Eukaryotic chromatin for structure, remodelling and modifications accompanying activation and silencing of genes
Eukaryotic cells contain a definite membrane-bound organelle, the nucleus, which carries genetic material, organized in the form of multiple DNA molecules in complex with a variety of proteins to form chromosomes. Chromosomes contain genes, their regulatory elements and other DNA bound proteins, which serve to package the DNA and control its functioning. The total length of the cellular DNA is upto hundred thousand times a cell's length, hence the entire chromosomal DNA is held in the nucleus, in the form of a nucleoprotein complex, called chromatin during a significant period of the cell cycle. Thus, the major components of chromatin are DNA and histone proteins, alongwith a variety of other chromosomal proteins, such as transcription factors which also play a significant role in the regulation of gene expression. Each human cell contains about 1.8 meters of DNA, but when wound on the histones, it has about 90 millimetres of chromatin, which results in about 120 micrometres of chromosomes after duplication and condensation. (Redon C et al, 2002). Nucleosomes are the structural units of chromatin formed by packaging of DNA by histones. They undergo a series of folding to eventually form a chromosome and eventually regulate gene expression. Recent research to identify post-translational chromatin modifications as key factors in gene expression has garnered interest in the interrelation between chromatin and transcription.
Chromatin Structure: The Basics
Histones are the Principal Structural Proteins of Eukaryotic Chromosomes:
Histones are relatively small, alkaline proteins with a very high proportion of positively charged amino acids (lysine and arginine). Compact binding of histones to the highly negatively charged DNA is possible due to this positive charge. Histones can be grouped into five major classes, H1/H5, H2A, H2B, H3 and H4. They are further organised into super classes: Core histones, H2A, H2B, H3 and H4 and linker histones, H1 and H5. The core histones, also known as nucleosomal proteins are small proteins (102-135 amino acids) and are responsible for coiling the DNA into nucleosomes. H3 and H4 are considered to be among the highly conserved proteins. This conservation suggests that all their amino acids are functional, such that a change in any position is deleterious to the cell. The linker or H1 proteins are large proteins (containing about 220 amino acids) and have been less conserved during evolution than the nucleosomal histones. (Bhasin M et al, 2006) (Nelson D et al, 2005).
The Nucleosome Core Particle:
The nucleosome, fundamental packaging unit, gives chromatin a 'beads-on-a string' appearance in electron micrographs. On digestion of the long DNA string with nuclease, only the DNA between the nucleosome beads is degraded. The rest is protected from digestion and remains as double-stranded DNA. Each nucleosomal bead is a disc-shaped particle of about 11nm diameter, containing two copies each of the four nucleosomal histones H2A, H2B, H3 and H4 bound to about 146 nucleotide base pairs of double-stranded DNA. Thus, the core of each nucleosomal particle is a wedge-shaped protein octamer around which two turns of DNA are wound in a left-handed super helical manner with about 0-80 nucleotide base-pairs per turn. The two turns are sealed by a molecule of the linker histone, H1.
