Chromatin-modifying Activities Research Articles

Epigenetic regulation of telomere length in mammalian cells by the Suv39h1 and Suv39h2 histone methyltransferases.

Telomeres are capping structures at the ends of eukaryotic chromosomes composed of TTAGGG repeats bound to an array of specialized proteins. Telomeres are heterochromatic regions. Yeast and flies with defects in activities that modify the state of chromatin also have abnormal telomere function, but the putative role of chromatin-modifying activities in regulating telomeres in mammals is unknown. Here we report on telomere length and function in mice null with respect to both the histone methyltransferases (HMTases) Suv39h1 and Suv39h2 (called SUV39DN mice). Suv39h1 and Suv39h2 govern methylation of histone H3 Lys9 (H3-Lys9) in heterochromatic regions. We show that primary cells derived from SUV39DN mice have abnormally long telomeres relative to wild-type controls. Using chromatin immunoprecipitation (ChIP) analysis, we found that telomeres were enriched in di- and trimethylated H3-Lys9 but that telomeres of SUV39DN cells had less dimethylated and trimethylated H3-Lys9 but more monomethylated H3-Lys9. Concomitant with the decrease in H3-Lys9 methylation, telomeres in SUV39DN cells had reduced binding of the chromobox proteins Cbx1, Cbx3 and Cbx5, homologs of Drosophila melanogaster heterochromatin protein 1 (HP1). These findings indicate substantial changes in the state of telomeric heterochromatin in SUV39DN cells, which are associated with abnormal telomere elongation. Taken together, the results indicate epigenetic regulation of telomere length in mammals by Suv39h1 and Suv39h2.

Differential transcription factor occupancy but evolutionarily conserved chromatin features at the human and mouse M-CSF (CSF-1) receptor loci.

The c-FMS gene encodes the macrophage colony-stimulating factor receptor (M-CSFR or CSF1-R), which is a tyrosine kinase growth factor receptor essential for macrophage development. We have previously characterized the chromatin features of the mouse gene; however, very little is known about chromatin structure and function of the human c-FMS locus. Here we present a side-by-side comparison of the chromatin structure, histone modification, transcription factor occupancy and cofactor recruitment of the human and the mouse c-FMS loci. We show that, similar to the mouse gene, the human c-FMS gene possesses a promoter and an intronic enhancer element (c-fms intronic regulatory element or FIRE). Both elements are evolutionarily conserved and specifically active in macrophages. However, we demonstrate by in vivo footprinting that both murine and human c-FMS cis-regulatory elements are recognised by an overlapping, but non-identical, set of transcription factors. Despite these differences, chromatin immunoprecipitation experiments show highly similar patterns of histone H3 modification and a similar distribution of chromatin modifying and remodelling activities at individual cis-regulatory elements and across the c-FMS locus. Our experiments support the hypothesis that the same regulatory principles operate at both genes via conserved cores of transcription factor binding sites.

The in vitro reconstitution of nucleosome and its binding patterns with HMG1/2 and HMG14/17 proteins.

Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociated from DNA at 1M NaCl. When the salt concentration was slowly reduced to 650 mM and 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic "beads-on-a-string" structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution of nucleosome, the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372 to -194 bp) DNA sequences in the 5 flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene expression.

M Phase-specific Phosphorylation of BRCA2 by Polo-like Kinase 1 Correlates with the Dissociation of the BRCA2-P/CAF Complex

