IEC-1 suppresses <i>frq</i> transcription and rhythmically binds to the <i>frq</i> promoter.

<p>(A) Western blot analysis showing the circadian oscillation of FRQ proteins in the wild-type and <i>iec-1</i>^<i>KO</i> strains. The strains were grown in 2% glucose liquid media. The asterisk indicates a nonspecific cross-reacted protein band recognized by our FRQ antiserum. The Coomassie Brilliant Blue-stained membranes (mem) represent the total protein in each sample and were used as a loading control. (B) Northern blot analysis of <i>frq</i> transcription in the wild-type and <i>iec-1</i>^<i>KO</i> strains. rRNA was used as a loading control. The strains were grown in 2% glucose liquid media. (C) Immunodetection of IEC-1 protein in the wild-type strain and the <i>iec-1</i>^<i>KO</i> mutant using antiserum that specifically recognizes the IEC-1 protein in the wild-type strain. The arrow notes the specific IEC-1 protein band detected by our IEC-1 antibody. The strains were grown in 2% glucose liquid media. (D) ChIP analysis showing the recruitment of IEC-1 at different regions of the <i>frq</i> locus in the wild-type and <i>iec-1</i>^<i>KO</i> strains at DD18. The strains were grown in 2% glucose liquid media. C-box, clock box; PLRE, proximal light-regulated element; TSS, transcription start site; ORF, open reading frame; UTR, untranslated region. (E) ChIP analysis showing the enrichment of IEC-1 at the C-box of the <i>frq</i> promoter in the wild-type and <i>iec-1</i>^<i>KO</i> strains at the indicated time points. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3).</p

INO80 is rhythmically recruited at the C-box by IEC-1 and WCC-driven <i>frq</i> expression.

<p>(A) Immunodetection of INO80 in the wild-type strain and the <i>ino80</i>^<i>KO</i> mutant using antiserum that specifically recognizes INO80 protein in the wild-type strain. The strains were grown in 2% glucose liquid media. The arrow indicates the INO80 specific band in the wild-type strains. (B) ChIP analysis showing the recruitment of INO80 at different regions of the <i>frq</i> locus in the wild-type and <i>ino80</i>^<i>KO</i> strains at DD18. The <i>ino80</i>^<i>KO</i> strain was used as a negative control in the ChIP assay. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3). (C) ChIP analysis showing the recruitment of INO80 at the C-box in the wild-type, <i>ino80</i>^<i>KO</i> and <i>iec-1</i>^<i>KO</i> strains at the indicated time points. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3). (D) ChIP analysis showing the enrichment of histone H3 at the C-box, PLRE or TSS of the <i>frq</i> promoter region in the wild-type, <i>wc-2</i>^<i>KO</i> (<i>bd</i>) and <i>frq</i>^<i>9</i> (<i>bd</i>) mutant strains. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3). (E) ChIP analysis showing the recruitment of INO80 at the C-box of the <i>frq</i> promoter in the wild-type, <i>ino80</i>^<i>KO</i>, <i>wc-2</i>^<i>KO</i>(<i>bd</i>), <i>frq</i>^<i>9</i>(<i>bd</i>), and <i>wc-2</i>^<i>KO</i> <i>frq</i>^<i>9</i> (<i>bd</i>) strains at the indicated time points. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3). (F) Western blot analysis showing the INO80 protein levels in the wild-type, <i>ino80</i>^<i>KO</i>, <i>wc-2</i>^<i>K</i>O (<i>bd</i>), <i>frq</i>^<i>9</i> (<i>bd</i>) and <i>wc-2</i>^<i>KO</i> <i>frq</i>^<i>9</i> (<i>bd</i>) strains. The strains were grown in 2% glucose liquid media.</p

IEC-1 is required for normal circadian clock function.