The core histone proteins contain a characteristic structural motif, 'the histone fold', which consists of three alpha helices (Î±1-3) separated by two loops(L1-2). On salt treatment, the histones form H2A-H2B heterodimers and H3-H4 heterotetramers. The H2A/H2B dimer binds onto the H3/H4 tetramer due to interactions between H4 and H2B, resulting in the formation of a hydrophobic cluster. The histone octamer thus formed contains a H3/H4 tetramer in the centre sandwiched between two H2A/H2B dimmers. The histone octamer is only stable in the presence of DNA or very high salt concentrations due to high alkalinity of all the four core histones. (Luger et al, 1997)
Each core histone contains two separate functional domains, the 'histone fold' motif responsible for both histone-histone and histone-DNA interactions within the nucleosome. Also, an amino-terminal and carboxyl-terminal tail domains that contain sites for post-translational modifications such as methylation, acetylation, phosphorylation and ubiquitination. Although these histone tails are mostly unresolved in the crystal structure of the nucleosome, they appear to be radiated from the nucleosome, conveniently positioned to associate with the linker DNA between nucleosomes or with neighbouring nucleosomes. (Hansen, J.C., 2001)
In addition to core histones, chromatin-associated proteins called linker histones bind to the exterior portion of nucleosomes and thus stabilise the highly condensed states of chromatin fibres. H1 binding protects about 20-30 nucleotide base-pairs of DNA from nuclease digestion at the core particle boundary. They contain a globular domain flanked by amino-terminal and carboxyl terminal tail domains. Each H1 molecule binds through its globular protein to a unique site on a nucleosome. Hence, although the linker histone globular domain is essential for binding to nucleosomes, the tail domains are believed to play an important role in chromatin folding.(Horn P.J. et al, 2002)
Higher order structures:
In eukaryotic cells, each nucleosome is linked to the next by small segments of linker DNA. Most chromatin is further condensed by winding in a polynucleosome fibre, which may be stabilized through the binding of histone H1 to each nucleosome and to the linker DNA. Although long strings of nucleosomes form on most chromosomal DNA, in the living cell chromatin rarely adopts the extended "beads-on-a-string" form. Instead the nucleosomes are stacked upon one another to create a structure in which the DNA is even more condensed. Most of the chromatin is seen to be in a compacted in the form of a fibre with a diameter of about 30 nm. The DNA is not only packaged with histones into regularly repeating nucleosomes that are packed into 30-nm fibres but it is also folded and organized by other proteins into a series of subdomains of distinct character that build an entire chromosome.
High concentration of chromatin-packing ions, magnesium and calcium, have been recently reported in metaphase chromosomes, further supporting the view of extremely high DNA compaction in these structures.(Bradbury, E.M et al.,2002).Although the structure of the basic subunit of chromatin, the nucleosome core is well known, the structure of the compacted 30nm fibre remains unresolved, as it is too compact to allow visualization of the spatial location of individual nucleosomes and the linker DNA. However, studies conducted have lead to two different models of its structure. The solenoid model, in which consecutive nucleosomes are next to each other in the fibre, which folds into a simple one-start helix. In the second model, nucleosomes are arranged in a zigzag manner such that two rows of nucleosomes are formed and the linker DNA criss-crosses between each stack of nucleosomes producing a double helical structure. (Bednar et al, 1998).
A recent study performed, solved the crystal structure of an array of four nucleosome cores (a tetranucleosome) to a resolution of 9 A (Schalch et al., 2005). Although this resolution is low, it was possible to define the positions of the linker DNA and nucleosomes and the structure solved by molecular replacement using the structure of the nucleosome core particle. The overall structure clearly showed two rows of two nucleosomes with the three-linker DNA segments crisscrossing between them, thus supporting the zigzag two-start helix model of the 30 nm fibre. Importantly, this zigzag conformation is in agreement with a previous cross-linking study performed in solution with longer nucleosomal arrays (12 nucleosome repeats). (Dorigo et al., 2004). Analysis of protein-protein cross-linked products revealed that rather than a single stack of 12 nucleosomes, two rows of 6 nucleosomes were produced as predicted for a zigzag fibre conformation.
Transcriptional activation and Remodelling:
Activation of gene transcription in vivo is accompanied by an alteration of chromatin structure. The specific binding of transcriptional activators disrupts nucleosomal arrays, suggesting that the primary steps leading to transcriptional initiation involve interactions between activators and chromatin. The affinity of transcription factors for nucleosomal DNA is determined by the location of recognition sequences within nucleosomes, and by the cooperative interactions of multiple proteins targeting binding sites contained within the same nucleosomes. Chromatin remodelling involves the disruption and re-formation of histone-DNA contacts. Chromatin remodelling factors comprise an ATPase subunit along with other polypeptides that are responsible for the regulation, efficiency, and functional specificity of each complex.
