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ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 1 Development Team Paper Coordinat or: Prof. Kuldeep K. Sharma Department of Zoology, University of Jammu Principal Investigator: Prof. Neeta Sehgal Department of Zoology, University of Delhi Content Write r: Dr. Renu Solanki , Deen Dayal Upadhyaya College, Delhi University Dr. Sudhida Gautam, Hansraj College, Delhi University Mr. K iran kumar Salam, Hindu College, Delhi University Content Reviewer: Prof. Rup Lal Department of Zoology, University of Delhi Co - Principal Investigator : Prof. D.K. Singh Department of Zoology, University of Delhi Paper : 15 Molecular Cell Biology Module : 26 Cell Regulatory Mechanisms: Chromatin Remodeling ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 2 Description of Module Subject Name ZOOLOGY Paper Name Molecular Cell Biology; Zool 015 Module Name/Title Cell Regulatory Mechanisms Module Id M 26 ; Chromatin Remodeling Keywords Chromatin, Histone, Nucleosome, Chromatosome, Chromatin re modeling, Histone ac

etylation, Histone methylation, Histone phosphorylation Contents 1. Learning Objectives 2. Introduction 3. Experimental Evidence for Deducing Nucleosome Model 4. ATP Dependent Chromatin Remodeling i. ATP Dependent Chromatin Remodeling Complexes (a) Ro les of Remodeling Complexes ii. Covalent Modifications of Histones (a) Histone Acetylation (b) Histone Methylation  Histone Methylation in X Chromosome Inactivation for Dosage Compensation (c) Histone Phosphorylation 5. Role of Remodelling Complexes in Nucleosome Organization at the Promoter 6. Role of Histone Modifications in DNA Repair 7. Chromatin Remodeling and Cancer 8. Summary ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 3 1. Learning Outcomes  Need for chromatin remodeling  Experimental evidence to prove packaging of DNA as chromatin.  Nucleosome structure  Outcomes of chromat in remodeling  ATP dependent chromatin remodeling  Roles of Remodeling complexes in Nucleosome Organization at the promoter  Histone covalent modifications: Purpose and functions 2. Introduction Chromatin is the complex of eukaryotic DNA and proteins which re sult in the formation of chromosomes in eukaryotic nucleus. Chromatin remodeling helps us to open up the chromatin and facilitate the expression of individual genes ( Figure 1). DNA is several meters long thus, with time it has developed to coil itself arou nd histone proteins to fit itself into the nucleus of a cell and this complex is refe

rred to as chromatin. Histones are major proteins of chromatin. Histones have high proportion of basic amino acids like arginine and lysine. These basic amino acids facil itate the winding of negatively charged DNA around histones. Therefore, nuclear DNA in eukaryotic nucleus does not exist as free linear strands but highly condensed. Figure 1 . Structure of Chromatin . Source : http://www.mdpi.com/biology/biology - 02 - 01378/article_deploy/html/ images/biology - 02 - 01378 - g001 - 1024.png ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 4 Chromatin contains two types of proteins histone and non - histone . There are f ive major types of histones including H1, H2A, H2B, H3, and H4 and it has been found that they are very similar among different species of eukaryotes. The basic structural unit of chromosome is nucleosome which was described by Roger Kornberg in 1974 ( Figu re 2). Roger D. Kornberg received Nobel Prize in chemistry 2006. Figure 2 . Roger D. Kornberg Source: https://med.stanford.edu/profiles/roger - kornberg 3. Experiments for Deducing Nucleosome M odel Chromatin is digested with microcococcal nuclease (An enzyme that degrades DNA). It was found that DNA fragments of approximately 200 base pairs were produced. In contrast, when naked DNA which was not associated with proteins was subjected to microc ococcal nuclease digestion, a continuous smear of different size fragments was produced. These results suggested that binding of proteins to DNA restricted the action of enzyme to a limited area

