Fig 2: PROSER1 regulates DNA demethylation on UTX/H3K4me1-dependent enhancers and CpG islands.(A) Dot blot analysis of genomic 5mC, 5hmC, 5fC, and 5caC in WT and PROSER1 KO HEK293 cells. 5mC is unchanged and 5hmC, 5fC, and 5caC are decreased in PROSER1 KO versus WT cells. Methylene Blue staining was performed to ensure that equal amounts of DNA were used from WT and PROSER1 KO cells. (B) Heat maps displaying whole genome bisulfite sequencing (WGBS) and hMeDIP-seq results centered on 4,421 UTX/H3K4me1-down-regulated regions in PROSER1 KO versus WT cells. (C) Heat maps showing WGBS and hMeDIP-seq results centered on 128 hypermethylated CpG islands in PROSER1 KO compared with WT cells. (D) Heat maps centered on hypermethylated DMR500 regions in PROSER1 KO compared with WT cells overlapping with at least one PROSER1, UTX, or H3K4me1 peak in WT cells. Occupancy of PROSER1, UTX, OGT, TET1, TET2, H3K4me1, H3K4me2, H3K4me3, and H3K27ac and WGBS results are shown.

Fig 3: Model of PROSER1 function at enhancers and CpG islands and broader implication for PROSER1 as a general mediator of OGT activity in chromatin signaling.(A) Model of PROSER1 function at enhancers and CpG islands. PROSER1 mediates OGT interaction with and O-GlcNAcylation of TET2 to control TET2 stabilization at enhancers and CGIs that are controlled by the activity of the MLL3/4 complexes. TET2 is involved in the recruitment of the MLL3/4 complexes via their complex-specific subunit UTX. In the absence of PROSER1 TET2 stabilization at enhancers and CGIs is impaired resulting in reduced recruitment of the MLL3/4 complexes. TF, transcription factor. (B) Western blot of IP with an O-GlcNAc–specific antibody from WT and PROSER1 KO HEK293 cells confirming strongly reduced O-GlcNAcylation of TET2 in PROSER1 KO compared with WT cells. Nuclear extracts were used as input. Actin was used as a loading control for the inputs. (C) IP with an O-GlcNAc–specific antibody followed by mass spectrometry identifies TET1 and OGT as differentially O-GlcNAcylated in WT versus PROSER1 KO HEK293 cells. Many components of the H3K4 methyltransferase and demethylase family also display reduced O-GlcNAcylation in PROSER1 KO compared with WT cells. Of note, QSER1, a protein just recently described as a factor that protects DNA methylation valleys from de novo methylation also showed reduced O-GlcNAcylation in PROSER1 KO versus WT cells.

Fig 4: The MLL3/4 complexes associate with PROSER1, a novel proline and serine rich protein, the DNA demethylase TET2 and the glycosyltransferase OGT.(A) FLAG-UTX immunoprecipitation (IP) followed by mass spectrometry identifies all known subunits of the MLL3/4 complexes along with PROSER1, a novel serine and proline rich protein, the DNA demethylase TET2 and the glycosyltransferase OGT. SC, spectral counts; TP, peptide counts; abundance = SC × 50 (kD)/protein size (kD). (B) Western blot of FLAG-UTX IP from HEK293 cells confirming interaction of UTX with PROSER1, TET2 and OGT. UTX interacts with the H3K4 methyltransferases MLL3 and MLL4, RBBP5 a core component of the MLL3/4 complexes, PROSER1, TET2, and OGT. HEK293 cells with a FLAG-tag expressing plasmid were used as an IP control. Nuclear extracts were used as input. Actin was used as a loading control for the inputs. (C) Glycerol gradient sedimentation after FLAG-UTX IP reveals co-fractionation of PROSER1 and TET2 with components of the MLL3/4 complexes (red box).

Fig 5: PROSER1 mediates TET2 O-GlcNAcylation and stability by promoting the interaction between OGT and TET2.(A) Top: Domain structure and constructs of the human TET2 double-stranded ß-helix 2 (DSBH2) domain. TET2 DSHB2-N: N-terminal 93 aa of DSHB2. TET2 DSHB2-C: C-terminal 66 aa of DSHB2. Bottom: HEK293 cells were transiently transfected with the FLAG-tagged constructs as shown on the top. Western blot of FLAG IPs showing interaction of PROSER1 and OGT with TET2 DSHB2-C. Total cell lysates were used as inputs. Actin was used as a loading control for the inputs. The asterisks indicate an unspecific band (upper panel) or the IgG heavy chain (lower panel). TET2 DSHB2-N and TET2 DSHB2-C could not be detected in the inputs. Failure to detect FLAG-TET2 DSBH2-C after FLAG IP might be due to its positive charge at the pH of the sample buffer. (B) WT and PROSER1 KO HEK293 cells were transiently transfected with FLAG-tagged TET2 DSHB2. WB of FLAG IPs depicts decreased interaction of OGT with TET2 DSHB2 in the absence of PROSER1. Total cell lysates were used as inputs. Actin was used as a loading control for the inputs. The asterisks indicate unspecific bands (middle panel) or the IgG heavy chain (lower panel). TET2 DSHB2 could not be detected in the inputs. (C) TET2 IP from nuclear extracts of WT and PROSER1 KO HEK293 cells. Approximately equal amounts of TET2 were IPed from WT and PROSER1 KO cells. A significant decrease in OGT binding to TET2 and a strong reduction of TET2 O-GlcNAcylation is observed in PROSER1 KO versus WT HEK293 cells. Nuclear extracts were used as inputs. Actin was used as a loading control for the inputs. The asterisks indicate unspecific bands, the arrow O-GlcNAcylated TET2. (D) WB for the indicated proteins from nuclear extracts of WT and PROSER1 KO HEK293 cells. Actin was used as a loading control. (E) FLAG IP from total cell lysates of FHNG-PROSER1 HEK293 cells treated with control or OGT siRNA. PROSER1 protein levels are decreased upon OGT knockdown. IPed PROSER1 from control and OGT siRNA-treated cells was adjusted to equal amounts to show that OGT catalyzes O-GlycNAcylation of PROSER1. Total cell lysates were used as inputs. Actin was used as a loading control for the inputs. The asterisk indicates the IgG heavy chain. (F) Model of PROSER1 function. PROSER1 mediates the recruitment of OGT to TET2 to facilitate O-GlcNAcylation of TET2 and PROSER1. In the absence of PROSER1, OGT association with TET2 is impaired resulting in decreased TET2 and PROSER1 O-GlcNAcylation and protein stability.