MNase Digestion Research Articles

Evidence for a Complex of Transcription Factor IIB (TFIIB) with Poly(A) Polymerase and Cleavage Factor 1 Subunits Required for Gene Looping

Gene looping, defined as the interaction of the promoter and the terminator regions of a gene during transcription, requires transcription factor IIB (TFIIB). We have earlier demonstrated association of TFIIB with the distal ends of a gene in an activator-dependent manner (El Kaderi, B., Medler, S., Raghunayakula, S., and Ansari, A. (2009) J. Biol. Chem. 284, 25015-25025). The presence of TFIIB at the 3' end of a gene required its interaction with cleavage factor 1 (CF1) 3' end processing complex subunit Rna15. Here, employing affinity chromatography and glycerol gradient centrifugation, we show that TFIIB associates with poly(A) polymerase and the entire CF1 complex in yeast cells. The factors required for general transcription such as TATA-binding protein, RNA polymerase II, and TFIIH are not a component of the TFIIB complex. This holo-TFIIB complex was resistant to MNase digestion. The complex was observed only in the looping-competent strains, but not in the looping-defective sua7-1 strain. The requirement of Rna15 in gene looping has been demonstrated earlier. Here we provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits Rna14 and Pcf11 are also required for loop formation of MET16 and INO1 genes. Accordingly, cross-linking of TFIIB to the 3' end of genes was abolished in the mutants of Pap1, Rna14, and Pcf11. We further show that in sua7-1 cells, where holo-TFIIB complex is not formed, the kinetics of activated transcription is altered. These results suggest that a complex of TFIIB, CF1 subunits, and Pap1 exists in yeast cells. Furthermore, TFIIB interaction with the CF1 complex and Pap1 is crucial for gene looping and transcriptional regulation.

Fragmentation of coding region chromatin of trp-dioxygenase (to) and tyr-aminotransferase (tat) genes in their active and repressed states by micrococcal nuclease

The blot-hybridization technique-assisted we have studied the pattern of fragmentation by mirococcal nuclease (MNase) of DNA tyr-aminotransferase (tat) and trp-dioxygenase (to) genes in active (in rat cell liver nuclei) and repressed (in brain nuclei) states. It was provided, over a wide range of enzyme concentration two types of fragments are mainly produced: near full-size to- and tat-transcription unit (19,000 and 11,000 bp, respectively) and their large (from 1500 bp) heterogeneous in length. To-and tat-fragments of both kinds are preserved in hydrolyzates at limit of MNase digestion of total chromatin DNA when nuclease breaks occur in nearly all accessible sites of chromatin. This means that these fragments originate from two distinct subsets of transcription units coexistent in liver nuclei. The first of them do not contain MNase accessible sites over their entire length, whereas in other resistant regions alternate with rare irregular located MNase-sensitive segments. We presume that resistance to MNase within transcription units is peculiar to the competent genes. As a result of transcription MNase-sensitive areas arise which possibly flank elongating RNA polymerases.

How Chromatin Is Remodelled during DNA Repair of UV-Induced DNA Damage in Saccharomyces cerevisiae

Global genome nucleotide excision repair removes DNA damage from transcriptionally silent regions of the genome. Relatively little is known about the molecular events that initiate and regulate this process in the context of chromatin. We've shown that, in response to UV radiation–induced DNA damage, increased histone H3 acetylation at lysine 9 and 14 correlates with changes in chromatin structure, and these alterations are associated with efficient global genome nucleotide excision repair in yeast. These changes depend on the presence of the Rad16 protein. Remarkably, constitutive hyperacetylation of histone H3 can suppress the requirement for Rad7 and Rad16, two components of a global genome repair complex, during repair. This reveals the connection between histone H3 acetylation and DNA repair. Here, we investigate how chromatin structure is modified following UV irradiation to facilitate DNA repair in yeast. Using a combination of chromatin immunoprecipitation to measure histone acetylation levels, histone acetylase occupancy in chromatin, MNase digestion, or restriction enzyme endonuclease accessibility assays to analyse chromatin structure, and finally nucleotide excision repair assays to examine DNA repair, we demonstrate that global genome nucleotide excision repair drives UV-induced chromatin remodelling by controlling histone H3 acetylation levels in chromatin. The concerted action of the ATPase and C3HC4 RING domains of Rad16 combine to regulate the occupancy of the histone acetyl transferase Gcn5 on chromatin in response to UV damage. We conclude that the global genome repair complex in yeast regulates UV-induced histone H3 acetylation by controlling the accessibility of the histone acetyl transferase Gcn5 in chromatin. The resultant changes in histone H3 acetylation promote chromatin remodelling necessary for efficient repair of DNA damage. Recent evidence suggests that GCN5 plays a role in NER in human cells. Our work provides important insight into how GG-NER operates in chromatin.

