Saturday, 22 December 2012

Chromatin I

Five major epigenetic mechanisms are capable of establishing and stabilizing open or closed chromatin structures, thereby regulating transcriptional activity (
Fig. 1).
1) Nucleosome remodelers can stimulate transcription by removing nucleosomes from promoter regions, allowing transcription factors to gain access to the underlying DNA (47). Additional functions of nucleosome remodelers include histone variant exchange and nucleosome sliding (93).
2) Mammalian cells express three histone H3 variants: H3.1, H3.2, and H3.3. Specific enrichment of H3.3 in transcriptionally active genes and regulatory regions, and H3.1 in repressed or inactive genetic elements suggest a regulatory function of these variants (60). Incorporation of histone variants is also important for many other chromatin-related processes. Faithful DNA damage repair, for example, depends on the presence of a histone H2A variant, H2A.X, which is phosphorylated on damage detection allowing binding of the DNA damage repair machinery (11).
3) Increasing evidence suggests important functions for long noncoding RNAs (ncRNAs) in transcriptional regulation. Direct interactions of chromatin-modifying enzymes with noncoding RNAs are speculated to facilitate targeting to specific genomic loci. A well-studied example is the histone methyltransferase Ezh2, which was shown to interact with different ncRNAs to induce X inactivation and repression of developmental genes (75101). Small ncRNAs, e.g., siRNAs and promoter antisense RNAs, can also trigger formation of repressive chromatin structures as will be discussed below.
4) Cytosine bases, preferably in the context of CpG dinucleotides, can be methylated to 5-methylcytosine by DNA methyltransferases. DNA methylation is a repressive modification that is enriched at promoter regions of genes and noncoding DNA sequences. MBD (methyl binding domain) proteins and MeCP2 can bind methylated DNA stretches and in turn recruit corepressor complexes to facilitate transcriptional silencing (78).
5) Histones can be posttranslationally modified. The major modifications include phosphorylation, acetylation, and methylation. Combinations of different histone modifications represent chromatin signals that are recognized by specific binding proteins that then mediate downstream effects. In the context of transcriptional regulation, histone lysine methylation has been particularly well characterized. This modification generates a high complexity of signals as each lysine position can be mono- (me1), di- (me2), or trimethylated (me3) and distinct binding proteins for each methylation state can mediate different functions. Transcriptionally active, euchromatic domains are characterized by histone H3 lysine 4 trimethylation (H3K4me3) at gene promoters and H3K36me3 across gene bodies (2). The two types of heterochromatin carry distinct modification patterns. Facultative heterochromatin is marked by high levels of H3K27me3 (86). In contrast, constitutive heterochromatin features the combinatorial mark H3K9me3 and H4K20me3 (52).

Bradley Cairns Nature 461 (2009)
a, Open promoters have a depleted proximal nucleosome adjacent to the transcription start site (TSS, black arrow), a feature common at constitutive genes. b, Covered promoters have a nucleosome adjacent to the TSS in their repressed state, a feature common at highly regulated genes. The figure depicts features more common in each contrasting promoter type, but most yeast genes blend the features shown to provide appropriate regulation. Green nucleosomes contain canonical H2A, whereas brown nucleosomes bear H2A.Z. Binding sites (BS) for transcriptional activators (ACT) are shown. These are mainly exposed for open promoters and mainly occluded by nucleosomes (in the repressed state) at covered promoters. Covered promoters typically have nucleosome positioning sequence elements of varying strength and locations that help define nucleosome positions (faded green) and promoter architecture. NDR, nucleosome-depleted region.

