Specific Chromatin Structure Research Articles

Centromere identity is specified by a single centromeric nucleosome in budding yeast.

Chromosome segregation ensures that DNA is equally divided between daughter cells during each round of cell division. The centromere (CEN) is the specific locus on each chromosome that directs formation of the kinetochore, the multiprotein complex that interacts with the spindle microtubules to promote proper chromosomal alignment and segregation during mitosis. CENs are organized into a specialized chromatin structure due to the incorporation of an essential CEN-specific histone H3 variant (CenH3) in the centromeric nucleosomes of all eukaryotes. Consistent with its essential role at the CEN, the loss or up-regulation of CenH3 results in mitotic defects. Despite the requirement for CenH3 in CEN function, it is unclear how CenH3 nucleosomes structurally organize centromeric DNA to promote formation of the kinetochore. To address this issue, we developed a modified chromatin immunoprecipitation approach to analyze the number and position of CenH3 nucleosomes at the budding yeast CEN. Using this technique, we show that yeast CENs have a single CenH3 nucleosome positioned over the CEN-determining elements. Therefore, a single CenH3 nucleosome forms the minimal unit of centromeric chromatin necessary for kinetochore assembly and proper chromosome segregation.

Protection of telomeres by a conserved Stn1–Ten1 complex

Telomeres are specialized chromatin structures that protect chromosome ends. Critical among telomere proteins are those that bind the telomeric single-strand DNA (ssDNA) overhangs. These proteins are thought to differ among eukaryotes. Three interacting proteins (Cdc13, Stn1, and Ten1) associate with the telomeric overhang in budding yeast, a single protein known as Pot1 (protection of telomeres-1) performs this function in fission yeast, and a two-subunit complex consisting of POT1 and TPP1 associates with telomeric ssDNA in humans. Cdc13 and Pot1 have related oligonucleotide/oligosaccharide-binding fold (OB-fold) domains that bind the telomeric ssDNA overhang. Here we show that Schizosaccharomyces pombe has Stn1- and Ten1-like proteins that are essential for chromosome end protection. Stn1 orthologs exist in all species that have Pot1, whereas Ten1-like proteins can be found in all fungi. Fission yeast Stn1 and Ten1 localize at telomeres in a manner that correlates with the length of the ssDNA overhang, suggesting that they specifically associate with the telomeric ssDNA. Unlike in budding yeast, in which Cdc13, Stn1, and Ten1 all interact, fission yeast Stn1 and Ten1 associate with each other, but not with Pot1. Our findings suggest that two separate protein complexes are required for chromosome end protection in fission yeast. Structural profiling studies detect OB-fold domains in Stn1 and Ten1 orthologs, indicating that protection of telomeres by multiple proteins with OB-fold domains is conserved in eukaryotic evolution.

Recruitment of factors linking transcription and processing of pre-rRNA to NOR chromatin is UBF-dependent and occurs independent of transcription in human cells.

Efficient ribosome biogenesis requires coordination of a highly complex series of events. Early events include pre-RNA transcription, processing, and modification. Analysis in yeast has demonstrated that t-UTPs, components of the U3 snoRNA-containing pre-rRNA processing complex, are required for efficient transcription of ribosomal genes (rDNA) by RNA polymerase I (pol I). Here, we characterize human t-UTPs and establish that their ability to link transcription and pre-rRNA processing is evolutionarily conserved. The pol I transcription factor UBF binds extensively across rDNA throughout the cell cycle, resulting in a specialized form of chromatin that is the hallmark of active nucleolar organizer regions (NORs). Transcriptionally silent pseudo-NORs are ectopic, chromosomally integrated, artificial arrays that mimic this specialized chromatin structure. Pseudo-NORs sequester t-UTPs and factors linking transcription with pre-rRNA modification (Nopp140 and Treacle). Recruitment is independent of transcription, the underlying DNA sequence, and location within the nucleolus. Previously, we have demonstrated that pseudo-NORs sequester every component of the pol I transcription machinery. Taken together, these results highlight the importance of the specialized chromatin structure at active NORs in coordinating early events in ribosome biogenesis and nucleolar formation.

Epigenetic Flexibility Underlying Lineage Choices in the Adaptive Immune System

Although fundamental models have emerged in recent years describing how chromatin and transcription regulation interface with one another in the developing immune system, the order of events and their biological impact are still being resolved. Recent advances have provided a flexible, rather than static, view of chromatin regulation to reveal how both positive and negative forces work concomitantly to establish specific chromatin structures and regulate gene expression. The challenge will now be to explore new epigenetic models and validate them during lymphocyte development, with the ultimate goal of unraveling the long-sought mechanisms that support the emerging complexity of the adaptive immune response.