Chromatin structure at active genes

For gene switching, chromatin must be manipulated, a process initiating at key cis-controlling elements, labelled operationally as DNaseI hypersensitive sites (DHS). This minireview series expands on how the remodelling of chromatin structure comes about and the consequences for gene activation and repression The conventional wisdom embodied (but not, in this age of rapid development, embalmed) in many textbooks has it that chromatin exists in cells either in a linker histone-containing condensed fibre of nucleosomes, diameter 30–40 nm, characteristic of heterochromatin, or as an open extended beads-on-a-string like structure, characteristic of all active genes. Even brief thought gives the lie to the second concept, since in very active tissues such as brain or stem cells in which, say, 40% of all genes are active, were they all fully extended throughout their transcribed length the nuclear volume could not possibly accommodate them. Since the chromatin of an active gene must be opened up at the moment of polymerase passage, it follows that continuous manipulation of chromatin structure must occur at active genes. Indeed, using the criterion of DNaseI accessibility we have recently shown that the overall state of certain active genes differs little from that of an immediately adjacent heterochromatin segment, experimentally demonstrated to be in the form of the 30 nm fibre. This time-averaged observation does not apply, however, to cis-controlling elements such as promoters and enhancers that, since the seminal observations of Hal Weintraub and Mark Groudine in 1976, have been shown to be locally hypersensitive to DNaseI digestion, i.e. in a more open conformation. Thus whereas the presence of bound histones, including the H1 linker, does not in general present a barrier to passage of the polymerase complex through an active gene, access to the DNA at cis elements requires a more open structure. It is worth emphasizing that DNaseI remains a valuable tool in studying local chromatin structure, not only for defining key control points as DNaseI hypersensitive sites, but also for locating the rare segments of open nucleosomal chromatin, i.e. genuine beads-on-a-string structures, sometimes found in the region of promoters or enhancers. This results from the fact that, whereas MNase digestion of nuclei always gives rise to conventional nucleosomal ladders, whether the underlying structure is the 30 nm fibre or open nucleosomal beads, DNaseI does not generate ladders from 30 nm chromatin fibres (the bulk of the chromatin) but it does from open beaded structures. Overall, the controlled manipulation of chromatin structure at such locations, in particular the displacement or even eviction of nucleosomes to permit the ordered recruitment of transcription factors, is a complex process requiring many interdependent components, in particular chromatin remodelling and histone modifying complexes. The following three minireviews address the problem of chromatin manipulation. The first, by Peter Cockerill, takes an overview of the chromatin structure at active genes and then illustrates this with the process of activating the human GM-CSF (granulocyte/macrophage colony stimulating factor) gene during T cell development, emphasizing how the binding of transcription factors is dependent on nucleosome displacement and is accompanied by the appearance of DNaseI hypersensitive sites. The paper by Gordon Hager and colleagues reviews how, following treatment of cells with steroid hormones, the chromatin structure of responsive genes changes in consequence of binding by the immediate hormone targets, the nuclear receptors. Finally, Stefan Dimitrov and his colleague Jan Bednar relate how the bare bone mechanics of chromatin manipulation could occur as nucleosomes and chromatin fibres are put under stress by pulling and tugging whilst held by optical and magnetic tweezers. In a field that can be confounded by the wide variation of experimental conditions, the authors very helpfully provide a summarizing table of published results.

