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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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Expression of SAFB1 from a transfected plasmid rescued SUMOylation of promoter-bound chromatin. (A) A second set of siRNAs targeting the 3′-UTR of SAFB1 and the coding region of SAFB2 along with plasmid vector or plasmid for the expression of SAFB1 as indicated were transfected into HeLa cells. ChIP using control IgG (black) or SUMO-1 (gray) specific antibody was probed by qPCR for the promoters of the indicated genes. (B) Immunoblots for SAFB (top) or the unaffected loading control, RNA helicase A (RHA; bottom) were analyzed for control siRNA plus empty vector (lane 1), SAFB siRNAs plus empty vector (lane 2), and SAFB siRNAs plus SAFB1 expression plasmid (lane 3).
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Figure 4: Expression of SAFB1 from a transfected plasmid rescued SUMOylation of promoter-bound chromatin. (A) A second set of siRNAs targeting the 3′-UTR of SAFB1 and the coding region of SAFB2 along with plasmid vector or plasmid for the expression of SAFB1 as indicated were transfected into HeLa cells. ChIP using control IgG (black) or SUMO-1 (gray) specific antibody was probed by qPCR for the promoters of the indicated genes. (B) Immunoblots for SAFB (top) or the unaffected loading control, RNA helicase A (RHA; bottom) were analyzed for control siRNA plus empty vector (lane 1), SAFB siRNAs plus empty vector (lane 2), and SAFB siRNAs plus SAFB1 expression plasmid (lane 3).

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.


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)

Expression of SAFB1 from a transfected plasmid rescued SUMOylation of promoter-bound chromatin. (A) A second set of siRNAs targeting the 3′-UTR of SAFB1 and the coding region of SAFB2 along with plasmid vector or plasmid for the expression of SAFB1 as indicated were transfected into HeLa cells. ChIP using control IgG (black) or SUMO-1 (gray) specific antibody was probed by qPCR for the promoters of the indicated genes. (B) Immunoblots for SAFB (top) or the unaffected loading control, RNA helicase A (RHA; bottom) were analyzed for control siRNA plus empty vector (lane 1), SAFB siRNAs plus empty vector (lane 2), and SAFB siRNAs plus SAFB1 expression plasmid (lane 3).
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Figure 4: Expression of SAFB1 from a transfected plasmid rescued SUMOylation of promoter-bound chromatin. (A) A second set of siRNAs targeting the 3′-UTR of SAFB1 and the coding region of SAFB2 along with plasmid vector or plasmid for the expression of SAFB1 as indicated were transfected into HeLa cells. ChIP using control IgG (black) or SUMO-1 (gray) specific antibody was probed by qPCR for the promoters of the indicated genes. (B) Immunoblots for SAFB (top) or the unaffected loading control, RNA helicase A (RHA; bottom) were analyzed for control siRNA plus empty vector (lane 1), SAFB siRNAs plus empty vector (lane 2), and SAFB siRNAs plus SAFB1 expression plasmid (lane 3).
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.

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