Fig 4. Bdf1 is required for the transcription of NDT80 and middle sporulation genes.

(A) List of over-represented transcription factors with binding sites in the promoters of the genes which are differentially downregulated at 4h and 8h when Bdf1 bromodomains are mutated (fold change < -2, adjusted p value < 0.05). A transcription factor was considered significantly over-represented when the p-value describing its enrichment was below 0.001. On the right, the normalized counts present the expression levels for NDT80, CUP9, CUP2, FKH1 and ROX1. (B) Pie chart representing the proportion of genes which are downregulated in the bdf1-Y187F-Y354F strain at 4 and 8h of sporulation induction (833 genes in total) and with putative Ndt80 binding sites in their promoter (531 genes, their list is available in S5 Table). (C) ROC-AUC plot representing the enrichment of the Ndt80 binding sequence in the promoter of the genes downregulated in the bdf1-Y187F-Y354F mutant strain. (D) Venn diagram illustrating the overlap between genes differentially downregulated in bdf1 bromodomain mutant at 4 and 8h of sporulation induction (833 genes) and genes previously identified as Ndt80-regulated in Chu et al. [35]. (E) Sporulation efficiency upon ectopic expression of NDT80 in WT and bdf1-Y187F-Y354F strains during sporulation. Here, the endogenous NDT80 promoter was replaced by an oestrogen-dependent promoter and its expression activated by the addition of oestradiol 6 hours after sporulation induction [44,45]. (F) Bdf1 chromatin immunoprecipitation. Bdf1 occupancy was monitored in the meiotic-specific promoters of the early genes IME1 and IME2 and middle genes AMA1 and NDT80 during sporulation. Mutation of Bdf1 bromodomains impairs the recruitment of Bdf1 to chromatin. IntV is an intergenic and transcriptionally inactive region of chromosome V between coordinates 9,762 and 9,812. (G) Sporulation efficiency when Bdf1 bromodomain mutations are combined with Sum1 and Hst1 deletions, two known repressors of NDT80 activation [41].