BRCA2 is a breast tumor susceptibility gene encoding a 390-kDa protein with functions in maintaining genomic stability and cell cycle progression. Evidence has been accumulated to support the concept that BRCA2 has a critical role in homologous recombination of DNA double-stranded breaks by interacting with RAD51. In addition, BRCA2 may have chromatin modifying activity through interaction with a histone acetyltransferase protein, p300/CBP-associated factor (P/CAF). To explore how the functions of BRCA2 may be regulated, the post-translational modifications of BRCA2 throughout the cell cycle were examined. We found that BRCA2 is hyperphosphorylated specifically in M phase and becomes dephosphorylated as cells exit M phase and enter interphase. This specific phosphorylation of BRCA2 was not observed in cells treated with DNA-damaging agents. Systematic mapping of the potential mitosis specific phosphorylation sites revealed the N-terminal 284 amino acids of BRCA2 (BR-N1) as the major region of phosphorylation and mass spectrometric analysis identified two phosphopeptides that contain "phosphorylation consensus motifs" for Polo-like kinase 1 (Plk1). Phosphorylation of BR-N1 with Plk1 recapitulated the electrophoretic mobility change as seen in BR-N1 isolated from M phase cells. Plk1 interacts with BRCA2 in vivo, and mutation of Ser193, Ser205/206, and Thr203/207 to Ala in BR-N1 abolished Plk1 phosphorylation, suggesting that BRCA2 is the substrate of Plk1. Furthermore, both the hyperphosphorylated and hypophosphorylated forms of BRCA2 bind to RAD51, whereas the M phase hyperphosphorylated form of BRCA2 no longer associates with the P/CAF, suggesting that the dissociation of P/CAF-BRCA2 complex is regulated by phosphorylation. Taken together, these results implicate a potential role of BRCA2 in modulating M phase progression.

Dynamic Chromatin Alterations Triggered by Natural and Synthetic Activation Domains

The activation domains (ADs) of transcription activators recruit a multiplicity of enzymatic activities to gene promoters. The mechanisms by which such recruitment takes place are not well understood. Using chromatin immunoprecipitation, we demonstrate dynamic alterations in the abundance of histones H2A, H3, and H4 at promoters of genes regulated by the HSF and Gal4 activators of Saccharomyces cerevisiae. Transcriptional activation of these genes, particularly those regulated by HSF, is accompanied by a significant reduction in both acetylated and unacetylated histones at promoters and may involve the transient displacement of histone octamers. To gain insight into the function of ADs, we conducted a genetic screen to identify polypeptides that could substitute for the 340-residue C-terminal activator of HSF and rescue the temperature sensitivity caused by its deletion. We found that the ts(-) phenotype of HSF(1-493) could be complemented by peptides as short as 11 amino acids. Such peptides are enriched in acidic and hydrophobic residues, and exhibit both trans-activating and chromatin-modifying activities when fused to the Gal4 DNA-binding domain. We also demonstrate that a previously identified 14-amino acid histone H3-binding module of human CTF1/NF1, which is similar to synthetic ADs, can substitute for the HSF C-terminal activator in conferring temperature resistance and can mediate the modification of promoter chromatin structure. Possible mechanisms of AD function, including one involving direct interactions with histones, are discussed.

Attenuation of a phosphorylation-dependent activator by an HDAC-PP1 complex.

The second messenger cAMP stimulates transcription with burst-attenuation kinetics that mirror the PKA-dependent phosphorylation and subsequent protein phosphatase 1 (PP1)-mediated dephosphorylation of the cAMP responsive element binding protein (CREB) at Ser133. Phosphorylation of Ser133 promotes recruitment of the co-activator histone acetylase (HAT) paralogs CBP and P300, which in turn stimulate acetylation of promoter-bound histones during the burst phase. Remarkably, histone deacetylase (HDAC) inhibitors seem to potentiate CREB activity by prolonging Ser133 phosphorylation in response to cAMP stimulus, suggesting a potential role for HDAC complexes in silencing CREB activity. Here we show that HDAC1 associates with and blocks Ser133 phosphorylation of CREB during pre-stimulus and attenuation phases of the cAMP response. HDAC1 promotes Ser133 dephosphorylation via a stable interaction with PP1, which we detected in co-immunoprecipitation and co-purification studies. These results illustrate a novel mechanism by which signaling and chromatin-modifying activities act coordinately to repress the activity of a phosphorylation-dependent activator.

The VP16 Activation Domain Interacts with Multiple Transcriptional Components as Determined by Protein-Protein Cross-linking in Vivo

Transcriptional activator proteins recruit the RNA polymerase II machinery and chromatin-modifying activities to promoters. Biochemical experiments indicate that activator proteins can associate with a large number of proteins, and many such proteins have been proposed to be direct targets of activators. However, there is great uncertainty about which biochemical interactions are physiologically relevant. Here, we develop a formaldehyde-based cross-linking procedure to identify protein-protein interactions that occur under physiological conditions. We show that the VP16 activation domain directly interacts with TATA-binding protein (TBP), TFIIB, and the SAGA histone acetylase complex in vivo.

