Abstract
MAF1 represses Pol III-mediated transcription by interfering with TFIIIB and Pol III. Herein, we found that MAF1 knockdown induced CDKN1A transcription and chromatin looping concurrently with Pol III recruitment. Simultaneous knockdown of MAF1 with Pol III or BRF1 (subunit of TFIIIB) diminished the activation and looping effect, which indicates that recruiting Pol III was required for activation of Pol II-mediated transcription and chromatin looping. Chromatin-immunoprecipitation analysis after MAF1 knockdown indicated enhanced binding of Pol III and BRF1, as well as of CFP1, p300, and PCAF, which are factors that mediate active histone marks, along with the binding of TATA binding protein (TBP) and POLR2E to the CDKN1A promoter. Simultaneous knockdown with Pol III abolished these regulatory events. Similar results were obtained for GDF15. Our results reveal a novel mechanism by which MAF1 and Pol III regulate the activity of a protein-coding gene transcribed by Pol II.
Highlights
Transcription by ribonucleic acid (RNA) polymerase III (Pol III) is regulated by MAF1, which is a highly conserved protein in eukaryotes (Pluta et al, 2001; Reina et al, 2006)
We carried out quantitative RT-PCR (qRT-PCR) to confirm whether CDKN1A expression was upregulated by MAF1 knockdown
Because the majority of short interspersed element (SINE) are transcribed by the type II internal Pol III promoter that contains an A-box and B-box (Okada and Ohshima, 1995), our model indicates that mutation of the Pol III promoter element in the promoter-associated SINE should abolish the enhancement of reporter expression after MAF1 knockdown
Summary
Transcription by RNA polymerase III (Pol III) is regulated by MAF1, which is a highly conserved protein in eukaryotes (Pluta et al, 2001; Reina et al, 2006). MAF1 represses Pol III transcription through association with BRF1, a subunit of initiation factor TFIIIB, which prevents attachment of TFIIIB onto DNA. This interaction inhibits Pol III from binding to BRF1, which in turn prevents recruitment of Pol III to Pol III promoters. Association of MAF1 with Pol III-transcribed genes has been detected genome-wide concomitant with an increase in occupation during repression; this indicates that direct interaction of MAF1 with Pol III genes is an important attribute of repression (Roberts et al, 2006). To investigate the potential regulatory role of MAF1 in Pol II genes, we carried out MAF1 knockdown coupled with microarray analysis. Microarray analysis showed that 124 genes were upregulated and 170 genes were downregulated more than twofold after MAF1 knockdown. CDKN1A ( known as p21) was significantly upregulated and the mechanism of induced transcription of this gene after MAF1 knockdown was further investigated
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