Tetrameric Structure of Centromeric Nucleosomes in Interphase Drosophila Cells

Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3). However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical “beads-on-a-string” appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity.

Different epigenetic layers engage in complex crosstalk to define the epigenetic state of mammalian rRNA genes

Eukaryotic cells contain several hundred ribosomal RNA (rRNA) genes (rDNA), a fraction of them being silenced by epigenetic mechanisms. The presence of two epigenetically distinct states of rRNA genes provides a unique opportunity to decipher the molecular mechanisms that establish the euchromatic, i.e. transcriptionally active, and the heterochromatic, i.e. transcriptionally silent, state of rDNA. This article summarizes our knowledge of the epigenetic mechanisms that control rDNA transcription and emphasizes how DNA methyltransferases and histone-modifying enzymes work in concert with chromatin-remodeling complexes and RNA-guided mechanisms to establish a specific chromatin structure that defines the transcriptional state of rRNA genes. These studies exemplify the mutual dependence and complex crosstalk among different epigenetic players in the alteration of the chromatin structure during the process of gene activation or silencing.

Osmotic Stress-dependent Repression Is Mediated by Histone H3 Phosphorylation and Chromatin Structure

Histone H3 phosphorylation has been linked to various environmental stress responses and specific chromatin structure. The role of H3 phosphorylation in the osmotic stress response was investigated on the mouse mammary tumor virus (MMTV) promoter in different chromatin configurations. Hormone-dependent transcription from the MMTV promoter is repressed by osmotic stress when the promoter is integrated and has a normal chromatin structure. However, when the MMTV promoter is transiently transfected, the chromatin structure is less organized, and hormone induction is not affected by osmotic stress. On the integrated MMTV promoter, phosphorylation of histone H3 serine 10 and 28 increases in response to osmotic stress, but the transient promoter shows no change. Hormone-dependent glucocorticoid receptor binding is reduced on the repressed promoter, and elevated H3 phosphorylation is temporally correlated with maximal MMTV repression Additionally, the protein kinase C inhibitor rottlerin, but not other kinase inhibitors, blocks both histone H3 phosphorylation and osmotic repression of MMTV transcription. Glucocorticoid receptor binding is inversely correlated with H3 phosphorylation, suggesting that displacement of the glucocorticoid receptor from the promoter is due to H3 phosphorylation and is the mechanism for the osmotic repression of hormone-dependent transcription.

RNA polymerase I in yeast transcribes dynamic nucleosomal rDNA

RNA polymerase (Pol) I-transcribed ribosomal genes of budding yeast exist as a tandem array (about 150 repeats) with transcription units separated by spacer sequences. Half of these rDNAs are inactivated by repressive chromatin structure, whereas the rest exist in an open conformation transcribed by closely spaced Pol I elongation complexes. Whereas previous studies have suggested that active rDNA is devoid of nucleosomal structure, we demonstrate that active rDNA has nucleosomal structure, according to chromatin immunoprecipitation and biochemical fractionation. Using a yeast strain with reduced numbers of all actively transcribed rDNA repeats, we show that rDNA exists in a dynamic chromatin structure of unphased nucleosomes. Furthermore, it is associated with chromatin-remodeling enzymes Chd1p, Isw1p and Isw2p, whose inactivation causes defects in transcription termination. We suggest that Pol I transcription, like that of Pol II, may be modulated by specific chromatin structures.

Proteolysis restricts localization of CID, the centromere-specific histone H3 variant of Drosophila, to centromeres

Centromere identity is determined by the formation of a specialized chromatin structure containing the centromere-specific histone H3 variant CENP-A. The precise molecular mechanism(s) accounting for the specific deposition of CENP-A at centromeres are still poorly understood. Centromeric deposition of CENP-A, which is independent of DNA replication, might involve specific chromatin assembly complexes and/or specific interactions with kinetochore components. However, transiently expressed CENP-A incorporates throughout chromatin indicating that CENP-A nucleosomes can also be promiscuously deposited during DNA replication. Therefore, additional mechanisms must exist to prevent deposition of CENP-A nucleosomes during replication and/or to remove them afterwards. Here, using transient expression experiments performed in Drosophila Kc cells, we show that proteasome-mediated degradation restricts localization of Drosophila CENP-A (CID) to centromeres by eliminating mislocalized CID as well as by regulating available CID levels. Regulating available CID levels appears essential to ensure centromeric deposition of transiently expressed CID as, when expression is increased in the presence of proteasome inhibitors, newly synthesized CID mislocalizes. Mislocalization of CID affects cell cycle progression as a high percentage of cells showing mislocalized CID are reactive against αPSer10H3 antibodies, enter mitosis at a very low frequency and show strong segregation defects. However, cells showing reduced amounts of mislocalized CID show normal cell cycle progression.

