Person:

McElroy, Kyle

Chromatin plays a critical role in faithful implementation of gene expression programs. Different post-translational modifications (PTMs) of histone proteins reflect the underlying state of gene activity, and many chromatin proteins write, erase, bind, or are repelled by, these histone marks. One such protein is UpSET, the Drosophila homolog of yeast Set3 and mammalian KMT2E (MLL5). Here, we show that UpSET is necessary for the proper balance between active and repressed states. Using CRISPR/Cas-9 editing, we generated S2 cells that are mutant for upSET. We found that loss of UpSET is tolerated in S2 cells, but that heterochromatin is misregulated, as evidenced by a strong decrease in H3K9me2 levels assessed by bulk histone PTM quantification. To test whether this finding was consistent in the whole organism, we deleted the upSET coding sequence using CRISPR/Cas-9, which we found to be lethal in both sexes in flies. We were able to rescue this lethality using a tagged upSET transgene, and found that UpSET protein localizes to transcriptional start sites (TSS) of active genes throughout the genome. Misregulated heterochromatin is apparent by suppressed position effect variegation of the wm4 allele in heterozygous upSET-deleted flies. Using nascent-RNA sequencing in the upSET-mutant S2 lines, we show that this result applies to heterochromatin genes generally. Our findings support a critical role for UpSET in maintaining heterochromatin, perhaps by delimiting the active chromatin environment.

Chromatin plays a vital role in the implementation of gene expression programs. Several disparate groups of regulatory proteins alter chromatin state through post-translational modification of histone proteins, nucleosome remodeling, and higher order chromatin structure in order to affect gene expression. Several of these key groups, such as the Male-Specific Lethal complex and Polycomb Group have been well characterized in Drosophila. Yet aspects of their biology at the molecular level, such as the means by which they are faithfully targeted to regulated loci throughout the genome and the molecular mechanisms they employ to alter transcriptional state, still remain unexplained. In this dissertation I explore how identifying protein-protein interactions on chromatin reveals insights into these unanswered questions critical to chromatin biology. My results highlight the importance of balancing active and repressive chromatin states for the proper maintenance of gene expression.

The Male-Specific Lethal complex is the dosage compensation complex in Drosophila, which upregulates gene expression on the male X chromosome approximately two-fold. The MSL complex catalyzes an acetyl mark which may create a uniquely permissive chromatin state to promote transcriptional elongation. A proteomic screen for MSL-interacting proteins identified UpSET, the Drosophila homolog of yeast SET3 and mammalian MLL5. Interestingly, SET3 and UpSET have been characterized to assemble into histone deacetylase complexes. I employed genetic, genomic, and proteomic techniques to assess whether UpSET plays a role in dosage compensation. UpSET appears to play a role in limiting the level of activation of the MSL complex. Surprisingly, UpSET appears to play a more important role in the maintenance of heterochromatin.

The Polycomb Group is comprised of a well characterized set of developmental repressors. The PcG assembles into several multiprotein complexes to maintain the repressed state. The PcG is opposed by a group of activators known as the Trithorax group. Although the PcG and TrxG often appear to be recruited to the same genomic elements in different tissues, whether they might interact directly was not known. In a collaboration with Dr. Hyuckjoon Kang, I characterized the TrxG protein Female sterile (1) homeotic and found that it interacts specifically with PRC1. The data support a model that bivalency, a poised state observed in mammalian stem cells, may be critical, perhaps transiently, in the developing Drosophila embryo. The mechanism of coordination amongst the various PcG complexes on chromatin is not well understood. We also identified the Sex comb on midleg protein, a known member of the PcG, as a potential physical bridge between PRC1 and PRC2.

In these sets of experiments, I have characterized instances of crosstalk between activating and repressing regulators which are critical for the proper maintenance of chromatin state. Perturbations of these interactions may lead to an imbalance of regulators on chromatin and aberrant transcriptional activity. These findings highlight the need for tuning gene expression state and suggest chromatin-based mechanisms by which this can be accomplished.

Chromatin-binding proteins must navigate the complex nuclear milieu to find their sites of action, and a constellation of protein factors and other properties are likely to influence targeting specificity. Despite considerable progress, the precise rules by which binding specificity is achieved have remained elusive. Here, we consider early targeting events for two groups of chromatin-binding complexes in Drosophila: the Male-Specific Lethal (MSL) and the Polycomb group (PcG) complexes. These two serve as models for understanding targeting, because they have been extensively studied and play vital roles in Drosophila, and their targets have been documented at high resolution. Furthermore, the proteins and biochemical properties of both complexes are largely conserved in multicellular organisms, including humans. While the MSL complex increases gene expression and PcG members repress genes, the two groups share many similarities such as the ability to modify their chromatin environment to create active or repressive domains, respectively. With legacies of in-depth genetic, biochemical and now genomic approaches, the MSL and PcG complexes will continue to provide tractable systems for understanding the recruitment of multiprotein chromatin complexes to their target loci.

Chromatin architecture is regulated through both enzymatic and non-enzymatic activities. For example, the Polycomb Group (PcG) proteins maintain developmental gene silencing using an array of chromatin-based mechanisms. The essential Drosophila PcG protein, Posterior Sex Combs (PSC), compacts chromatin and inhibits chromatin remodeling and transcription through a non-enzymatic mechanism involving nucleosome bridging. Nucleosome bridging is achieved through a combination of nucleosome binding and self-interaction. Precisely how PSC interacts with chromatin to bridge nucleosomes is not known and is the subject of this work. We determine the stoichiometry of PSC-chromatin interactions in compact chromatin (in which nucleosomes are bridged) using Scanning Transmission Electron Microscopy (STEM). We find that full compaction occurs with one PSC per nucleosome. In addition to compacting chromatin, we show that PSC oligomerizes nucleosome arrays. PSC-mediated oligomerization of chromatin occurs at similar stoichiometry as compaction suggesting it may also involve nucleosome bridging. Interactions between the tail of histone H4 and the acidic patch of histone H2A are important for chromatin folding and oligomerization, and several chromatin proteins bind the histone H2A acidic patch. However, mutation of the acidic patch of histone H2A does not affect PSC’s ability to inhibit chromatin remodeling or bridge nucleosomes. In fact, PSC does not require nucleosomes for bridging activity but can bridge naked DNA segments. PSC clusters nucleosomes on sparsely assembled templates, suggesting it interacts preferentially with nucleosomes over bare DNA. This may be due to the ability of PSC to bind free histones. Our data are consistent with a model in which each PSC binds a nucleosome and at least one other PSC to directly bridge nucleosomes and compact chromatin, but also suggest that naked DNA can be included in compacted structures. We discuss how our data highlight the diversity of mechanisms used to modify chromatin architecture.