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The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing.

Liu HW, Banerjee T, Guan X, Freitas MA, Parvin JD - Nucleic Acids Res. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Informatics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.

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SUMOylation facilitates RNAPII recruitment on the active promoters. (A-C) Chromatin was isolated from control siRNA transfected cells (black) or UBC9 siRNA transfected cells (gray), and immunopurified using antibody specific for unphosphorylated RNAPII (8WG16, panel A), p-Ser-5 RNAPII (panel B) or SUMO-1 (panel C). Enrichment of the promoters of the indicated genes was detected by ChIP-qPCR, and to control for transfection efficiency, reactions were normalized to the control siRNA. Each column represents the mean ± SEM. T-test using the data from four biological replicates of ChIP–qPCR was conducted. The asterisk indicates statistical significance (*P ≤ 0.05; **P ≤ 0.01). Non-normalized results are presented in Supplementary Figure S1. (D) Immunoblots from the cells used in panels (A)–(C) were stained for UBC9 (top) and the loading control α-tubulin (bottom).
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Figure 1: SUMOylation facilitates RNAPII recruitment on the active promoters. (A-C) Chromatin was isolated from control siRNA transfected cells (black) or UBC9 siRNA transfected cells (gray), and immunopurified using antibody specific for unphosphorylated RNAPII (8WG16, panel A), p-Ser-5 RNAPII (panel B) or SUMO-1 (panel C). Enrichment of the promoters of the indicated genes was detected by ChIP-qPCR, and to control for transfection efficiency, reactions were normalized to the control siRNA. Each column represents the mean ± SEM. T-test using the data from four biological replicates of ChIP–qPCR was conducted. The asterisk indicates statistical significance (*P ≤ 0.05; **P ≤ 0.01). Non-normalized results are presented in Supplementary Figure S1. (D) Immunoblots from the cells used in panels (A)–(C) were stained for UBC9 (top) and the loading control α-tubulin (bottom).

Mentions: We have previously shown that SUMO-1 is enriched on the chromatin proteins bound to promoter regions of some of the most active genes during interphase, such as ribosomal protein (RP) encoding genes (8), which are highly abundant and constitutively transcribed by RNA polymerase II (RNAPII). Depletion of SUMO-1 caused a decrease in mRNA production, suggesting that the presence of SUMO-1 on the active promoter regions had a positive role for SUMOylation in transcriptional regulation (8). It had not been shown in human cells whether the decrease in mRNA abundance following depletion of SUMO-1 was due to a decrease in RNAPII binding. In yeast, which have only one SUMO isoform, SUMO protein is required for RNAPII recruitment on the constitutive genes during transcription initiation (9). We tested whether the SUMO-1 mark on the chromatin bound to a housekeeping promoter stimulates active RNAPII recruitment to promoters in mammalian cells. We investigated RNAPII occupancy under defective SUMOylation in HeLa cells by siRNA transfection targeting UBC9, the only SUMO-specific E2 ligase found in cells, and testing for RNAPII binding to promoter regions of ribosomal protein genes such as RPL3, RPL7A, RPL10A, RPL26, which were found enriched with SUMO-1 (8). In addition, we analyzed the β-actin promoter, which was not bound by SUMO-1, as a negative control. The enrichment of SUMO-1, RNAPII with phosphorylated serine-5 (pSer-5) of the carboxy-terminal domain (CTD) and RNAPII that is mainly unphosphorylated (8WG16) bound to promoters, was analyzed by ChIP-qPCR. The results were normalized to the control siRNA in each experiment to correct for a modest amount of variation in the percent of input obtained in each quantitative PCR assay. The results showed that upon UBC9 siRNA depletion, the occupancy of SUMO-1 on the promoters of RP genes significantly decreased 4- to 5-fold compared to controls (Figure 1C). We found that depletion of UBC9 and consequent defect in SUMOylation caused a decrease in the occupancy at these promoters of both unphosphorylated and phosphorylated form of RNAPII. Binding of the unphosphorylated RNAPII was decreased 2.5- to 5-fold. Binding of the pSer-5 form of RNAPII was decreased 4- to 5-fold compared to controls (Figure 1A and B). By contrast, the binding of either form of RNAPII to the β-actin promoter was not affected by depletion of UBC9 (Figure 1A and B). It is noteworthy that the effect on the pSer-5 form of RNAPII was of somewhat higher magnitude than the unphosphorylated form of RNAPII, suggesting that SUMOylation impacted the initiation process. The same results, shown in Supplementary Figure S1 but as individual experiments, clearly indicate the reduction in RNAPII and phospho-RNAPII associated with these promoters after depletion of the UBC9.


The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing.