Cooperativeness of the Higher Chromatin Structure of the β-Globin Locus Revealed by the Deletion Mutations of DNase I Hypersensitive site 3 of the LCR

High-level transcription of the globin genes requires the enhancement by a distant element, the locus control region (LCR). Such long-range regulation in vivo involves spatial interaction between transcriptional elements, with intervening chromatin looping out. It has been proposed that the clustering of the HS sites of the LCR, the active globin genes, as well as the remote 5′ hypersensitive sites (HSs) (HS-60/-62 in mouse, HS-110 in human) and 3′HS1 forms a specific spatial chromatin structure, termed active chromatin hub (ACH). Here we report the effects of the HS3 deletions of the LCR on the spatial chromatin structure of the β-globin locus as revealed by the chromatin conformation capture (3C) technology. The small HS3 core deletion (0.23 kb), but not the large HS3 deletion (2.3 kb), disrupted the spatial interactions among all the HS sites of the LCR, the β-globin gene and 3′HS1. We have previously demonstrated that the large HS3 deletion barely impairs the structure of the LCR holocomplex, while the structure is significantly disrupted by the HS3 core deletion. Taken together, these results suggest that the formation of the ACH is dependent on a largely intact LCR structure. We propose that the ACH indeed is an extension of the LCR holocomplex.

Effect of hypermethylation of CCWGG sequences in DNA of Mesembryanthemum crystallinum plants on their adaptation to salt stress

Under salt stress conditions, the level of CpNpG-methylation (N is any nucleoside) of the nuclear genome of the facultative halophyte Mesembryanthemum crystallinum in the CCWGG sequences (W = A or T) increases two-fold and is coupled with hypermethylation of satellite DNA on switching-over of C3-photosynthesis to the crassulacean acid metabolism (CAM) pathway of carbon dioxide assimilation. The methylation pattern of the CCWGG sequences is not changed in both the 5'-promoter region of the gene of phosphoenolpyruvate carboxylase, the key enzyme of C4-photosynthesis and CAM, and in the nuclear ribosomal DNA. Thus, a specific CpNpG-hypermethylation of satellite DNA has been found under conditions of expression of a new metabolic program. The functional role of the CpNpG-hypermethylation of satellite DNA is probably associated with formation of a specialized chromatin structure simultaneously regulating expression of a large number of genes in the cells of M. crystallinum plants on their adaptation to salt stress and switching-over to CAM metabolism.

Chromatin domain activation via GATA-1 utilization of a small subset of dispersed GATA motifs within a broad chromosomal region

Cis elements that mediate transcription factor binding are abundant within genomes, but the rules governing occupancy of such motifs in chromatin are not understood. The transcription factor GATA-1 that regulates red blood cell development binds with high affinity to GATA motifs, and initial studies suggest that these motifs are often unavailable for occupancy in chromatin. Whereas GATA-2 regulates the differentiation of all blood cell lineages via GATA motif binding, the specificity of GATA-2 chromatin occupancy has not been studied. We found that conditionally active GATA-1 (ER-GATA-1) and GATA-2 occupy only a small subset of the conserved GATA motifs within the murine beta-globin locus. Kinetic analyses in GATA-1-null cells indicated that ER-GATA-1 preferentially occupied GATA motifs at the locus control region (LCR), in which chromatin accessibility is largely GATA-1-independent. Subsequently, ER-GATA-1 increased promoter accessibility and occupied the betamajor promoter. ER-GATA-1 increased erythroid Krüppel-like factor and SWI/SNF chromatin remodeling complex occupancy at restricted LCR sites. These studies revealed three phases of beta-globin locus activation: GATA-1-independent establishment of specific chromatin structure features, GATA-1-dependent LCR complex assembly, and GATA-1-dependent promoter complex assembly. The differential utilization of dispersed GATA motifs therefore establishes spatial/temporal regulation and underlies the multistep activation mechanism.

Functional elements residing within satellite DNAs

Satellite DNAs represent a fast-evolving portion of the eukaryotic genome whose evolution is proposed to be driven by the stochastic process of molecular drive. Recent results indicate that satellite DNAs are subject to certain structural constraints, which are probably related to their interaction with proteins involved in the establishment of specific chromatin structures. The evolutionary persistence and high sequence conservation of some satellites, as well as the presence of stage- or tissue-specific, differentially expressed transcripts in several species, are consistent with the hypothesis that satellite DNA could have a regulatory role in eukaryotic organisms. Although the role of most transcripts is not known, some act as precursors of small interfering RNAs, which are now recognized as having an important role in chromatin modulation and the control of gene expression. Furthermore, some transcripts are involved in the cellular response to stress.

