The assembly of chromatin in vivo is mediated by a concerted action of core histone chaperones and ATP-dependent nucleosome assembly factors. The chromatin assembly is an important reaction that interfaces all major processes of the nuclear DNA metabolism. Our long-term goal is to understand how eukaryotic chromosomes are assembled, reproduced and regulated. Drosophila CHD1 and ISWI-containing complexes ACF, NoRC/ToRC and RSF can mediate nucleosome assembly in an ATP-dependent manner in vitro and in vivo. The specific hypothesis is that ACF and ACF-like factors mediate reconstitution of oligonucleosome filament as well as higher-order chromatin and can regulate nuclear reactions, such as transcription, DNA replication and repair. In the early embryo, chromatin assembly in the male pronucleus is preceded by sperm chromatin remodeling (SCR) and eviction of paternal protamines. This reaction is mediated by a separate set of factors that we recently identified. The specific hypothesis is that, similar to nucleosome assembly, SCR is mediated by a concerted action of protamine chaperones and ATP-dependent motor proteins.
The specific aims are to: 1. Investigate mechanisms of ATP-dependent chromatin assembly in vitro. We will determine the molecular basis for intrinsic nucleosome assembly activity of ISWI and CHD1. We will analyze precursors of nucleosome assembly and formation of higher-order chromatin fiber. We will study the interplay between ATP-dependent factors and histone chaperones during chromatin assembly. 2. Examine activities and targets of ACF and ACF-like factors in vivo in Drosophila. We will examine genomic, subcellular and cell-cycle dependent localization of chromatin assembly factors. We will explore the function of ACF-like factors in the deposition of core and linker histones into chromosomes. We will analyze specific biological functions of a novel Drosophila assembly factor NoRC/ToRC. 3. Analyze biochemical and biological functions of factors that mediate SCR. We will characterize factors that mediate SCR and molecular mechanisms of their action. We will elucidate biological functions of SCR-mediating factors in vivo. Our work will add to the fundamental understanding of chromatin assembly and related nuclear processes (transcription, replication and repair). It will help to design methods of diagnosis and treatment of human diseases that involve defects in these nuclear processes. Furthermore, our studies will contribute to the development of molecular techniques to reconstitute functional metazoan chromosomes.

Public Health Relevance

Chromatin is the native state of nuclear DNA in human cells. Packaging of DNA into chromatin is essential for the maintenance of genome integrity and regulation of DNA metabolism. The goal of this project is to understand the function of factors that mediate the assembly of chromatin. Our work will help to design methods of diagnosis and treatment of human diseases that involve defects in nuclear reactions.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics B Study Section (MGB)
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Carter, Anthony D
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Albert Einstein College of Medicine
Anatomy/Cell Biology
Schools of Medicine
United States
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Emelyanov, Alexander V; Rabbani, Joshua; Mehta, Monika et al. (2014) Drosophila TAP/p32 is a core histone chaperone that cooperates with NAP-1, NLP, and nucleophosmin in sperm chromatin remodeling during fertilization. Genes Dev 28:2027-40
Lu, Xingwu; Wontakal, Sandeep N; Kavi, Harsh et al. (2013) Drosophila H1 regulates the genetic activity of heterochromatin by recruitment of Su(var)3-9. Science 340:78-81
Emelyanov, Alexander V; Vershilova, Elena; Ignatyeva, Maria A et al. (2012) Identification and characterization of ToRC, a novel ISWI-containing ATP-dependent chromatin assembly complex. Genes Dev 26:603-14
Morettini, Stefano; Tribus, Martin; Zeilner, Anette et al. (2011) The chromodomains of CHD1 are critical for enzymatic activity but less important for chromatin localization. Nucleic Acids Res 39:3103-15
Podhraski, Valerie; Campo-Fernandez, Beatriz; W├Ârle, Hildegard et al. (2010) CenH3/CID incorporation is not dependent on the chromatin assembly factor CHD1 in Drosophila. PLoS One 5:e10120
Lu, Xingwu; Wontakal, Sandeep N; Emelyanov, Alexander V et al. (2009) Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure. Genes Dev 23:452-65
Konev, Alexander Y; Tribus, Martin; Park, Sung Yeon et al. (2007) CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo. Science 317:1087-90