i.e. approximately at a space of 200 base pairs. Electron mi croscopy also revealed that chromatin fibers exist as beaded structure in which beads are present at a space interval of approximately 200 base pairs. Therefore, it can be suggested on the basis of nuclease digestion experiment and electron microscopy that chromatin is composed of repeating 200 - base - pair units, which were called nucleosomes ( Figure 3). ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 5 Figure 3 . Structure of nucleosome Source : http://www.nature.com/nri/journal/v2 /n3/fig_tab/nri747_F1.html When chromatin is subjected to extensive digestion with micrococcal nuclease, fragments corresponding to 146 base pairs were found called nucleosome core particles; these were observed in the form of beads by electron microscopy ( Figure 4). A detailed analysis has shown that these particles contain 146 base pairs of DNA wrapped 1.65 times around a histone core consisting of two molecules each of H2A, H2B, H3, and H4. There is a single molecule of fifth histone, H1, also called li nker histone which bound to DNA as it enters each nucleosome core particle. The chromatin subunit having 166 base pairs of DNA wrapped around the histone core and held in place by H1 is called chromatosome ( Figure 3). ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 6 Figure 4 . Digestion of chromatin with micrococcal nuclease . Source : Molecular Biology of the Gen

e by Watson et al.) Binding of H1 stabilizes higher - order chromatin structures. First of all, DNA wraps around the histone proteins forming nucleosomes and gives an ap pearance of beads on a string. Further DNA is packaged into 30nm fibre, which forms loops averaging 300 nanometers in length. The 300 nm fibres are further compressed and folded to produce 250nm wide fibre, which is tightly coiled into the chromatid of a c hromosome ( Figure 5). Chromatin remodeling helps to uncoil the DNA and expose the transcription sites having the promoters and terminators . ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 7 Figure 5 . Chromatin remodeling Source : http://america.pink/images/9/8/6/0/6/3/en/2 - chromatin - remodeling.jpg The extent of chromatin condensation varies during different phases of cell. In interphase (non - dividing) cells, most of the chromatin which called euchromatin is in relatively d econdensed and distributed throughout the nucleus . During this period of the cell cycle, DNA is replicated and genes are transcribed for preparing cell for division. Most of the euchromatin in interphase nuclei appears to be in the form of 30 - nm fibers and about 10% of the euchromatin, containing the genes that are actively transcribed, is in a more decondensed state (the 10 - nm conformation) that is required for transcription ( Figure 5). Chromatin structure is thus directly linked to the control of gene exp ression in eukaryotes. Chromatin remodeling is the dynamic modification of chromatin architecture to

allow access of condensed genomic DNA to the regulatory transcription machinery proteins, and thereby control gene expression. In simple words, it can be d efined as t he general process of inducing changes in chromatin structure. There are several alternative outcomes of chromatin remodeling 1. Sliding of nucleosome: Histone octamers may slide along DNA, changing the relationship between the nucleic acid and pr otein. It alters the position of a particular sequence on the nucleosomal surface. ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 8 2. Reorganization of space between nucleosome: The spacing between histone octamers may be changed, again with the result the positions of individual sequences are altered rela tive to protein. It results in alteration of the position of a particular sequence on the nucleosomal surface. 3. Displacement of nucleosome from DNA: The most extensive change is that an octamer(s) may be displaced entirely from DNA to generate a nucleosome free gap. Alternatively, one or both H2A - H2B dimers can be displaced ( Figure 6). Figure 6. Changes in nucleosome organization by remodeling complexes With the help of this process DNA accessibility to various proteins or factors alters and for this spec ialized proteins are required sequentially. Chromatin remodeling is required to change the architecture or organization of nucleosomes at the promoter of the gene that is to be transcribed so that the transcription machinery can get access to the promoter to start the transcription. It can also prevent transcr