The SWI2/SNF2 group:
The SWI2/SNF2 group includes yeast SWI/SNF (ySWI/SNF), yeast RSC, the Drosophila Brahma complex, and the human BRM (hBRM) and BRG1 (hBRG1) complexes. All of these contain a highly conserved ATPase subunit, which belongs to the Swi2/Snf2 subfamily of proteins: Swi2/Snf2, Sth1, Brm, hBRM, and BRG1, respectively. The homology of these proteins extends beyond the ATPase domain, as they all contain a bromo-domain in the C-terminal region and two other conserved regions of unknown function called domains 1 and 2 (Laurent et al., 1993). ySWI/SNF complex was the first remodeling complex to be described. It contains 11 known subunits, including Swi2/Snf2. Several of the subunits were initially identified genetically as gene products involved in the regulation of either the HO endonuclease gene or the SUC2 gene, which encodes invertase. HO is required for mating type switching, hence SWI, while SUC2 mutants are classified as sucrose nonfermenters, thus SNF. The SWI/SNF genes were subsequently shown to be involved in the transcriptional regulation of a wider subset of yeast genes (Holstege F .C. P.et al.) Additionally, genetic studies provided a connection between the functions of the SWI/SNF complex and chromatin. Several mutations that suppressed SWI/SNF phenotypes corresponded to genes encoding histones and other chromatin proteins (Recht J.et al, 1999). The relationship between the function of the ySWI/SNF complex and chromatin was strengthened when the complex was purified and found to alter nucleosome structure in an ATP-dependent mechanism. (Cote J., et al, 1994)
The highly related RSC complex contains many proteins that are homologues of SWI/SNF subunits. Actually, the two complexes share at least two identical subunits .The RSC complex was initially identified by these sequence homologies and subsequently purified. The biochemical activities of the RSC complex that have been observed thus far are similar to those of ySWI/SNF (see below). However, the RSC complex is far more abundant than SWI/SNF in the yeast cell (thousands of molecules compared to 100 to 200 molecules of SWI/SNF). In addition, it contains several subunits that are essential for viability whereas none of the SWI/SNF subunits is essential (Cairns et al, 1996). In addition to these, SWI/SNF and RSC contain the same two actin-related proteins, Arp7 and Arp9 (Cairns et al., 1998). Arp7 is identical to Swp61 of the SWI/SNF complex and Rsc11 of the RSC complex, while Arp9 is identical to Swp59 and Rsc12. Yeast cells have many other known actin-related proteins (Arp1 to Arp10), many of which remain uncharacterized. It has been suggested that these actin-related proteins may link remodelling complexes to either actin-binding proteins or to nuclear proteins not previously thought to be associated with actin, such as components of the nuclear matrix or chromatin itself. Moreover, Baf53, an actin-related protein, and actin itself have been reported to be components of the mammalian SWI/SNF complexes (Wang et al., 1996).
The ISWI group:
The second group of ATP-dependent remodelling complexes contains the ISWI protein as the ATPase subunit. The most extensively studied members of this group, ACF (ATP-utilizing chromatin assembly and remodelling factor), NURF (nucleosome-remodelling factor), and CHRAC (chromatin accessibility complex), were purified from Drosophila extracts using biochemical methods based on their ability to disrupt and/or generate regularly spaced nucleosomal arrays (Tsukiyama et al, 1995). All of these complexes contain the nucleosome-dependent ATPase, ISWI, which has homology with Swi2/Snf2 exclusively over the region of the ATPase domain (Elfring et al, 1994). The ISWI-containing complexes are smaller and have fewer subunits than their SWI/SNF counterparts. NURF has a molecular mass of approximately 500 kDa and contains four subunits, including ISWI, p215, and the WD repeat protein Nurf-55, a protein identical to the 55-kDa subunit of Drosophila chromatin assembly factor dCAF-1 (Tsukiyama et al., 1998). The smallest subunit of NURF, Nurf-38, was reported to be inorganic pyrophosphatase. Both recombinant Nurf-38 and purified NURF complex have inorganic pyrophosphatase activity; however, inhibition of this activity does not affect the ability of NURF to remodel chromatin (Gdula et al., 1998). CHRAC has a molecular mass of approximately 670 kDa and contains five subunits, two of which were identified as ATPases, ISWI and topoisomerase II ( Becker et al, 1997). ACF has a molecular mass of approximately 220 kDa and contains ISWI as the catalytic subunit . Recently, the protein Acf1 was described as a component of ACF . Purification of ACF from Drosophila revealed that it exists as two complexes. Both of them contain ISWI plus one of the two Acf1 forms, p170 or p185. By contrast, Acf1 did not copurify with NURF or CHRAC. Since the predicted size of ACF is only 220 kDa, it is believed that ACF exists as heterodimers of either form of Acf1 and ISWI (Ito et al., 1998).
