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Regulation of chromatin structure

Regulation of chromatin structure


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Recently, I reviewed the different levels of chromatin structure. The primary level is nucleosomes, where DNA is bound to histones, and has structural similarity to "beads on a string." The secondary level is a 30nm fiber, and the tertiary level is formed by radially looping the fibers.

I've also been learning about the histone code and how different modifications to the core histones relate to transcriptional regulation. Are these modifications the primary regulation mechanism for chromatin structure? In other words, does chromatin assume the most compact structure possible until histone modifications are made to enable transcription? Or have other regulatory mechanisms unrelated to transcription been discovered and characterized?


"Are these modifications the primary regulation mechanism for chromatin structure?"

It depends on how you define primary, we might currently think of histone modifications as primary because other regulatory mechanisms have not yet been well studied. Something else you can think of are the various regulatory proteins that interact with histone marks to modify chromatin.

I don't think you should imagine chromatin as assuming "the most compact structure possible until histone modifications are made to enable transcription" but rather histone modifications being a dynamic process with various transcription factors (a class of proteins) coming in and adding/removing histone marks as well as 'remodeling chromatin' (adding/removing nucleosomes)

As a sidenote, I don't believe much is known about the tertiary stage you initially mentioned so maybe that could play a huge role in the regulatory mechanisms of chromatin structure, it has just not been well explored.


Regulation of Chromatin Structure and gene expression

The long term objective of my laboratory is to gain a molecular understanding of epigenetic processes that regulate chromatin structure and gene expression. Towards this end we have identified a novel tandem kinase in Drosophila, JIL-1, that localizes specifically to the gene-active interband regions of the larval polytene chromosomes, phosphorylates histone H3S10, and is enriched almost two-fold on the transcriptionally hyperactive male larval polytene X chromosome. In JIL-1 hypomorphs orderly interband regions of polytene chromosomes are disrupted and the chromosome arms highly condensed. Position effect variegation (PEV) in Drosophila has served as a major paradigm for the identification and genetic analysis of evolutionarily conserved determinants of epigenetic regulation of chromatin structure and gene silencing and we provide evidence that loss-of-function alleles of the JIL-1 histone H3S10 kinase can act either as suppressors or enhancers of PEV depending on the chromatin environment of the reporter locus. These effects on PEV were correlated with the spreading of the major heterochromatin markers dmH3K9 and HP1 to ectopic locations on the chromosome arms with the most pronounced upregulation found on the male and female X chromosomes. Based on these findings we propose a model where JIL-1 kinase activity and phosphorylation of histone H3S10 at interphase functions to antagonize heterochromatization by regulating a dynamic balance between factors promoting repression and activation of gene expression.

Gene silencing is a critical developmental process relevant to many human health problems that include cancer. Furthermore, JIL-1 is the Drosophila homolog of the mammalian MSK1 kinase which also functions as a regulator of chromatin structure by phosphorylating the histone H3S10 residue. At present the mammalian studies have been directed towards analyzing histone phosphorylation in the context of immediate early gene transcription. However, our results suggest that the concept of histone phosphorylation should be expanded to be considered in the context of the regulation of gene silencing as well. Thus, our studies will serve to provide general insights into the molecular mechanisms of how kinase activity modulates chromatin structure and gene regulation that are directly relevant to humans.


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Principles of the metabolic link to epigenetics

Despite the challenges in completely understanding the context-dependent roles of the metabolism𠄾pigenetics axis and the complexity in metabolic pathways and chromatin modifications regulated by them, there are several universal principles underlying this cross-talk, that demonstrate the evolutionary emergence of specific molecular mechanisms that facilitate epigenomic dynamics under metabolic alterations.

The ability of epigenetic modifications to respond to fluctuations in metabolic activities is a consequence of the intrinsic thermodynamic and kinetic parameters of chromatin-modifying enzymes (Box 1). Addition and removal of most of these modifications are catalyzed by enzymes (i.e. ‘writers’ and 𠆎rasers’) that utilize metabolites as substrates or cofactors (i.e. chromatin-modifying metabolites ( Figure 1 )). Chromatin-modifying enzymes that use metabolite substrates whose physiological concentrations are close to or lower than the enzymes’ intrinsic Km and Kd values [G] are more susceptible to metabolic pathway alterations than those whose substrates are present in excess amounts 13 . This property thus enables metabolic fluctuations to influence the activities of certain chromatin-modifying enzymes and modulate the levels of specific epigenetic modifications, and the difference in substrate availabilities and Km values may determine relative sensitivities of epigenetic modifications to metabolic alterations 24,25 (Box 1). On the other hand, chromatin modifications can also be added non-enzymatically, of which the detailed kinetic and thermodynamic properties are less well characterized but are influenced to some extent by the law of mass action.

