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The Drosophila melanogaster CHD1 Chromatin Remodeling Factor Modulates Global Chromosome Structure and Counteracts HP1a and H3K9me2  [PDF]
Lakshmi Bugga, Ivy E. McDaniel, Liana Engie, Jennifer A. Armstrong
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0059496
Abstract: CHD1 is a conserved chromatin remodeling factor that localizes to active genes and functions in nucleosome assembly and positioning as well as histone turnover. Mouse CHD1 is required for the maintenance of stem cell pluripotency while human CHD1 may function as a tumor suppressor. To investigate the action of CHD1 on higher order chromatin structure in differentiated cells, we examined the consequences of loss of CHD1 and over-expression of CHD1 on polytene chromosomes from salivary glands of third instar Drosophila melanogaster larvae. We observed that chromosome structure is sensitive to the amount of this remodeler. Loss of CHD1 resulted in alterations of chromosome structure and an increase in the heterochromatin protein HP1a, while over-expression of CHD1 disrupted higher order chromatin structure and caused a decrease in levels of HP1a. Over-expression of an ATPase inactive form of CHD1 did not result in severe chromosomal defects, suggesting that the ATPase activity is required for this in vivo phenotype. Interestingly, changes in CHD1 protein levels did not correlate with changes in the levels of the euchromatin mark H3K4me3 or elongating RNA Polymerase II. Thus, while CHD1 is localized to transcriptionally active regions of the genome, it can function to alter the levels of HP1a, perhaps through changes in methylation of H3K9.
A Key Role for Chd1 in Histone H3 Dynamics at the 3′ Ends of Long Genes in Yeast  [PDF]
Marta Radman-Livaja equal contributor,Tiffani K. Quan equal contributor,Lourdes Valenzuela,Jennifer A. Armstrong,Tibor van Welsem,TaeSoo Kim,Laura J. Lee,Stephen Buratowski,Fred van Leeuwen,Oliver J. Rando ,Grant A. Hartzog
PLOS Genetics , 2012, DOI: 10.1371/journal.pgen.1002811
Abstract: Chd proteins are ATP–dependent chromatin remodeling enzymes implicated in biological functions from transcriptional elongation to control of pluripotency. Previous studies of the Chd1 subclass of these proteins have implicated them in diverse roles in gene expression including functions during initiation, elongation, and termination. Furthermore, some evidence has suggested a role for Chd1 in replication-independent histone exchange or assembly. Here, we examine roles of Chd1 in replication-independent dynamics of histone H3 in both Drosophila and yeast. We find evidence of a role for Chd1 in H3 dynamics in both organisms. Using genome-wide ChIP-on-chip analysis, we find that Chd1 influences histone turnover at the 5′ and 3′ ends of genes, accelerating H3 replacement at the 5′ ends of genes while protecting the 3′ ends of genes from excessive H3 turnover. Although consistent with a direct role for Chd1 in exchange, these results may indicate that Chd1 stabilizes nucleosomes perturbed by transcription. Curiously, we observe a strong effect of gene length on Chd1's effects on H3 turnover. Finally, we show that Chd1 also affects histone modification patterns over genes, likely as a consequence of its effects on histone replacement. Taken together, our results emphasize a role for Chd1 in histone replacement in both budding yeast and Drosophila melanogaster, and surprisingly they show that the major effects of Chd1 on turnover occur at the 3′ ends of genes.
Transgenerational Propagation and Quantitative Maintenance of Paternal Centromeres Depends on Cid/Cenp-A Presence in Drosophila Sperm  [PDF]
Nitika Raychaudhuri,Raphaelle Dubruille,Guillermo A. Orsi,Homayoun C. Bagheri,Benjamin Loppin,Christian F. Lehner
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1001434
Abstract: In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3), named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles during meiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamine exchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid is present in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion, paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric Cid levels throughout development. Hence, the amount of Cid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses.