All eukaryotes analyzed, including yeast, plants, nematodes, flies, bovines, mice, and humans, have putative ISWI homologs. (Eisen et al, 1995). This suggests that these proteins have an important function conserved in evolution. It is also remarkable that all three of the species studied in detail to date (flies, humans, and yeast), contain several ATP-dependent remodelling complexes. The abundance of these complexes seems to be highly variable: some of them are present in a high concentration, such as yeast RSC, while others, such as ySWI/SNF, are present in much lower amounts. Finally, while some complexes seem to be dispensable, others contain essential proteins, suggesting that they are not totally redundant.
The Mi-2 group: chromatin-remodelling and deacetylase complexes:
Similar complexes that possess both chromatin-remodelling and deacetylase activities were recently purified from human cells by several different groups (Xue et al., 1998). The minor differences reported in their compositions might reflect the purification protocols used in the different cases. The complex, which we will generically refer to as hNURD, contains the retinoblastoma protein (Rb)-associated proteins RbAp46 and -48, and the Swi2/Snf2 ATPase homologue CHD4, also known as Mi-2Î². These CHD/Mi-2 proteins are known to be self antigens in the human disease Mi-2 dermatomyositis, and they have, besides the Snf2 ATPase motif, two zinc fingers and two chromo domains. The complex was also shown to contain MTA1 and MTA2, proteins that are found in metastatic cells. Recently obtained data suggest that MTA2 modulates the deacetylase activity of the hNURD complex (Zhang et al., 1998)
The functional significance of these multifunctional complexes could be very interesting. While remodelling complexes are usually associated with de-repression of transcription, deacetylases are related to repression. In this respect, it was proposed that the ATP-dependent remodelling activity of the NURD complexes might facilitate the deacetylation of the target histones. Furthermore, the presence of methyl-CpG-binding proteins in both the human and Xenopus Mi-2 complexes supports the idea that these activities might be specifically directed to methylated regions of the genome. In turn, this would lead to repression either via compaction of the chromatin structure or by allowing the binding of repressor proteins. Finally, it has been suggested that Mi-2 could be recruited to specific genes by repressors. Thus, these discoveries establish links among chromatin remodelling, deacetylation, methylation, silencing, and cancer progression (Kehle et al., 1998).
A subset of cellular genes specifically requires the function of the SWI/SNF complexes for complete activation. One possibility is that the strength of a particular promoter plays a role in its dependence on chromatin-modifying complexes. An otherwise weak promoter may require the complex for full activity, while a strong promoter may not. In support of this hypothesis, the removal of two of the four Gal4-binding sites in the Gal1-Gal10 upstream activation sequence causes the normally SWI/SNF-independent Gal1 promoter to become SWI/SNF-dependent (Burns et al., 1998). Another possibility is that remodelling complexes are only required for the transcription of promoters that possess positioned nucleosomes, which leave transcription factor sites unavailable for binding.
How are chromatin-remodelling complexes recruited to the specific promoters of the genes they regulate in living cells? Since there are only about 100 SWI/SNF molecules per yeast cell, the concentration of the complex near a target gene needs to be increased to allow remodelling of nucleosomes at specific promoters. An early report suggested that gene-specific activators might interact with components of the hSWI/SNF complex. In the presence of hSWI/SNF, Gal4-VP16 bound more strongly to nucleosomal templates than either the DNA-binding domain of Gal4 [Gal4 (1-94)] or Gal4-AH (the AH domain is a weaker activation domain than that of VP16). Since the DNA-binding domain of each of these fusion proteins is the same, Kwon et al. implied that the activation domain might play a role in the enhanced binding.(Kwon et al., 1996)
In the two primary models of SWI/SNF targeting that exist, (i) remodelling complexes are targeted to promoters via interactions with sequence-specific transcription factors and (ii) the SWI/SNF complex is recruited to promoters through association with RNA polymerase II. Studies showing association of SWI/SNF with the yeast RNA polymerase II holoenzyme and mammalian RNA polymerase II support the second model. SWI/SNF might be recruited to a promoter with RNA polymerase II, and/or SWI/SNF could recruit the transcription machinery to promote and enhance the transcription of a gene via its interaction with the holoenzyme. The Swi2/Snf2, Swi3, Snf5, and Snf11 proteins have been reported to be integral components of the mediator complex, which is tightly associated with the C-terminal domain of RNA polymerase II (Yoshinaga et al., 1992).