The abundance of chromatin-modifying metabolites is intracellularly regulated by several mechanisms. Metabolites that are taken up by cells can passively or actively diffuse through the plasma and nuclear membrane in order to modify chromatin. Alternatively, metabolites can be processed internally by the activity of metabolic enzymes that convert them into substrates or co-factors for chromatin-remodelling enzymes. These enzymes can also translocate to the nucleus where they can locally produce substrates for chromatin modification. The resultant consequence of metabolite abundance on the rate of chromatin modification is dependent upon the kinetic and thermodynamic parameters of the enzyme. Enzymes with initial [S]/Km ratios on the highlighted linear part of the displayed curve are more susceptible to perturbations to substrate concentrations — these include methyltransferases and acetyltransferases, among others. Finally, once the modifications have been deposited, effector proteins can recognize and bind them using specific protein-binding modules, upon which they determine a variety of intracellular fates including the regulation of homeostasis, development, immune regulation and tumorigenesis.

Box 1:

Kinetic and thermodynamic properties of chromatin-modifying enzymes

Rates of enzyme reactions depend on many factors including intrinsic enzymatic parameters such as turnover number and Michaelis–Menten constant (Km) values, enzyme abundance, substrates, cofactors and allosteric activators or inhibitors, and other environmental factors such as pH, temperature, and local viscosity. These variables together determine the overall reaction rate through a quantitative relationship that depends on the molecular mechanism of the enzymatic reaction 236 . Studies describing the biochemistry and structural biology of chromatin-modifying enzymes have provided crucial insights about their catalytic mechanisms and regulators. Kinetic mechanisms of enzymes involved in DNA methylation, histone acetylation and histone methylation have been extensively investigated particularly when issues around drug development are concerned, showing that the exact catalytic mechanism for each chromatin-modifying enzyme is diverse and largely context-dependent (see the figure). For example, the histone acetyltransferases PCAF and GCN5 exhibit an ordered ternary catalytic mechanism in which acetyl-CoA binds to the enzyme before the histone peptide, which is followed by the release of acetylated histone and finally, the CoA molecule 237� . By contrast, p300 and HAT8 function through a ping-pong mechanism that starts with the binding of acetyl-CoA and ends with the release of acetylated histone 240,241 . Finally, random ternary kinetics was observed in the yeast histone acetyltransferase Rtt109 242 .

Given the diversity in catalytic mechanisms of chromatin-modifying enzymes and the variability in the specific enzymatic kinetic parameters, epigenetic modifications are likely to exhibit specificity due to these different mechanisms. The thermodynamic and kinetic properties of epigenetic modifications depend on the type of modification, the corresponding chromatin-modifying enzyme, the specific genomic locus, and the abundance of allosteric regulators and cofactors. This variability results in distinct dynamics of deposition and turnover in response to perturbations to metabolism. Directly targeting these epigenetic modifications has shown that the dynamics of histone acetylation is in general faster than changes in that of DNA and histone methylation 243 , and that acetylation and deacetylation occur at the timescale of minutes in vivo 244,245 . Histone and DNA methylation, on the other hand, are more stable compared to acetylation, which might constitute a type of epigenetic memory in response to stronger but transient perturbations 243 . An additional layer of complexity arises from the heterogenous affinity and activity of chromatin modifiers towards different chromatin modifications 246 and different genomic loci 247,248 . Variation in the abundances of chromatin-modifying metabolites and apparent Km values in different cells and tissues might therefore lead to heterogeneity in the sensitivity of epigenetic marks to metabolic alterations in different contexts.

There are several additional mechanisms that enable the efficient and precise regulation of enzyme-catalyzed chromatin modifications by metabolic activity. Metabolic enzymes involved in the synthesis of chromatin-modifying metabolites, such as acetyl-CoA and S-adenosylmethionine (SAM), may be able to localize in the nucleus and interact with nucleosomes and chromatin-modifying enzymes to efficiently produce metabolites at specific genomic loci 26� . Levels of chromatin-modifying metabolites, such as SAM, are controlled by multiple mechanisms including both environmental inputs such as nutrient availability and intracellular methyl group sinks [G] that consume SAM 33� . Methyl group sinks are mediated by enzymes that metabolize SAM, allowing them to divert methyl groups away from enzymes such as histone methyltransferases, thus affecting their activity. These mechanisms provide avenues for control of metabolite levels and thus for chromatin to sense intracellular metabolic status.

