduced lifespan.

Neurodegeneration in transgenic SCA17 mice
Immunogold labelling showed that TBP-105 sformed nuclear inclusions in cerebellar granule neurons. Degenerating Purkinje cells and degenerating axons.

Alteration of TBP function caused by polyQ expansion.
TBP forms transcriptionally incompetent dimers to prevent unregulated gene expression. Length of polyQ domain was inversely related to relative amount of TBP dimers. Dimer-to-monomer ratio was lower for polyQ-expanded TBPs than normal TBP in transfected HEK293 cells and HT22 hippocampal cells.

They generated polyQ-expanded TBPs with an internal deletion spanning part of carboxy-terminal dimerisation domain. Double mutants could not dimerise but formed aggregates.

After cotransfection of TBP-31Q ant TBP-71G, a heterodimer was detected on western blots between 2 homodimers. Incrasing expression of TBP-71Q depleted level of TBP-31homodimer.  PolyQ formation can reduced formation of TBP homodimerisation in cis and in trans. Decreased dimerisation by mutant TBP may precede and facilitate its aggregation.

Enhanced interaction of polyQ-expanded TBP with TFIIB
Almost all TBP inclusions in TBP-105Q cerebellar sections contained TFIIB. GST-TFIIB pulled down more TBP-71Q than TBP-31Q. TFIIB pulled down more solutble TBP-105Q than TBP-13Q.

If mutant TBP abnormally binds TFIIB to affect gene transcription, TFIIB overexpression should reduce mutant TBP-induced toxicity  TFIIB was overexpressed in cultured cerebellar granule cells from TBP-105Q mice.  GFP transfection visualised integrity of neurites of individual neurons. More neurons expressing only GFP showed disrupted neurites than those cotransfected with TFIIB. TFIIB overexpression had protective effect.

Polyglutamine expansion reduces the association of TBP with DNA and induces DNA binding-independent neurotoxicity by Friedman
PolyQ expansion reduces in vitro binding of TBP to TATA box DNA.

N-terminal TBP fragments harbour the expanded polyQ tract. It lacks an intact C-terminal DNA-binding domain. They are present in transgenic SCA17 mouse brains.

polyQ-expanded TBP cannot bind DNA. It formed nuclear inclusions and caused severe neurological phenotype in transgenic mice.

PolyQ-expanded TBP is inhibitory to TATA_dependent transcription when it cannot bind DNA productively. It can induce neurotoxicity independent of DNA binding.

PolyQ expansion inhibits TBP binding to DNA
TBP N-terminus is normally antagonists to formation of stable TBP-TATA box complex. They compared in vitro interaction of normal and polyQ-expanded TBP with TATA box DNA. Recombinant TBPs containing either 31 or 71 Qs were incubated with a radiolabelled probe containing TATA box. Mutant TBP shifted less probe than normal TBP.

Truncated, polyQ-expanded TBP forms nuclear aggregates that sequester TFIIB
Double immunolabelling showed that truncated TBPs with expanded polyQ sequestered endogenous TFIIB in nuclear aggergates. Truncated polyQ-expanded TBP could still interact aberrantly with TFIIB.

Discussion
As expanded polyQ can inhibit association of TBP with TATA, it is possible that interaction of TBP C-terminus with DNA may be regulated by length of N-terminal polyQ domain.

It is expected that expanded polyQ suppresses gene expression. Full-length mutant TBP stimulates a TATA-dependent transcription reporter in culture cells. Reducing intrinsic DNA binding ability of TBP may not be sufficient to inhibit TBP-mediated transcription.

Truncated polyQ-expanded TBP which is unable to bind DNA is more toxic than full-lenth polQ TBP. Truncatedd TBP inhibited TATA-dependent transcriptional reporter activity. Level of inhibition increased with polyQ tract length.

Truncated polyQ TBP formed more nuclear aggregates than fulllength mutant TBP in transfected cells. As aggregates formed by truncated mutant TBP can also sequester TFIIB, it is likely that mutant TBP becomes more toxic when it cannot bind DNA productively but can still associate with other TFs. Continued interaction with TFIIB, which is strengthened by poly-expansion, might undermine assembly of PIC and inhibit transcription activity.