Remodellers use ATP hydrolysis to alter nucleosomes and are specialized for certain tasks. Most remodellers of the ISWI family (except NURF and Isw1b) help conduct chromatin assembly and organization and provide consistent spacing of nucleosomes. This organization can cover a binding site (red) for a transcriptional activator (ACT). SWI/SNF-family remodellers provide access to binding sites in nucleosomal DNA, mainly through nucleosome movement or ejection. SWR1-family remodellers reconstruct nucleosomes by inserting the histone variant H2A.Z into nucleosomes, specializing their composition. This can create an unstable nucleosome in certain compositional and temporal contexts, and might lead to ejection, sliding or reconstruction at promoters.
Constitutive genes have open promoters
Competition between nucleosomes and TFs at many promoters. Constitutive genes favour binding of TFs. These promoters are open. Constitutive genes have a nucleosome depleted region (NDR) upstream of TSS containing key cis-regulatory sequences.  NDR has poly(dA:dT) tracts. They resist bending and deter nucleosome formation and stability  AA/TT dinucleotides repeat every 10 bp. They impose curvature favouring nucleosome formation and stability. Open promoters have a central poly (dA:dT) tract, deters nucleosome binding.  Flanked by 2 NPS (nulceosome positioning sequence) elements which fix positions of 2 flanking nucleosomes, called -1 and +1 nucleosomes in yeast. Creates a nucleosome boundary, Prevents nucleosomes from encroaching into NDR.

In open promoters, binding sites for transcriptional ativators are often in NDR itself. Not buried under nucleosomes upstream. Exposure in NDR promotes activator binding and gene expression. Open promoters in yeast are often linked to essential genes. 

Regulated genes have covered promoters

At regulated genes in repressed state, nucleosomes often cover TSS, regions flanking TSS and most of binding sites for transcriptional activators  They are covered promoters or closed promoters.  

Nucleosomes compete with TFs for occupanc of c-s reulgatory binding sites. Covered promoters rely more than open promoters on chromatin remodelling. Helps modify enqymes to help uncover cis sites and allow activity. 

At least one binding site is exposed in linker DNA between nucleosomes. This exposed site allows a TF access to promoter. Chromatin modification and remodelling may be required to expose extra sites under nucleosomes.

TBP is present at al Pol II promoters. Required for initiation at TATA-containg and TATA-less promoters.  TATA box is more enriched at covered than open promoters.  More enriched at highly regulated than constitutive genes.   

TATA-containing genes use TFIID. TATA-less promoters use SAGA in yeast and pCAF.GCN5 in humans.  

ISWI remodellers organise nucleosomes
ISWI remodellers promote repression. They remodel nucleosomes that lack acetylation. Confine activity to nucleosomes at transcriptionally inactive regions. They space nucleosomes by measuring DNA linker between nucleosomes and slide nucleoeoms until linker DNA reaches a fixed distance. Uniform spacing. Yeast Isw2 pevents antisense transcription in intergenic reiong.s

SWI/SNF remodellers disorganise nucleosomes
Can slide and eject nucloeomes. Help activate promoters. Domains bind acetylated tails.  May be needed in open promoters to remove nucleosomes from poly (dA:dT) sequenes or at covered promoters to slide or eject nucleosomes. In yeast, normally at -1 nucleosome. 

SWR1 and properties of H2A.Z nucleosomes
Histone H2A variant H2A.Z differs from H2A in amino-terminal tail sequence and key internal residues. It is assembled into spec promoter nucleosomes. Replaces H2A. This is performed by sWR1 remodellers. 

It is highly enriched at open TATA-less promoters. Not exclusive to open promtoers.

H2A.Z is lost from genes as transcription increases. Presence of acetylated H2A.Z increases with activation.  In yeast and humans, inactive or basal genes have high level. Acetylation and loss of H2A.Z accompanies activation. They are less stable when assembled into nucleosomes with H3.3. Instability of nucleosome mayt promote initiation. All yeasts contain H3.3 so all yeast H2A.Z nucleosomes are unstable. This may cause it to be sensitive to movement or ejection by chromatin remodellers. Expose promoters. 

Functions of DNA methylation: islands, start sites, gene bodies and beyond by Jones 2012

