mately the impact on transcription may be similar. 10 / 20 hnRNP A1/A2 as Transcription Elongation Factors hnRNP A1/A2 proteins affect the distribution of RNA polymerase II on the Kitlg gene Our model postulates that the increased association of pol II with the mycUP1 gene when A1/ A2 are depleted results from a problem in transcription elongation affecting many P-TEFb-dependent genes. Expression and ChIP-PCR assays suggest that Kitlg belongs to this latter class and that it may have experienced such an elongation defect. The impact of DRB on pol II binding to Kitlg is indicative of promoter-proximal stalling. For sorbitol, while a decrease in pol II occupancy in the gene body of Kitlg was seen 8 hours post-treatment, the situation at promoter-proximal positions indicated a slight increase at position +254 and a slight decrease at position +854. Monitoring pol II occupancy on Kitlg at earlier times indicated a drop in occupancy after 1 hour, followed by restoration of pol II occupancy only at positions +254 and +854, consistent with defective P-TEFb activity. In contrast, no change in pol II occupancy was observed on the mycUP1 gene 1 hour post-treatment, but occupancy increased afterwards, indicating that the increase in pol II occupancy on mycUP1 might be occurring only after the initial drop in pol II occupancy on Kitlg is detected. The impact of depleting A1/A2 on pol II occupancy on Kitlg was not as clear, possibly reflecting the weaker effect of siA1/A2 on Kitlg expression. With the hope of improving sensitivity, we performed a pol II-ChIP assay followed by deep sequencing. The ChIPseq data confirmed that both DRB and the RNAi-mediated knockdown of A1/A2 stimulated RNA pol II occupancy on Egr1. For Kitlg, the decreased expression imposed by DRB was associated with a redistribution of pol II at more downstream positions, suggesting promoter-proximal pausing. The depletion of A1/A2 also provoked a slight redistribution in pol II occupancy in favor of more downstream positions, in addition to an overall decrease in pol II occupancy. Thus, these results suggest that the siRNA-mediated depletion of A1/A2 alters the promoter-proximal occupancy of pol II on Kitlg. The siRNA-mediated depletion of A1 and A2 modifies the rate of elongation To evaluate more directly if low levels of A1/A2 elicit an elongation defect on Kitlg, we relied on a quantitative RT-PCR-based PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19668086 elongation rate assay that measures the recovery of transcription at different positions of a gene following the release of an elongation block. Under transfected conditions with a control siRNA, transcription signals MedChemExpress Nigericin (sodium salt) reappear gradually with time throughout Kitlg yielding an elongation rate of approximately 2.3 Kbp/min, consistent with previous values. Upon releasing the elongation block in A1/A2-depleted cells, transcription output at the proximal position +854 closely followed that of the control transfection. In contrast however, the recovery of transcription at all downstream positions was significantly reduced compared to the control situation, consistent with impaired elongation in A1/A2-depleted cells. The siRNA-mediated depletion of hnRNP A1/A2 has a broad impact on gene expression and polymerase II occupancy To assess how generalized the knockdown of A1/A2 affects gene expression, we carried out next-generation transcriptome sequencing using untreated, A1/A2-depleted and DRB-treated HCT116 cells. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19667050 The read coverage for the complete set of genes is available at GEO accessi
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