• Molecules and Cells
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• v.44 no.2
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• pp.79-87
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• 2021

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Schizosaccharomyces pombe Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1-DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.6
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• pp.791-797
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• 2018

Objective: Trimethylation of histone 3 (H3) at 4th lysine N-termini (H3K4me3) in gene promoter region was the universal marker of active genes specific to cell lineage. On the contrary, coexistence of trimethylation at 27th lysine (H3K27me3) in the same loci-the bivalent H3K4m3/H3K27me3 was known to suspend the gene transcription in germ cells, and could also be inherited to the developed stem cell. In galline species, throughout example of H3K4m3 and H3K27me3 ChIP-seq analysis was still not provided. We therefore designed and demonstrated such procedures using ChIP-seq and mRNA-seq data of chicken follicular mesenchymal cells and male germ cells. Methods: Analytical workflow was designed and provided in this study. ChIP-seq and RNA-seq datasets of follicular mesenchymal cells and male germ cells were acquired and properly preprocessed. Peak calling by Model-based analysis of ChIP-seq 2 was performed to identify H3K4m3 or H3K27me3 enriched regions ($Fold-change{\geq}2$, $FDR{\leq}0.01$) in gene promoter regions. Integrative genomics viewer was utilized for cellular retinoic acid binding protein 1 (CRABP1), growth differentiation factor 10 (GDF10), and gremlin 1 (GREM1) gene explorations. Results: The acquired results indicated that follicular mesenchymal cells and germ cells shared several unique gene promoter regions enriched with H3K4me3 (5,704 peaks) and also unique regions of bivalent H3K4m3/H3K27me3 shared between all cell types and germ cells (1,909 peaks). Subsequent observation of follicular mesenchyme-specific genes-CRABP1, GDF10, and GREM1 correctly revealed vigorous transcriptions of these genes in follicular mesenchymal cells. As expected, bivalent H3K4m3/H3K27me3 pattern was manifested in gene promoter regions of germ cells, and thus suspended their transcriptions. Conclusion: According the results, an example of chicken H3K4m3/H3K27me3 ChIP-seq data analysis was successfully demonstrated in this study. Hopefully, the provided methodology should hereby be useful for galline ChIP-seq data analysis in the future.

• BMB Reports
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• v.42 no.3
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• pp.154-159
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• 2009

JARID1B (jumonji AT rich interactive domain 1B) is a large nuclear protein that is highly expressed in breast cancers and is proposed to function as a repressor of gene expression. In this paper, a phage display screen using the N-terminus of JARID1B as bait identified one of the JARID1B interacting proteins, namely PcG protein (Polycomb group) hPc2. We demonstrated that the C-terminal region, including the COOH box, was required for the interaction with the N-terminus of JARID1B. In a reporter assay system, co-expression of JARID1B with hPc2 significantly enhanced the transcriptional repression. These results support a role for hPc2 acting as a transcriptional co-repressor.

• Molecules and Cells
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• v.45 no.9
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• pp.622-630
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• 2022

Colorectal cancer (CRC) has a high mortality rate among cancers worldwide. To reduce this mortality rate, chemotherapy (5-fluorouracil, oxaliplatin, and irinotecan) or targeted therapy (bevacizumab, cetuximab, and panitumumab) has been used to treat CRC. However, due to various side effects and poor responses to CRC treatment, novel therapeutic targets for drug development are needed. In this study, we identified the overexpression of EHMT1 in CRC using RNA sequencing (RNA-seq) data derived from TCGA, and we observed that knocking down EHMT1 expression suppressed cell growth by inducing cell apoptosis in CRC cell lines. In Gene Ontology (GO) term analysis using RNA-seq data, apoptosis-related terms were enriched after EHMT1 knockdown. Moreover, we identified the CHOP gene as a direct target of EHMT1 using a ChIP (chromatin immunoprecipitation) assay with an anti-histone 3 lysine 9 dimethylation (H3K9me2) antibody. Finally, after cotransfection with siEHMT1 and siCHOP, we again confirmed that CHOP-mediated cell apoptosis was induced by EHMT1 knockdown. Our findings reveal that EHMT1 plays a key role in regulating CRC cell apoptosis, suggesting that EHMT1 may be a therapeutic target for the development of cancer inhibitors.

• BMB Reports
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• v.53 no.12
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• pp.634-639
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• 2020

In prostate cancer, the androgen receptor (AR) transcription factor is a major regulator of cell proliferation and metastasis. To identify new AR regulators, we focused on Mixed lineage leukemia 5 (MLL5), a histone-regulating enzyme, because significantly higher MLL5 expression was detected in prostate cancer tissues than in matching normal tissues. When we expressed shRNAs targeting MLL5 gene in prostate cancer cell line, the growth rate and AR activity were reduced compared to those in control cells, and migration ability of the knockdown cells was reduced significantly. To determine the molecular mechanisms of MLL5 on AR activity, we proved that AR physically interacted with MLL5 and other co-factors, including SET-1 and HCF-1, using an immunoprecipitation method. The chromatin immunoprecipitation analysis showed reduced binding of MLL5, co-factors, and AR enzymes to AR target gene promoters in MLL5 shRNA-expressing cells. Histone H3K4 methylation on the AR target gene promoters was reduced, and H3K9 methylation at the same site was increased in MLL5 knockdown cells. Finally, xenograft tumor formation revealed that reduction of MLL5 in prostate cancer cells retarded tumor growth. Our results thus demonstrate the important role of MLL5 as a new epigenetic regulator of AR in prostate cancer.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.12
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• pp.1680-1688
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• 2013

Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly ($88.9{\rightarrow}114.4$). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT.

