Chromatin Dynamics: Chromatin Remodeler, Epigenetic Modification and Diseases

The gene transcription patterns are regulated in response to extracellular stimuli and intracellular development programs. Recent studies have shown that chromatin dynamics which include nucleosome dynamics and histone modification play a crucial role in gene expression. Chromatin dynamic is regulated by chromatin modification enzymes including chromatin remodeling complex and histone posttranslational modifications. Multiple studies have shown that chromatin dynamics dysregulation and aberrant and histone modifications resulted in the occurrence of various diseases and cancers. Moreover, frequent mutations and chromosomal aberrations in the genes associated with subunits of the chromatin remodeling complexes have been detected in various cancer types. In this review, we highlight the current understanding of orchestration of nucleosome position, histone modification, and the importance of these properly regulated dynamics. We also discuss the consequences of aberrant chromatin dynamic which results in disease progression and provides insights for potential clinic applications

The highly expressed methionine synthase gene of Neurospora crassa is positively regulated by its proximal heterochromatic region

In Neurospora crassa, the methionine synthase gene met-8 plays a key role in methionine synthesis. In this study, we found that MET-8 protein levels were compromised in several mutants defective in proper heterochromatin formation. Bioinformatics analysis revealed a 50-kb AT-rich region adjacent to the met-8 promoter. ChIP assays confirmed that trimethylated H3K9 was enriched in this region, indicating that heterochromatin may form upstream of met-8. In an H3K9R mutant strain, the output of met-8 was dramatically reduced, similar to what we observed in mutant strains that had defective heterochromatin formation. Furthermore, the production of ectopically expressed met-8 at the his-3 locus in the absence of a normal heterochromatin environment was inefficient, whereas ectopic expression of met-8 downstream of two other heterochromatin domains was efficient. In addition, our data show that the expression of mig-6 was also controlled by an upstream 4.2-kb AT-rich region similar to that of the met-8 gene, and we demonstrate that the AT-rich regions adjacent to met-8 or mig-6 are required for their peak expression. Our study indicates that met-8 and mig-6 may represent a novel type of gene, whose expression relies on the proper formation of a nearby heterochromatin region

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

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

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

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

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

The highly expressed methionine synthase gene of Neurospora crassa is positively regulated by its proximal heterochromatic region

In Neurospora crassa, the methionine synthase gene met-8 plays a key role in methionine synthesis. In this study, we found that MET-8 protein levels were compromised in several mutants defective in proper heterochromatin formation. Bioinformatics analysis revealed a 50-kb AT-rich region adjacent to the met-8 promoter. ChIP assays confirmed that trimethylated H3K9 was enriched in this region, indicating that heterochromatin may form upstream of met-8. In an H3K9R mutant strain, the output of met-8 was dramatically reduced, similar to what we observed in mutant strains that had defective heterochromatin formation. Furthermore, the production of ectopically expressed met-8 at the his-3 locus in the absence of a normal heterochromatin environment was inefficient, whereas ectopic expression of met-8 downstream of two other heterochromatin domains was efficient. In addition, our data show that the expression of mig-6 was also controlled by an upstream 4.2-kb AT-rich region similar to that of the met-8 gene, and we demonstrate that the AT-rich regions adjacent to met-8 or mig-6 are required for their peak expression. Our study indicates that met-8 and mig-6 may represent a novel type of gene, whose expression relies on the proper formation of a nearby heterochromatin region