Nucleosome fragility reveals novel functional states of chromatin and poises genes for activation

The structural complexity of nucleosomes underlies their functional versatility. Here we report a new type of complexity-nucleosome fragility, manifested as high sensitivity to micrococcal nuclease, in contrast to the common presumption that nucleosomes are similar in resistance to MNase digestion. Using differential MNase digestion of chromatin and high-throughput sequencing, we have identified a special group of nucleosomes termed "fragile nucleosomes" throughout the yeast genome, nearly 1000 of which were at previously determined "nucleosome-free" loci. Nucleosome fragility is broadly implicated in multiple chromatin processes, including transcription, translocation, and replication, in correspondence to specific physiological states of cells. In the environmental-stress-response genes, the presence of fragile nucleosomes prior to the occurrence of environmental changes suggests that nucleosome fragility poises genes for swift up-regulation in response to the environmental changes. We propose that nucleosome fragility underscores distinct functional statuses of the chromatin and provides a new dimension for portraying the landscape of genome organization.

Sequence determinants of histone–DNA binding preferences: Comment on “Cracking the chromatin code: Precise rule of nucleosome positioning” by Edward N. Trifonov

DNA in eukaryotic cells is packaged into compact chromatin state. The fundamental unit of chromatin is a nucleosome, a highly conserved protein-DNA complex in which ~ 147 basepairs (bp) of DNA are wrapped around a histone octamer in a left-handed superhelix [1]. Histone-DNA binding affinity depends on the nucleotide sequence of the nucleosomal site: indeed, the ability of the DNA molecule to bend into the superhelix is mostly governed by dinucleotide base-stacking energies. The review by Trifonov [2] emphasizes the role of deformational properties of DNA in nucleosome positioning and energetics, proposing a novel sequence motif CGRAAATTTYCG that favors nucleosome formation. The motif is inferred from a large-scale map of C.elegans nucleosomes [3]. In building up the case for the 10–11 bp-periodic nucleosome positioning motif shown above, the author has chosen to focus mostly on his own work and the work of his colleagues. However, I find myself intrigued by how well the proposed pattern stacks up against some of the other models and datasets (nucleosome positioning determinants and the idea of the “nucleo-some code” have recently garnered a lot of interest in the chromatin field). The author argues that due to steric exclusion between neighboring particles only single-nucleosome conformations can be used to compare experiment with theory [4]. However, techniques similar to dynamic programming in computer science and transfer matrices in physics can be used to convert histone-DNA binding energies into probabilities of nucleosome formation at every bp, without any approximations related to the finite particle size (see e.g. [5]). Furthermore, because the DNA bendability matrix proposed by the author is capable of placing nucleosomes with 1 bp resolution, only seven nucleosomes whose positions are known precisely from experiment have been chosen to test the model, with impressive success [4]. This seems to be too restrictive – certainly a high-resolution algorithm can predict lower-resolution data. Besides, the vast majority of the algorithms proposed in the literature also have 1 bp resolution and have nonetheless been used to predict genome-wide occupancy profiles for nucleosomes mapped with ~ 10–20 bp precision by micrococcal nuclease (MNase). Moreover, C.elegans data from which the model was inferred in the first place employed MNase digestion followed by 454 pyrosequencing [3], and therefore has the usual ~ 10 – 20 bp accuracy. It would be especially interesting to see whether the DNA bendability model proposed by Trifonov and colleagues has greater predictive power against large-scale nucleosome maps than simple models that assign the same scores to mono- and dinucleotides of the same type, regardless of their position with respect to the DNA helical repeat [6, 7]. Finally, nucleosomes on which the analysis by Trifonov and colleagues is based come from in vivo chromatin in a mixture of C.elegans cells. The nucleosome positions in this sample are averaged over cell types and may have been affected, among other things, by chromatin remodeling enzymes and competition with other DNA-binding proteins. It would be reassuring to see the proposed motif also appear in two recent large-scale maps of nucleosomes positioned in vitro on genomic sequences from S.cerevisiae and E.coli [8, 9].