Epigenetic modifications influence transcriptional programs through various mechanisms (Box 2). All of these outcomes can potentially be influenced by the metabolic regulation of the epigenome. Additionally, recent studies have found that chromatin compartments with differing transcriptional activity can segregate into membrane-less organelles through liquid–liquid phase separation [G] in response to chromatin modifications, establishing distinct chromatin domains with distinct patterns of regulation. Transcriptionally inactive heterochromatin can form phase-separated liquid droplets by interacting with heterochromatin protein 1 (HP1) which recognizes and binds to the histone modification H3K9me, allowing chromatin to stably condense inside these droplets 36� . On the other hand, active chromatin regions, such as those containing histone acetylation, enhancers [G] and super-enhancers [G] , are also able to phase separate through interacting with binding proteins such as bromodomain [G] -containing proteins 39� . Similar effects promoting phase separation have also been found to be mediated by the interaction between N 6 -methyladenosine (m 6 A) in mRNA and the m 6 A-binding YTHDF proteins 42 . Although it is an open question as to whether chromatin phase separation is regulated by metabolism, these findings suggest that the ability of chromatin to phase-separate within cells may be regulated by epigenetic modifications derived from metabolites and might be sensitive to cellular metabolism. Furthermore, phase separation of other biomolecules has been shown to concentrate molecules in a certain phase to activate biochemical signalling processes 43 . Whether chromatin phase separation also results in localization of metabolites and activation or inhibition of chromatin-modifying reactions in a specific phase remains unknown, but it potentially serves as an additional mechanism for the precise control of local metabolite levels and chromatin modifications.

Box 2:

Influences of epigenetic modifications on chromatin structure and gene expression

The earliest clues suggesting the functional consequences of particular epigenetic modifications are their locus-specific enrichment and association with gene expression levels and gene regulation. Although not conclusively implying causality, these findings have been used to theorize on the existence of a ‘histone code’, which postulates that the presence and combination of specific DNA and histone modifications link to the regulation of transcriptional programs and gene expression events 249 . Studies over the past few decades have revealed two major ways for epigenetic modifications to regulate gene expression: by changing the local chromatin structure, or by influencing the recruitment of non-histone protein effectors to chromatin 250 . Chromatin can be roughly classified into two categories based on their structural and biochemical properties: condensed, transcriptionally silenced heterochromatin, and decondensed, actively transcribed euchromatin. Formation of heterochromatin and euchromatin is dependent on the existence of epigenetic modifications. Histone acetylation, typically enriched in euchromatin, is able to neutralize the positive charge of the modified lysine residue, thus disrupting the interaction between histone and DNA and resulting in an open chromatin structure that facilitates active transcription. Heterochromatin is typically enriched of trimethylation of histone 3 lysine 9 (H3K9me3), which is bound by heterochromatin protein 1 (HP1) that promotes compaction and spreading of heterochromatin 251 and triggers liquid–liquid phase separation by forming oligomers 38 .

The chromatin-binding affinities of a plethora of proteins, including transcription factors, chromatin remodellers and components of the transcription machinery, can be modulated by these modifications. This is best demonstrated by the presence of chromatin-binding ‘reader’ modules in these proteins, such as the chromodomains and PHD fingers that recognize and bind to methylated histones, and the bromodomains and YEATS domains that bind to acetylated histones 252,253 . It has been hypothesized that the association between H3K4me3 and active transcription is largely mediated by the binding of H3K4me3 by the PHD finger of the TAF3 subunit of TFIID, a component of the RNA polymerase pre-initiation complex 254 . DNA methylation has recently been shown to reduce the binding affinity of most transcription factors while promoting the binding of PHD-containing proteins through hydrophobic interactions with the methylated cytosine 255 . Readers of some of the emerging, non-canonical histone modifications, such as succinylation 116 and crotonylation 256,257 , have also been identified recently 258 .


Chromatin

Germ cells in prophase of meiosis I from a him-8 hermaphrodite. The unsynapsed X chromosomes are enriched for H3K9me2 (arrowheads) H3K9ac is enriched on autosomes.

