Abstract 1049 O-GlcNAc crosslinking of Chromodomain Helicase DNA Binding Protein 4 (CHD4) reveals novel functions for the Nucleosome Chromatin Remodeling Complex

CHD4 (Chromodomain Helicase DNA Binding Protein 4) is a DNA helicase and an essential component of the NuRD (Nucleosome Remodeling and Deacetylase) complex. CHD4 can interact with numerous proteins to form unique repressor or activator complexes to control gene expression. The control of CHD4 protein interactions is not well understood. We hypothesized that CHD4 O-GlcNAcylation allows CHD4 to interact with and form multi-protein complexes with numerous proteins. O-GlcNAc is a single N-accetylglucosamine sugar (O-GlcNAc) modification found on intracellular proteins. To identify novel O-GlcNAcylated CHD4 interacting proteins, we treated a gene-edited erythroleukemia cell line (UAP1:F383G K-562) with a sugar analogue containing a photo-crosslinkable diazirine group (GlcNDAz). O-GlcNAc transferase (OGT) adds the sugar analogue to proteins. Thus, after crosslinking we used CHD4 immunoprecipitation (IP) coupled with mass spectrometry to identify CHD4 interacting proteins. Interestingly, in the control IP, OGT was associated with unmodified CHD4; while, O-GlcNDAzylated CHD4 was associated as expected with proteins involved in gene regulation. Next, we induced gene expression and terminal differentiation in the UAP1:F383G K-562 cells and identified GlcNDAz CHD4 interacting proteins. We found greater than 150 novel interacting proteins. GlcNDAz CHD4 interacted preferentially with proteins in the mini-chromosome complex (MCM), mitotic spindle, and with specific NuRD complex members. Using cell cycle synchronized HeLa cells, we validated an S phase interaction of CHD4 with MCM7 and a M phase interaction with the mitotic spindle. Together, these data show robust and novel functions for CHD4 and suggest these functions are preferentially generated by O-GlcNAcylation of CHD4.