• BMB Reports
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• v.44 no.8
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• pp.535-540
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• 2011

Reprogramming errors, which appear frequently in cloned animals, are reflected by aberrant gene expression. We previously reported the aberrant expression of TIMP-2 and PBEF in cloned placenta and differential expression of PBEF genes during pregnancy. To examine the epigenetic modifications that regulate dynamic gene expression in developing placentae, we herein analyzed the mRNA and protein expression levels of PBEF and TIMP-2 in the placentae of normal mice during pregnancy and then examined potential correlations with epigenetic modifications. DNA methylation pattern analysis revealed no difference, but ChIP assays using antibodies against H3-K9/K14 and H4-K5 histone acetylation revealed that the H3-K9/K14 acetylation levels, but not the H4-K5 acetylation levels, of the TIMP-2 and PBEF loci were significantly correlated with their gene expression levels during placentation in normal mice. These results suggest that epigenetic changes may regulate gene expression level in the developing placentae of normal mice and that inappropriate epigenetic reprogramming might be one cause of the abnormal placentae seen in cloned animals.

• Journal of Life Science
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• v.22 no.8
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• pp.999-1008
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• 2012

The circadian clock is a self-sustaining 24-hour timekeeper that allows organisms to anticipate daily-changing environmental time cues. Circadian clock genes are regulated by a transcriptional-translational feedback loop. In Arabidopsis, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) transcripts are highly expressed in the morning. Translated LHY and CCA1 proteins repress the expression of the TIMING OF CAB EXPRESSION 1 (TOC1) transcripts, which peaks in the evening. The TOC1 protein elevates the expression of the LHY and CCA1 transcripts, forming a negative feedback loop that is believed to constitute the oscillatory mechanism of the clock. In mammals, the transcription factor protein CLOCK, which is a central component of the circadian clock, was reported to have an intrinsic histone acetyltransferase (HAT) activity, suggesting that histone acetylation is important for core clock mechanisms. However, little is known about the components necessary for the histone acetylation of the Arabidopsis clock-related genes. Here, I report that DET1 (De-Etiolated1) functions as a negative regulator of a key component of the Arabidopsis circadian clock gene LHY in constant dark phases (DD) and is required for the down-regulation of LHY expression through the acetylation of histone 3 (H3Ac). However, the HATs directly responsible for the acetylation of H3 within LHY chromatin need to be identified, and a link connecting the HATs and DET1 protein is still absent.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.6
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• pp.1023-1033
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• 2020

Objective: The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 recombinase (RAD51) expression were associated with increased knock-in efficiency on the β-casein (bCSN2) gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system. Methods: MAC-T cells were treated with HDAC inhibitors, valproic acid, trichostatin A, or sodium butyrate for 24 h, then transfected with a knock-in vector, RAD51 expression vector and TALEN to target the bCSN2 gene. After 3 days of transfection, the knock-in efficiency was confirmed by polymerase chain reaction and DNA sequencing of the target site. Results: The level of HDAC 2 protein in MAC-T cells was decreased by treatment with HDAC inhibitors. The knock-in efficiency in MAC-T cells treated with HDAC inhibitors was higher than in cells not treated with inhibitors. However, the length of the homologous arm of the knock-in vector made no difference in the knock-in efficiency. Furthermore, DNA sequencing confirmed that the precision of the knock-in was more efficient in MAC-T cells treated with sodium butyrate. Conclusion: These results indicate that chromatin modification by HDAC inhibition and RAD51 expression enhanced the homologous recombination efficiency on the bCSN2 gene locus in MAC-T cells.

• Environmental Mutagens and Carcinogens
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• v.24 no.4
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• pp.171-179
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• 2004

Cytochrome P4503A4(CYP3A4) is the most abundnat CYPs in human liver, comparising approximately 30% of the total liver CYPs contents ans is involbed in the metabolism of more than 60% of currently used therapeutic drugs. The expression of CYP3A4 is induced by a variety of structurally unrelated xonobiotics including the antibiotic rifampicin and endogenous hormones, and might be mediated through steroid and xenobiotic receptor(SXR) system. The molecular mechanisms underlying regulation of CYP3A4 gene expression hae not been understood. In order to gain the insight of the molecular mechanism of CYP3A4 gene expression, study has been undertaken to investigate if the histone deacelylation is involved in the regulation of CYP3A4 gene expression by proximal promoter or not. Also SXR was investigated to see if they were involved in the regulation of CYP3A4 proximal promoter activity. HepG2 or Hena-I cells were transfected with a plasmid containing~1kb of the CYP3A4 proximal promoter region (-863 to +64bp) cloned in front of a reporter gene, luciferase, in the presence or absence of SXR or hER. Transfected cells were treated with CYP3A4 inducers such as rifampicin, PCN and RU 486, or with estradiol, in order to exmine to regulation of CYP3A4 gene expression in the presence or absence of trichostatin A (TSA). In HepG2 cells, CYP3A4 inducers and estradiol increased significantly the luciferase activity by CYP3A4 proximal promoter, only when TSA was co-treated after SXR cotransfection. In the case of Hepa-I cells CYP3A4 inducers and estradiol incressed modestly the luciferase activity when TSA was co-treated, but this increment was not enhanced by SXR cotransfection in contrast to HepG2 cells. Taken together, these results indicated that the inhibition of histone deacetylation was required to SXR-mediated increase in CYP3A4 proximal promoter region when rifampicin, or PCN was treated. Futher a trans-activation by SXR may demand other species-specific transcription factors.