Meiotic silencing of unpaired chromatin is a widespread phenomenon that has been described in many vertebrate and invertebrate organisms. In C. elegans, as in other species, unpaired chromosomes accumulate a high level of repressive chromatin modifications that are not observed on synapsed chromosomes. For example, the male X chromosome accumulates a high level of histone H3 lysine 9 dimethylation (H3K9me2), as do hermaphrodite Xs that fail to synapse due to mutation (see figure). This widely conserved histone modification is associated with assembly of closed chromatin structure and transcriptional repression. Our goals are to understand the mechanisms by which unpaired chromatin is recognized and silenced during meiosis and to identify the chromosomal targets of meiotic silencing.

We have demonstrated that a branch of the C. eleganssmall RNA network functions in regulating meiotic H3K9me2 accumulation and distribution. Components of this pathway include the RNA-directed RNA polymerase, EGO-1, and the Argonaute/Slicer protein, CSR-1. MET-2 is the histone methyltransferase responsible for H3K9me2 accumulation in the germ line. We are working to understand how activity of the EGO-1/CSR-1 small RNA pathway influences MET-2 activity to direct it toward unsynapsed chromosomes.

Germ cells in prophase of meiosis I from wildtype and ego-1 mutant males. H3K9me2 is enriched on the X chromosome in wildtype (arrows) but not ego-1 male.

C. elegans provides an unparalleled system for studying the mechanism of meiotic silencing in the context of germline development. The C. elegans germ line has proven an excellent model for studying genetic regulation and developmental questions. Extensive genetic tools available in C. elegans allow us study the meiotic silencing machinery as well as address larger questions about the role of meiotic silencing in germline development and function. One focus of our current work is to identify additional factors acting together with MET-2 and/or the EGO-1/CSR-1 pathway to regulate H3K9me2 accumulation. Another focus is to identify the sites where H3K9me2 modifications are deposited on unpaired chromosomes. By understanding the mechanism and targets of H3K9me2 accumulation, we will be in a position to investigate the developmental implications of this process. We expect our results to facilitate the study of meiotic silencing in more complex animals, including mammals.


PERSPECTIVES AND CONCLUSION

Our 3D cryo-EM structures at 11 Å resolution have provided the fundamental structural features of the elusive 30-nm chromatin fiber and a solid foundation for understanding the basic principle of chromatin compaction, whereas higher-resolution structures of chromatin fiber are needed to uncover much more structural details for the nucleosome–nucleosome, nucleosome–H1, and H1–H1 interactions in chromatin fibers. In addition, our cryo-EM structures clearly imply the presence of three important interaction interfaces in the 30-nm fiber, whereas it still remains unclear how these interfaces are regulated by different chromatin factors. Regarding the variation of NRLs in vivo, reconstituted chromatin fibers with a combination of different NRLs will also be good candidates for single-molecule and cryo-EM studies in the future. These further studies will not only enhance our understanding of the diversity of chromatin structures in vivo but also provide structural basis for how different combinations of DNA sequences, NRLs, histone variants, chromatin modifications, and chromatin architectural proteins can be coordinated to precisely regulate the biological function of genomic DNA in the nucleus.

It is still a puzzle as to whether the structural results from the in vitro studies can represent the actual structure of chromatin fibers in situ. Therefore, the 3D organization of chromatin fibers in the intact nuclei needs to be further studied by using newly developed techniques. Well-characterized chromatin fibers reconstituted in vitro, including compacted 30-nm fibers and open nucleosomal arrays, would be perfect structural references for analyzing the 3D organization of chromatin fibers in situ in these studies. The combination of cryo-EM with super-resolution fluorescence imaging techniques has been recently developed to visualize and quantify the ultrastructure of cryo-preserved cells (Chang et al. 2014 Liu et al. 2015). The combination of genomic approaches (such as micro-C and RICC-seq) and CRISPR (clustered regularly interspaced short palindromic repeat)-based imaging techniques may enable us to probe the ultrastructure and 3D organization of chromatin fiber at defined genomic regions in the intact nuclei. Undoubtedly, more structural detail for the 3D organization of chromatin fibers in situ can be obtained by the application of these advanced imaging techniques in the future.


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Current Topics in Developmental Biology. Vol. 56 2003. p. 55-83 (Current Topics in Developmental Biology Vol. 56).