iption by moving away the essential promoter sequences. Chromatin remodeling also plays an important role in other manipulations of chromatin, including repair of damaged DNA. It is required for DNA recombination including immunoglobulin gene rearrangement and DNA replication. 4. ATP Dependent Chromatin Remodeling Alteration in Chromatin structure is accomplished by - (i) ATP Dependent Chromatin Remodeling Complexes Chromatin remodeling consists of me chanisms for displacing histones and it is energy dependent. Histones can be released from chromatin when there would be disruption of many protein - protein and ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 9 protein - DNA interactions. This could be possible by providing energy. This is accomplished by AT P - dependent chromatin remodeling complexes, which use ATP hydrolysis to provide the energy for remodeling. Remodeling complexes range from small heterodimeric complexes which have the ATPase subunit with a single partner to large complexes consisting of te n or more subunits. Among different nucleosome remodeling complexes, ATP hydrolyzing subunit is similar which catalyzes DNA movement, whereas the other subunits associated with each complex modulate their function. For example, some of these complexes can have subunits that target them to particular chromosomal locations and this targeting is mediated by interactions between subunits of the remodeling complex and DNA bound transcription factors. On the other hand, some nucleosome remodeling complexes have s ubunits that localized them to

specific modifications of the histone amino terminal tails (via chromodomains or bromodomains). These complexes may vary in their remodeling activities. Multiple types of nucleosome remodeling complexes are present in any giv en cell. The most important component of remodeling complex is its ATPase subunit (Table: 1). ATPase subunits of all remodeling complexes are related members of a large protein superfamily which is further subdivided into subfamilies. Remodeling complexes classification is based on the subfamily of ATPase that they contain as their catalytic subunit. Major subfamilies are SWI/SNF, ISWI, CHD and INO80/ SWR1 Table 1 . ATP dependent Nucleosome Remodeling Complexes Type Number of subunits Histone Binding Domain s Slide DNA on histone octamer Transfer of histone octamer from one DNA helix to another Examples SWI/SNF 8 - 11 Bromodomain Yes Yes Yeast: SWI/SNF, RSC (Remodels the structure of chromatin) Fly: dSWI/SNF (Brahma) Human: hSWI/SNF ISWI 2 - 4 Bromodomain, SANT domain, PHD finger Yes No Yeast: ISW1a, ISW1b, ISW2 Fly: NURF (nucleosome - remodeling factor), CHRAC (chromatin ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 10 accessibility complex), ACF ACF (ATP - utilizing chromatin assembly and remodeling factor) Human: RSF, hACF/WCFR, hCHRAC Frog: WICH, CHRAC, ACF C HD 8 - 10 Chromodomain, PHD finger Yes No Yeast: CHDI Fly: JMIZ Human: NuRD Frog: Mi - 2 SWR1 12 - 14 Bromodomain, SANT domain Yes Nd Yeast: SWRI Human: SRCAP INO80

10 - 12 None Yes Nd Yeast: INO80 Fly: Tip60 Human: SRCAP Nd: not determined SWI/SNF (“switch sni ff”) complex in yeast was the first described remodeling complex which has homologs in all eukaryotes. The chromatin remodeling superfamily is large and diverse, and most species have multiple complexes in different subfamilies. SWI/SNF is the prototypic r emodeling complex. Many of its subunits are encoded by genes originally identified in swi or snf mutants of Saccharomyces cerevisiae ( swi mutants cannot switch mating type, and snf sucrose non fermenting mutants cannot use sucrose as a carbon source), as its name indicated. SWI/SNF acts catalytically in vitro, and there are only about 150 complexes per yeast cell. All of the genes encoding the SWI/SNF subunits are nonessential, which implies that yeast must also have other ways of remodeling chromatin. Th e related RSC (remodels the structure of chromatin) complex is more abundant and also is essential. It acts at about 700 target loci. 2% of Saccharomyces cerevisiae genes require SWI/SNF complex to remodel chromatin at their promoters. Different subfamilie s of remodeling complexes show distinct modes of remodeling which indicates differences in their ATPase subunits as well as effects of other proteins present in individual remodeling complexes. For instance, SWI/SNF complexes can remodel chromatin by slidi ng DNA on ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 11 the surface of histone octamer or by displacement of octamer to a different DNA molecule. On the