Many in vivo studies involving nuclear hormone receptors favor the first model, i.e., targeting of chromatin-modifying complexes via gene-specific activators. Using co-immunoprecipitation studies, an interaction between a region of the glucocorticoid receptor (GR) and Swi3 (a component of the SWI/SNF complex) was detected. However, the interaction does not occur in Î”swi1 or Î”swi2 strains, suggesting that the GR interacted with Swi3 in the context of the SWI/SNF complex. Additional studies that do not involve nuclear hormone receptors also support the activator-mediated targeting model. An association of the NURD complex (containing the Mi-2 DNA-dependent ATPase) with the DNA-binding proteins in lymphoid cells was demonstrated. In addition, the erythroid transcription factor EKLF has been shown to require E-RC1 (a SWI/SNF-related complex) to activate transcription in vitro. Finally, it has been shown that heat shock factor 1 can direct chromatin disruption in the transcribed region of the hsp70 gene (Kingston et al, 1999).
Direct evidence of an association between activator and remodelling complexes has been limited until recently. Several laboratories have recently demonstrated that the ySWI/SNF complex can directly interact with acidic transcriptional activators. Mutation of the acidic activation domains of Herpes virus VP16 and yeast Gcn4 affects their interaction with SWI/SNF. By contrast, glutamine-rich and proline-rich activation domains do not interact directly with ySWI/SNF. The acidic activators were able to interact with SWI/SNF in the context of yeast whole-cell extract. Furthermore, using an immobilized DNA template assay, it was shown that Gal4-AH and Gal4-VP16 were able to recruit SWI/SNF out of yeast nuclear extracts in a manner that was independent of RNA polymerase II holoenzyme and TATA-binding protein. Acidic activators can also recruit SWI/SNF remodelling activity to increase restriction enzyme accessibility and mononucleosome disruption by DNase I, while a proline-rich activator cannot. Targeting of the SWI/SNF complex has also been linked to gene regulation. Activator-dependent transcriptional stimulation of nucleosomal arrays upon ySWI/SNF targeting to specific promoters was demonstrated in vitro. An in vivo function for activator interactions was shown as well (.Neely et al., 1999)
All of the above studies support a positive role for SWI/SNF in transcriptional regulation via its interaction with activator proteins. However, a recent study suggests a role for gene-specific repressors in the targeting of the SWI/SNF complex. Yeast SWI/SNF components co-immunoprecipitated with the co-repressors Hir1p and Hir2p, which negatively regulate the transcription of the yeast histone HTA1-HTB1 gene locus (Dimova et al, 1999). Another possible connection between a SWI/SNF complex and repression was suggested previously. The human proteins hBRM and BRG-1 interact with Rb, which in turn interacts with E2F1.The binding of hbrm to E2F1 via Rb represses the activity of E2F1 and induces complete cell cycle arrest. (Guha et al, 1994).
Various chromatin structures are commonly divided into euchromatin and heterochromatin. Euchromatin corresponds, in general, to genome regions that possess actively transcribed genes (or potentially active ones), and that are decondensed during interphase. The regulatory sequences in these regions are accessible to nucleases and have characteristically; unmethylated CpG islands and the core histones H3 and H4 are hyper-acetylated on their N-terminal lysine residues. In general, euchromatic domains replicate early in S phase By contrast, heterochromatin refers to the transcriptionally inactive and highly condensed regions of the genome. Within heterochromatin, the DNA renders itself inaccessible to nucleases, it is usually methylated in the dinucleotide CpG and histones are markedly hypoacetylated.
Depending on whether heterochromatin is established in every cell type or limited to a particular lineage, heterochromatic domains may be further divided into constitutive or facultative heterochromatin, respectively. (Arney et al., 2004)Constitutive heterochromatin is genetically poor and forms mainly on repetitive sequences, such as satellite centromeric and peri-centromeric repeats. These regions replicate late in S-phase. Histones H3 and H4 are typically tri-methylated on lysine residues K9 and K20, respectively. In human and mouse cells, the chromatin in peri-centromeric regions is enriched in histone methyltransferases and HP1 (heterochromatin protein 1) proteins, which bind specifically to tri-methylated H3-K9( Banister et al., 2004).