About 60% of human genes have CpG islands (CGIs) at their promoters and frequently have nucleosome-depleted regions (NDRs) at the transcriptional start site (TSS). The nucleosomes flanking the TSS are marked by trimethylation of histone H3 at lysine 4 (H3K4me3), which is associated with active transcription, and the histone variant H2A.Z, which is antagonistic to DNA methyltransferases (DNMTs). Downstream of the TSS, the DNA is mostly CpG-depleted and is predominantly methylated in repetitive elements and in gene bodies. CGIs, which are sometimes located in gene bodies, mostly remain unmethylated but occasionally acquire 5-methylcytosine (5mC) in a tissue-specific manner (not shown). Transcription elongation, unlike initiation, is not blocked by gene body methylation, and variable methylation may be involved in controlling splicing. Gene bodies are preferential sites of methylation in the context CHG (where H is A, C or T) in embryonic stem cells5, but the function is not understood (not shown). DNA methylation is maintained by DNMT1 and also by DNMT3A and/or DNMT3B, which are bound to nucleosomes containing methylated DNA99. Enhancers tend to be CpG-poor and show incomplete methylation, suggesting a dynamic process of methylation or demethylation occurs, perhaps owing to the presence of ten-eleven translocation (TET) proteins in these regions, although this remains to be shown. They also have NDRs, and the flanking nucleosomes have the signature H3K4me1 mark and also the histone variant H2A.Z32, 100. The binding of proteins such as CTCF to insulators can be blocked by methylation of their non-CGI recognition sequences, thus leading to altered regulation of gene expression, but the generality of this needs further exploration. The sites flanking the CTCF sites are strongly nucleosome-depleted, and the flanking nucleosomes show a remarkable degree of phasing. The figure does not show the structure of CpG-depleted promoters or silenced CGIs, although in both cases the silent state is associated with nucleosomes at the TSS. LMR, low-methylated region.
Active promoters and enhancers have nucleosome-depleted regions (NDRs) that are often occupied by transcription factors and chromatin remodellers. Loss of factor binding — for example, during differentiation — leads to increased nucleosome occupancy of the regulatory region, providing a substrate for de novo DNA methylation. DNA methylation subsequently provides added stability to the silent state and is likely to be a mechanism for more accurate epigenetic inheritance during cell division. The example given is for the OCT4 and NANOG genes45, and its generality is not yet known, but inactive genes are often more susceptible to de novo methylation than their more active counterparts (Refs 36,40,41,42,43,51,52). In the figure, OCT4 binding is shown and NANOG binding is not shown, although its expression is required. Recent experiments have demonstrated that the methylation must be removed by active and/or passive processes to reactivate the gene. DNMT3A, DNA methyltransferase 3A; siRNA, small interfering RNA.

Landscapes and archipelagoes: spatial organisation of gene regulation in vertebrates by Monatavon and Duboule

Figure 2. Long-range regulation at selected genetic loci. Genes are represented by rectangles and regulatory elements by ovals. Arrows indicate interactions controlling gene activation; curved lines without arrowheads represent physical associations with unknown functional consequences. Regulations occurring in different tissues or cell types are depicted using different colors. Grey boxes represent genes that are not affected by the described long-range regulations. Note the different scales used for each panel. (a) β-Globin (Hbb) locus. The locus control region (LCR) contacts and activates either embryonic or adult globin genes at different developmental stages in erythrocytes. Distal sites (open ovals) contact the LCR in both erythroid progenitors and mature erythrocytes (orange lines), yet these sequences are not required for gene activation. (bSonic hedgehog (Shh) locus. Candidate enhancers located within the upstream gene desert recapitulate Shh expression in specific regions of the central nervous system (CNS) or epithelial linings. An enhancer located within Lmbr1 contacts Shh in the developing limb bud and is required for its expression in this structure. (c) The HoxD regulatory archipelago. An array of regulatory ‘islands’ dispersed within the centromeric gene desert coordinately activates Hoxd13Hoxd10, as well as Lnp and Evx2 transcription in developing digits. These multiple elements are brought into the vicinity of the HoxD cluster in developing digits and each contribute, in a partially redundant manner, to gene activation. Global regulation controlling Hoxd genes in different structures relies on control elements located on either side of the gene cluster.
β-globin genes
Locus control region (LCR) controls β-globin genes. LCR selectively contacts embryonic or adult genes at diff developmental stages. Do not occur in cell lineages in which globin genes are inactive.

Sonic hedgehog
Encodes a signalling protein for developmental patterning. Several enhancers regulate different aspects of Shh transcription in CNS when isolated as transgenes. 

Hox gene clusters and regulatory archipelagoes
Hox genes encode TFs essential for patterning animal body plan. A group of Hoxd genes transcribed in developing digits establishes long range interactions with sequences dispersed in centromeric gene desert  Sites of contacts are clustered into islands. DEcorated with histone marks associated with enhancer sequences. 