CHD1 Contributes to Intestinal Resistance against Infection by P. aeruginosa in Drosophila melanogaster  [PDF]
Johanna Sebald, Stefano Morettini, Valerie Podhraski, Cornelia Lass-Fl?rl, Alexandra Lusser
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0043144
Abstract: Drosophila SNF2-type ATPase CHD1 catalyzes the assembly and remodeling of nucleosomal arrays in vitro and is involved in H3.3 incorporation in viin vivo during early embryo development. Evidence for a role as transcriptional regulator comes from its colocalization with elongating RNA polymerase II as well as from studies of fly Hsp70 transcription. Here we used microarray analysis to identify target genes of CHD1. We found a fraction of genes that were misregulated in Chd1 mutants to be functionally linked to Drosophila immune and stress response. Infection experiments using different microbial species revealed defects in host defense in Chd1-deficient adults upon oral infection with P. aeruginosa but not upon septic injury, suggesting a so far unrecognized role for CHD1 in intestinal immunity. Further molecular analysis showed that gut-specific transcription of antimicrobial peptide genes was overactivated in the absence of infection in Chd1 mutant flies. Moreover, microbial colonization of the intestine was elevated in Chd1 mutants and oral infection resulted in strong enrichment of bacteria in the body cavity indicating increased microbial passage across intestinal epithelia. However, we did not detect enhanced epithelial damage or alterations of the intestinal stem cell population. Collectively, our data provide evidence that intestinal resistance against infection by P. aeruginosa in Drosophila is linked to maintaining proper balance of gut-microbe interactions and that the chromatin remodeler CHD1 is involved in regulating this aspect.
CHD1 Remodels Chromatin and Influences Transient DNA Methylation at the Clock Gene frequency  [PDF]
William J. Belden,Zachary A. Lewis,Eric U. Selker,Jennifer J. Loros,Jay C. Dunlap
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002166
Abstract: Circadian-regulated gene expression is predominantly controlled by a transcriptional negative feedback loop, and it is evident that chromatin modifications and chromatin remodeling are integral to this process in eukaryotes. We previously determined that multiple ATP–dependent chromatin-remodeling enzymes function at frequency (frq). In this report, we demonstrate that the Neurospora homologue of chd1 is required for normal remodeling of chromatin at frq and is required for normal frq expression and sustained rhythmicity. Surprisingly, our studies of CHD1 also revealed that DNA sequences within the frq promoter are methylated, and deletion of chd1 results in expansion of this methylated domain. DNA methylation of the frq locus is altered in strains bearing mutations in a variety of circadian clock genes, including frq, frh, wc-1, and the gene encoding the frq antisense transcript (qrf). Furthermore, frq methylation depends on the DNA methyltransferase, DIM-2. Phenotypic characterization of Δdim-2 strains revealed an approximate WT period length and a phase advance of approximately 2 hours, indicating that methylation plays only an ancillary role in clock-regulated gene expression. This suggests that DNA methylation, like the antisense transcript, is necessary to establish proper clock phasing but does not control overt rhythmicity. These data demonstrate that the epigenetic state of clock genes is dependent on normal regulation of clock components.
Assembly of Drosophila Centromeric Chromatin Proteins during Mitosis  [PDF]
Barbara G. Mellone ,Kathryn J. Grive equal contributor,Vladimir Shteyn equal contributor,Sarion R. Bowers,Isaac Oderberg,Gary H. Karpen
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002068
Abstract: Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. We analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and CAL1, which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation.
The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C  [PDF]
Elaine M. Dunleavy equal contributor,Nicole L. Beier equal contributor,Walter Gorgescu,Jonathan Tang,Sylvain V. Costes,Gary H. Karpen
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1001460
Abstract: CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote.
