PROSER1 mediates TET2 O-GlcNAcylation to regulate DNA demethylation on UTX-dependent enhancers and CpG islands

Abstract

DNA methylation at enhancers and CpG islands usually leads to gene repression, which is counteracted by DNA demethylation through the TET protein family. However, how TET enzymes are recruited and regulated at these genomic loci is not fully understood. Here, we identify TET2, the glycosyltransferase OGT and a previously undescribed proline and serine rich protein, PROSER1 as interactors of UTX, a component of the enhancer-associated MLL3/4 complexes. We find that PROSER1 mediates the interaction between OGT and TET2, thus promoting TET2 O-GlcNAcylation and protein stability. In addition, PROSER1, UTX, TET1/2, and OGT colocalize on many genomic elements genome-wide. Loss of PROSER1 results in lower enrichment of UTX, TET1/2, and OGT at enhancers and CpG islands, with a concomitant increase in DNA methylation and transcriptional down-regulation of associated target genes and increased DNA hypermethylation encroachment at H3K4me1-predisposed CpG islands. Furthermore, we provide evidence that PROSER1 acts as a more general regulator of OGT activity by controlling O-GlcNAcylation of multiple other chromatin signaling pathways. Taken together, this study describes for the first time a regulator of TET2 O-GlcNAcylation and its implications in mediating DNA demethylation at UTX-dependent enhancers and CpG islands and supports an important role for PROSER1 in regulating the function of various chromatin-associated proteins via OGT-mediated O-GlcNAcylation.

The MLL3/4 complexes and MiDAC act antagonistically as genome-wide regulators of H4K20ac to control a specific gene expression program

“…We show that UTX, a complex-specific subunit of the MLL3/4 complexes, acts as an important hub to ensure the physical association with MiDAC via its scaffolding subunit ELMSAN1 (Figure 1). Indeed, apart from mediating the interaction with MiDAC, UTX might generally function as a unique docking site within the MLL3/4 complexes to recruit other chromatin-associated protein complexes, including the previously reported TOP (TET2, OGT, and PROSER1) complex (Wang et al, 2022). Interestingly, UTX is often mutated in the same cancer types as MLL3 and/or MLL4 linking the function of the MLL3/4 complexes directly to carcinogenesis via UTX (Martinez-Jimenez et al 2020).…”

“…Glycerol gradient fractionation was conducted as previously described (Wang et al 2022). 34 fractions of approximately 325 μl each were collected and analyzed by western blotting for UTX, MLL3, MLL4, RBBP5, ELMSAN1, DNTTIP1, HDAC1 and HDAC2.…”

“…Following the procedure described before (Wang et al, 2022) Subsequently, MACS2 program (Zhang et al 2008) was used to call peaks in narrow mode, with --nomodel -q 0.05 flags (high confidence peaks). Separately, narrow peaks were also called with more relaxed criteria, setting the -q flag to 0.5, which are here referred to as FDR50 peaks.…”

“…Following the approach used previously (Wang et al, 2022), genomic regions were assigned to their genomic contexts with an in-house script based on pybedtools (Dale et al 2011) (v0.8.1), such that each region could only be assigned to one feature. For this purpose, genomic regions were successively overlapped with predefined genomic contexts in the following prioritization order:…”

“…MLL3+/+ MLL4+/+ HCT116 cells and MLL3-/-MLL4-/-HCT116 cells were generated from MLL3-/-MLL4+/+ HCT116 cells by the CAGE at St. Jude Children's Research Hospital using CRISPR/Cas9-mediated gene editing (FigureS3A). Isogenic tetracycline-inducible FLAG-UTX and FLAG control HEK293 cells were previously described by our lab(Wang et al 2022). Isogenic tetracycline-inducible FLAG-DNTTIP1, FLAG-ELMSAN1, FLAG-TRERF1 and FLAG control HEK293 cells were generated by using Flp recombinase-mediated integration in ELMSAN1 TRERF1 DKO HEK293 cells.HEK293 and HCT116 cells were cultured in Dulbecco's Modified Eagle Medium (Gibco, 11995065) with 10% FBS (VWR, 97068-085) and 1% Penicillin/Streptomycin (Gibco, 15140122).…”