Regions and DNA sequences that are subject to a developmentally regulated transcriptional silencing constitute the facultative heterochromatin. Large-scale hetero-chromatinization of the genome is frequently observed in terminally differentiated cells. Examples of sequences subject to hetero-chromatinization include genes silenced during cell differentiation, the inactive chromosome X in female mammal cells (cytologically observed as a dense nuclear structure referred to as the Barr body), and the inactive alleles of genes with monoallelic expression (such as those subject to imprinting). Lysine 27 of histone H3 is typically methylated within these regions, a mark set and recognized by proteins of the Polycomb group (PcG) (Lachner et al., 2001).
Post-translational modifications in nucleosomal histones, either at the local or the genomic level, seem to be related to transcriptional states. This is the hypothesis of the so-called 'histone code'( B.D. Strahl and C.D. Allis, The language of covalent histone modifications, Nature 403 (2000), pp. 41-45. View Record in Scopus | Cited By in Scopus (2522)Strahl et al., 2000), whereby "distinct histone amino-terminal modifications can generate synergistic or antagonistic interaction affinities for chromatin-associated proteins, which in turn regulate access to the underlying DNA(Allis et al., 2001)
More than 30 residues within each of the four octameric histone partners comprising a nucleosome are described as sites that can bemodified in the context of chromatin. These covalent modifications include acetylation, methylation, phosphorylation and ubiquitination. Particularly on their N-terminal tails, individual histones may acquire a series of modification marks in close proximity to each other. The way these modifications interact with each other and the way they correlate with the transcriptional states are currently object of significant research efforts. The emerging view is that enzymes that catalyze histone modifications and proteins that are able to read the "code" act in a concerted and highly interdependent fashion. These translators or effector proteins bind to specific modifications and recruit other regulatory or remodelling factors which, in turn, will help to maintain a particular chromatin structure, thus dictating transcriptional activity. (Fischle et al., 2003)
Chromatin modulators possess a set of conserved domains (including bromo and chromo domains) that catalize or recognize histone modifications. These protein modules bind specifically to different lysine modifications and can thus act as starting transmission points of appropriate regulatory signals. Specifically, the bromo domain interacts selectively with acetylated lysines and is in general linked to transcriptional activity, whereas the chromo domain may work as a recognition module for methylated marks and is typically associated with gene silencing and assembly of heterochromatic domains. Additionally, within lysine modifications, the protein domains may be specific depending on the position of the residue in the histone. For instance, the chromo domain from HP1 is selective for H3-K9, and only poorly binds to H3 peptides with methylated lysine K4. Moreover, lysine residues may be mono-, di- or tri-methylated, adding even more complexity to the signalling cues generated by this mark.Not all methylated marks correlate with gene silencing, and some acetylated marks repress instead of activate transcription. For instance, H3-K4 methylation seems to constitute an euchromatic mark, and methylation of arginines in histones H3 and H4 synergistically lead to trancriptional activation. By contrast, acetylation of H4-K12 seems to reinforce a silent chromatin state.(Rice et al, 2004) Banister et al., 2001)
Histone modifications are interdependent and can favour or repress other modifications. In histone H3, phosphorylation of serine 10 inhibits methylation of K9, and may act in a synergistic manner with acetylations of K9 and K14, or methylation of K4. On the other hand, deacetylation of H3-K14 facilitates the subsequent methylation of K9.
DNA Methylation as an Epigenetic Mechanism:
In the nucleus of mammal cells, the stable silencing of a gene, i.e. maintained in a hereditary manner, is frequently correlated with DNA methylation in its promoter, along with specific modifications in the N-terminal tails of nucleosomal histones. DNA methylation in mammals occurs in the cytosine of the CpG dinucleotide via a reaction catalysed by proteins named DNA methyltransferases (DNMT). In mammals, there are three of these proteins whose presence is crucial to embryonic development: DNMT1, DNMT3A and DNMT3B and. DNMT1 is referred to as the maintenance methyltransferase, as it possesses the capacity to reproduce the methylation pattern of a DNA sequence during replication, due to its preference to hemi-methylated substrates. The proteins DNMT3A and DNMT3B are mainly involved in de novo methylation. They are therefore important for the establishment of new methylation patterns of the genome (Bird et al., 1999).
