Saturday, 22 December 2012

Activation of transcription

Some transcription factors ("Enhancer-binding protein") bind to regions of DNA that are thousands of base pairs away from the gene they control. Binding increases the rate of transcription of the gene.

Enhancers can be located upstream, downstream, or even within the gene they control.
How does the binding of a protein to an enhancer regulate the transcription of a gene thousands of base pairs away?

One possibility is that enhancer-binding proteins — in addition to their DNA-binding site, have sites that bind to transcription factors ("TF") assembled at the promoter of the gene.

This would draw the DNA into a loop (as shown in the figure).

Recent evidence shows that these loops are stabilized by cohesin — the same protein complex that holds sister chromatids together during mitosis and meiosis.

Visual evidence

Michael R. Botchan (who kindly supplied these electron micrographs) and his colleagues have produced visual evidence of this model of enhancer action. They created an artificial DNA molecule with
  • several (4) promoter sites for Sp1 about 300 bases from one end. Sp1 is a zinc-finger transcription factor that binds to the sequence 5' GGGCGG 3' found in the promoters of many genes, especially "housekeeping" genes.
  • several (5) enhancer sites about 800 bases from the other end. These are bound by an enhancer-binding protein designated E2.
  • 1860 base pairs of DNA between the two.
When these DNA molecules were added to a mixture of Sp1 and E2, the electron microscope showed that the DNA was drawn into loops with "tails" of approximately 300 and 800 base pairs.
At the neck of each loop were two distinguishable globs of material, one representing Sp1 (red), the other E2 (blue) molecules. (The two micrographs are identical; the lower one has been labeled to show the interpretation.)
Artificial DNA molecules lacking either the promoter sites or the enhancer sites, or with mutated versions of them, failed to form loops when mixed with the two proteins.

Significance of "Looping"

The looping of chromosomes that brings enhancers close to promoters (and promoters close to other promoters) seems to be a mechanism to ensure the expression (or inhibition) of groups of genes that must perform together. The response of a cell to the arrival of a signal (e.g., a hormone) may involve turning on (or off) hundreds of different genes whose products must be produced in a coordinated way for the cell to respond appropriately. The dynamic movement of portions of the chromosome carrying the appropriate gene loci into a "transcription factory" may be a mechanism to accomplish this [Link]. If so, we are seeing the eukaryotic equivalent of the coordinated gene expression provided by operons in bacteria. [Link]


Silencers are control regions of DNA that, like enhancers, may be located thousands of base pairs away from the gene they control. However, when transcription factors bind to them, expression of the gene they control is repressed.


A problem:
As you can see above, enhancers can turn on promoters of genes located thousands of base pairs away. What is to prevent an enhancer from inappropriately binding to and activating the promoter of some other gene in the same region of the chromosome?
One answer: an insulator.
Insulators are
  • stretches of DNA (as few as 42 base pairs may do the trick)
  • located between the
    • enhancer(s) and promoter or
    • silencer(s) and promoter
    of adjacent genes or clusters of adjacent genes.
Their function is to prevent a gene from being influenced by the activation (or repression) of its neighbors.
Example:The enhancer for the promoter of the gene for the delta chain of the gamma/delta T-cell receptor for antigen (TCR) is located close to the promoter for the alpha chain of the alpha/beta TCR (on chromosome 14 in humans). A T cell must choose between one or the other. There is an insulator between the alpha gene promoter and the delta gene promoter that ensures that activation of one does not spread over to the other

All insulators discovered so far in vertebrates work only when bound by a protein designated CTCF ("CCCTC binding factor"; named for a nucleotide sequence found in all insulators). CTCF has 11 zinc fingers. [View another example of a zinc-finger protein]
Another example: In mammals (mice, humans, pigs), only the allele for insulin-like growth factor-2 (IGF2) inherited from one's father is active; that inherited from the mother is not — a phenomenon called imprinting.The mechanism: the mother's allele has an insulator between the IGF2 promoter and enhancer. So does the father's allele, but in his case, the insulator has been methylated. CTCF can no longer bind to the insulator, and so the enhancer is now free to turn on the father's IGF2 promoter.