Distinct influences of tandem repeats and retrotransposons on CENH3 nucleosome positioning
Jonathan I Gent, Kevin L Schneider, Christopher N Topp, Carmen Rodriguez, Gernot G Presting, R Kelly Dawe
Epigenetics & Chromatin , 2011, DOI: 10.1186/1756-8935-4-3
Abstract: In order to investigate what influence the DNA sequence exerts on CENH3 chromatin structure, we examined CENH3 nucleosome footprints on maize centromere DNA. We found a predominant average nucleosome spacing pattern of roughly 190-bp intervals, which was also the dominant arrangement for nucleosomes genome-wide. For CENH3-containing nucleosomes, distinct modes of nucleosome positioning were evident within that general spacing constraint. Over arrays of the major ~156-bp centromeric satellite sequence (tandem repeat) CentC, nucleosomes were not positioned in register with CentC monomers but in conformity with a striking ~10-bp periodicity of AA/TT dimers within the sequence. In contrast, nucleosomes on a class of centromeric retrotransposon (CRM2) lacked a detectable AA/TT periodicity but exhibited tightly phased positioning.These data support a model in which general chromatin factors independent of both DNA sequence and CENH3 enforce roughly uniform centromeric nucleosome spacing while allowing flexibility in the mode in which nucleosomes are positioned. In the case of tandem repeat DNA, the natural bending effects related to AA/TT periodicity produce an energetically-favourable arrangement consistent with conformationally rigid nucleosomes and stable chromatin at centromeres.Centromeres are the regions of chromosomes where kinetochores form and microtubules attach to guide chromosomes to opposite poles during cell division. A wide variety of centromere forms are observed in the diverse cellular contexts of different organisms, from the small point centromeres of Saccharomyces cerevisiae to the diffuse, holocentric centromeres of Caenorhabditis elegans. Even within a single individual, centromeres must meet the distinct requirements of chromosome dynamics in both meiotic and mitotic cell division. From the outset, one would guess that centromere DNA elements would specify binding sites for structural and regulatory proteins. Indeed, specific, functionally important
Mis17 Is a Regulatory Module of the Mis6-Mal2-Sim4 Centromere Complex That Is Required for the Recruitment of CenH3/CENP-A in Fission Yeast  [PDF]
Yoshiharu Shiroiwa,Takeshi Hayashi,Yohta Fujita,Alejandro Villar-Briones,Nobuyasu Ikai,Kojiro Takeda,Masahiro Ebe,Mitsuhiro Yanagida
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0017761
Abstract: The centromere is the chromosome domain on which the mitotic kinetochore forms for proper segregation. Deposition of the centromeric histone H3 (CenH3, CENP-A) is vital for the formation of centromere-specific chromatin. The Mis6-Mal2-Sim4 complex of the fission yeast S. pombe is required for the recruitment of CenH3 (Cnp1), but its function remains obscure.
Single-epitope recognition imaging of native chromatin
Hongda Wang, Yamini Dalal, Steven Henikoff, Stuart Lindsay
Epigenetics & Chromatin , 2008, DOI: 10.1186/1756-8935-1-10
Abstract: Here we apply a recognition mode of AFM imaging to directly identify CenH3 within histone core particles released from native centromeric chromatin. More than 90% of these particles were found to be tetrameric in height. The specificity of recognition was confirmed by blocking with a CenH3 peptide, and the strength of the interaction was quantified by force measurements. These results imply that the particles imaged by AFM are indeed mature CenH3-containing hemisomes.Efficient and highly specific recognition of CenH3 in histone core particles isolated from native centromeric chromatin demonstrates that tetramers are the predominant form of centromeric nucleosomes in mature tetramers. Our findings provide proof of principle that this approach can yield insights into chromatin biology using direct and rapid detection of native nucleosomes in physiological salt concentrations.Eukaryotic genomes are packaged with octameric protein particles, consisting of two copies each of histones H2A, H2B, H3 and H4, which wrap nearly two turns of DNA to form nucleosomes [1]. Since the discovery of nucleosomes in the early 1970s, a variety of techniques have been applied to their study. However, both ultrastructural technologies (e.g. crystallography and electron microscopy) and biochemical analyses (e.g. nuclease assays and sedimentation) have been limited in their scope, because they cannot simultaneously assay structure and dynamics. In recent years, progress has been made in applying new technologies that have the potential to bridge the gap between static ultrastructural features and dynamic physiological processes in the study of chromatin. These technologies, which include scanning confocal fluorescence microscopy [2], molecular tweezers [3,4] and atomic force microscopy (AFM) [5,6], have provided remarkable insights into the behavior of individual nucleosomes. The combination of single-molecule resolution, solution biochemistry and observation of native macromolecular complex
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