other hand, ISWI family primarily affects nucleosome positioning by a sliding reaction in which histone octamer moves and there is no displacement of octamers. The activity of ISWI requires the histone H4 tail as well as binding to linker DNA. The ATPase subunits are distantly related to helicases but they do not have any unwinding activity as present in helicases. It is assumed that in SWI/SNF and IS WI classes of remodeling complexes, there is hydrolysis of ATP which is used to twist DNA on the nucleosomal surface. As a result of twisting, mechanical force is generated that allows a small region of DNA to be released from the surface of octamer and t hen repositioned. Consequently, there is formation of transient loops of DNA on the surface of the octamer. These loops are themselves accessible to interact with other factors or they can propagate along the nucleosome, resulting in nucleosome sliding. (a ) Roles of Remodeling Complexes 1. SWI/SNF complexes are generally involved in transcriptional activation. 2. Some of ISWI complexes act as repressors. They use their remodeling activity to slide nucleosomes onto promoter regions and prevent transcription. 3. Memb ers of the CHD (Chromodomain helicase DNA binding) family also act as repressors, particularly the Mi - 2/NuRD complexes which contain both chromatin remodeling and deacetylase activities. 4. Remodelers in the SWRI/INO80 class have a unique activity: Some memb ers of this class also have histone exchange capability in addition to their normal remodeling capabilities. They are responsible for replacement of individual histones (

usually H2A/H2B dimers) typically with a histone variant. (ii) Covalent Modifications of Histones All histones are subjected to various covalent modifications which occur mostly in the histone tails. All histones can be modified at numerous sites by methylation, acetylation, phosphorylation, mono - ubiquitylation, sumoylation, and ADP - ribos ylation. Most common target for covalent modifications in histone tails are lysine residues. ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 12 Figure 7. Histone tails are the sites of many modifications. Histones H3 and H4 N terminal tails can be acetylated, methylated or phosphorylated. Source: Genes IX by Benjamin Lewin Acetylation, methylation, ubiquitylation and sumoylation all occur on the free epsilon amino group of lysine ( Figure 7). There is neutralization of positive charge that is present on NH 3 + - amino group by acetylation, whereas positive c harge is retained in case of lysine methylation and lysine can be mono - ,di - , or trimethylated ( Figure 8). Phos - phorylation occurs on the hydroxyl group of serine and threonine and it introduces a negative charge in the form of the phosphate group. ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 13 Fig ure 8 . Lysine and serine residues in Histone tails are the targets for modifications. Overall positive charge is reduced by acetylation of lysine and phosphorylation of serine. Source: Genes IX by Benjamin Lewin All these modifications are transient and br ing abou

t change in the charge of the protein molecule, and the functional properties of the histone octamers. Histone modifications are associated with structural changes in chromatin that are required for DNA replication and transcription, and specific m odifications also facilitate DNA repair. Modifications at specific positions on specific histones are associated with different functions of chromatin. Combinations of specific histone modifications define the function of local regions of chromatin; that is known as histone code. Protein interacts with chromatin by recognizing differently modified sites. For example, Proteins interact with chromatin with the help of bromodomain by recognizing acetylated lysines. Different acetylated targets are recognized by different bromodomain containing proteins. On the other hand, different domains of proteins recognize methylated lysine and arginines and also able to distinguish between mono, di or trimethylatedlysines. The domains present in chromatin associated pr oteins which can recognize methylated lysines are chromodomain, PHD (plant home - odomain) and Tudor domains. (a) Histone Acetylation All the core histones are subject to multiple covalent modifications. Different modifications are required for different fu nctions. Histone acetylation and deacetylation is one of the important and extensive modifications. In this modification, lysine residues within N terminal tail protruding from the histone core of the nucleosome are acetylated and deacetylated which is a p art of gene regulation also. Acetylation and deacetylation of histones is reversible and ca

