Regions Of Silent Chromatin Research Articles

SOX9 reprograms endothelial cells by altering the chromatin landscape.

The transcription factor SOX9 is activated at the onset of endothelial-to-mesenchymal transition (EndMT) during embryonic development and in pathological conditions. Its roles in regulating these processes, however, are not clear. Using human umbilical vein endothelial cells (HUVECs) as an EndMT model, we show that SOX9 expression alone is sufficient to activate mesenchymal genes and steer endothelial cells towards a mesenchymal fate. By genome-wide mapping of the chromatin landscape, we show that SOX9 displays features of a pioneer transcription factor, such as opening of chromatin and leading to deposition of active histone modifications at silent chromatin regions, guided by SOX dimer motifs and H2A.Z enrichment. We further observe highly transient and dynamic SOX9 binding, possibly promoted through its eviction by histone phosphorylation. However, while SOX9 binding is dynamic, changes in the chromatin landscape and cell fate induced by SOX9 are persistent. Finally, our analysis of single-cell chromatin accessibility indicates that SOX9 opens chromatin to drive EndMT in atherosclerotic lesions in vivo. This study provides new insight into key molecular functions of SOX9 and mechanisms of EndMT and highlights the crucial developmental role of SOX9 and relevance to human disease.

Spreading-dependent or independent Sir2-mediated gene silencing in budding yeast.

In the budding yeast Saccharomyces cerevisiae, a silent chromatin structure is formed at three distinct loci, including telomeres, rDNA, and mating-type loci, which silence the expression of genes within their structures. Sir2 is the only common factor, regulating the three silent chromatin regions. S. cerevisiae has 32 telomeres, but studies on gene silencing in budding yeast have been performed using some reporter genes, artificially inserted in the telomeric regions. Therefore, insights into the global landscape of Sir-dependent silencing of genes within the silent chromatin regions are required. This study aimed to obtain global insights into Sir2-dependent gene silencing on all silent chromatin regions in budding yeast. RNA-sequencing was performed to identify genes that are silenced by Sir2. By comparing with the chromatin immunoprecipitation-sequencing (ChIP-seq) of Sir2 in the wild-type strain, we confirmed Sir2-regulated genes. Using Sir2 ChIP-seq data, we identified that the Sir2 binding domain length caused by Sir2 spreading from the chromosomal end is different in each telomere in budding yeast. Expression of most subtelomeric genes increased in the ∆sir2 strain. Some Sir2-regulated subtelomeric genes were positioned within the telomeric Sir2-binding domain, while the others were outside the Sir2-binding domain. In addition, Sir2 was bound to the mating-type loci and rDNA region, and gene expression increased in the ∆sir2 strain. We concluded that S. cerevisiae has two modes of Sir2-mediated gene silencing: one is dependent on chromatin binding and spreading of Sir2, and the other is independent of spreading of Sir2.

OCT2 pre-positioning facilitates cell fate transition and chromatin architecture changes in humoral immunity.

During the germinal center (GC) reaction, B cells undergo profound transcriptional, epigenetic and genomic architectural changes. How such changes are established remains unknown. Mapping chromatin accessibility during the humoral immune response, we show that OCT2 was the dominant transcription factor linked to differential accessibility of GC regulatory elements. Silent chromatin regions destined to become GC-specific super-enhancers (SEs) contained pre-positioned OCT2-binding sites in naive B cells (NBs). These preloaded SE 'seeds' featured spatial clustering of regulatory elements enriched in OCT2 DNA-binding motifs that became heavily loaded with OCT2 and its GC-specific coactivator OCAB in GC B cells (GCBs). SEs with high abundance of pre-positioned OCT2 binding preferentially formed long-range chromatin contacts in GCs, to support expression of GC-specifying factors. Gain in accessibility and architectural interactivity of these regions were dependent on recruitment of OCAB. Pre-positioning key regulators at SEs may represent a broadly used strategy for facilitating rapid cell fate transitions.

Large-scale reconstruction of chromatin structures of maize temperate and tropical inbred lines.