Targeted recruitment of Rpd3 histone deacetylase represses transcription by inhibiting recruitment of Swi/Snf, SAGA, and TATA binding protein.

Certain DNA-binding repressors inhibit transcription by recruiting Rpd3 histone deacetylase complexes to promoters and generating domains of histone deacetylation that extend over a limited number of nucleosomes. Here, we show that the degree of Rpd3-dependent repression depends on the activator and the level of activation, not the extent of histone deacetylation. In all cases tested, activator binding is unaffected by histone deacetylation. In contrast, Rpd3-dependent repression is associated with decreased occupancy by TATA binding protein (TBP), the Swi/Snf nucleosome-remodeling complex, and the SAGA histone acetylase complex. Transcriptional repression is bypassed by direct recruitment of TBP and several TBP-associated factors, but not by natural activation domains or direct recruitment of polymerase II holoenzyme components. These results suggest that the domain of localized histone deacetylation generated by recruitment of Rpd3 mediates repression by inhibiting recruitment of chromatin-modifying activities and TBP.

Histone H3 specific acetyltransferases are essential for cell cycle progression.

Longstanding observations suggest that acetylation and/or amino-terminal tail structure of histones H3 and H4 are critical for eukaryotic cells. For Saccharomyces cerevisiae, loss of a single H4-specific histone acetyltransferase (HAT), Esa1p, results in cell cycle defects and death. In contrast, although several yeast HAT complexes preferentially acetylate histone H3, the catalytic subunits of these complexes are not essential for viability. To resolve the apparent paradox between the significance of H3 versus H4 acetylation, we tested the hypothesis that H3 modification is essential, but is accomplished through combined activities of two enzymes. We observed that Sas3p and Gcn5p HAT complexes have overlapping patterns of acetylation. Simultaneous disruption of SAS3, the homolog of the MOZ leukemia gene, and GCN5, the hGCN5/PCAF homolog, is synthetically lethal due to loss of acetyltransferase activity. This key combination of activities is specific for these two HATs because neither is synthetically lethal with mutations of other MYST family or H3-specific acetyltransferases. Further, the combined loss of GCN5 and SAS3 functions results in an extensive, global loss of H3 acetylation and arrest in the G(2)/M phase of the cell cycle. The strikingly similar effect of loss of combined essential H3 HAT activities and the loss of a single essential H4 HAT underscores the fundamental biological significance of each of these chromatin-modifying activities.

Epigenetic aspects of X-chromosome dosage compensation.

The X chromosomes of mammals and fruit flies exhibit unusual properties that have evolved to deal with the different dosages of X-linked genes in males (XY) and females (XX). The X chromosome dosage-compensation mechanisms discovered in these species are evolutionarily unrelated, but exhibit surprising parallels in their regulatory strategies. These features include the importance of noncoding RNAs, and epigenetic spreading of chromatin-modifying activities. Sex chromosomes have posed a fascinating puzzle for biologists. The dissimilar organization, gene content, and regulation of the X and Y chromosomes are thought to reflect selective forces acting on original pairs of identical chromosomes (1-3). The result in many organisms is a male-specific Y chromosome that has lost most of its original genetic content, and a difference in dosage of the X chromosome in males (XY) and females (XX).

Histone acetyltransferases.

Transcriptional regulation in eukaryotes occurs within a chromatin setting and is strongly influenced by nucleosomal barriers imposed by histone proteins. Among the well-known covalent modifications of histones, the reversible acetylation of internal lysine residues in histone amino-terminal domains has long been positively linked to transcriptional activation. Recent biochemical and genetic studies have identified several large, multisubunit enzyme complexes responsible for bringing about the targeted acetylation of histones and other factors. This review discusses our current understanding of histone acetyltransferases (HATs) or acetyltransferases (ATs): their discovery, substrate specificity, catalytic mechanism, regulation, and functional links to transcription, as well as to other chromatin-modifying activities. Recent studies underscore unexpected connections to both cellular regulatory processes underlying normal development and differentiation, as well as abnormal processes that lead to oncogenesis. Although the functions of HATs and the mechanisms by which they are regulated are only beginning to be understood, these fundamental processes are likely to have far-reaching implications for human biology and disease.

