The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing.
Bottom Line: In this study, we found that SUMO-1 marks the promoters of ribosomal protein genes via modification of the Scaffold Associated Factor B (SAFB) protein, and the SUMOylated SAFB stimulated both the binding of RNA polymerase to promoters and pre-mRNA splicing.Depletion of SAFB decreased RNA polymerase II binding to promoters and nuclear processing of the mRNA, though mRNA stability was not affected.This study reveals an unexpected role of SUMO-1 and SAFB in the stimulatory coupling of promoter binding, transcription initiation and RNA processing.
Affiliation: Department of Biomedical Informatics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.Show MeSH
Mentions: To determine if SAFB was responsible for the recruitment of SUMO-1 binding on the specific promoters, we tested whether depletion of SAFB affected the recruitment of SUMO-1 to promoters that we had previously characterized to be SUMO-1 bound (8). Since there are two highly related isoforms of SAFB, we depleted both homologs with siRNAs targeting SAFB1/2. Following siRNA transfection, immunoblot analysis showed that SAFB protein was depleted by >90% (Figure 3C). Consistent with earlier results, ChIP-qPCR analysis showed that under control conditions SUMO-1 and RNAPII were enriched on the RP gene promoter regions analyzed; IL2 was included as a negative control since it is not expressed in HeLa cells and its promoter had no detected SUMO-1. SAFB depletion caused a significant decrease in the SUMO-1 marks on the RP gene promoters, down to 40–50% compared to the controls (Figure 3A). Depletion of SAFB also caused a decrease in RNAPII occupancy on these promoters (Figure 3B). By contrast, when testing active genes that are not labeled by SUMO-1, such as β-actin, depletion of SAFB did not affect RNAPII occupancy on its promoter (Figure 3B), suggesting that SAFB facilitates RNAPII binding on the SUMO-1 labeled active genes. We further asked whether this phenomenon was caused by SAFB1. To this end, a second set of siRNAs for depletion of SAFB targeted the 3′UTR of SAFB1 and a second site in the ORF of SAFB2. Transfection of this second set of siRNAs also decreased ChIP specific for SUMO-1 at RP gene promoters, and expression of SAFB1 from a cotransfected plasmid rescued SUMO-1 binding to these promoters (Figure 4). These results clearly indicate that SAFB1 is a functionally relevant SUMO-1 target bound to the promoters, and these results support a model whereby the SUMOylation of SAFB stimulates RNAPII binding to target gene promoters.
Affiliation: Department of Biomedical Informatics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.