talyzed by specific type of enzymes. Histone acetytransferases (HATs) are responsible for acetylation of histone ( Figure 9). During acetylation , HATs transfer an ac etyl group from a molecule of Acetyl Coenzyme - A (Acetyl - CoA) to the NH 3 + group on Lysine. There are two classes of HAT enzymes: enzyme of group A are involved in transcription regulation or gene expression and act on histones in chromatin, enzymes in group B are involved in nucleosome assembly and therefore act on newly synthesized histones during DNA synthesis in S phase. Group A HATs enzymes are typically found in large complexes, like ATP - dependent remodeling enzymes. These can be targeted to DNA - bindin g factors. ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 14 Acetyl groups are removed by histone deacetylases (HDACs). There is a decrease in positive charge because of acetylation which decreases the interaction of the N termini of histones with the negatively charged phosphate groups of DNA. Consequ ently, condensed chromatin is transformed into a more relaxed structure that is associated with greater levels of gene transcription. This can be correlated because it has been found that histone acetylation is increased in active genes, and acetylated chr omatin is more sensitive to DNAse I. On the other hand, the relaxed chromatin is changed into condensed chromatin by HDAC activity which is associated with transcription repression. Modifications of nucleosome can be restricted to a small area (local event ) for example, at the promoter region or exten

d over large domains or even to an entire chromosome as in the case of sex chromosomes. Acetylation is the mechanism involved in dosage compensation of 2X chromosomes in females and 1X chromosome in males in ma mmals. In females, the inactive X chromosome has underacetylated histones. In Drosophila males, super active X chromosome has increased acetylation of H4. This indicates that acetylation is prerequisite for a less condensed, active structure. In male Droso phila , K16 of histone H5 is specifically acetylated by MOF (males absent on the first) enzyme. This enzyme is recruited as part of large a large protein complex and introduces changes mainly acetylation in the X chromosome that enable it to be more highly expressed. Therefore, X chromosome in male Drosophila shows high level of expression because of its acetylation. Trichostatin and butyric acid inhibit histone deacetylases, and cause accumulation of acetylated nucleosomes. Therefore, use of these inhibitor s has been found useful in analyzing acetylation and its correlation with gene expression . It is known that acetylation is linked to activation and deacetylation is linked to transcriptional repression. Therefore, site - specific activators recruit coactiva tors with HAT activity whereas site - specific repressor proteins can recruit corepressor complexes, which often contain HDAC activity. ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 15 Figure 9 . Acetylation and deacetylation of Histones (b) Histone Methylation It is a process by which methyl groups are t ransferr

ed to amino acids of histone proteins. Transcriptional level of genes can either increase or decrease, depending on the specific site of methylation , i.e., which and how many amino acids in the histones are methylated. It has been found that such m ethylation events increase transcriptional level only if they are involved in weakening the attraction forces between histone tails and DNA as they enable the uncoiling of DNA from nucleosomes and increase accessibility of DNA to transcription factor prote ins and RNA polymerase. This process is mainly responsible for differential expression of genes in different cells. Histones can be methylated on lysine (K) and arginine (R) residues. There are numerous sites of lysine methylation in the tail and core of h istone H3, and a singly lysine in the tail of H4. In addition, three arginines in H3 and one in H4 are also methylated. Di or trimethylation of H3K4 is associated with transcriptional activation, and trimethylated H3K4 occurs around the start site of acti ve genes ( Figure 10). In contrast, H3 methylated at K9 or K27 is feature of transcriptionally silent regions of chromatin, including heterochromatin and smaller regions containing one or more silent genes. Histone methylation is catalyzed by histone methyl transferases which transfer groups from S - Adenosyl methionine onto the lysine or arginine residues of the H3 and H4 histones. The histone methyltransferases are specific to either lysine or arginine. The lysine - specific transferases are further classified depending on the presence of SET domain or a non - SET domain. Like acetylati

