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THOC5 controls 3'end-processing of immediate early genes via interaction with polyadenylation specific factor 100 (CPSF100).

Tran DD, Saran S, Williamson AJ, Pierce A, Dittrich-Breiholz O, Wiehlmann L, Koch A, Whetton AD, Tamura T - Nucleic Acids Res. (2014)

Bottom Line: Furthermore, THOC5 depletion does not influence the expression of the most rapidly induced IEGs, e.g. Fos and Jun.THOC5 is required for recruitment of CPSF100 to 3'UTR of THOC5 target genes.These data suggest the presence of a novel mechanism for the control of IEG response by THOC5 via 3'end-processing.

View Article: PubMed Central - PubMed

Affiliation: Institut fuer Biochemie, OE4310, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30623 Hannover, Germany.

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THOC5 is required for recruiting of the CPSF100 to 3′end of THOC5 target gene. pCTAP (cTAP) and pCTAP carrying THOC5 cDNA (TAP-THOC5) were transfected into mouse NIH3T3 cells (Bait: THOC5 (A)), or ERT2 THOC5 (flox/flox) MEF cells were treated with or without tamoxifen for 2 days (Baits: CPSF100 (B), RNApolymerase II (C), or CFIm68 (D)). The cells were then incubated for 24 h in the presence of 20% FCS. After serum starvation for 24 h, cells were stimulated with (+) or without (−) serum for 1 h. After cross-linking by adding formaldehyde, protein and DNA were extracted and the chromatin was sheared by sonication. Cell extracts and binding fractions with streptavidin Sepharose or immunoprecipitates using CPSF100, RNApolymerase II or CFIm68 antibodies or control IgG were analyzed by Id3 (promoter region (-314- -199)and 3′UTR in exon 3 (1433-1571)) and Ier2 (promoter region (-701- -535) and 3′UTR region (1349-1522))-specific PCR (Table 1; ChIP). The promoter region of each gene was described by Zhao etal. (24). Numbers represent nucleotide numbers from the initiation site for each gene. Data represent% input of each PCR reaction. Three independent experiments were performed.
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Figure 6: THOC5 is required for recruiting of the CPSF100 to 3′end of THOC5 target gene. pCTAP (cTAP) and pCTAP carrying THOC5 cDNA (TAP-THOC5) were transfected into mouse NIH3T3 cells (Bait: THOC5 (A)), or ERT2 THOC5 (flox/flox) MEF cells were treated with or without tamoxifen for 2 days (Baits: CPSF100 (B), RNApolymerase II (C), or CFIm68 (D)). The cells were then incubated for 24 h in the presence of 20% FCS. After serum starvation for 24 h, cells were stimulated with (+) or without (−) serum for 1 h. After cross-linking by adding formaldehyde, protein and DNA were extracted and the chromatin was sheared by sonication. Cell extracts and binding fractions with streptavidin Sepharose or immunoprecipitates using CPSF100, RNApolymerase II or CFIm68 antibodies or control IgG were analyzed by Id3 (promoter region (-314- -199)and 3′UTR in exon 3 (1433-1571)) and Ier2 (promoter region (-701- -535) and 3′UTR region (1349-1522))-specific PCR (Table 1; ChIP). The promoter region of each gene was described by Zhao etal. (24). Numbers represent nucleotide numbers from the initiation site for each gene. Data represent% input of each PCR reaction. Three independent experiments were performed.

Mentions: We have previously shown that THOC5 is recruited to the last exon, but not the promoter region of the Ets gene induced by stimulation with macrophage-colony stimulating factor (M-CSF) (12). In agreement with these data, after stimulation with serum THOC5 was recruited to 3′UTR of the Id3 gene, but not the promoter region of Id3 (Figure 6A) (24). THOC5 was not recruited to the THOC5-independent IEG, Ier2 (Figure 6A). In control cells, CPSF100 was recruited to 3′UTR of the Id3 and Ier2 genes. In THOC5-depleted cells, CPSF100 was recruited to the Ier2 gene to a similar extent as in control cells, while it was not recruited to the Id3 gene (Figure 6B), suggesting that THOC5 plays a role in recruitment of CPSF100 to 3′UTR of its target gene. RNA polymerase II was equally recruited to both genes in the presence or absence of THOC5 (Figure 6C), suggesting that THOC5 participates in 3′end-processing, but not elongation of THOC5 target genes.