Synergistic and Additive Properties of the Beta-Globin Locus Control Region (LCR) Revealed by 5′HS3 Deletion Mutations: Implication for LCR Chromatin Architecture

Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5'HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (beta-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5'HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5'HS3 deletion abolished histone acetylation throughout the beta-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5' DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5'HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5' DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5'HS3 and HS3 core deletions.

MBD3L2 Interacts with MBD3 and Components of the NuRD Complex and Can Oppose MBD2-MeCP1-mediated Methylation Silencing

MBD2 and MBD3 are two proteins that contain methyl-CpG binding domains and have a transcriptional repression function. Both proteins are components of a large CpG-methylated DNA binding complex named MeCP1, which consists of the nucleosome remodeling and histone deacetylase complex Mi2-NuRD and MBD2. MBD3L2 (methyl-CpG-binding protein 3-like 2) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG-binding domain. Unlike MBD3L1, which is specifically expressed in haploid male germ cells, MBD3L2 expression is more widespread. MBD3L2 interacts with MBD3 in vitro and in vivo, co-localizes with MBD3 but not MBD2, and does not localize to methyl-CpG-rich regions in the nucleus. In glutathione S-transferase pull-down assays, MBD3L2 is found associated with several known components of the Mi2-NuRD complex, including HDAC1, HDAC2, MTA1, MBD3, p66, RbAp46, and RbAp48. Gel shift experiments with nuclear extracts and a CpG-methylated DNA probe indicate that recombinant MBD3L2 can displace a form of the MeCP1 complex from methylated DNA. MBD3L2 acts as a transcriptional repressor when tethered to a GAL4-DNA binding domain. Repression by GAL4-MBD3L2 is relieved by MBD2 and vice versa, and repression by MBD2 from a methylated promoter is relieved by MBD3L2. The data are consistent with a role of MBD3L2 as a transcriptional modulator that can interchange with MBD2 as an MBD3-interacting component of the NuRD complex. Thus, MBD3L2 has the potential to recruit the MeCP1 complex away from methylated DNA and reactivate transcription.

Central role of Ifh1p–Fhl1p interaction in the synthesis of yeast ribosomal proteins

The 138 genes encoding the 79 ribosomal proteins (RPs) of Saccharomyces cerevisiae form the tightest cluster of coordinately regulated genes in nearly all transcriptome experiments. The basis for this observation remains unknown. We now provide evidence that two factors, Fhl1p and Ifh1p, are key players in the transcription of RP genes. Both are found at transcribing RP genes in vivo. Ifh1p, but not Fhl1p, leaves the RP genes when transcription is repressed. The occupancy of the RP genes by Ifh1p depends on its interaction with the phospho-peptide recognizing forkhead-associated domain of Fhl1p. Disruption of this interaction is severely deleterious to ribosome synthesis and cell growth. Loss of functional Fhl1p leads to cells that have only 20% the normal amount of RNA and that synthesize ribosomes at only 5-10% the normal rate. Homeostatic mechanisms within the cell respond by reducing the transcription of rRNA to match the output of RPs, and by reducing the global transcription of mRNA to match the capacity of the translational apparatus.

The Histone Fold Domain of Cse4 Is Sufficient for CEN Targeting and Propagation of Active Centromeres in Budding Yeast

Centromere-specific H3-like proteins (CenH3s) are conserved across the eukaryotic kingdom and are required for packaging centromere DNA into a specialized chromatin structure required for kinetochore assembly. Cse4 is the CenH3 protein of the budding yeast Saccharomyces cerevisiae. Like all CenH3 proteins, Cse4 consists of a conserved histone fold domain (HFD) and a divergent N terminus (NT). The Cse4 NT contains an essential domain designated END (for essential N-terminal domain); deletion of END is lethal. To investigate the role of the Cse4 NT in centromere targeting, a series of deletion alleles (cse4DeltaNT) were analyzed. No part of the Cse4 NT was required to target mutant proteins to centromere DNA in the presence of functional Cse4. A Cse4 degron strain was used to examine targeting of a Cse4DeltaNT protein in the absence of wild-type Cse4. The END was not required for centromere targeting under these conditions, confirming that the HFD confers specificity of Cse4 centromere targeting. Surprisingly, overexpression of the HFD bypassed the requirement for the END altogether, and viable S. cerevisiae strains in which the cells express only the Cse4 HFD and six adjacent N-terminal amino acids (Cse4Delta129) were constructed. Despite the complete absence of the NT, mitotic chromosome loss in the cse4Delta129 strain increased only 6-fold compared to a 15-fold increase in strains overexpressing wild-type Cse4. Thus, when overexpressed, the Cse4 HFD is sufficient for centromere function in S. cerevisiae, and no posttranslational modification or interaction of the NT with other kinetochore component(s) is essential for accurate chromosome segregation in budding yeast.