Many of the commercially-important varieties of pigs have been bred to contain a gene that increases the ratio of skeletal muscle to fat. This gene has been sequenced and turns out to be an allele of IGF2, which contains a single point mutation in one of its introns. Pigs with this mutation produce higher levels of IGF2 mRNA in their skeletal muscles (but not in their liver).
This tells us that:
  • Mutations need not be in the protein-coding portion of a gene in order to affect the phenotype.
  • Mutations in non-coding portions of a gene can affect how that gene is regulated (here, a change in muscle but not in liver).
The complex transcription regulatory landscape of our genome: control in 3 dimensions by Splinter and de Laat
Regulatory DNA sequences in genome can be tested using property defining assays in vitro. Isolated sequence element is placed in plasmids carrying a reporter gene. When transfected into cells they may activate or repress transcription or neutralise transcriptional activaction when placed between an activator and gene promoter. They are classified as an enhancer, silencer or insulator. 

Rules of engagement in a complex regulatory landscape, 1) linear proximity
Enhancers usually act on nearest genes in cis. Proximity on linear DNA template or genomic order is major determinant of selectivity. First come, first served.  

However order is no longer important when 2 competing genes are positioned close together at a large distance from a shared enhancer (Heuchel 1989, Dillon 1997). 

While linear proximity to a regulatory site is often a good predictor of target genes, many examples of enhancers ignore nearest genes and specifically act on genes further away. e.g. limb bud-specific enhancer of SHH gene, present in an intron of Lmbr gene but acts on SHH gene 1Mb away. Why act on distal genes?

The three rules of engagement that dictate enhancer–promoter interactions. I: Proximity. (a) When multiple genes (cylinders) are compatible with (open lock) and relatively close to a shared enhancer (E), the most proximal gene is preferentially activated over the distal gene (represented by the number of transcripts originating from the gene). (b) This competitive advantage disappears when both genes are located far away from the shared enhancer. II: Compatibility. Enhancers ignore the ‘first-come, first-served’ rule when the proximal promoter is incompatible (closed lock) with the enhancer. Result: activation of the distal gene. III: Insulation. The presence of CTCF can block enhancer function across its binding site and prevent a compatible gene from being activated by the enhancer.

Rules of engagement in a complex regulatory landscape 2: 2) promoter specificity
Many enhancers interact with just a subset ofequally nearby target promoters. An enhancer is thought to ignore a gene because its promoter is not accessible in the tissue where the enhancer is active. In β-globin locus, LCR exclusively acts on β-globin genes and totally ignores nearby olfactory receptor genes.  even when potentially interfering insulator sites are disrupted. LCR also ignores nearby fetal globin genes to exclusively act on more distal adult β-globin genes at later stages of development. Inactivity or promoter inaceesbility may be a reason.

Selective gene activation can be explained by promoter competition. Activation of one promoter precludes activation of another equidistant promoter. In Drosophila transgenic embryo assays, different enhancers were shown to prefer distinct classes of promoters, depending on presence of core promoter elements (Ohtsuki 1998, Butler and Kadonaga 2001). 

Promoter competition occurs between mammalian genes. It manifests when deletion of one or more genes affects expression of remaining neighbouring genes, or when deletion of a regulator causes down-regulation of multiple genes. This phenomena was found in Hox and globin gene clusters.

Regulatory sites exist in genome that can act on multiple endogenous genes. Number of genes controlled by a  regulatory site may depend on its chromosomal context. At its natural location, β-globin LCR activates maximally 2 or 3 β-globin genes at any given development stage. When tested without globin genes ata defined location in genome, it activated 6-7 genes up to 150kb away in cis and 2 genes in trans.

Enhancer-promoter interactions are mutually exclusive for some time. Experiments that measured ongoing transcriptional activity of LCR-competing &beta-globin genes in cells with traceable transcriptional history. LCR activated only a single gene at the time. But over time it dynamiocally flip-flopped between competing globin genes. (Wigerde 1995).

Regulators sometimes activate more than their presumed target gene. B-cell-specific human Igβ gene is highly expressed but may not be functional, due to its linear proximity to LCR that acts on more distal growth hormone gene in this tissue.

Rules of engagement: 3) insulators can block enhancer-promoter interactions
Genome may be partitioned in physically separated chromatin comains that each has their own independent regulatory activities  2 assays were developed to test this. One investigated ability of sequence elements flanking a reporter gene to overcome gene repression when integrated into heterochromatin.  Another analysed whether an element can block enhancer activity when positioned in between enhancer and gene promoter.

