The main focus of the group is the role of chromatin structure in gene regulation during normal development and in disease. We are also interested in evolutionary aspects of chromatin organization. The fundamental unit of chromatin organization – nucleosome – comprises about 150 bp of DNA and eight histone proteins of four types. There are two major roles that nucleosomes play in gene regulation: (i) nucleosomes control accessibility of DNA for interaction with transcription factors and (ii) they are subject to epigenetic modifications recognized by chromatin modifiers.
We study both these aspects of nucleosome organization. In the area of nucleosome positioning we focus on sequence-directed nucleosome positioning and the role of chromatin remodelers in establishing specific patterns of nucleosome occupancy at genomic regulatory regions such as gene starts and enhancers. In the area of nucleosome composition our focus is on histone variants, which replace major histones in a replication independent manner and often are essential for development.
Here are some of our current and recently completed projects:
Histone variants: where, what, why?
where do they work in the genome, what do they do exactly, why have they emerged during evolution
Probing chromatin with MNase: from static nucleosome positioning to dynamic response.
Swi/Snf complex: a link between chromatin remodeling and cancer suppression.
Histone H2A variants: one family, different roles in the human genome
Readout of genomic information is controlled and modulated by chromatin structure, which at the basic level is represented by the DNA wrapped around the histone core. Although chromatin structure in human cells has been extensively investigated in recent years, the biological role and genomic distribution of the replacement histone variants remain poorly understood. Using publicly available and newly generated data, we focus on the variants of histone H2A, one of the most diverse histone families (Tolstorukov, Goldman, et al., and Kingston, Park, Mol Cell, 2012). In particular, we produced genome-wide profiles of the variants H2A.Z, macroH2A and H2A.Bbd using HeLa cell lines that stably express affinity-tagged versions of the corresponding histones. We report that nucleosomes bearing variant H2A.Bbd protect less DNA and are enriched inside actively transcribed genes. This is in contrast to macroH2A nucleosomes, which are enriched in repressed genes. At the same time, H2A.Bbd and macroH2A are not mutually exclusive and a detectable fraction of the genome is enriched for both variants. To further investigate the role of the recently discovered variant H2A.Bbd we preformed a comparative analysis of the transcription products in the cells where H2A.Bbd was depleted with shRNA and in control. This analysis showed that the H2A.Bbd depletion results in the 'net' down-regulation of gene expression and in the disruption of mRNA splicing pathways. Thus, our analysis suggests that H2A.Bbd may be involved in the formation of a specific chromatin structure that facilitates transcription elongation and initial mRNA processing. We also observe that specific chromatin organization involving histone variants may affect the level of conservation of the underlying DNA sequence. For instance, we report that the loci preferentially occupied by the nucleosomes bearing H2A.Z variant as well as such histone modifications as H3K4me3 show decreased frequency of SNPs as compared to the loci associated with 'bulk' nucleosomes. Taken together, our results demonstrate that H2A variants play highly specialized roles in human chromatin and that their distribution is evolutionary conserved.
Unraveling intricate relationship between chromatin remodeling, transcription, and cancer
Tolstorukov, Sansam, Ping, et al., and Park, Roberts, PNAS, 2013:
It is becoming increasingly clear that dynamic regulation of nucleosome positioning serves a key role in the control of mammalian development and further that mutation of nucleosome remodeling complexes is a frequent occurrence in cancer. Thus, understanding the mechanisms by which the nucleosome landscape is established as well as its contribution to transcriptional control is of great interest. We have studied the role of the Swi/Snf (Baf) chromatin remodeling complex, which is capable of utilizing the energy of ATP hydrolysis to mobilize nucleosomes in vitro. We conditionally inactivated two of its core components, Snf5 and Brg1, in primary mouse cells and profiled nucleosome positioning and occupancy genome-wide. We performed detailed bioinformatic analysis of both changes in chromatin structure and the effects upon gene expression. Our main observation is that the Swi/Snf complex is required for establishment of specific patterns of nucleosomes at the transcriptional start sites (TSS). In particular, inactivation of either of the studied subunits resulted in loss in the density of highly accessible nucleosomes around gene starts and changes in the phasing of nucleosomes downstream of TSS. We find that these effects influence transcription in an intricate way, ultimately promoting cell proliferation. This is consistent with the emerging role of the Swi/Snf complex as a tumor suppressor specifically mutated in a variety of human cancers. Collectively our findings reveal a complex relationship between chromatin remodelers, chromatin structure, and transcriptional regulation.
Altered transcription profile
with net up-regulation effect, promoting cell proliferation