Balancing transcriptional activity in Drosophila through protein-protein interactions on chromatin

Abstract

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.