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.




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