The Effect of Micrococcal Nuclease Digestion on Nucleosome Positioning Data

Eukaryotic genomes are packed into chromatin, whose basic repeating unit is the nucleosome. Nucleosome positioning is a widely researched area. A common experimental procedure to determine nucleosome positions involves the use of micrococcal nuclease (MNase). Here, we show that the cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. This strongly affects nucleosome positioning data and especially sequence-dependent models for nucleosome positioning. As a consequence we see a need to re-evaluate whether the DNA sequence is a major determinant of nucleosome positioning in vivo. More generally, our results show that data generated after MNase digestion of chromatin requires a matched control experiment in order to determine nucleosome positions.

Chromatin particle spectrum analysis: a method for comparative chromatin structure analysis using paired-end mode next-generation DNA sequencing

Microarray and next-generation sequencing techniques which allow whole genome analysis of chromatin structure and sequence-specific protein binding are revolutionizing our view of chromosome architecture and function. However, many current methods in this field rely on biochemical purification of highly specific fractions of DNA prepared from chromatin digested with either micrococcal nuclease or DNaseI and are restricted in the parameters they can measure. Here, we show that a broad size-range of genomic DNA species, produced by partial micrococcal nuclease digestion of chromatin, can be sequenced using paired-end mode next-generation technology. The paired sequence reads, rather than DNA molecules, can then be size-selected and mapped as particle classes to the target genome. Using budding yeast as a model, we show that this approach reveals position and structural information for a spectrum of nuclease resistant complexes ranging from transcription factor-bound DNA elements up to mono- and poly-nucleosomes. We illustrate the utility of this approach in visualizing the MNase digestion landscape of protein-coding gene transcriptional start sites, and demonstrate a comparative analysis which probes the function of the chromatin-remodelling transcription factor Cbf1p.

Differential Histone Modification and Protein Expression Associated with Cell Wall Removal and Regeneration in Rice (Oryza sativa)

The cell wall is a critical extracellular structure that provides protection and structural support in plant cells. To study the biological function of the cell wall and the regulation of cell wall resynthesis, we examined cellular responses to enzymatic removal of the cell wall in rice (Oryza sativa) suspension cells using proteomic approaches. We find that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Nucleus DAPI stain and MNase digestion further show that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses reveal that substantial histone modification changes, particularly H3K18(AC) and H3K23(AC), are associated with the removal and regeneration of the cell wall. Label-free quantitative proteome analyses further reveal that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, along with cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.

Abstract 3946: Stem cell protein piwil2 enhances nucleotide excision repair through regulating chromatin remodeling following DNA damage

Abstract The piwi family gene, which are defined by conserved PAZ and Piwi domains, play important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms. Three piwi homologs have been identified in the mouse genome. Amongst these, mili (piwil2) protein expression appears to be associated with tumorigenesis as evidenced by the facts that piwil2 stably expresses in pre-cancerous stem cells and also appears at variable levels in different human and animal tumor cell lines. Based on our preliminary data showing that piwil2 is induced in normal cultured human fibroblasts in vitro by physical and chemical genotoxic agents, we hypothesized that piwil2 might function in DNA damage response. Here, we demonstrated that mammalian cells lacking piwil2 are hypersensitive to cisplatin and UV irradiation. In addition, nucleotide excision repair (NER) efficiency is significantly decreased in the absence of piwil2 as assessed by the removal of cisplatin-induced intra-strand cross-links (Pt-GG) and UV-induced cyclobutane pyrimidine dimers (CPDs) from genomic DNA. Although quantitative real-time PCR analysis indicated higher transcript levels of various NER proteins in piwil2 knockout mouse embryo fibroblasts (MEFs), the Western blotting did not show an obvious difference in the NER protein levels between mili+/+ and mili−/− MEFs. Since piwil2 is believed to orchestrate chromatin remodeling, we reasoned that low NER efficiency in mili−/− MEFs may result from disrupted chromatin remodeling in response to UV or cisplatin induced DNA damage. Thus, chromatin relaxation and histone acetylation was analyzed in these MEFs following treatment with UV or cisplatin. MEFs lacking piwil2 showed less sensitivity to micrococcal nuclease (MNase) digestion compared with piwil2-proficient MEFs, indicating a compact chromatin structure in these cells. Moreover, in contrast to mili+/+ MEFs, mili−/− MEFs exhibited decreased acetylated Histone H3 upon both UV and cisplatin treatment. This decrease could be blocked by histone deacetylase (HDAC) inhibition indicating that piwil2 may function in damage-induced chromatin remodeling via inhibiting the HDAC activity. In summary, our data suggests that piwli2 can participate in the NER through regulating chromatin relaxation which could be a pivotal factor in genotoxin-induced tumorigenesis as well as efficacy of cancer therapeutics. (Supported by NIH grants CA93413, ES2388 and ES12991 to AAW). Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3946.