Friedman proposes a model for molecular pathogenesis of SCA17. When TBP is soluble, a long polyQ tract may antogonise dimerisation, a mech of negative autoregulation.  It may enhance interactions with other TFs eg TFIIB. This facilitates TBP recruitment to promoter DNA. Enahnced protein-protein interations compensate for reduced DNA binding ability of TBP. However, polyQ causes TBP to misofld and aggregate over time. TBP aggregates presumably cannot bind promoter DNA but can still interact with TFIIB and/or other TFs. This could negatively affect gene transcription by reducing promoter occupancy of affected proteins.
Model for mutant TBP-mediated transcriptional dysregulation in SCA17. Increased interaction of soluble polyQ-expanded TBP with TFIIB and/or other transcription factors may allow for recruitment of the former to certain TATA-containing promoters. Because this soluble form of TBP is not inherently defective, its recruitment can stimulate TATA-dependent transcriptional activity (upper panel). However, mutant TBP can no longer productively interact with promoter DNA after proteolytic processing and/or misfolding. TBP aggregates can sequester particular transcription factors, such as TFIIB, and thereby reduce their availability at certain promoters. Decreased transcriptional activity is the likely consequence of aberrant interaction of misfolded TBP with basal transcription factors or activator proteins (lower panel).


TBP, a polyglutamine tract containing protein, accumulates in Alzheimer's disease by Reid
AD is characterised by protein cleavage, protein misfolding and protein accumulation. Results in abnormal deposition of βA plaques and NFTs throughout brain. Major component ofNFT is filamentous aggregates of hyper-phosphorylation Tau protein.

Aberrant TBP associates with neurofibrillary structures in AD brain.  Polyclonal TBP antibody showed disease specific staining in cortical regions of MTG sections.

Insoluble βA was detected in all AD samples and at low levels in 3 of controls. Insoluble Tau was detected in disease samples.

TBP and Tau colocalise in most tangles studied.

Western analysis showed a rapidly migrating form of TBP at high levels in some disease brains. This may be truncated form of TBP. As the epitope of pTBP is at N-terminus, C-terminal cleavage might occur. Smaller product might however be caused by conformational change causing rapid migration through gel.

A splice variant of TBP encoding polyQ-containing N-terminal domain that accumulates in Alzheimer's disease by Reid
Search of human EST databases identified TBP splice variants with C-terminal truncated ORFs. A variant ORF encodes N-terminal domain of TBP including polyQ tract and terminates at an alternative stop codon conserved in most tetrapods. This splice variant encodes TBP fragment observed in disease brain.

Vertebrate TBP N-terminal domain is encoded by exons 2 and 3 including polyQ domain. Identified splice transcript encodes an ORF which skips exon3 3 splice acceptor site. Terminates at variant stop site in intron 3 immediately following splice acceptor site.

Fig. 1. Schematic diagram of the human full-length TBP and splice variant TBPv3 transcripts. The predominant TBP isoform is encoded by eight exons and is 339 aa in length, assuming that the polymorphic polyglutamine (polyQ) region has 38 glutamine residues. A black box denotes the N-terminal polyQ tract and arrows represent the C-terminal domain direct repeat. The location of a putative nuclear localisation signal (NLS) in the TBP mRNA is indicated by a cross-striped box. TBPex3 encodes a C-terminal truncated isoform of 165 amino acids. Exons and introns are not drawn to scale.

Fig. 2. Vertebrate TBP exon three splice acceptor sites and predicted truncated proteins. (A) Nucleotide and deduced amino acid sequences of conserved the 3′ splice acceptor sites of TBP exon three in vertebrate genomes in the current public database. Exonic nucleotides are shown in upper case and intronic nucleotides in lower case. The splice acceptor sites are underlined and amino acids encoded by potential intron-skipping transcripts are shown in bold text. (B) Alignment of deduced amino acid sequences of TBP variant TBPv3 from human, mouse and bovine (verified) and other vertebrates (predicted). Sequence accession numbers/loci: Homo sapiens (human), NM_003194; Pan troglodytes(chimp), NM_001104607; Equus caballus (horse), LOC100049846; Macaca mulatta (macaque), LOC696258; Canis familiaris (dog), LOC611193; Bos taurus (bovine), NM_001075742; Rattus norvegicus (rat), NM_001004198;Ornithorhynchus anatinus (platypus), LOC100091633; Mus musculus (mouse), NM_013684; Monodelphis domestica(opossum) LOC100028736; Gallus gallus (chicken), NM_205103; Danio rerio (zebrafish), NM_200096.
Human TBPex3 variant is transcribed in diverse adult tissues.