on, methylation is reversible, and two different families of lysine demethylases (KDMs) have been ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 16 identified: LSD1 (lysine - specific demethylase 1, also known as KDM1) family and the Jumonji family. Different classes of enzymes demethylatearginines. Figure 10 . Histone Methylation and Demethylation . Source: http://pharmacy.wisc.edu/jiang - lab/research  Histone Methylation in X C hromosome Inactivation for Dosage Compensation In female organisms, there are two copies of X chromosome and if both X chromosomes would be transcriptionally active it would double the amount of protein products transcribed. Therefore, one X chromosome is inactivated to compensate the protein products transcribed by X chromosomes. This inactivation is brought about by the condensation of X chromosome and mediated by methylation of the different lysine residues of different histones. X chromosome inactivatio n is a random process and mediated by the non - coding RNA XIST. Although methylation of lysine residues occurs on many different histones, the most characteristic of Xi occurs on the ninth lysine of the third histone (H3K9). While a single methylation of th is region allows for the genes bound to remain transcriptionally active, in heterochromatin this lysine residue is often methylated twice or three times, H3K9me2 or H3K9me3 respectively, to ensure that the DNA bound is inactive. ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 17

Figure 11 . Three types o f modifications affect chromatin: Acetylation of histones activates chromatin whereas methylation of DNA and histones inactivates chromatin. Source: Genes IX by Benjamin Lewin (c) Histone Phosphorylation Histone phosphorylation is linked to transcription, repair, chromosome condensation and cell cycle progression Histones are phosphorylated cyclically during the cell cycle, in association with chromatin remodeling during transcription and during DNA repair. In the cell cycle histone phosphorylation is like ly to be a signal for condensation. Its effect in transcription and repair appears to be opposite, where it contributes to open chromatin structures compatible with transcription activation and repair processes. Covalent modifications of chromatin are res ponsible for various functions Most sites in histones have a single and specific type of modification, but some sites can have more than one type of modification. Individual functions can be assigned to some of the modifications as depicted in table (2): ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 18 Table 2 . Specific Histone Modifications and functions Histone Site Modification Function H3 K - 4 Methylation Transcription activation H3 K - 9 Methylation Chromatin condensation K - 9 Methylation Required for DNA methylation K - 9 Acetylation Transcription activation H3 S - 10 Phosphorylation Transcription activation H3 K - 14 Acetylation Prevents methylation at Lys - 9 H3 K - 79 Methylation Te

lomeric silencing H4 R - 3 Methylation H4 K - 5 Acetylation Nucleosome assembly H4 K - 12 Acetylation Nucleosome assembly H4 K - 16 Acetylation Nucleosome assembly K - 16 Acetylation Fly X activation 5. Role of Remodeling Complexes in Nucleosome Organization at the Promoter Remodeling complexes are recruited to specific sites on chromatin with the help of activators or someti mes by repressors. For example, yeast HO gene (gene involved in mating type switching) is activated by transcription factor Swi 5. This transcription factor enters the nucleus near the end of mitosis, binds to HO promoter and recruits SWI/SNF remodeling co mplex to the promoter. It is then released leaving behind SWI/SNF complex at the promoter. It means that a transcription factor can activate a promoter by a “hit and run” mechanism, in which its function is fulfilled once the remodeling complex has bound. The SWI/SNF complex is also recruited to promoters through association with RNA polymerase II. There is involvement of remodeling complexes in gene activation as they are essential for certain transcription factors to activate their target genes. For exam ple, GAGA factor need NURF remodeling complex for activation of Drosophila hsp70 p romoter. GAGA factor binds to four (CT ) n rich sites ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 19 near the promoter disrupts the nucleosomes and creates a hypersensitive region. It also causes rearrangement of nucleosom es so that they occupy preferential instead of random positions. Di