Research output : Chapter in Book/Report/Conference proceeding › Chapter

T1 - Regulation of Chromatin Structure and Gene Activity by Poly(ADP-Ribose) Polymerases

N2 - Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear protein that plays an important role in repairing DNA and responding to infection. Here we review recent evidence that PARP1 and related proteins also carry out crucial functions regulating genes during normal development. Genetic studies in mammals and Drosophila have implied that PARPs mediate rapid responses to environmental stimuli, including infection, stress, hormones, and growth signals. In addition, these polymerases may control fundamental processes that differentially mold and remodel chromatin within the many cell types of a developing embryo. We discuss a unified mechanism of PARP action during DNA repair, gene transcription, and chromatin modulation.

AB - Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear protein that plays an important role in repairing DNA and responding to infection. Here we review recent evidence that PARP1 and related proteins also carry out crucial functions regulating genes during normal development. Genetic studies in mammals and Drosophila have implied that PARPs mediate rapid responses to environmental stimuli, including infection, stress, hormones, and growth signals. In addition, these polymerases may control fundamental processes that differentially mold and remodel chromatin within the many cell types of a developing embryo. We discuss a unified mechanism of PARP action during DNA repair, gene transcription, and chromatin modulation.


RNA Directed DNA Methylation

Steven E. Jacobsen is an Investigator at the Howard Hughes Medical Institute and Professor of Molecular Cell and Developmental Biology at the University of California, Los Angeles, USA. His research is focused on mechanisms of epigenetic inheritance in plants, including the genetics and genomics of DNA methylation, histone methylation as well as small RNA driven silencing pathways. With over 130 epigenetic related publications he made fundamental contributions to this emerging field of research.

Jacobsen lectured on the RNA dependent DNA Methylation (RdDM) pathway that Arabidopsis thaliana utilizes to maintain and probably establish cytosine DNA methylation in order to silence genes and transposons. While the well-studied DNA methyltransferases MET1, CMT3 and DRM2 are responsible for maintaining pre-established methylation patterns via classical mechanisms, apparently DRM2 – a homolog of mammalian Dnmt3 – is the only one to mediate RdDM. DRM2 recruitment to the respective DNA site is thereby mediated by a complex interplay of Polymerase IV (PolIV), Polymerase V (PolV), the RNA species produced by them and numerous other factors.

Since PolIV and PolV DNA binding does not show any significant sequence specificity, epigenetic marks are most likely to depict their recognition sites. PolIV was shown to interact with SHH1, which binds to H3K9me, a silencing mark to be frequently found at RdDM sites, while PolV´s indirect interaction with SUVH2 targets it to methylated DNA. By the fusion of SUVH2 with a zinc finger domain targeted to an unmethylated epiallele of the homeodomain transcription factor FWA, Jacobsen´s lab demonstrated that SUVH2 is sufficient to recruit PolV, establish DNA methylation and ultimately cause gene silencing.

Mechanistically, PolV and PolV associated with RdDM sites produce 24 nucleotide siRNAs and lncRNAs, respectively. These RNAs interact with a number of further factors like AGO4 to establish a very complex network that ultimately results in the recruitment of DRM2 to preserve existing methylation patterns. Interestingly, there is a close interdependence between such methylation marks and other epigenetic modifications like for example histone methylation, which generates robust reinforcement loops. Nevertheless, it remains unclear how the initial establishment of RdDM takes place.


Abstract

Pioneer transcription factors play a primary role in establishing competence for gene expression and initiating cellular programming and reprogramming, and their dysregulation causes severe effects on human health, such as promoting tumorigenesis. Although more than 200 transcription factors are expressed in each cell type, only a small number of transcription factors are necessary to elicit dramatic cell-fate changes in embryonic development and cell-fate conversion. Among these key transcription factors, a subset called “pioneer transcription factors” have a remarkable ability to target nucleosomal DNA, or closed chromatin, early in development, often leading to the local opening of chromatin, thereby establishing competence for gene expression. Although more key transcription factors have been identified as pioneer transcription factors, the molecular mechanisms behind their special properties are only beginning to be revealed. Understanding the pioneering mechanisms will enhance our ability to precisely control cell fate at will for research and therapeutic purposes.

  • Biological Mechanisms > Cell Fates
  • Biological Mechanisms > Regulatory Biology
  • Developmental Biology > Lineages

8.4: Genes and Chromatin in Eukaryotes

  • Contributed by Gerald Bergtrom
  • Professor Emeritus (Biosciences) at University of Wisconsin-Milwaukee

Chromosomes and chromatin are a uniquely eukaryotic association of DNA with more or less protein. Bacterial DNA (and prokaryotic DNA generally) is relatively &lsquonaked&rsquo &ndash not visibly associated with protein.

