Associate Professor, Department of Cell Biology
BIOCHEMISTRY AND GENETICS OF CHROMATIN ASSEMBLY
In the eukaryotic nucleus, hundreds of millions of base pairs of DNA are packed into chromosomes. Chromatin, the central nucleoprotein filament of a chromosome, has many forms and organization levels. Chromatin is the natural state of DNA in the nucleus and the native substrate for nuclear reactions, such as DNA replication, recombination, repair and transcription. The assembly of DNA into chromatin and dynamic conversion between its different forms are critical steps in the maintenance and regulation of the eukaryotic genome. The ultimate goal of our research is to understand how chromosomes are assembled and how chromatin assembly regulates the structure and activity of eukaryotic chromosomes. The crucial first step in this direction is a systematic study of factors that mediate this process. To this end, we use biochemical approaches to analyze mechanisms of chromatin assembly by histone chaperones and ATP-dependent enzymes. We also dissect their function in vivo by methods of Drosophila genetics. Thus, we are trying to uncover the network of chromatin assembly factors and to elucidate their roles in hierarchical organization of the chromosome.
1. Molecular mechanisms of nucleosome assembly
ACF (ATP-utilizing chromatin assembly factor) was identified on the basis of its ability to mediate ATP-dependent reconstitution of chromatin in vitro. ACF consists of two subunits, a SNF2-like ATPase ISWI and another evolutionary conserved polypeptide, termed Acf1. In the presence of a core histone chaperone NAP-1, ACF mediates deposition of histone octamers onto DNA and forms arrays of regularly spaced nucleosomes. We study ACF and ISWI as prototype factors to elucidate elementary molecular events that take place during ATP-dependent formation of nucleosomes. Upon reaction initiation, ACF commits to the DNA template and assembles nucleosomes as a processive, ATP-driven, DNA-translocating motor. Multiple conserved domains of Acf1 and ISWI are required for this activity.
2. Biological functions of chromatin assembly factors
ACF is the major ATP-dependent chromatin assembly factor in Drosophila. To expose its biological functions, we study fly mutants that do not express ACF. ACF-deficient animals have multiple defects of chromatin organization. However, ACF is not essential for fly viability due to the presence of additional, redundant ACF-like factors. We discovered novel ISWI-containing complexes ToRC (comprising Tou, ISWI and CtBP) and RSF (comprising Rsf1 and ISWI) that can functionally substitute ACF in vivo. Our genetic and cytological analyses implicate the network of ATP-dependent, ISWI-containing chromatin assembly factors in diverse, partially redundant pathways of regulation of chromatin structure and activity.
SNF2-like protein CHD1 is another ATP-dependent nucleosome assembly factor. We disrupted Chd1 in flies and discovered that CHD1 is required for replication-independent deposition of histones into chromatin in vivo. Specifically, CHD1 is essential during early embryonic development for deposition of replacement histone H3.3 into paternal chromatin.
3. Higher-order chromatin forms
ACF can mediate deposition of both core and linker histones (H1) in vitro. Thus, it can assemble the 30 nm chromatin fiber in a defined system. To reconstitute other higher-order chromatin structures, we incorporate modified core histones, histone variants and heterochromatin proteins. In vitro assembled chromatin vectors can turn into useful tools in research and therapy. Among other outcomes, these studies will eventually lead to the discovery of techniques to reconstitute functional metazoan chromosomes.
To better understand the biology of H1 deposition into chromatin, we decided to first analyze the processes that are associated with elimination of H1 from chromatin in vivo. In collaboration with the lab of Dr. A. Skoultchi, we began to examine phenotypes of animals in which H1 is depleted in vivo by RNAi or genetic approaches. We discovered that H1 is the major component of heterochromatin and is required to establish its biochemical identity and functional properties. For instance, H1 recruits histone methyltransferase Su(var)3-9, which mediates dimethylation of lysine 9 of histone H3, a signature heterochromatin-specific epigenetic mark.