sruption of nucleosome is an energy dependent process and is facilitated by the NURF remodeling complex, a complex in the ISWI subfamily. Figure 12: Steps involved in the activation of pr omoter (i) Binding of transcription factors to specific sequence (ii) Binding of Remodeling complex via factors (iii) Relea se of transcription factor (iv) Changing of nucleosome organization by remodeling complexes (v) Binding of Acetylase complex via remo deling complexes and (vi) Finally acetylation of histones. ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 20 6. Role of Histone Modification in DNA Repair Chromatin in the vicinity of DNA damage must be modified and remodeled to increase the accessibility to the repair machinery. The original chromatin st ate must be restored after repair is completed. Both histone modification and chromatin remodeling are essential for repair of DNA damage in chromatin. H2A phosphorylation (γ - H2AX) is a conserved double - strand break - dependent modification that recruits chromatin modifying activities and facilitates assembly of repair factors. Different patterns of histone modifications may distinguish stages of repair or diff erent pathways of repair. Remodelers and chaperones are required to reset chromatin structure after completion of repair. A number of chromatin - remodeling enzymes act at double - strand breaks. Subfamilies of chromatin remodeling complexes which are involved in double - strand break repair are the SWI/SNF and RSC complexes of theSNF2 subfamily, the INO80 and SWRI complexes of the INO80 gro

up, and RAd54 and Rdh54 of the Rad54 subfamily. Chaperones Asf1 and CAF - 1 are required to restore chromatin structure on th e newly repaired region and allow recovery from the DNA damage checkpoint. 7. Chromatin Remodeling and Cancer It is now widely recognized that epigenetic events because of chromatin remodeling complexes and covalent modifications of histones are importan t mechanisms underlying cancer development and progression. Both of these mechanisms work in a coordinated and ordered fashion to regulate cellular processes such as DNA replication, gene transcription and DNA repair. Alterations in the functions of either chromatin remodeling complexes or covalent modifications of histones may result in impairments of above mentioned cellular processes which ultimately lead to oncogenic transformation and the development of cancer. For example, in humans disruption in the regulation of histone deacetylase leads to the uncontrolled proliferation of immature blood cells known as myelocytes and to acute myeloid leukemia (AML) and acute promyelocytic leukemia (PML). 8. Summary Chromatin is the complex of DNA and proteins in euk aryotes and result in the formation of chromosomes. Major proteins of chromatin are histones. There are five major types of histones H1, H2A, H2B, H3 and H4. Nucleosome is the structural unit of chromosome formed by DNA and five histone proteins. One nucle osome has a histone core and 200bp of DNA. Histone core consists of two ZOO LOGY Molecular Cell Biology Cell Regulatory Mechanisms : Chromatin Remodeling 21 molecules of each histone proteins H2

A, H2B, H3 and H4, around which 146 bp of DNA is wrapped and remaining base pairs act as linker DNA. The fifth histone protein is present in linker DNA. Structure of nucleosome is deduced by digestion of chromatin DNA with micrococcal nuclease. Initially, DNA wraps around histone proteins forming nucleosome and further packaged into 30nm fibre, 300nm fibre and finally into chromatid of chromosomes. Chromatin condensation and de - condensation varies in different phases of a cell. Therefore, chromatin structure alters to allow the expression of genes. This structural alteration of chromatin is called chromatin remodeling. Chromatin remodeling may result in nucleosome sliding, variations in space between nucleosome and displacement of nucleosomes from DNA. Chromatin structure may be altered by ATP dependent chromatin remodeling complexes. All remodeling complexes have a common ATPase subunit and other sub units number may vary from 1 to more subunits. Some of the ATP dependent nucleosome remodeling complexes is SWI/SNF, ISWI, CHD, SWRI and INO80. These remodeling complexes are important for transcriptional activation and repression. Histones are also coval ently modified by methylation, acetylation and phosphorylation. Most common targets for histone acetylation and methylation are lysine residues in histone tails. Phosphorylation occurs on the hydroxyl group of serine and threonine. All these modifications are transient and bring about changes in the charge of the protein molecule and functional properties of histone octamers. These modifications play an important role in DNA replication, transcriptio