DPPA4 modulates chromatin structure via association with DNA and core histone H3 in mouse embryonic stem cells

Developmental pluripotency associated 4 (DPPA4) is one of the uncharacterized genes that is highly expressed in embryonic stem (ES) cells. DPPA4 is associated with active chromatin and involved in the pluripotency of mouse ES cells. However, the biological function of DPPA4 remains poorly understood. In this study, we performed fluorescence recovery after photobleaching (FRAP) analysis to examine the dynamics of DPPA4 in ES cells. FRAP analysis showed that the mobility of DPPA4 is similar to that of histone H1. In addition, biochemical analysis with purified proteins and immunoprecipitation analysis showed that DPPA4 directly binds to both DNA and core histone H3. The analysis using truncated proteins indicated that DPPA4 is associated with DNA via the N-terminal region and histone H3 via the C-terminal region. In vitro assembled chromatin showed resistance to micrococcal nuclease (MNase) digestion in the presence of DPPA4. Moreover, MNase assay and FRAP analysis with the truncated proteins implies that DPPA4 binding to both DNA and histone H3 is necessary for the chromatin structure resistant to MNase and for the proper localization of DPPA4 in ES cell nuclei. These results suggest that DPPA4 modulates the chromatin structure in association with DNA and histone H3 in ES cells.

Histone H1 subtype preferences of DFF40 and possible nuclear localization of DFF40/45 in normal and trichostatin A-treated NB4 leukemic cells

A major hallmark of the terminal stages of apoptosis is the internucleosomal DNA fragmentation. The endonuclease responsible for this type of DNA degradation is the DNA fragmentation factor (DFF). DFF is a complex of the endonuclease DFF40 and its chaperone/inhibitor, DFF45. In vitro work has shown that histone H1 and HMGB1/2 recruit/target DFF40 to the internucleosomal linker regions of chromatin and that histone H1 directly interacts with DFF40 conferring DNA binding ability and enhancing its nuclease activity. The histone H1 family is comprised of many subtypes, which recent work has shown may have distinct roles in chromatin function. Thus we studied the binding association of DFF40 with specific H1 subtypes and whether these binding associations are altered after the induction of apoptosis in an in vivo cellular context. The apoptotic agent used in this study is the histone deacetylase inhibitor, trichostatin A (TSA). We separated the insoluble chromatin-enriched fraction from the soluble nuclear fraction of the NB4 leukemic cell line. Using MNase digestion, we provide evidence which strongly suggests that the heterodimer, DFF40-DFF45, is localized to the chromatin fraction under apoptotic as well as non-apoptotic conditions. Moreover, we present results that show that DFF40 interacts with the all H1 subtypes used in this study, but preferentially interacts with specific H1 subtypes after the induction of apoptosis by TSA. These results illustrate for the first time the association of DFF40 with individual H1 subtypes, under a specific apoptotic stimulus in an in vivo cellular context.

