Proteomic Investigation of Chromatin Biology in Cultured Mammalian Cells

Abstract

Multiprotein complexes are known to catalyse histone methylation reactions. An important evolutionally conserved histone mthyltransferase (HMT) is SET/MLL complex. In humans, there are six homologs of the main catalytic SET-domain-containing enzyme including SET1A, SET1B, MLL1, MLL2, MLL3, and MLL4, forming at least six distinct complexes. All six share core components including ASH2L, RBBP5, WDR5, and DPY30, while other proteins are specific to individual complexes. In this thesis, SETD1A/COMPASS was studied. This complex methylates histone H3K4, resulting in an epigenetic mark that is associated with increased transcription. The goal was to study the composition of the complex in different cell lines (HEK293T and NT2) in order to improve understanding of its function. A combination of immunoprecipitation using antibodies (SETD1A, RBBP5, and ASH2L) against SETD1A/COMPASS proteins and label-free quantitative mass spectrometry was performed. All previous known interactors with SETD1A were identified such as RBBP5, ASH2L, and DPY30, as well as a novel interactor RAD18, which is a ubiquitin ligase involved in DNA repair pathways. Using label-free proteomics to evaluate relative stoichiometry, and co immunoprecipitation experiments, RAD18 was found to interact with SETD1A independently of other members of SETD1A complex such as RBBP5 and ASH2L. Further investigation of the nature of this interaction was performed using size exclusion chromatography to resolve complexes of differing size. These experiments were suggested that the STD1A-RAD18 interaction is independent from SETD1A/COMPASS and the expected molecular size of this complex is around 250 KDa at least in HEK293T cells. However, in NT2 cells the results of size exclusion chromatography were inconclusive. Therefore, further investigation using biophysical experiments will be needed. Next, the molecular function of SETD1A and RAD18 was investigated. Depletion of both SETD1A and RAD18 led to reduced mRNA expression of both proteins, suggesting some form of mutual regulation at the level of transcription. Loss of both SETD1A and RAD18 causes reduced cell survival in the presence of the DNA damage agent mitomycin C. It also found a synergistic effect between the two genes, whereby the effect of perturbation of both genes was greats than either alone. Depletion of SETD1A or RAD18 results in a defect in the monoubiquitylation of PCNA in the presence of mitomycin C. In addition, in NT2 cells, disruption of SETD1A causes decreased in the VI

expression of the pluripotency markers NANOG and OCT4, while loss of RAD18 causes decreased only of OCT4. Disruption of SETD1A or RAD18 have no effect on differentiation markers such as HOXD9. These results suggest that SETD1A and RAD18 contribute to control the expression of OCT4 and that lead to regulate the pluripotent state of the cells. Overall, this work identifies a new link between an epigenetic enzyme and a DNA damage repair protein.