Fig. 7: HSF5 binds to the gene promoter regions.
a HSF5-bound peaks were commonly identified among the 4 replicates of HSF5 CUT&Tag (rabbit anti-HSF5-N1, rabbit anti-HSF5-N2, rabbit anti-HSF5-C antibodies), but not overlapped with control IgG CUT&Tag. HSF5 binding sites were classified by the genomic locations as indicated. b Heat map of the common HSF5 binding sites of HSF5 CUT&Tag at the positions −3.0 kb upstream to +3.0 kb downstream relative to the TSS. Average distributions of HSF5- ChIP-seq binding peak are shown on the top. c Genomic view of the CUT&Tag peaks revealed by HSF5-N1, HSF5-N2, HSF5-C (rep1, rep2) antibodies and control IgG over representative gene loci. Genomic coordinates were obtained from RefSeq. RefSeq IDs for mRNA isoforms are indicated. To specify testis specific transcription, binding peaks of RNA polymerase II in the testis are shown48. d Gene expression patterns of HSF5 bound genes during spermatogenesis were shown as Heatmap. Heatmaps show the hierarchical relationship of the expression patterns of the HSF5-bound genes across the developmental direction. Expression pattern of the HSF5-bound genes was assessed by scRNA-seq data of spermatogenic cells as described in Fig. 6k. The cluster number is indicated on the top. The order of clusters from left to right corresponds to developmental direction of spermatogenesis (undifferentiated spermatogonia to pachytene spermatocyte). The expression patterns of the HSF5-bound genes were classified (Class1-Class3). On the right, top3 gene enrichment terms ranked by Enrichment score, log(q-value), are shown. e The average expression levels of the HSF5-target genes (Class 1, 2, and 3) are shown in violin plots with a median. The expression levels were compared between WT and Hsf5 KO mice using the scRNA-seq data for pachytene sub-populations that were pooled from clusters 9, 3, and 2. Note that scRNA-seq data of cluster 10 was excluded since the cluster 10 sub-population was already eliminated in Hsf5 KO mice. Statistical significance is shown by p-value (Class1; p = 1.145 × 10−10, Class 2; p < 2.2 × 10−16, Class 3; p < 2.2 × 10−16, two-sided Wilcoxon rank sum test). See also Supplementary Data 6 for a complete list of the HSF5-bound genes and the Gene enrichment analyzes.