The CpG islands, regions with more than 500bp and a G+C content larger than 55%, are localized in the promoter regions of 40% of all the genes in mammals and are normally maintained in the non-methylated form and. The stable silencing of tumour suppressor genes in several human cancers, as well as of lineage specific genes during cell differentiation, frequently involves methylation of CpG islands, but these modifications seem to be preceded by modifications of nucleosomal histones. A complex interplay between histone and DNA marks may then stabilize the repressive chromatin structure and thereby manifest to transcriptional inactivity (Croce et al, 2004).
Dynamics of Histone Modifications:
The dynamic nature of histone modifications determines the stability of a given chromatin structure. The concerted relationship between histone acetyltransferases and deacetylases determines the level of acetylation. By contrast, the methylated marks are e considered as stable modifications that might contribute to epigenetic memory at long course. Methylation of H3-K9 is mostly associated with the assembly of heterochromatin and to the stable silencing of genes. By contrast, methylation of H3-K4 and of some arginines in histones H3 and H4 are related with transcriptional activation. In these cases, for the dynamic regulation of gene expression, the methylated mark has to be actively and dynamically removed. Only recently two different classes of enzymes were described that are capable of removing the methylation of lysines via an oxidative reaction , or of antagonising arginine methylation by conversion into citrulline and. Recently, a new family of proteins containing the JmjC domain with histone demethylases activity has been described. (Katan-Khaykovich et al., 2006)
One of the first evidences for a functional relationship between DNA and histone methylation came from work in Neurospora crassa, where it was shown that mutations in the HMT that methylates H3-K9 severely compromises genomic DNA methylation. A recently described functional link between proteins of the Polycomb group and DNA methyltransferases further suggest that heritable patterns of gene silencing may be established and sustained by the interconnection of these major silencing pathways. This interaction is achieved through a mechanism that involves the direct recruitment of DNMTs (DNMTs 1, 3A and/or 3B) to regulatory regions of PcG-repressed genes by the H3-K27 methyltransferase EZH2, and the resulting methylation of local CpG dinucleotides, thus implying for the presence of a self-reinforced set of chromatin modifications working in concert to establish and propagate the structure of a silenced state, potentially all through different cell generations. This might work as a platform for the deposition of linker histones and for the binding of additional epigenetic factors, such as methyl-binding proteins, components of the polycomb repressive complex 1 (PRC1), and HDACs and H3-K9 HMT (Hernandez-Munoz et al, 2005) (Sewalt et al, 2002)
Chromatin structure affects gene expression as well as replication, recombination and DNA repair. Several human diseases are linked to or are even based on defects in the machinery maintaining and/or modifying chromatin structure. For instance, DNA methylation patterns are severely altered in tumors with a bias for overall hypo-methylation of the genome and hypermethylation of specific CpG rich regions. Locally restricted hypermethylation can lead to gene repression, while genomic hypo-methylation particularly of repetitive sequences was suggested to relate to increased genomic instability. So far hypo-methylation is known to coincide with loss of acetylation at K16 and trimethylation at K20 of histone H4, strengthening the notion of a co-operation between DNA and histone modifications in the establishment of both normal and abnormal patterns of chromatin structures. (Gaudet et al, 2003).
Research performed in recent years, however, has shed some light on the mechanism leading to locally restricted hypermethylation of some promoters and the consequential silencing of their genes. Such a hypermethylation can result from the abnormal recruitment of DNA methyltransferases by transcription factors which confer locus specificity. Deregulated transcription factors are considered to play a dominant role in the development of leukemias. This idea is supported by analysis of gene-knockout mice, which uncovered crucial roles of several transcription factors in normal haematopoiesis, and of individuals with leukemia, in whom transcription factors are frequently miss-expressed or mutated. Several chromosomal translocations, which are associated with specific forms of leukemia, generate fusion genes that encode altered transcription factors. (Look et al, 2004)
Combinatorial treatment of retinoic acid and epigenetic drugs (such as HDAC/HMTs inhibitors and/or DNA demethylating agents) is more likely able to revert these chromatin modifications thus regaining transcription of the silenced gene. (Croce et.al, 2005)
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