The electron micrograph of an interphase cell (below) reveals that the chromatin can itself exist in various states of condensation.

Chromatin is maximally condensed during mitosis, forming chromosomes. During interphase, chromatin exists in more or less condensed forms, called Heterochromatinand euchromatinrespectively. Transition between these chromatin forms involve changes in the amounts and types of proteins bound to the chromatin, and can that can occur during gene regulation, i.e., when genes are turned on or off. Active genes tend to be in the more dispersed euchromatin so that enzymes of replication and transcription have easier access to the DNA. Genes that are inactive in transcription are heterochromatic, obscured by additional chromatin proteins present in heterochromatin. We&rsquoll be looking at some experiments that demonstrate this in a later chapter.

We can define three levels of chromatin organization in general terms:

1. DNA wrapped around histone proteins form nucleosomes in a "beads on a string" structure.

2. Multiple nucleosomes coil (condense), forming 30 nm fiber (solenoid) structures.

3. Higher-order packing of the 30 nm fiber leads to formation of metaphase chromosomes seen in mitosis & meiosis.

The levels of chromatin structure were determined in part by selective isolation and extraction of interphase cell chromatin, followed by selective chemical extraction of chromatin components. The steps are:

· Nuclei are first isolated from the cells.

· The nuclear envelope gently ruptured so as not to physically disrupt chromatin structure.

· the chromatin can be gently extracted by one of several different chemical treatments (high salt, low salt, acid. ).

The levels of chromatin structure are illustrated below.

Salt extraction dissociates most of the proteins from the chromatin. When a low salt extract is centrifuged and the pellet resuspended, the remaining chromatin looks like beads on a string. DNA-wrapped nucleosomes are the beads, which are in turn linked by uniform lengths of metaphorical DNA &ldquostring&rsquo ( # 1 in the illustration above). A high salt chromatin extract appears as a coil of nucleosomes, or 30 nm solenoid fiber (# 2 above). Other extraction protocols revealed other aspects of chromatin structure shown in #s 3 and 4 above. Chromosomes seen in metaphase of mitosis are the &lsquohighest order&rsquo, most condensed form of chromatin.

The 10 nm filament of nucleosome &lsquobeads-on-a-string&rsquo remaining after a low salt extraction can be seen in an electron microscope as shown below.

When these nucleosome necklaces were digested with the enzyme deoxyribonuclease (DNAse), the DNA between the &lsquobeads&rsquo was degraded, leaving behind shortened 10nm filaments after a short digest period, or just single beads the beads after a longer digestion (below).

Roger Kornberg (son of Nobel Laureate Arthur Kornberg who discovered the first DNA polymerase enzyme of replication) participated in the discovery and characterization of nucleosomes while he was still a post-doc! Electrophoresis of DNA extracted from these digests revealed nucleosomes separated by a &ldquolinker&rdquo DNA stretch of about 80 base pairs. DNA extracted from the nucleosomes was about 147 base pairs long. This is the DNA that had been wrapped around the proteins of the nucleosome.

After separating all of the proteins from nucleosomal DNA, five proteins were identified (illustrated below).

Histones are basic proteins containing many lysine and arginine amino acids. Their positively charged side chains enable these amino acids bind the acidic, negatively charged phosphodiester backbone of double helical DNA. The DNA wraps around an octamer of histones (2 each of 4 of the histone proteins) to form the nucleosome. About a gram of histones is associated with each gram of DNA. After a high salt chromatin extraction, the structure visible in the electron microscope is the 30nm solenoid, the coil of nucleosomes modeled in the figure below.

As shown above, simply increasing the salt concentration of an already extracted nucleosome preparation will cause the &lsquonecklace&rsquo to fold into the 30nm solenoid structure.

As you might guess, an acidic extraction of chromatin should selectively remove the basic histone proteins, leaving behind an association of DNA with non-histone proteins. This proved to be the case. An electron micrograph of the chromatin remnant after an acid extraction of metaphase chromosomes is shown on the next page.

DNA freed of the regularly spaced histone-based nucleosomes, loops out, away from the long axis of the chromatin. Dark material along this axis is a protein scaffolding that makes up what&rsquos left after histone extraction. Much of this protein is topoisomerase, an enzyme that prevents DNA from breaking apart under the strain of replication.


Watch the video: Histone modifications Introduction (May 2022).