A Deficiency Screen for Dominant Suppressors of Telomeric Silencing in Drosophila

Heterochromatin is a specialized chromatin structure in chromosomal regions associated with repeated DNA sequences and low concentrations of genes. Formation of heterochromatin is determined in large part by enzymes that modify histones and structural proteins that bind to these modified histones in a cooperative fashion. In Drosophila, mutations in genes that encode heterochromatic proteins are often dominant and increase expression of genes placed into heterochromatic positions. To find components of telomeric heterochromatin in Drosophila, we screened a collection of autosomal deficiencies for dominant suppressors of silencing of a transgene at the telomere of chromosome 2L. While many deficiency chromosomes are associated with dominant suppressors, in the cases tested on chromosome 2 the suppressor mapped to the 2L telomere, rather than the deficiency. We infer that background effects may hamper the search for genes that play a role in telomeric heterochromatin formation and that either very few genes participate in this pathway or mutations in these genes are not dominant suppressors of telomeric position effect. The data also suggest that the 2L telomere region plays a major role in telomeric silencing.

Kinetochores prevent repair of UV damage in Saccharomyces cerevisiae centromeres.

Centromeres form specialized chromatin structures termed kinetochores which are required for accurate segregation of chromosomes. DNA lesions might disrupt protein-DNA interactions, thereby compromising segregation and genome stability. We show that yeast centromeres are heavily resistant to removal of UV-induced DNA lesions by two different repair systems, photolyase and nucleotide excision repair. Repair resistance persists in G(1)- and G(2)/M-arrested cells. Efficient repair was obtained only by disruption of the kinetochore structure in a ndc10-1 mutant, but not in cse4-1 and cbf1 Delta mutants. Moreover, UV photofootprinting and DNA repair footprinting showed that centromere proteins cover about 120 bp of the centromere elements CDEII and CDEIII, including 20 bp of flanking CDEIII. Thus, DNA lesions do not appear to disrupt protein-DNA interactions in the centromere. Maintaining a stable kinetochore structure seems to be more important for the cell than immediate removal of DNA lesions. It is conceivable that centromeres are repaired by postreplication repair pathways.

Recruitment of SWI/SNF to the Human Immunodeficiency Virus Type 1 Promoter

Following human immunodeficiency virus type 1 (HIV-1) integration into the host cell's genome, the 5' long terminal repeat (LTR) is packaged into a highly specific chromatin structure comprised of an array of nucleosomes positioned with respect to important DNA sequence elements that regulate the transcriptional activity of the provirus. While several host cell factors have been shown to be important for chromatin remodeling and/or basal transcription, no specific mechanism that relieves the transcriptional repression imposed by nuc-1, a positioned nucleosome that impedes the start site of transcription, has been found. Since phorbol esters cause the rapid disruption of nuc-1 and markedly stimulate HIV-1 transcription, we looked for protein factors that associate with this region of the HIV-1 promoter in a phorbol-ester-dependent manner. We report here that ATF-3, JunB, and BRG-1 (the ATPase subunit of the 2-MDa human chromatin remodeling machine SWI/SNF) are recruited to the 3' boundary of nuc-1 following phorbol myristate acetate stimulation in Jurkat T cells. Analysis of the recruitment of BRG-1 in nuclear extracts prepared from Jurkat T cells and reconstitution of an in vitro system with purified components demonstrate that ATF-3 is responsible for targeting human SWI/SNF (hSWI/SNF) to the HIV-1 promoter. Importantly, this recruitment of hSWI/SNF required HMGA1 proteins. Further support for this conclusion comes from immunoprecipitation experiments showing that BRG-1 and ATF-3 can exist together in the same complex. Although ATF-3 clearly plays a role in the specific targeting of BRG-1 to the HIV-1 promoter, the maintenance of a stable association between BRG-1 and chromatin appears to be dependent upon histone acetylation. By adding BRG-1 back into a BRG-1-deficient cell line (C33A cells), we demonstrate that trichostatin A strongly induces the 5'-LTR-driven reporter transcription in a manner that is dependent upon BRG-1 recruitment.