THOC5 controls 3'end-processing of immediate early genes via interaction with polyadenylation specific factor 100 (CPSF100).

Tran DD, Saran S, Williamson AJ, Pierce A, Dittrich-Breiholz O, Wiehlmann L, Koch A, Whetton AD, Tamura T - Nucleic Acids Res. (2014)

THOC5 is required for recruiting of the CPSF100 to 3′end of THOC5 target gene. pCTAP (cTAP) and pCTAP carrying THOC5 cDNA (TAP-THOC5) were transfected into mouse NIH3T3 cells (Bait: THOC5 (A)), or ERT2 THOC5 (flox/flox) MEF cells were treated with or without tamoxifen for 2 days (Baits: CPSF100 (B), RNApolymerase II (C), or CFIm68 (D)). The cells were then incubated for 24 h in the presence of 20% FCS. After serum starvation for 24 h, cells were stimulated with (+) or without (−) serum for 1 h. After cross-linking by adding formaldehyde, protein and DNA were extracted and the chromatin was sheared by sonication. Cell extracts and binding fractions with streptavidin Sepharose or immunoprecipitates using CPSF100, RNApolymerase II or CFIm68 antibodies or control IgG were analyzed by Id3 (promoter region (-314- -199)and 3′UTR in exon 3 (1433-1571)) and Ier2 (promoter region (-701- -535) and 3′UTR region (1349-1522))-specific PCR (Table 1; ChIP). The promoter region of each gene was described by Zhao etal. (24). Numbers represent nucleotide numbers from the initiation site for each gene. Data represent% input of each PCR reaction. Three independent experiments were performed.
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Figure 6: THOC5 is required for recruiting of the CPSF100 to 3′end of THOC5 target gene. pCTAP (cTAP) and pCTAP carrying THOC5 cDNA (TAP-THOC5) were transfected into mouse NIH3T3 cells (Bait: THOC5 (A)), or ERT2 THOC5 (flox/flox) MEF cells were treated with or without tamoxifen for 2 days (Baits: CPSF100 (B), RNApolymerase II (C), or CFIm68 (D)). The cells were then incubated for 24 h in the presence of 20% FCS. After serum starvation for 24 h, cells were stimulated with (+) or without (−) serum for 1 h. After cross-linking by adding formaldehyde, protein and DNA were extracted and the chromatin was sheared by sonication. Cell extracts and binding fractions with streptavidin Sepharose or immunoprecipitates using CPSF100, RNApolymerase II or CFIm68 antibodies or control IgG were analyzed by Id3 (promoter region (-314- -199)and 3′UTR in exon 3 (1433-1571)) and Ier2 (promoter region (-701- -535) and 3′UTR region (1349-1522))-specific PCR (Table 1; ChIP). The promoter region of each gene was described by Zhao etal. (24). Numbers represent nucleotide numbers from the initiation site for each gene. Data represent% input of each PCR reaction. Three independent experiments were performed.
Mentions: We have previously shown that THOC5 is recruited to the last exon, but not the promoter region of the Ets gene induced by stimulation with macrophage-colony stimulating factor (M-CSF) (12). In agreement with these data, after stimulation with serum THOC5 was recruited to 3′UTR of the Id3 gene, but not the promoter region of Id3 (Figure 6A) (24). THOC5 was not recruited to the THOC5-independent IEG, Ier2 (Figure 6A). In control cells, CPSF100 was recruited to 3′UTR of the Id3 and Ier2 genes. In THOC5-depleted cells, CPSF100 was recruited to the Ier2 gene to a similar extent as in control cells, while it was not recruited to the Id3 gene (Figure 6B), suggesting that THOC5 plays a role in recruitment of CPSF100 to 3′UTR of its target gene. RNA polymerase II was equally recruited to both genes in the presence or absence of THOC5 (Figure 6C), suggesting that THOC5 participates in 3′end-processing, but not elongation of THOC5 target genes.

Bottom Line: Furthermore, THOC5 depletion does not influence the expression of the most rapidly induced IEGs, e.g. Fos and Jun.THOC5 is required for recruitment of CPSF100 to 3'UTR of THOC5 target genes.These data suggest the presence of a novel mechanism for the control of IEG response by THOC5 via 3'end-processing.

View Article: PubMed Central - PubMed

Affiliation: Institut fuer Biochemie, OE4310, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30623 Hannover, Germany.

Show MeSH