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Dmitry Fyodorov, Ph.D.

Dmitry Fyodorov, Ph.D.

Area of Research: Packaging DNA into chromatin maintains & compartmentalizes the genome. We examine how nucleosome remodeling factors, histone chaperones & modifying enzymes establish chromatin compartments and shape gene expression patterns.

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718.430.4021dmitry.fyodorov@einsteinmed.edu

Albert Einstein College of Medicine
Jack and Pearl Resnick Campus
1300 Morris Park Avenue
Chanin Building, Room 414a
Bronx, NY 10461

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BIOCHEMISTRY AND GENETICS OF CHROMATIN TRANSITIONS IN DROSOPHILA

Hundreds of millions of base pairs of nuclear DNA are packed into chromosomes. Chromatin, the nucleoprotein filament of a chromosome, has many organization levels. It is the natural state of DNA in the nucleus and the native substrate for DNA-directed 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 goal of our research is to understand how chromosomes are assembled and how this process 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-driven 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 facilitate reconstitution of chromatin in vitro. It consists of two subunits, a SNF2-like ATPase ISWI and a polypeptide termed Acf1. In conjunction with a core histone chaperone NAP-1, ACF mediates deposition of histones onto DNA and forms arrays of regularly spaced nucleosomes. We study ACF as a prototype factor to elucidate molecular events that take place during ATP-dependent formation of nucleosomes. During assembly, ACF commits to the DNA template and forms 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 assembly factor in Drosophila. To expose its biological functions, we produced fly mutants that do not express ACF. ACF-deficient animals have multiple defects of chromatin organization. However, ACF is not essential for viability due to the presence of redundant ACF-like factors. We discovered novel ISWI-containing complexes ToRC (comprising Tou, ISWI and CtBP) and RSF (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

To reconstitute higher-order chromatin structures, we supplement the in vitroassembly system with modified core histones, histone variants, linker histone (H1) and heterochromatin proteins, such as Drosophila HP1a.Chromatin vectors can turn into useful tools in research and therapy. These studies will also eventually lead to the discovery of techniques to reconstitute functional metazoan chromosomes.

In collaboration with A. Skoultchi, we began to examine flies in which H1 is depleted 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 HMT Su(var)3-9, which mediates methylation of lysine 9 of histone H3 (H3K9), a signature heterochromatin-specific epigenetic mark. We have also demonstrated that H1 is essential for the faithful regulation of DNA endoreplication timing in Drosophila larval cells. We are now extending these studies to normal, mitotically dividing cells.

A prevalent view of heterochromatic silencing is that its physical compaction results in steric exclusion of regulatory proteins, such as RNA polymerases. In collaboration with G. Karpen (LBNL), we have recently shown that the formation of heterochromatin domains is also mediated by liquid-liquid phase separation that gives rise to a non-membrane-bound nuclear compartment. We demonstrated that HP1a and H1 undergo demixing in vitro and nucleate into foci that display liquid properties during heterochromatin domain formation in early Drosophila embryos. We propose that biophysical properties associated with phase-separated systems are critical to understanding the behavior of heterochromatin and, potentially, other chromatin forms that regulate essential nuclear functions.

4. Sperm chromatin assembly and remodeling

In sperm, DNA is compacted with cysteine-rich protamines and protamine-like sperm nuclear basic proteins (SNBPs) to form enzymatically static sperm “chromatin”. We have begun to analyze protein factors that mediate SNBP 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.

Upon deposition on sperm DNA, protamines/SNBPs are extensively crosslinked via interchain disulfide bonds. After fertilization, the egg has to reverse the crosslinks for efficient eviction of SNBPs. This nuclear reaction is mediated by specific thioredoxin (TRX) and thioredoxin reductase (TRXR) molecules. Thus, we are investigating biological roles of the evolutionary conserved thioredoxin system in sperm chromatin metabolism and female fertility. A number of chemical compounds are known to specifically inhibit the function of TRX and TRXR proteins. We are studying their ability to suppress fertilization in the egg in vivo and testing their utility as novel, non-hormone agents for female contraception.

5. Non-coding RNA

Epigenetic regulation is dependent in part on non-coding RNAs that affect gene expression post-transcriptionally or by interfacing chromatin via tethering DNA- and histone-modifying enzymes in a sequence-specific manner. This tethering involves a formation of RNA-DNA hybrids (R-loops), which encompass a large fraction of eukaryotic genomes. Despite their apparent importance, biochemical details of R-loop formation and the identities of factors that mediate this reaction remain enigmatic. We have designed an in vitroassay to look for enzymes that mediate sequence-specific RNA-DNA pairing in an ATP-dependent fashion in trans. By chromatographic fractionation of Drosophila extracts, we purified to near homogeneity a novel, evolutionary conserved factor termed R-loop enzymatic complex, RLEC. We are now characterizing biochemical properties and biological functions of Drosophila RLEC.

Selected Publications

Fyodorov, D.V.,† Zhou, B.-R., Skoultchi, A.I., and Bai, Y.† (2018). Emerging roles of linker histones in regulating chromatin structure and function. Nature Rev. Mol. Cell Biol. 19, 192-206.

Strom, A.R., Emelyanov, A.V., Mir, M., Fyodorov, D.V., Darzacq, X., and Karpen, G.H. (2017). Phase separation drives heterochromatin domain formation. Nature 547, 241-245.

Andreyeva, E.N., Bernardo, T.J., Kolesnikova, T.D., Lu, X., Yarinich, L.A., Bartholdy, B.A., Guo, X., Posukh, O.V., Healton, S., Willcockson, M.A., Pindyurin, A.V., Zhimulev, I.F., Skoultchi, A.I., and Fyodorov, D.V. (2017). Regulatory functions and chromatin loading dynamics of linker histone H1 during endoreplication in Drosophila. Genes Dev. 31, 603-616.

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.

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.

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.

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