CAC3 (MSI1) Suppression ofRAS2G19V Is Independent of Chromatin Assembly Factor I and Mediated by NPR1

Cac3p/Msi1p, the Saccharomyces cerevisiae homolog of retinoblastoma-associated protein 48 (RbAp48), is a component of chromatin assembly factor I (CAF-I), a complex that assembles histones H3 and H4 onto replicated DNA. CAC3 overexpression also suppresses the RAS/cyclic AMP (cAMP) signal transduction pathway by an unknown mechanism. We investigated this mechanism and found that CAC3 suppression of RAS/cAMP signal transduction was independent of either CAC1 or CAC2, subunits required for CAF-I function. CAC3 suppression was also independent of other chromatin-modifying activities, indicating that Cac3p has at least two distinct, separable functions, one in chromatin assembly and one in regulating RAS function. Unlike Cac1p, which localizes primarily to the nucleus, Cac3p localizes to both the nucleus and the cytoplasm. In addition, Cac3p associates with Npr1p, a cytoplasmic kinase that stablizes several nutrient transporters by antagonizing a ubiquitin-mediated protein degradation pathway. Deletion of NPR1, like overexpression of Cac3p, suppressed the RAS/cAMP pathway. Furthermore, NPR1 overexpression interfered with the ability of CAC3 to suppress the RAS/cAMP pathway, indicating that extra Cac3p suppresses the RAS/cAMP pathway by sequestering Npr1p. Deletion of NPR1 did not affect the quantity, phosphorylation state, or localization of Ras2p. Consistent with the idea that Npr1p exerts its effect on the RAS/cAMP pathway by antagonizing a ubiquitin-mediated process, excess ubiquitin suppressed both the heat shock sensitivity and the sporulation defects caused by constitutive activation of the RAS/cAMP pathway. Thus, CAC3/MSI1 regulates the RAS/cAMP pathway via a chromatin-independent mechanism that involves the sequestration of Npr1p and may be due to the increased ubiquitination of an Npr1p substrate.

Histone Acetylation and hSWI/SNF Remodeling Act in Concert to Stimulate V(D)J Cleavage of Nucleosomal DNA

The ordered assembly of immunoglobulin and TCR genes by V(D)J recombination depends on the regulated accessibility of individual loci. We show here that the histone tails and intrinsic nucleosome structure pose significant impediments to V(D)J cleavage. However, alterations to nucleosome structure via histone acetylation or by stable hSWI/SNF-dependent remodeling greatly increase the accessibility of nucleosomal DNA to V(D)J cleavage. Moreover, acetylation and hSWI/SNF remodeling can act in concert on an individual nucleosome to achieve levels of V(D)J cleavage approaching those observed on naked DNA. These results are consistent with a model in which regulated recruitment of chromatin modifying activities is involved in mediating the lineage and stage-specific control of V(D)J recombination.

Chromatin-related properties of CBP fused to MLL generate a myelodysplastic-like syndrome that evolves into myeloid leukemia.

As a result of the recurring translocation t(11;16) (q23;p13.3), MLL (mixed-lineage leukemia) is fused in frame to CBP (CREB binding protein). This translocation has been documented almost exclusively in cases of acute leukemia or myelodysplasia secondary to therapy with drugs that target DNA topo isomerase II. The minimal chimeric protein that is produced fuses MLL to the bromodomain, histone acetyltransferase (HAT) domain, EIA-binding domain and steroid-receptor coactivator binding domains of CBP. We show that transplantation of bone marrow retrovirally transduced with MLL-CBP induces myeloid leukemias in mice that are preceded by a long preleukemic phase similar to the myelodysplastic syndrome (MDS) seen in many t(11;16) patients but unusual for other MLL translocations. Structure-function analysis demonstrated that fusion of both the bromodomain and HAT domain of CBP to the amino portion of MLL is required for full in vitro transformation and is sufficient to induce the leukemic phenotype in vivo. This suggests that the leukemic effect of MLL-CBP results from the fusion of the chromatin association and modifying activities of CBP with the DNA binding activities of MLL.

