Date of Graduation
5-2016
Document Type
Dissertation (PhD)
Program Affiliation
Molecular Carcinogenesis
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Mark Bedford
Committee Member
David Johnson
Committee Member
Richard Wood
Committee Member
Taiping Chen
Committee Member
Mark McArthur
Abstract
Arginine residues can be modified in three different ways to produce asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and monomethylarginine (MMA). These modifications are catalyzed by a family of nine protein arginine methyltransferases (PRMT1-9), which are of three types (I, II, and III). The majority of Type I enzymes asymmetrically dimethylate Glycine- and Arginine-rich (GAR) motifs, except for PRMT4, which methylates Proline-, Glycine-, and Methionine-rich (PGM) motifs. The same substrates (GAR or PGM motifs) can also be dimethylated by PRMT5 in a symmetric fashion. However, it is not clear whether there are dedicated residues within these motifs for ADMA and SDMA, or if the two enzyme types (I and II) compete for the same arginine residue. In addition, very little is known about MMA, which commonly occurs as an intermediate in the pathway to ADMA and SDMA generation. But, occasionally some substrates are solely monomethylated due to the Type III activity of PRMT7. This project aimed at clarifying the dynamics of different methylation types using methylarginine-specific antibodies and PRMT null cell lines. By performing methyl-specific antibody Western and amino acid analysis, we were able to show that loss of PRMT1, which removes 90% of ADMA, causes a global rise in MMA and SDMA levels. Hence, we concluded that there is a dynamic interplay among the three types of methylation, and that ADMA acts as a dominant mark preventing the occurrence of MMA and SDMA on the same substrates.
The second project also involved methyl-specific antibodies, however, it was aimed at discovering novel PRMT4 substrates. PRMT4, also referred as coactivator-associated arginine methyltransferase (CARM1), functions as a regulator of transcription and splicing by methylating a diverse array of substrates. In order to broaden our understanding of CARM1’s mechanistic actions, we generated CARM1 substrate motif antibodies, and used immunoprecipitation coupled with mass spectrometry to identify novel cellular targets for CARM1, including Mediator Complex Subunit 12 (MED12) and the lysine methyltransferase KMT2D/MLL2. Both of these proteins are implicated in enhancer function. We identified the primary CARM1-mediated MED12 methylation site as arginine 1899. Using methyl-specific antibodies to this site, we found that MED12 methylation positively correlates with CARM1 levels. ChIP-seq studies reveal that CARM1 and its activity are tightly associated with ERα-specific enhancers and positively modulate transcription of estrogen (E2)-regulated genes. Using a cell-free biotinylated DNA pulldown assay, we demonstrated that CARM1 and MED12 are co-recruited to a GREB1 estrogen response element (ERE), and in cells, methylated MED12 efficiently assembles on a multicopy engineered ERE array in response to E2 treatment. Additionally, we show that MED12 interacts with the Tudor domain-containing effector molecule, TDRD3, in a CARM1-dependent fashion. These findings reveal an arginine methylation regulatory node on the Mediator complex that may facilitate the communication between DNA-bound transcription factors and RNA polymerase II.
Keywords
Arginine methylation, PRMT1, CARM1, MED12, ER, Monomethylation, asymmetric dimethylation, CARM1 substrates