Chromatin structure and the inheritance of epigenetic information by Margueron and Reinberg
Schematic depicting modifications that define different chromatin domains. The range of factors that can contribute to the characteristics of a domain are shown in the shaded boxes. The dashed lines represent the separation between two adjacent domains. PTM, post-translational modification.

Chromatin is a composite of various domains. They are enriched with spec combination of histone post-translational modifications (PTMs), histone variants, nucleosome occupancy, DNA methylation patterns and possibly nuclear localisation. 

The complex language of chromatin regulation during transcription by Berger
Chromatin marking mechanisms in transcription
2 types of chromatin modification regulate transcription.

DNA methylation occurs at C residues in CG dinucleotides or CNG trints. Opposes transcription. Localised at repeated regions and transposons in many eukaryotes. Promoters are normally not methylated because they have CpG clusters refractory to methylation.

DNA hypermethylation occurs in certain human cancers. Aberrant repression of tumour suppressor genes through methylation of CpG islands in promoters

Histone PTMs include acetylation, phosphorylation, methylation, ubiqiutylation and SUMOylation. They decorate canonical histones (H2A, H2B, H3, H4) and variants histones (H3.1, H3.3, HTZ.1). 

Lysine is key substrate residue. Modified by acetlyation, methylation, ubiquitylation and sumoylation. Acetylation is activating, sumoylation is repressing. 

All histone PTMs are removable. HDACs remove acetyls. Ser/thr phosphatases remove phosphates. Ubiquitin proteases removes mono-ubiquitin from H2B. Arginine metylation is altered by deiminases. 

Histone PTMs change structure of chromatin or act through recruitment of effector proteins. Histone PTMs are binding surfaces for association of effector protein. Acetyl lysine associates with bromodomains. Acetylated H3 stabilises binding of HAT, GCN5 through its bromodomain.  Methyl H3K9 associates with chromodomain of HP1 to promote its binding to heterochromatin.

H3K4 methylation recruits multiple effectors. These stabilised nucleosomes during transcriptional initiation and elongation. It can also recruit repressing effector proteins that stabilised nucleosomes during transcription attenuation or repression. 

CpG islands and the regulation of transcription by Deaton and Bird
Vertebrate CpG islands (CGIs) are short interspersed DNA sequences that deviate from average genome pattern. They are GC-rich, CpG-rich and predominantly nonmethylated. They are sites of transcription initiation. 
The genomic distribution of CGIs. (A) CGIs can be located at annotated TSSs, within gene bodies (Intragenic), or between annotated genes (Intergenic). Intragenic and intergenic CGIs of unknown function are classed as “orphan” CGIs. (Empty circles) Unmethylated CpG residues. (Filled circles) Methylated CpG residues. (B) The genomic distribution of CGIs in the human and mouse genome as determined by Illingworth and colleagues (2010). The total number of CGIs is given at the top of each graph.
Half of CGIs contain TSSs. They coincide with promoters. CGIs are often marked by trimethyltaion on H3 (H3K4me3) a signature of active promoters.  

CGI promoters often lack TATA boxes and have heterogenous TSSs.  CGI promoters adopt a transcriptionally permissive state. Initiation can occur at several locations. TATA boxes tend to be associated with focused transcriptional initiation. CGIs tend to lack these. Dispersed initiation patterns.

TF binding at CGIs
CGI share little long range sequence conservation except elevated CpG densityt and GC content. Lack core promoter elements eg TATA box.  GC richness increases probability that TFs will bind. Mammalian TF bindig sites are more GC-rich than bulk genome. Many contain CpG in recognition sequence. Eg Sp1, a general TF, which recruits TBP to promoters lacking a TATA box. 

Chromatin signature of CGIs
Nonmethylated CGIs are arranged in a characteristic chromatin structure that favours promoter activity. CGIs are nucleosome deficient. Primary response genes are classfieid into those that require SWI/SNF remodellers for activation adn those that do not. These corresponded with non-CGI and CGI promoters respectively. DNA in CGI chromatin may be accessible without need for nucleosome displacement. 

Characteristic histone modifications
CGI chromatin shows High levels of H3 and H4 acetylation, characteristic of transcriptional active chromatin. H3K4me3 is histone mark of CGI promoters. 