Maize is a model plant species often used for genetics and genomics research because of its genetic diversity. There are prominent morphological, genetic, and epigenetic variations between tropical and temperate maize lines. However, the genome-wide chromatin conformations of these two maize types remain unexplored. We applied a Hi-C approach to compare the genome-wide chromatin interactions between temperate inbred line D132 and tropical line CML288. A reconstructed maize three-dimensional genome model revealed the spatial segregation of the global A and B compartments. The A compartments contain enriched genes and active epigenome marks, whereas the B compartments are gene-poor, transcriptionally silent chromatin regions. Whole-genome analyses indicated that the global A compartment content of CML288 was 3.12% lower than that of D132. Additionally, global and A/B sub-compartments were associated with differential gene expression and epigenetic changes between two inbred lines. About 25.3% of topologically associating domains (TADs) were determined to be associated with complex domain-level modifications that induced transcriptional changes, indicative of a large-scale reorganization of chromatin structures between the inbred maize lines. Furthermore, differences in chromatin interactions between the two lines correlated with epigenetic changes. These findings provide a solid foundation for the wider plant community to further investigate the genome-wide chromatin structures in other plant species.

The Effects of Histone Variant H2A.Z on Chromatin Structure and Kinetics-A Single Molecule Optical Tweezers Study

Understanding how histone variants affect chromatin structure is essential for understanding their role in gene regulation and chromatin maintenance in vivo. The basic unit of chromatin is a nucleosome, which consists of 146 base pairs of DNA wrapped around a histone octamer, made up of two of each of the four core histones-H2A, H2B, H3, H4. Often in cells, however, these canonical histones get exchanged for histone variants so that certain regions of the genome become enriched with non-wild-type histones. For example, histone variant H2A.Z has been found to replace canonical H2A near the transcription start site of active genes, suggesting that H2A.Z promotes gene transcription. Conversely, H2A.Z has also been associated with regions of silent chromatin. In general, the mechanism by which H2A.Z affects chromatin structure and kinetics is not understood. Here, we use optical tweezers to mechanically stretch individual nucleosome arrays and quantitatively compare the behavior of nucleosomes containing wild-type histones and nucleosomes in which histone H2A has been replaced with H2A.Z. We find that H2A.Z decreases the nucleosome inner-turn rewinding rate compared to wild-type H2A, promoting an open conformation. Our results reveal the affect of H2A.Z on nucleosome kinetics and on chromatin structure.

Chromatin affinity-precipitation using a small metabolic molecule: its application to analysis of O-acetyl-ADP-ribose

In the cell, many small endogenous metabolic molecules are involved in distinct cellular functions such as modulation of chromatin structure and regulation of gene expression. O-acetyl-ADP-ribose (AAR) is a small metabolic molecule that is generated during NAD-dependent deacetylation by Sir2. Sir2 regulates gene expression, DNA repair, and genome stability. Here, we developed a novel chromatin affinity-precipitation (ChAP) method to detect the chromatin fragments at which small molecules interact with binding partners. We used this method to demonstrate that AAR associated with heterochromatin. Moreover, we applied the ChAP method to whole genome tiling array chips to compare the association of AAR and Sir2. We found that AAR and Sir2 displayed similar genomic binding patterns. Furthermore, we identified 312 potential association cluster regions of AAR. The ChAP assay may therefore be a generally useful strategy to study the small molecule association with chromosomal regions. Our results further suggest that the small metabolic molecule AAR associates with silent chromatin regions in a Sir2-dependent manner and provide additional support for the role of AAR in assembly of silent chromatin.

Histone Demethylation and Timely DNA Replication

It is well-established that silent regions of the genome replicate late during S phase. In this issue of Molecular Cell, Black et al. (2010) uncover a conserved role for the JMJD2 family of histone demethylases in promoting replication within silent chromatin regions that contain histone H3 lysine 9 methylation and HP1.

The role of protein arginine methylation in the formation of silent chromatin

Establishment and maintenance of silent chromatin in the Saccharomyces cerevisiae involves a step-wise assembly of the SIR complex. Here we demonstrate a role for the protein arginine methyltransferase Hmt1 in this process. In the absence of catalytically active Hmt1, yeast cells display increased transcription from silent chromatin regions and increased mitotic recombination within tandem repeats of rDNA. At the molecular level, loss of Hmt1's catalytic activity results in decreased Sir2 and dimethylated Arg-3 histone H4 occupancy across silent chromatin regions. These data suggest a model whereby protein arginine methylation affects the establishment and maintenance of silent chromatin.