In mammals, CTCF protein is associated with insulator activity.  In in vitro reporter assays, cTCF bound to DNA acts as an enhancer blocker. In vivo, CTCF binding sites are found next to genes that are active in an otherwise repressive chromatin surruonding eg human and mouse β-globin genes.

CTCF also acts as an allele-specific enhancer blocker to mediate imprinted gene expression at H19-Igf2 locus. 

A single CTCF site can function as an insulator. But introducing a 2nd CTCF site in between an enhancer and promoter often alleviates enhancer blocking effect.

20 000 -87 000 CTCF binding sites are in human genome. Most of these sites are conserved between different cell types. They are enriched at boundaries between repressive and active chromatin.  Genes separated by a CTCF site show reduced gene expression.

While not all CTCF sites act as insulators, their location must be taken into account.

Regulation of two pair-rule stripes by a single enhancer in the Drosophila Embryo by Small et al
Segmentation gene even-skipped is expressed in a series of 7 transverse stripes in precelllular embryos.  Eve promoter has a series of enhancers tat control expression of individual stripes.

One model of stripe 2 regulation is that stripe borders are established by transcriptional repressors. Maternal bicoid (bcd) gradient triggers expression of hunchback (hb). 2 proteins work synergestically to activate stripe 2 enhancer. Study undertaken to determine if these rules apply to regulation of other eve stripes.

In previous studies stripe 3 regulation was characterised. Small eve-lacZ fusion genes containing first 8kb of eve promoter region are expressed in limits of stripes 2,3 and 7.  Eve stripe enhancer is between -l.6 and -1.1 kb upstream of TSS.  A 900bp region between -3.8 and -2.9 kb is essential for stripe 3 expression. eve-lacZ fusion genes with deletions in this interval show defective patterns of stripe 3 expression.

Small et al characterised a 500bp enhancer element that regulates expression of stripe 3. It also drives expression of stripe 7. Thus it is the stripe 3+7 enhancer.

eve-lacZ fusion genes containing this enhancer were examined in segmentation mutants.  Results suggest regulation of two stripes is by single enhancer.  Activation is mediated by a ubiquitously distributed activator, which switches on enhancer on entire length of early embryo.  2-stripe pattern is defined by multiple tiers of repression mediated by terminal system, anterior morphogen bud and gap proteins hb and kni.

eve-lacZ gene fusions were constructed by fusing different portions of eve promoter to reporter gene eg. lacZ.  Fusions were cloned into vector.  These constructs are used to create transgenic Drosophila flies. Embryos were collected, fixed and stained. Antisense lacZ RNA was used to hybridise. Eve protein and lacZ RNA detection with double staining protocol using anti-eve antibody to verify positions of stripes 3 and 7.

Identification of a minimal enhancer that drives expression of eve stripes 3 and 7
Several DNA fragments were fused with minimal eve-lacZ fusion gene.  Fusion genes were inserted into transformation vector and expressed in transgenic embryos.  Expression patterns were visualised by hybridising transgenic embryos wit a digoxigenin-labelled lacZ antisense RNA probe.

A 500bp fragment between -3.8 and 3.3kb directs expression of a strong stripe near centre of embrryo and a weaker stripe in posterior regions.

Colocalisation of endogenous eve protein and lacZ RNA indicates that stripes produced by transgene coincide with endogenous stripes 3 and 7. Expression for stripe 7 is weaker  Other regions of promoter may be required for optimal stripe 7 expression. Consistent with previous promoter fusion assyas which showed other regions of eve promoter can direct weak expression of stripe 7.
FIG. 2. The eve stripe 3 +7 enhancer directs an authentic pattern of expression  Embryos are oriented with anterior to left and dorsal up.  Staining patterns were visualised by in situ hybridisation using digoxigenin labelled antisense RNA probes.  A) wild type cellularising embryo hybridised with eve antisense RNA probe. Staining pattern is 7 transverse stripes along anteroposterior axis.  B) Transgenic, cellularised embryo carrying a minimal eve stripe 2-lacZ gene. Staining was visualised with a lacZ antisense probe to detect expression mediated by transgene. Staining is observed within the limits of stripe 2.  An anterior dorsal patch of expression is due to vector sequences in P-transposon used in this analysis. C) Transgenic cellularised embryo carrying 500bp eve stripe 3+7 enhancer attached to the eve-lacZ fusion gene. Staining is observed in central regions and near the posterior pole. Anterior patch of expression is due to vector sequences in P-transposon. D) Transgenic embryo that was double stained with anti-eve antibodies and a lacZ antisense RNA probe.  Endogenous eve protein is stained brown. RNA expressed by stripe 3+7-lacZ transgene is blue. 2 lacZ stripes are expressed within limits of endogenous stripes 3 and 7.