CGI promoter silencing by DNA methylation
CGIs are normally in nonmethylated state in a methylated genome. CGIs that are methylated cause stable silencing of promoter.  This may be due to inhibition of TF binding.

During X chromosome inactivation, X-linked CGIs do not become methylted until after gene silencing and silencing chromatin modifications eg H3K27me3. 

CGIs and polycomb mediated silencing
CGI promoters can be silenced by polycomb group proteins. 
Schones and Zhao (2008) Nature Genetics

The interaction of DNA methylation, histone modification, nucleosome positioning and other factors such as small RNAs contribute to an overall epigenome that regulates gene expression and allows cells to remember their identity. Chromosomes are divided into accessible regions of euchromatin and poorly accessible regions of heterochromatin. Heterochromatic regions are marked with histone H3 lysine 9 di- and trimethylation (H3K9me2 and H3K9me3), which serve as a platform for HP1 (heterochromatic protein 1) binding. Small RNAs have been implicated in the maintenance of heterochromatin. DNA methylation is persistent throughout genomes, and is missing only in regions such as CpG islands, promoters and possibly enhancers. The H3K27me3 modification is present in broad domains that encompass inactive genes. Histone modifications including H3K4me3, H3K4me2, H3K4me1 as well as histone acetylation and histone variant H2A.Z mark the transcription start site regions of active genes. The monomethylations of H3K4, H3K9, H3K27, H4K20 and H2BK5 mark actively transcribed regions, peaking near the 5' end of genes. The trimethylation of H3K36 also marks actively transcribed regions, but peaks near the 3' end of genes.

Bannister and Kouzamides 2011 Cell Res
Domains binding modified histones. Examples of proteins with domains that specifically bind to modified histones as shown (updated from reference 53).

Sarma and Reinberg 2005. Nature Rev Mol Cell Biol 6
The major core histones contain a conserved histone-fold domain (HFD). In addition, they contain N- and C-terminal tails that harbour sites for various post-translational modifications. For simplicity, only well-established sites for lysine methylation (red flags) and serine phosphorylation (green circles) are shown (other types of modifications, such as ubiquitylation, are not shown). In the histone H3.3 variant, the residues that differ from the major histone H3 (also known as H3.1) are highlighted in yellow. Three of these residues are contained in the globular domain and one resides in the N terminus. This N-terminal residue (Ser31) has been speculated to be a potential site for phosphorylation on H3.3. The centromeric histone CENPA has a unique N terminus, which does not resemble other core histones. Two sites of phosphorylation have been identified in this region, of which Ser7 phosphorylation has been shown to be essential for completion of cytokinesis. The region in the globular domain that is required for targeting CENPA to the centromere is highlighted in light blue. Histone H2A variants differ significantly from the major core H2A in their C terminus. The C terminus of H2AX harbours a conserved serine residue (Ser139), the phosphorylation of which is an early event in response to DNA double-strand breaks. A short region in the C terminus of H2AZ is essential for viability inDrosophila melanogaster. MacroH2A has an extended C-terminal macro domain, the function of which is unknown. Finally, the H2ABBD is the smallest of the H2A variants and contains a distinct N terminus, which lacks all of the conserved modification sites that are present in H2A. The C terminus is also truncated and lacks the docking domain that is found in other H2A species. The histones H4 and H2B are also shown, including their known methylation and phosphorylation sites. The proposed functions of the variants are listed.

The major histones can be replaced by their variants to allow for a more transcriptionally competent chromatin state. Here, we show a model for the synergy between the H2AZ and H3.3 exchange complexes with FACT ('facilitates chromatin transcription'), which disassembles and reassembles chromatin during transcription. In one situation, displacement of an H2A–H2B dimer by the SPT16 subunit of FACT could allow exchange of the displaced H2A with H2AZ by SWR1 (a member of the ATP-dependent SWI/SNF family of chromatin-remodelling factors), which leads to an altered nucleosome that is homogeneous in its composition of H2AZ (see main text). In a second situation, the SSRP1 subunit of FACT could coordinate with the elongation factor SPT6 and the histone chaperone HIRA to replace H3 with H3.3. Both of these events would result in the formation of chromatin that is more amenable to transcription, either on the basis of the intrinsic structure of the variant nucleosome or by the presence of post-translational modifications on the variant histones.

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