Nuclear pore association confers optimal expression levels for an inducible yeast gene

The organization of the nucleus into subcompartments creates microenvironments that are thought to facilitate distinct nuclear functions. In budding yeast, regions of silent chromatin, such as those at telomeres and mating-type loci, cluster at the nuclear envelope creating zones that favour gene repression. Other reports indicate that gene transcription occurs at the nuclear periphery, apparently owing to association of the gene with nuclear pore complexes. Here we report that transcriptional activation of a subtelomeric gene, HXK1 (hexokinase isoenzyme 1), by growth on a non-glucose carbon source led to its relocalization to nuclear pores. This relocation required the 3' untranslated region (UTR), which is essential for efficient messenger RNA processing and export, consistent with an accompanying report. However, activation of HXK1 by an alternative pathway based on the transactivator VP16 moved the locus away from the nuclear periphery and abrogated the normal induction of HXK1 by galactose. Notably, when we interfered with HXK1 localization by either antagonizing or promoting association with the pore, transcript levels were reduced or enhanced, respectively. From this we conclude that nuclear position has an active role in determining optimal gene expression levels.

A Trans-tail Histone Code Defined by Monomethylated H4 Lys-20 and H3 Lys-9 Demarcates Distinct Regions of Silent Chromatin

The specific post-translational modifications of the histone proteins are associated with specific DNA-templated processes, such as transcriptional activation or repression. To investigate the biological role(s) of histone H4 lysine 20 (H4 Lys-20) methylation, we created a novel panel of antibodies that specifically detected mono-, di-, or trimethylated H4 Lys-20. We report that the different methylated forms of H4 Lys-20 are compartmentalized within visually distinct, transcriptionally silent regions in the mammalian nucleus. Interestingly, direct comparison of methylated H4 Lys-20 with the different methylated states of histone H3 lysine 9 (H3 Lys-9) revealed significant overlap and exclusion between the specific groups of methyl modifications. Trimethylated H4 Lys-20 and H3 Lys-9 were both selectively enriched within pericentric heterochromatin. Similarly, monomethylated H4 Lys-20 and H3 Lys-9 partitioned together and the dimethylated forms partitioned together within the chromosome arms; however, the mono- and dimethylated modifications were virtually exclusive. These findings strongly suggest that the combinatorial presence or absence of the different methylated states of H4 Lys-20 and H3 Lys-9 define particular types of silent chromatin. Consistent with this, detailed analysis of monomethylated H4 Lys-20 and H3 Lys-9 revealed that both were preferentially and selectively enriched within the same nucleosome particle in vivo. Collectively, these findings define a novel trans-tail histone code involving monomethylated H4 Lys-20 and H3 Lys-9 that act cooperatively to mark distinct regions of silent chromatin within the mammalian epigenome.

Repairing subtelomeric DSBs at the nuclear periphery

Nuclear organization creates microenvironments favoring distinct nuclear functions. In budding yeast, silent chromatin regions such as telomeres are clustered at the nuclear periphery, creating zones of transcriptional repression. Recently, in the Journal of Cell Biology, Therizols et al. report that "telomere tethering at the nuclear periphery is essential for DNA double strand break repair in subtelomeric regions". Here, we discuss these results and their functional implications.

Proteomic and genomic characterization of chromatin complexes at a boundary

We have dissected specialized assemblies on the Saccharomyces cerevisiae genome that help define and preserve the boundaries that separate silent and active chromatin. These assemblies contain characteristic stretches of DNA that flank particular regions of silent chromatin, as well as five distinctively modified histones and a set of protein complexes. The complexes consist of at least 15 chromatin-associated proteins, including DNA pol ɛ, the Isw2-Itc1 and Top2 chromatin remodeling proteins, the Sas3-Spt16 chromatin modifying complex, and Yta7, a bromodomain-containing AAA ATPase. We show that these complexes are important for the faithful maintenance of an established boundary, as disruption of the complexes results in specific, anomalous alterations of the silent and active epigenetic states.

Targeting of the Yeast Ty5 Retrotransposon to Silent Chromatin Is Mediated by Interactions between Integrase and Sir4p

The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.

CDNA of the yeast retrotransposon Ty5 preferentially recombines with substrates in silent chromatin.

