As the repression of miR-34a contributed to the upregulation of HSP70 in HSSca-1+ cells, we then investigated the underlying factors for miR-34a repression. First, we ruled out the possibility that the miR-34a genomic loci were deleted in HSSca-1+ cells, since equal amounts were detected in the same amounts of genomic DNA extracted from HSSca-1+ cells and non-HSSca-1+ cells using real-time PCR (Supporting Information Fig. S2). Although previous studies showed that DNA methylation on miR-34a promoter was one of the most common cause for its downregulation [9, 10], we found the CpG island on miR-34a promoter was hypomethylated in both HSSca-1+ cells and non-HSSca-1+ cells through bisulfate sequencing (Fig. 4A and Supporting Information Fig. S3), thus indicating DNA methylation is less likely to regulate their expression in HSSca-1+ cells. While trimethylation of histone H3 at Lys4 (H3K4me3) is linked to transcriptional activation, trimethylation of H3 at Lys 27 (H3K27me3) is linked to transcriptional repression, next we performed ChIP-qPCR to detect the relative occupancy of H3K4me3, H3K27me3, and Pol II (the RNA polymerase responsible for miR-34a transcription) on the genomic region extracted from ChIP-seq data from ENCODE project in university of california santa cruz (UCSC) genome browser. The results showed a reduction in enrichment of H3K4me3, Pol II as opposed to increased enrichment of H3K27me3 on miR-34a promoter in HSSca-1+ cells (Fig. 4B, 4C). These results indicate that the repression of miR-34a in HSSca-1+ cells is mediated by transcriptional repression via histone methylation instead of DNA methylation. Then we further investigated the mechanism of transcriptional repression for miR-34a. By searching the GenomeTrafac database, we found a putative HSF1 responsive element (HSF1-RE) in the mouse miR-34a promoter region (Fig. 4D). Deeper homology analysis found the putative HSF1 responsive element is highly conserved in multiple species (Fig. 4E). Furthermore, the putative element is near the previously reported p53 response element (p53-RE), which has been shown to be responsible for transactivation of miR-34 [11, 12]. The putative HSF1-RE along with the CpG islands, pol II, H3K4me3, and H3K27me3 peaks in miR-34a promoter was displayed using the UCSC genome browser (Fig. 4F). Based on the computational analysis and the inverse relationship of HSF1 and miR-34a (Fig. 4G), we determined whether HSF1 regulates the expression of miR-34a. To test our hypothesis, we cloned the WT and mutated construct (HSF1-RE deletion) of promoter region of mouse miR-34a upstream of a luciferase gene in a reporter plasmid (pGL4), respectively. Interestingly, transfection of reporter plasmid with mutated construct into HSSca-1+ cells led to significant upregulation of luciferase activity as compared to wide type, indicating HSF1-RE is a repressor element for miR-34a transcription (Fig. 4H). Moreover, knockdown of HSF1 by siRNA in HSSca-1+ cells led to upregulation of miR-34a (Fig. 4I) and miR-34a inhibitor rescued the loss of HSP70 caused by knockdown of HSF1. Finally, ChIP-qPCR validated the binding of HSF1 on the putative HSF1-RE and found HSF1 was enriched on this cis element in HSSca-1+ cells as compared to non-HSSca-1+ cells (Fig. 4M). Taken together, we concluded that miR-34a repression by chromatin remodeling is attributed to direct binding of HSF1 on miR-34a promoter.
Figure 4. miR-34a repression in HSSca-1+ cells is attributed to HSF1-mediated chromatin remodeling. (A): Bisulfite sequencing showing no hypermethylation on miR-34a promoter in HSSca-1+ cells. Genomic DNA derived from HSSca-1+ and non-HSSca-1+ cells was subjected to bisulfite sequencing and examined at the region on CpG island of miR-34a, as indicated in the map. Methylated CpG are presented by black circles and unmethylated sites by open circles. (B): ChIP qRT-PCR analysis of RNA polymerase II (Pol II), H3K4me3, and H3K27me3 on the promoter region of miR-34a in HSSca-1+ and non-HSSca-1+ cells. Data are normalized to inputs from three experiments (mean ± SEM). (*) denotes p < .05 for significant differences related to non-HSSca-1+ cells (C). (D): Mouse-human-conserved HSF1 responsive element (red circle) in miR-34a promoter region (downloaded from the GenomeTrafac database). (E): Sequence logo showing high conservation of the putative HSF1-RE. The logo was constructed from 12 mammal species. The height of each letter is proportional to the frequency of the base, and the height of the letter stack shows the conservation at that position. (F): AUCSC genome browser screenshot showing the location of validated p53 response element (p53-RE), the putative HSF1 response element (HSF1-RE), CpG island, Pol II, H3K4me3, and H3K27me3 tracks on miR-34a promoter in mouse genome. (G): Western blot (upper panel) and PCR (lower panel) showing inverse correlation between HSF1 and miR-34a expression. Nuclear protein and total RNA were extracted from the same batch of cells. (H): Luciferase (Luc) reporter constructs contain the miR-34a promoter with the potential HSF1-RE upstream of a luciferase gene (WT), or the miR-34a promoter with the deletion of potential HSF1-RE (HSF1-RE deletion). Red indicates the potential HSF1-RE (chr4:149,423,492-149,423,516). Sca-1+ cells were transfected with WT or HSF1-RE deletion plasmids for 24 hours, followed by heat shock treatment. Luciferase activity was measured 14 hours after heat shock. Luciferase activity is expressed as the ratio of luciferase activity of HSF1-RE deletion to WT luciferase activity and Renilla luciferase as a normalizing control. Data represent mean ± SEM (n = 3). (*) denotes p < .05, relative to WT. (I): Knockdown of HSF1 by siRNA as shown by Western blot significantly upregulated miR-34a expression detected by real-time PCR. Data represent mean ± SEM (n = 3). (*) denotes p < .05, relative to Scramble-transfected HSSca-1+ cells. (J): Western blot showing knockdown of HSF1 by siRNA downregulated HSP70 expression which was rescued by miR-34a inhibitors. (K): ChIP-qPCR verified the binding of HSF1 on the putative HSF1-RE, showing HSF1 was significantly enriched on the promoter region of miR-34a in HSSca-1+ cells as compared to non-HSSca-1+ cells. Data represent mean ± SEM (n = 3). (*) denotes p < .05, relative to non-HSSca-1+ cells. Abbreviations: HSF, heat shock factor; HSP, heat shock proteins; RLU, relative light units.
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