Histone H1 binding is inhibited by histone variant H3.3

Linker histones are involved in the formation of higher-order chromatin structure and the regulation of specific genes, yet it remains unclear what their principal binding determinants are. We generated a genome-wide high-resolution binding map for linker histone H1 in Drosophila cells, using DamID. H1 binds at similar levels across much of the genome, both in classic euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites for active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes; rather, all regions with low binding of H1 show enrichment of the histone variant H3.3. Knockdown of H3.3 causes H1 levels to increase at these sites, with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1, providing a mechanism to keep diverse genomic sites in an open chromatin conformation.

In Vivo DNase I, MNase, and Restriction Enzyme Footprinting via Ligation-Mediated Polymerase Chain Reaction (LM-PCR): Figure 1.

INTRODUCTIONGenomic footprinting and related methods can reveal important protein-DNA interactions not detected using other methods. This protocol presents procedures for DNase I genomic footprinting, micrococcal nuclease (MNase) mapping of nucleosome positioning, and monitoring nucleosome remodeling via restriction enzyme accessibility. These procedures rely on a technique known as ligation-mediated polymerase chain reaction (LM-PCR), which can be used in concert with a variety of agents that cleave or modify genomic DNA within an intact cell or nucleus, with each agent providing unique insight into the regulation of a gene. LM-PCR is a powerful technique for detecting DNA strand breaks within a complex sample because of its extreme sensitivity and specificity. This general method has proven to be invaluable for several purposes, in particular, in analysis of the properties of an endogenous DNA locus. Coupling LM-PCR to DNase I digestion results in assays that are analogous to in vitro DNase I footprinting or methylation protection. Coupling to MNase digestion allows for the high-resolution analysis of nucleosome positioning. Coupling to restriction enzyme digestion in isolated nuclei provides evidence of nucleosome remodeling. For each of these methods, nuclei must first be isolated by lysis of the cells with a non-ionic detergent, followed by centrifugation. The isolated nuclei are then treated with the appropriate nuclease, followed by purification of the genomic DNA. LM-PCR is then performed on the cleaved genomic DNA.

Native Chromatin Preparation and Illumina/Solexa Library Construction

INTRODUCTIONHigh-throughput whole-genome analysis has become a practical and important technique to understand nuclear processes, such as transcription, replication, and genome structure. Though microarrays have been the preferred genome-scale analysis method for over a decade, new technologies, referred to as next-generation sequencing, offer distinct advantages over microarrays in both sensitivity and scale. Several next-generation sequencing platforms are currently available, including the Genome Analyzer (Solexa/Illumina), 454 (Roche), and ABI-SOLiD (Applied Biosystems). This protocol describes sample preparation for sequencing of chromatin-immunoprecipitated DNA (ChIP-Seq) to analyze histone modification patterns using native chromatin and the Genome Analyzer. One advantage of using native chromatin as compared to cross-linked chromatin is that it provides single-nucleosome-level resolution and avoids nonspecific modification signals from different nucleosomes carried over through protein-protein interactions. The protocol includes purification of human CD4+ T cells from lymphocytes and chromatin fragmentation using micrococcal nuclease (MNase) digestion, followed by chromatin immunoprecipitation (ChIP) and construction of a library for sequencing.

Chromatin structure and DNA methylation of the IL-4 gene in human TH2 cells

Human T(H)2 cell differentiation results in the selective demethylation of several specific CpG dinucleotides in the IL-4 and IL-13 genes, which are expressed in activated T(H)2, but not T(H)1, cells. This demethylation is accompanied by the appearance of six DNase I hypersensitive sites within 1.4 kb at the 5'-end of the IL-4 gene. Micrococcal nuclease (MNase) digestion revealed that in both T(H)1 and T(H)2 cells nine nucleosomes with a repeat length of 201 bp are identically positioned around the 5'-end of the IL-4 gene. However, only in T(H)2 cells are six out of the eight intervening linkers exposed to DNase I. This suggests that a major perturbation of the higher-order chromatin structure occurs above the level of the nucleosome in vivo. It is observed in cells that are poised for expression but which are not actively expressing the gene (i.e. resting T(H)2 cells). Notably, all the demethylated CpGs in T(H)2 cells are found in DNA that is accessible to DNase I. This may suggest that the opening of the chromatin structure allows binding of specific trans-acting factors that prevent de novo methylation.