<p>(A) Race tube assays of the wild-type and <i>iec-1</i>^<i>KO</i> strains. (B) Amino acid sequence alignment of the zf-C2H2 domains of IEC-1 from <i>Neurospora crassa</i>, <i>Penicillium brasilianum</i>, <i>Aspergillus fumigates</i> and <i>Nectria haematococca</i>. (C) Race tube assays of the wild-type strain, <i>iec-1</i>^<i>KO</i> strain, and <i>iec-1</i>^<i>KO</i>, qa-Myc-IEC-1 transformants in a race tube with or without QA. Growth media on the race tubes did not consist of glucose. (D) Luciferase reporter assay showing the <i>frq</i> promoter activity in the <i>wt</i>, <i>frq-luc</i> and <i>iec-1</i>^<i>KO</i>, <i>frq-luc</i> strains grown in DD for several days. Raw data were normalized to subtract the baseline calculated by the LumiCycle analysis software.</p

The INO80 complex is required for the suppression of WC-independent <i>frq</i> transcription.

<p>(A) ChIP analysis showing WC-2 enrichment at the C-box in the wild-type, <i>ino80</i>^<i>KO</i>, <i>iec-1</i>^<i>KO</i>, and <i>wc-2</i>^<i>KO</i> (<i>bd</i>) strains. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3). (B) Western blot analysis showing the phosphorylation of WC-1 in the wild-type, <i>ies-1</i>^<i>KO</i>, <i>ino80</i>^<i>KO</i> and <i>iec-1</i>^<i>KO</i> strains. The numbers indicate the ratio of acrylamide/bisacrylamide used in the SDS-PAGE gel. The strains were grown in 2% glucose liquid media. (C) Western blot analysis showing the levels of WC-1 in the wild-type, <i>ies-1</i>^<i>KO</i>, <i>ino80</i>^<i>KO</i> and <i>ies-1</i>^<i>KO</i> strains. The strains were grown in 2% glucose liquid media. (D) Western blot analysis showing the phosphorylation of WC-2 in the wild-type, <i>ies-1</i>^<i>KO</i>, <i>ino80</i>^<i>KO</i> and <i>ies-1</i>^<i>KO</i> strains. The strains were grown in 2% glucose liquid media. (E) Western blot analysis showing the levels of WC-2 in the wild-type, <i>ies-1</i>^<i>KO</i>, <i>ino80</i>^<i>KO</i> and <i>ies-1</i>^<i>KO</i> strains. The strains were grown in 2% glucose liquid media. (F) Western blot analysis of FRQ or WC-1 in the wild-type, <i>ies-1</i>^<i>KO</i>, <i>wc-1</i>^<i>RIP</i> (<i>bd</i>), and <i>ies-1</i>^<i>KO</i> <i>wc-1</i>^<i>RIP</i> strains. The strains were grown in 2% glucose liquid media. (G) Northern blot analysis showing the levels of <i>frq</i> mRNA in the wild-type, <i>ies-1</i>^<i>KO</i>, <i>wc-1</i>^<i>RIP</i> (<i>bd</i>), and <i>ies-1</i>^<i>KO</i> <i>wc-1</i>^<i>RIP</i> strains. The strains were grown in 2% glucose liquid media.</p

The establishment of nucleosomal barriers at the <i>frq</i> promoter by the INO80 complex prevents RNA pol II initiation.

<p>(A) ChIP analysis showing H3 density at the C-box, TSS or ORF middle regions of the <i>frq</i> locus in the wild-type, <i>ies-1</i>^<i>KO</i>, and <i>ino80</i>^<i>KO</i> strains. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3). (B) ChIP analysis showing the recruitment of SET-2 and enrichment of H3K36me3 at the ORF 3’ of <i>frq</i> in the wild-type and <i>ino80</i>^<i>KO</i> strains. The strains were grown in 2% glucose liquid media. Significance was assessed by two-tailed t-test. *P<0.05, **P<0.01. Error bars show the mean ±S.D. (n = 3).</p