The yeast retrotransposon Ty5 preferentially integrates into regions of silent chromatin. Ty5 cDNA also recombines with homologous sequences, generating tandem elements or elements that have exchanged markers between cDNA and substrate. In this study, we demonstrate that Ty5 integration depends upon the conserved DD(35)E domain of integrase and cis-acting sequences at the end of the long terminal repeat (LTR) implicated in integrase binding. cDNA recombination requires Rad52p, which is responsible for homologous recombination. Interestingly, Ty5 cDNA recombines at least three times more frequently with substrates in silent chromatin than with a control substrate at an internal chromosomal locus. This preference depends upon the Ty5 targeting domain that is responsible for integration specificity, suggesting that localization of cDNA to silent chromatin results in the enhanced recombination. Recombination with a telomeric substrate occasionally generates highly reiterated Ty5 arrays, and mechanisms for tandem element formation were explored by using a plasmid-based recombination assay. Point mutations were introduced into plasmid targets, and recombination products were characterized to determine recombination initiation sites. Despite our previous observation of the importance of the LTR in forming tandem elements, recombination cannot simply be explained by crossover events between the LTRs of substrate and cDNA. We propose an alternative model based on single-strand annealing, where single-stranded cDNA initiates tandem element formation and the LTR is required for strand displacement to form a looped intermediate. Retrotransposons are increasingly found associated with chromosome ends, and amplification of Ty5 by both integration and recombination exemplifies how retroelements can contribute to telomere dynamics.

Silent chromatin determines target preference of the Saccharomyces retrotransposon Ty5.

The HML and HMR mating loci of Saccharomyces cerevisiae are bound in silent chromatin, which is assembled at the flanking E and I transcriptional silencers. The retrotransposon Ty5 preferentially integrates into regions of silent chromatin, and Ty5 insertions near the HMR-E silencer account for approximately 2% of total transposition events. Most Ty5 insertions occur within 800 bp on either side of the autonomously replicating consensus sequence within HMR-E. Ty5 target preference is determined by silent chromatin, because integration near HMR-E is abolished in strains with silencer mutations that alleviate transcriptional repression. The recognition of specific DNA sequences per se does not direct integration, rather, it is the protein complex assembled at the silencers. As demonstrated here for Ty5, recognition of specific chromatin domains may be a general mechanism by which retrotransposons and retroviruses determine integration sites.

The Saccharomyces retrotransposon Ty5 integrates preferentially into regions of silent chromatin at the telomeres and mating loci.

The nonrandom integration of retrotransposons and retroviruses suggests that chromatin influences target choice. Targeted integration, in turn, likely affects genome organization. In Saccharomyces, native Ty5 retrotransposons are located near telomeres and the silent mating locus HMR. To determine whether this distribution is a consequence of targeted integration, we isolated a transposition-competent Ty5 element from S. paradoxus, a species closely related to S. cerevisiae. This Ty5 element was used to develop a transposition assay in S. cerevisiae to investigate target preference of de novo transposition events. Of 87 independent Ty5 insertions, approximately 30% were located on chromosome III, indicating this small chromosome (approximately 1/40 of the yeast genome) is a highly preferred target. Mapping of the exact location of 19 chromosome III insertions showed that 18 were within or adjacent to transcriptional silencers flanking HML and HMR or the type X subtelomeric repeat. We predict Ty5 target preference is attributable to interactions between transposition intermediates and constituents of silent chromatin assembled at these sites. Ty5 target preference extends the link between telomere structure and reverse transcription as carried out by telomerase and Drosophila retrotransposons.

20] Mapping DNA-protein interactions by cross-linking

DNA- protein interactions attract much attention for they are involved in the structural organization of genomic DNA as well as in directing and regulating DNA function. This chapter describes here an experimental procedure for induction and analysis of DNA-protein cross-links. The chapter present the protocols for cross-linking proteins and DNA by dimethyl sulfate (DMS) methylation depurination- reduction and by UV light. Then, methods for recovery of the cross-linked complexes by depleting them of the uncross-linked DNA and proteins are explained. Methods for analysis of cross-links by gel electrophoresis diagonal techniques, DNA hybridization, immunological reactions, and peptide chemistry are described in this chapter. To characterize components of the cross-linked complex, hybridization with cloned DNA probes should be used for recognizing DNA sequences, whereas gel electrophoresis and/or specific antibodies are useful for identification of proteins. Gel electrophoresis is the method of choice for proteins that are present in the complex in a sufficient amount, for example, for histones. Thus, two-dimensional gel electrophoresis and hybridization with various probes, as described below, was used to demonstrate the reversible displacement of histones from transcribed gene regions, as well as an essential similarity in the arrangement of histones on DNA in core nucleosomes within transcribed and silent chromatin regions.