Investigating the Effect of Histone H4 Sumoylation on Chromatin Structure and Function

Thesis (Ph.D.)--University of Washington, 2016-06

The packaging of DNA within the nucleus of a eukaryotic organism is facilitated by the formation of a nucleoprotein complex known as chromatin. There are various DNA binding proteins that play an important part in this process, but the four core histone proteins namely, H2A, H2B, H3 and H4 are essential for forming the scaffolding for DNA packaging. The basic repeating unit of chromatin is called a nucleosome and is formed when 147 bp of DNA wrap around an octamer of histone proteins. The N-terminal regions of all four histones, known as histone tails, protrude out from the nucleosome and are accessible to various chromatin binding complexes. These complexes are capable of modifying histone tails with post-translational modifications (PTMs) that range from small chemical groups such as methyl, acetyl and phosphoryl to small proteins including ubiquitin and different isoforms of the small ubiquitin-like modifier (SUMO). Dysregulation of histone PTMs has been observed in many disease states, which makes the study of these PTMs essential for understanding a variety of pathologies. We focused on the poorly understood modification of histones by SUMO protein, because several components of the sumoylation and desumoylation pathway have been implicated in cancers of the breast, colon, ovaries and prostate. H4 sumoylation has been indirectly linked to transcriptional repression, but the precise effect of histone sumoylation on chromatin structure and function is still unknown. We utilized our expertise in chemical biology to synthesize homogeneous sumoylated histone H4 and incorporated it into chromatin arrays. Through analytical ultracentrifugation experiments we discovered that sumoylation inhibits chromatin compaction and aggregation. To our knowledge, this is the first reported repressive mark that favors a euchromatic state of chromatin. We further corroborated these results with single-molecule FRET studies and uncovered an intrinsic effect of SUMO in preventing inter- and intra-array interactions. Mechanistic studies into the effect of sumoylation revealed that SUMO’s steric bulk plays an important role in preventing the compaction of chromatin arrays. This unexpected result, namely, that a repressive mark favors the euchromatic state led us to further examine the biochemical pathways by which sumoylation might mediate transcriptional repression. We explored the effect of sumoylation of H4 on the activity of the LSD1/CoREST/HDAC complex, which has been known to mediate gene repression through its deacetylation and demethylation activities. We tested the hypothesis that SUMO can mediate repression by localizing the LSD1/CoREST complex by binding to the SUMO interaction motif found in CoREST. We focused on the demethylation of methylated Lys4 of H3 (H3 K4me2), which is a mark associated with transcriptionally active promoter regions. To this end, we performed the first reported synthesis of the native full-length H3 K4me2, which was incorporated into nucleosomes along with sumoylated H4. On comparing the rate of demethylation of H3 K4me2 by the LSD1/CoREST complex, in sumoylated and non-sumoylated nucleosomes, we discovered that sumoylation can facilitate the removal of this activating mark. This novel cross talk between sumoylation and methylation presents one possible pathway by which histone sumoylation can mediate transcriptional repression.