TBPex3 encodes a protein with low electrophoretic mobility. Western analysis shows that MW for full-length uman TBP with 37 repeat polyQ tract is 37.5kDa. Under SDS PAGE it travels at 42 kDa. TBPex has a predicted MW of 17.9kDa, but travels at 29kDa.

TBPEx protein accumulates in AD.  It was detected at higher levels in AD middle temporal gyrus tissue than in control middle MTG tissue.

Cellular distribution and localisation of TBPex3 protein.
Immunocytochemistry showed diffuse nuclear TBP labelling in all full-length TBP transfected cells. TBPex3 expression resulted in extranuclear and perinuclear inclusions. In many cases cell nucleus was distorted.

This indicates that TBPex3 has a greater tendency to aggregate in vivo and that TBPex3 can cross nuclear envelope and pass through nuclear pore.

Rational design of a super core promoter that enhances gene expression by Juven-Gershon
Super core promoter 1 (SPC1) directs high amounts of transcription by RNAPII in metazoans.  SPC1 contains TATA box, Inr, MTE and DPE in a single promoter.  It is stronger than CMV IE1 and AdML core promoters in vitro and invvo. Each of 4 promoter motifs needed for full SCP1 activity.

Strong synergy between MTE and TATA and between MTE and DPE.

SPC1 comprises sequences form -36 to +45 relative to +1 Adenine in Inr.

TATA box is from CMV IE1 core promoter. A cp,[psote Onr based on sequences on AdML and D. melanogaster G retrotransposon core promoters. MTE from D. melanogaster Tolllo core promoter. DPE from Drosophila G core promoter. BRE was not included as it has been reported to have positive and negative effects on transcription.

Figure 1. SCP1 is stronger than CMV and AdML core promoters in vitro.
(a) Diagram of SCP1. KrD. melanogaster Krüppel gene; G, Drosophila G retrotransposon. The nucleotide sequence is given in Methods. (b) SCP1, CMV and AdML core promoters (each of which contains their respective sequences from -36 to +45 relative to the +1 start site cloned into pUC119) were subjected to in vitro transcription analysis (in duplicate reactions) with a standard HeLa transcription extract21. The resulting transcripts were detected by primer extension. To test whether transcription was catalyzed by RNA polymerase II, alpha-amanitin (4 mug/ml) was included, as indicated. (c) Transcription reactions were performed as in b with the indicated amounts of template DNA in a volume of 50 mul.
SCP1 is stronger than CMV core promoter or AdML core promoter in vitro. Transcription from SCP1 is inhibited by α-amanatin so transcription from SCP1 is catalysed by RNAPII.

SCP1 is transcribed more efficiently than other core promoters. Rate of PIC assembly was higher in SPC1 than in other core promoters. Higher template usage (40%) in SCP1 than in CMV (15%) or AdML (6%) core promoters.

Purified TFIID binds to SCP1 with high affinity
DNase I footprinting showed TFIID binds SCP1 with at least 10-fold higher affinity than to CMV or AdML core promoters. Unlike TBP, TFIID binds TATA box weakly. Mutations in TATA box, Inr, MTE or DPE in SCP1 variants decreased binding of TFIID. All core promoter motifs contribute to TFIID interaction with SCP1.

SCP1 is a strong core promoter in vivo
SCP1, CMV and AdML core promoters were subcloned into a promoterless, enhancerless vector with a leciferase (luc) reporter gene. Constructs were transfected into HeLaS3 cells. Luciferase activity was measured. SCP1 showed several-fold higher levels of luciferase than either the CMV or AdML core promoters.

SCP1 increases level of enhancer-driven transcription
SCP1, CMV and AdML core promoters were inserted intopGL3-enhancer luc reporter vector which contains SV40 enhancer   SCP1 is 3fold more actibe than CMV or AdML core promoters with SV40 enhancer.

SCP1 directs accurate initiation of transcription in vivo
HeLa cells were transfected with pGL3-enhancer constructs containing SPC1, CMW and AdML core promoters. RNA was isolated from cells. RNA was subjected to primer extension analysis. Cells were co-transfected with a β-galatosidase (lacZ) reporter plasmid. SCP1 mediates transcription from A+1 site in Inr motif. Amount of RNA producd in SCP1 was higher than from CMV or AdML core promoters.