Systematic Functional Proteomic Investigation of Mammalian Histone Methylation-related Protein Complexes

Epigenetic regulation of chromatin structure, which involves site-specific methylation of histone proteins, is critical for regulating gene expression during development. While the enzymatic components of histone methylation-related protein complexes have been discovered, little is generally known regarding the associated tissue-specific co-factors that regulate complex activity in vertebrate/mammalian embryos. To better understand the composition and control of histone methylation-related complexes in mammals, I used an affinity purification and mass spectrometry (AP-MS) approach to identity stably interacting proteins of histone methylation-related proteins in human embryonic kidney (HEK) 293 cells. My AP-MS approach included replicate isolation and characterization of 33 histone methylation- and/or transcription-related proteins, resulting in an interaction network encompassing 573 protein-protein interactions (PPIs). Among the findings were a novel set of RNA polymerase II (RNAPII) binding proteins, known as the "RPRD" proteins, which recognize phosphorylated RNAPII, and several putative new components of the evolutionarily conserved gene silencing machinery. Most notably, I identified and characterized ZNF644, a protein that is putatively causally linked to high-grade myopia, as a novel interacting partner for the paralogous histone methyltransferases (HMTs) G9a and GLP and as a new co-regulator of histone H3 at lysine 9 dimethylation (H3K9me2). Using a zebrafish embryo model system, I characterized the roles of zebrafish g9a and two orthologues of ZNF644, termed znf644a and znf644b, in the forming retina and midbrain. I found that even modest disruption of g9a activity caused widespread apoptosis in progenitor and differentiating cell populations in the retina. I also found that znf644a and znf644b have common retina-specific roles in suppressing progenitor gene expression (i.e., vsx2 and ccnd1) by regulating promoter H3K9me2. However, despite the overlapping roles in progenitor gene suppression, the two znf644 paralogues controlled distinct aspects of retinal differentiation; specifically, I observed that disruption of znf644b caused retina-specific developmental delays, resulting in the delayed differentiation of RPCs, whereas disruption of znf644a caused the mislocalized expression of H3K9me2 marked genes (i.e., znf644a and znf644b) during differentiation, resulting in the impaired viability of differentiated neurons. Importantly, I used complementation assays as well as "genetic cooperativity" assays to demonstrate that, despite the disparate phenotypes of g9a, znf644a and znf644b morphant retinas, the regulatory roles of znf644a and znf644b are critically dependent on their ability to functionally and physically interact with g9a. In addition, I provided evidence that the functions of znf644a and znf644b are encompassed by mammalian ZNF644 and that the retinal roles of g9a, znf644a and znf644b are recapitulated in progenitor cells of the developing midbrain. Collectively, my thesis provides both a high-confidence global molecular interaction map that furthers understanding of the physical and functional associations underlying the mammalian histone methylation machinery and highlights the unexpected role of a previously unappreciated co-factor, ZNF644 as a co-regulator of H3K9me2-mediated gene silencing and a multifaceted regulator of neural differentiation.

Keywords: Histone methylation
Protein Complexes
Retinal Development
Advisor: Emili, Andrew