In sperm, DNA is compacted with protamines to form enzymatically static sperm “chromatin”. We have begun to analyze protein factors that mediate protamine deposition during spermatogenesis and their removal from DNA after fertilization. It turns out that sperm chromatin assembly and remodeling is mediated by a group of factors that are similar to core histone chaperones.
Emelyanov, A.V., and Fyodorov, D.V. (2016). Thioredoxin-dependent disulfide bond reduction is required for protamine eviction from sperm chromatin. Genes Dev. 30, 2151-2156.
Xu, N., Lu, X., Kavi, H., Emelyanov, A.V., Bernardo, T.J., Vershilova, E., Skoultchi, A.I., and Fyodorov, D.V. (2016). BEN domain protein Elba2 can functionally substitute for linker histone H1 in Drosophila in vivo. Sci. Rep. 6, 34354.
Kavi, H., Lu, X., Xu, N., Bartholdy, B.A., Vershilova, E., Skoultchi, A.I., and Fyodorov, D.V. (2015). A genetic screen and transcript profiling reveal a shared regulatory program for Drosophila linker histone H1 and chromatin remodeler CHD1. G3 (Bethesda) 5, 677-687.
Emelyanov, A.V., Rabbani, J., Mehta, M., Vershilova, E., Keogh, M.C., and Fyodorov, D.V. (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, 2027-2040.
Lu, X., Wontakal, S.N., Kavi, H., Kim, B.J., Guzzardo, P.M., Emelyanov, A.V., Xu, N., Hannon, G.J., Zavadil, J., Fyodorov, D.V. †, and Skoultchi, A.I. † (2013). Drosophila H1 regulates the genetic activity of heterochromatin by recruitment of Su(var)3-9. Science 340, 78-81.
Emelyanov, A.V., Vershilova, E., Ignatyeva, M.A., Pokrovsky, D.K., Lu, X., Konev, A.Y., and Fyodorov, D.V. (2012). Identification and characterization of ToRC, a novel ISWI-containing ATP-dependent chromatin assembly complex. Genes Dev. 26, 603-614.
Morettini, S., Tribus, M., Zeilner, A., Sebald, J., Campo-Fernandez, B., Scheran, G., Wörle, H., Podhrask,i V., Fyodorov, D.V., and Lusser, A. (2011).The chromodomains of CHD1 are critical for enzymatic activity but less important for chromatin localization. Nucleic Acids Res. 39, 103-115.
Emelyanov, A.V., Konev, A.Y., Vershilova, E., and Fyodorov, D.V. (2010). Protein complex of Drosophila ATRX/XNP and HP1a is required for the formation of pericentric beta-heterochromatin in vivo. J. Biol. Chem. 285, 15027-15037.
Lu, X., Wontakal, S.N., Emelyanov, A.V., Morcillo, P., Konev, A.Y., Fyodorov, D.V. †, and Skoultchi, A.I. † (2009). Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure. Genes Dev. 23, 452-465.
Konev, A.Y., Tribus, M., Park, S.Y., Podhraski, V., Lim, C.Y., Emelyanov, A.V., Vershilova, E., Pirrotta, V., Kadonaga, J.T., Lusser, A., and Fyodorov, D.V. (2007). CHD1 motor protein is required for deposition of histone H3.3 into chromatin in vivo. Science 317, 1087-1090.
Fyodorov, D.V., Blower, M.D., Karpen, G.H., and Kadonaga, J.T. (2004). Acf1 confers unique activities to ACF/CHRAC and promotes the formation rather than disruption of chromatin in vivo. Genes Dev. 18, 170-183.
Fyodorov, D.V., and Kadonaga, J.T. (2003). Chromatin assembly in vitro with purified recombinant ACF and NAP-1 . Meth. Enzymol. 371, 499-515.
Fyodorov, D.V., and Kadonaga, J.T. (2002). Dynamics of ATP-dependent chromatin assembly by ACF. Nature 418, 896-900.
Fyodorov, D.V., and Kadonaga, J.T. (2001). The many faces of chromatin remodeling: SWItching beyond transcription. Cell 106, 523-525.
More Information About Dr. Dmitry Fyodorov
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Albert Einstein College of Medicine
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