Liu HW, Banerjee T, Guan X, Freitas MA, Parvin JD - Nucleic Acids Res. (2015)

SUMOylation facilitates RNAPII recruitment on the active promoters. (A-C) Chromatin was isolated from control siRNA transfected cells (black) or UBC9 siRNA transfected cells (gray), and immunopurified using antibody specific for unphosphorylated RNAPII (8WG16, panel A), p-Ser-5 RNAPII (panel B) or SUMO-1 (panel C). Enrichment of the promoters of the indicated genes was detected by ChIP-qPCR, and to control for transfection efficiency, reactions were normalized to the control siRNA. Each column represents the mean ± SEM. T-test using the data from four biological replicates of ChIP–qPCR was conducted. The asterisk indicates statistical significance (*P ≤ 0.05; **P ≤ 0.01). Non-normalized results are presented in Supplementary Figure S1. (D) Immunoblots from the cells used in panels (A)–(C) were stained for UBC9 (top) and the loading control α-tubulin (bottom).
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Figure 1: SUMOylation facilitates RNAPII recruitment on the active promoters. (A-C) Chromatin was isolated from control siRNA transfected cells (black) or UBC9 siRNA transfected cells (gray), and immunopurified using antibody specific for unphosphorylated RNAPII (8WG16, panel A), p-Ser-5 RNAPII (panel B) or SUMO-1 (panel C). Enrichment of the promoters of the indicated genes was detected by ChIP-qPCR, and to control for transfection efficiency, reactions were normalized to the control siRNA. Each column represents the mean ± SEM. T-test using the data from four biological replicates of ChIP–qPCR was conducted. The asterisk indicates statistical significance (*P ≤ 0.05; **P ≤ 0.01). Non-normalized results are presented in Supplementary Figure S1. (D) Immunoblots from the cells used in panels (A)–(C) were stained for UBC9 (top) and the loading control α-tubulin (bottom).
Mentions: We have previously shown that SUMO-1 is enriched on the chromatin proteins bound to promoter regions of some of the most active genes during interphase, such as ribosomal protein (RP) encoding genes (8), which are highly abundant and constitutively transcribed by RNA polymerase II (RNAPII). Depletion of SUMO-1 caused a decrease in mRNA production, suggesting that the presence of SUMO-1 on the active promoter regions had a positive role for SUMOylation in transcriptional regulation (8). It had not been shown in human cells whether the decrease in mRNA abundance following depletion of SUMO-1 was due to a decrease in RNAPII binding. In yeast, which have only one SUMO isoform, SUMO protein is required for RNAPII recruitment on the constitutive genes during transcription initiation (9). We tested whether the SUMO-1 mark on the chromatin bound to a housekeeping promoter stimulates active RNAPII recruitment to promoters in mammalian cells. We investigated RNAPII occupancy under defective SUMOylation in HeLa cells by siRNA transfection targeting UBC9, the only SUMO-specific E2 ligase found in cells, and testing for RNAPII binding to promoter regions of ribosomal protein genes such as RPL3, RPL7A, RPL10A, RPL26, which were found enriched with SUMO-1 (8). In addition, we analyzed the β-actin promoter, which was not bound by SUMO-1, as a negative control. The enrichment of SUMO-1, RNAPII with phosphorylated serine-5 (pSer-5) of the carboxy-terminal domain (CTD) and RNAPII that is mainly unphosphorylated (8WG16) bound to promoters, was analyzed by ChIP-qPCR. The results were normalized to the control siRNA in each experiment to correct for a modest amount of variation in the percent of input obtained in each quantitative PCR assay. The results showed that upon UBC9 siRNA depletion, the occupancy of SUMO-1 on the promoters of RP genes significantly decreased 4- to 5-fold compared to controls (Figure 1C). We found that depletion of UBC9 and consequent defect in SUMOylation caused a decrease in the occupancy at these promoters of both unphosphorylated and phosphorylated form of RNAPII. Binding of the unphosphorylated RNAPII was decreased 2.5- to 5-fold. Binding of the pSer-5 form of RNAPII was decreased 4- to 5-fold compared to controls (Figure 1A and B). By contrast, the binding of either form of RNAPII to the β-actin promoter was not affected by depletion of UBC9 (Figure 1A and B). It is noteworthy that the effect on the pSer-5 form of RNAPII was of somewhat higher magnitude than the unphosphorylated form of RNAPII, suggesting that SUMOylation impacted the initiation process. The same results, shown in Supplementary Figure S1 but as individual experiments, clearly indicate the reduction in RNAPII and phospho-RNAPII associated with these promoters after depletion of the UBC9.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Informatics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.

Show MeSH
Related in: MedlinePlus