Stripe 3+7 enhancer contains kni-bindings sites
DNase I footprint assays were performed to determine if kni might directly repress the tsripe 3 enhancer.  use bacterial GST-fusion protein that contains N-terminal 342 aa residues of kni. Includes both putative cys/cys zinc fingers that mediate DNA binding

Binding assays were performed with 2 overlapping fragments from 500bp enhancer that directs expression of stripes 3 and 7 in transgenic embryos. 5 kni-binding sites were identified. Recognition sequences of these binding sites show some similarity to previously identified consensus sequence for kni-binding sites.

Anterior repression of a Drosophila stripe enhancer requires 3-position-specific mechanisms by Andrioli
Striped expression pattern of pair-rule gene even skipped (eve) is established by 5 stripe-specific enhancers. Each responds in a unique way to gradients of position info in early Drosophila embryo.  Enhancer for eve stripe 2 (eve 2) is activated by morphogens bicoid (Bcd) and Hunchback (Hb).  As proteins are distributed throughout anterior half of embryo, formation of single stripe requires that enhancer activation is prevented in all nuclei anterior to the stripe 2 position.

Gap gene giant (gt) is involved in a repression mechanism that sets anterior stripe border. Genetic removal of gt causes stripe expansion only in anterior subregion adjacent to stripe border.

A map of the eve locus is shown at the top. The positions of five enhancers that control early stripe formation (1-7) are shown. Two other enhancers that control the refinement of the initial stripes (LE), and later expression in inter-stripe regions (ftz-like) are also indicated. A map of the eve 2 minimal stripe element (MSE) is shown in the middle with positions of defined binding sites for transcription factors. Activator and repressor sites are closely linked, especially in two clusters, each of which contains two pairs of overlapping sites. Regions tested by deletion analyses are marked (D1-D5). A model for eve 2 regulation is presented at the bottom. Activation is mediated by Bcd and Hb, while Gt and Kr are involved in repression mechanisms that form the stripe borders.
To identify sequences important for eve 2 regulation,  a series of mutants containing deletions (D1-D5) of regions between known binding sites was constructed. Each deletion was tested incontext of eve 2 lacZ fusion gene  All 5 deletions disrupt normal function of eve 2 enhancer. 4 (D1, D3, D4, D5) reduces level of stripe activation. D2 seems to strengthen stripe 2 activation and ectopic expression in more anterior regions.  D2 region may contain sequences required for repression in region of ectopic stripe.

Kr4 and Bcd2 sites on eve 2 enhancer sequences are well-conserved. The best-conserved sequence block in the intervening region is a 16 bp sequence consisting of  4 repeats of GTTT.  Deletion and mutagenesis cause severe anterior derepressions. A deetion that removes rest of D2 sequence but leaves (GTTT)4 sequence intact does not cause detectable change in enhancer activity. (GTTT)4 sequence is major binding site for repressive activity that prevents expression of eve2 enhancer in specific anterior region.

Region-specific repression of eve2
3 distinct mechanisms are required for anterior repression of eve2. Each activity functions in a psecific subregion.

In subregion III, Gt-binding sites are crucial for repression. Deletion of sites causes anterior expansion of stripe. Gt does not act alone.

In subregion  II, repression of eve2 is mediated by (GTTT)4 site. A candidate protein is Slp1. It binds specifically to this place in yeast 1-hybrid experiment.

In subregion I, eve 2 repression is controlled by Tor. Tor may downregulate Bcd-dependent activation.

Eve2 enhancer is directly activated by Bcd, but activation is prevented near anterior pole by Tor. Anterior expression patterns of defined repressors of eve2 (Slp1 and Gt) are activated by Bcd.

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