Comparative analysis of H2A.Z nucleosome organization in the human and yeast genomes

Eukaryotic DNA is wrapped around a histone protein core to constitute the fundamental repeating units of chromatin, the nucleosomes. The affinity of the histone core for DNA depends on the nucleotide sequence; however, it is unclear to what extent DNA sequence determines nucleosome positioning in vivo, and if the same rules of sequence-directed positioning apply to genomes of varying complexity. Using the data generated by high-throughput DNA sequencing combined with chromatin immunoprecipitation, we have identified positions of nucleosomes containing the H2A.Z histone variant and histone H3 trimethylated at lysine 4 in human CD4(+) T-cells. We find that the 10-bp periodicity observed in nucleosomal sequences in yeast and other organisms is not pronounced in human nucleosomal sequences. This result was confirmed for a broader set of mononucleosomal fragments that were not selected for any specific histone variant or modification. We also find that human H2A.Z nucleosomes protect only approximately 120 bp of DNA from MNase digestion and exhibit specific sequence preferences, suggesting a novel mechanism of nucleosome organization for the H2A.Z variant.

Reconstitution of Yeast Silent Chromatin: Multiple Contact Sites and O-AADPR Binding Load SIR Complexes onto Nucleosomes In Vitro

At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.

ATM Signaling Facilitates Repair of DNA Double-Strand Breaks Associated with Heterochromatin

Ataxia Telangiectasia Mutated (ATM) signaling is essential for the repair of a subset of DNA double-strand breaks (DSBs); however, its precise role is unclear. Here, we show that < or =25% of DSBs require ATM signaling for repair, and this percentage correlates with increased chromatin but not damage complexity. Importantly, we demonstrate that heterochromatic DSBs are generally repaired more slowly than euchromatic DSBs, and ATM signaling is specifically required for DSB repair within heterochromatin. Significantly, knockdown of the transcriptional repressor KAP-1, an ATM substrate, or the heterochromatin-building factors HP1 or HDAC1/2 alleviates the requirement for ATM in DSB repair. We propose that ATM signaling temporarily perturbs heterochromatin via KAP-1, which is critical for DSB repair/processing within otherwise compacted/inflexible chromatin. In support of this, ATM signaling alters KAP-1 affinity for chromatin enriched for heterochromatic factors. These data suggest that the importance of ATM signaling for DSB repair increases as the heterochromatic component of a genome expands.

Quantitative differences in chromatin accessibility across regulatory regions can be directly compared in distinct cell-types

Transcriptional activation in eukaryotes is often accompanied by alterations to chromatin structure at specific regulatory sites while other genomic regions may remain unchanged. In this study, we have examined the correlation between expression and chromatin accessibility of the human CR2 gene in a panel of cell lines (U937, REH, Ramos, and Raji) using the CHART-PCR assay with the accessibility agent micrococcal nuclease (MNase). To validate the use of this assay for comparing multiple cell-types, we first tested a series of genomic regions to determine if we could observe consistent, site-specific levels of MNase chromatin accessibility. Promoter regions of the ubiquitously expressed genes GAPDH and β-actin were similar and showed high accessibility to MNase digestion in each of the cell lines, while on the other hand, promoter regions of developmentally restricted genes PAX-7 and SP-A2 showed consistently reduced chromatin accessibility. Since CHART-PCR detected site-specific differences in chromatin accessibility in a manner that could be compared between cell-types, we next examined chromatin accessibility over the CR2 core promoter in the panel of cell lines representing either CR2 expressing or CR2 non-expressing cell-types. Our data revealed significantly enhanced accessibility over the −289 to −101 and the −115 to −12 regions of the CR2 promoter in expressing B-cells (Ramos, Raji) compared to non-expressing cells (U937, REH). Thus, CHART-PCR assays detected a correlation between chromatin accessibility and expression of the human CR2 gene, while the accessibility of other genomic regions was site-specific, but not altered between cell-types.