Fig. 4: ∆brd-8 has reduced acetyl-histone H4 and Pol II levels at frq.
From: A crucial role for dynamic expression of components encoding the negative arm of the circadian clock
a The Integrative Genomics Viewer (IGV) visualization of ChIP-sequencing data. Antibodies against histone H4 or acetyl histone H4 (AcH4) were applied in ChIP experiments using cultures collected at DD20 for WT and DD26 for ∆brd-8 to compensate the 3-h period lengthening and 3-h phase delay seen in the mutant. Only the ChIP-sequencing data are shown. Red bars represent coding regions of the genes: ORFs are in bold while UTRs or introns are in thin bars and arrows designate the direction of transcription. Bottom, diagram of the frq gene showing regions amplified by primer sets used in ChIP-qPCRs (Fig. 4b-J) as derived from a previous publication94 with removal of unrelated information. To validate the data in Fig. 4a, independent Neurospora samples were collected in the same way as in Fig. 4a (WT at DD20 while ∆brd-8 at DD26), and then ChIP-qPCRs were carried out using histone H4 (b) acetyl-histone H4 (c), or Pol II (d) antibody and frq-specific primer sets in (a) with WT and ∆brd-8. In another set of biologically independent experiments (e-h) to rescue the decreased level of WC-1 in ∆brd-8, the promoter of wc-1 in ∆brd-8 was replaced by an inducible qa-2 promoter. Two strains, WT and qa-2 promoter-driven wc-1 in ∆brd-8, were cultured in the presence of 10-2 M QA throughout and afterwards both harvested at DD16. e ChIP-qPCRs were carried out using WC-2 antibody with primers “frq1”, “frq2”, “C-box”, and “pLRE” in WT and qa-2:wc-1; ∆brd-8 at DD16. ChIP-qPCRs were performed with histone H4 (f), acetyl-histone H4 (g), or Pol II (h) antibody and frq-specific primer sets in (a) with WT and qa-2:wc-1; ∆brd-8 at DD16. ChIP-qPCRs were done using V5 antibody with primers “C-box” and “pLRE” (i) and “frq1-8” (j) in WT, brd-8V5, and brd-8V5; ∆wc-1 at DD16. For ChIP-qPCR assays, three technical replicates were performed, and the bars represent average values plotted as a percentage of the input, with error bars representing the SEM (n = 3). Source data were supplied in the Source Data file.