Retinoid receptors in health and disease: co-regulators and the chromatin connection

Retinoid receptors behave as ligand-dependent transcriptional regulators, repressing transcription in the absence of ligand and activating transcription in its presence. The different effects on transcription are carried out through recruitment of co-regulators: unliganded receptors bind corepressors (NCoR and SMRT) that are found within a complex containing histone deacetylase (HDAC) activity, whereas liganded receptors recruit coactivators with histone acetylase activity (HATs). Chromatin remodeling activities have also shown to be required, suggesting a hierarchy of promoter structure modifications in RA target genes carried out by multiple coregulatory complexes. In this review, we examine the experimental evidence for the model just sketched. We focus on recent findings highlighting new molecular details in receptor*b1coregulator interactions, including the discovery and initial characterisation of novel complexes with multiple chromatin modifying activities. Finally, we look at the role of aberrant recruitment of the NCoRHDAC complex by altered retinoid receptors in the pathogenesis of acute promyelocytic leukemia. These results point to a crucial role for control of transcription factor coregulator interactions in the regulation of cellular processes, and suggest new molecular targets for cancer therapy.

Histone acetyltransferase and protein kinase activities copurify with a putative Xenopus RNA polymerase I holoenzyme self-sufficient for promoter-dependent transcription.

Mounting evidence suggests that eukaryotic RNA polymerases preassociate with multiple transcription factors in the absence of DNA, forming RNA polymerase holoenzyme complexes. We have purified an apparent RNA polymerase I (Pol I) holoenzyme from Xenopus laevis cells by sequential chromatography on five columns: DEAE-Sepharose, Biorex 70, Sephacryl S300, Mono Q, and DNA-cellulose. Single fractions from every column programmed accurate promoter-dependent transcription. Upon gel filtration chromatography, the Pol I holoenzyme elutes at a position overlapping the peak of Blue Dextran, suggesting a molecular mass in the range of approximately 2 MDa. Consistent with its large mass, Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels reveal approximately 55 proteins in fractions purified to near homogeneity. Western blotting shows that TATA-binding protein precisely copurifies with holoenzyme activity, whereas the abundant Pol I transactivator upstream binding factor does not. Also copurifying with the holoenzyme are casein kinase II and a histone acetyltransferase activity with a substrate preference for histone H3. These results extend to Pol I the suggestion that signal transduction and chromatin-modifying activities are associated with eukaryotic RNA polymerases.

Chromatin deacetylation by an ATP-dependent nucleosome remodelling complex.

The dynamic assembly and remodelling of eukaryotic chromosomes facilitate fundamental cellular processes such as DNA replication and gene transcription. The repeating unit of eukaryotic chromosomes is the nucleosome core, consisting of DNA wound about a defined octamer of histone proteins. Two enzymatic processes that regulate transcription by targeting elements of the nucleosome include ATP-dependent nucleosome remodelling and reversible histone acetylation. The histone deacetylases, however, are unable to deacetylate oligonucleosomal histones in vitro. The protein complexes that mediate ATP-dependent nucleosome remodelling and histone acetylation/deacetylation in the regulation of transcription were considered to be different, although it has recently been suggested that these activities might be coupled. We report here the identification and functional characterization of a novel ATP-dependent nucleosome remodelling activity that is part of an endogenous human histone deacetylase complex. This activity is derived from the CHD3 and CHD4 proteins which contain helicase/ATPase domains found in SWI2-related chromatin remodelling factors, and facilitates the deacetylation of oligonucleosomal histones in vitro. We refer to this complex as the nucleosome remodelling and deacetylating (NRD) complex. Our results establish a physical and functional link between the distinct chromatin-modifying activities of histone deacetylases and nucleosome remodelling proteins.