• 대한약침학회지
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• 제16권4호
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• pp.7-13
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• 2013

Objectives: Whether the ultra-highly-diluted remedies used in homeopathy can effectively bring about modulations of gene expressions through acetylation/deacetylation of histones has not been explored. Therefore, in this study, we pointedly checked if the homeopathically-diluted anti-cancer remedy Condurango 30C (ethanolic extract of Gonolobus condurango diluted $10^{-60}$ times) was capable of arresting the cell cycles in cervical cancer cells HeLa by triggering an epigenetic modification through modulation of the activity of the key enzyme histone deacetylase 2 vis-a-vis the succussed alcohol (placebo) control. Methods: We checked the activity of different signal proteins (like $p21^{WAF}$, p53, Akt, STAT3) related to deacetylation, cell growth and differentiation by western blotting and analyzed cell-cycle arrest, if any, by fluorescence activated cell sorting. After viability assays had been performed with Condurango 30C and with a placebo, the activities of histone de-acetylase (HDAC) enzymes 1 and 2 were measured colorimetrically. Results: While Condurango 30C induced cytotoxicity in HeLa cells in vitro and reduced HDAC2 activity quite strikingly, it apparently did not alter the HDAC1 enzyme; the placebo had no or negligible cytotoxicity against HeLa cells and could not alter either the HDAC 1 or 2 activity. Data on $p21^{WAF}$, p53, Akt, and STAT3 activities and a cell-cycle analysis revealed a reduction in DNA synthesis and G1-phase cell-cycle arrest when Condurango 30C was used at a 2% dose. Conclusion: Condurango 30C appeared to trigger key epigenetic events of gene modulation in effectively combating cancer cells, which the placebo was unable to do.

• Genomics & Informatics
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• 제18권4호
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• pp.42.1-42.9
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• 2020

Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-Seq) is a powerful technology to profile the location of proteins of interest on a whole-genome scale. To identify the enrichment location of proteins, many programs and algorithms have been proposed. However, none of the commonly used peak calling programs could accurately explain the binding features of target proteins detected by ChIP-Seq. Here, publicly available data on 12 histone modifications, including H3K4ac/me1/me2/me3, H3K9ac/me3, H3K27ac/me3, H3K36me3, H3K56ac, and H3K79me1/me2, generated from a human embryonic stem cell line (H1), were profiled with five peak callers (CisGenome, MACS1, MACS2, PeakSeq, and SISSRs). The performance of the peak calling programs was compared in terms of reproducibility between replicates, examination of enriched regions to variable sequencing depths, the specificity-to-noise signal, and sensitivity of peak prediction. There were no major differences among peak callers when analyzing point source histone modifications. The peak calling results from histone modifications with low fidelity, such as H3K4ac, H3K56ac, and H3K79me1/me2, showed low performance in all parameters, which indicates that their peak positions might not be located accurately. Our comparative results could provide a helpful guide to choose a suitable peak calling program for specific histone modifications.

• Genes and Genomics
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• 제40권12호
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• pp.1301-1308
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• 2018

Background Adipocyte differentiation is completed by changing gene expression. Chromatin is closely related to gene expression. Therefore, its structure might be changed for adipocyte differentiation. Mouse 3T3-L1 preadipocytes have been used as a cell model to study molecular mechanisms of adipogenesis. Objective To examine changes of chromatin modification and expression of histone modifying enzymes during adipocyte differentiation. Methods Microscopic analysis and Oil Red O staining were performed to determine distinct phenotype of adipocyte differentiation. RT-PCR and Western blot analysis were used to examine expression levels of histone modifying enzymes during adipocyte differentiation. Histone modifications were examined by immunostaining analysis. Results Expression levels of P300 and cbp were increased during adipocyte differentiation. However, acetylation of histones was not quantitatively changed postdifferentiation of 3T3-L1 cells compared to that at pre-differentiation. RT-PCR and Western blot analyses showed that expression levels of hdac2 and hdac3 were increased during adipocyte differentiation, suggesting histone acetylation at chromatin level was homeostatically controlled by increased expression of both HATs and HDACs. Tri-methylation level of H3K9 (H3K9me3), but not that of H3K27me3, was significantly decreased during adipocyte differentiation. Decreased expression of setdb1 was consistent with reduced pattern of H3K9me3. Knock-down of setdb1 induced adipocyte differentiation. This suggests that setdb1 is a key chromatin modifier that modulates repressive chromatin. Conclusion These results suggest that there exist extensive mechanisms of chromatin modifications for homeostatic balance of chromatin acetylation and deconstruction of repressive chromatin during adipocyte differentiation.

• BMB Reports
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• 제50권4호
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• pp.162-163
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• 2017

RNA polymerase II-interacting the Set2 methyltransferase co-transcriptionally methylates histone H3 at lysine 36 within the body of genes. This modification facilitates histone deacetylation by Rpd3S HDAC in 3' transcribed regions to suppress cryptic initiation and slow elongation. Although this pathway is important for global deacetylation, no strong effects have been seen on genome-wide transcription under optimized laboratory conditions. In contrast, this pathway slows the kinetics of mRNA induction when target genes are induced upon environmental changes. Interestingly, a majority of Set2-repressed genes are overlapped by a lncRNA transcription that targets H3K36 methylation and deacetylation by Rpd3S HDAC to mRNA promoters. Furthermore, this pathway delays the induction of many cryptic transcripts upon environmental changes. Therefore, the Set2-Rpd3S HDAC pathway functions to fine-tune expression dynamics of mRNAs and ncRNAs.

• BMB Reports
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• 제50권1호
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• pp.49-54
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• 2017

Previously, we reported that vitamin C facilitates the CpG demethylation of Foxp3 enhancer in $CD4^+Foxp3^+$ regulatory T cells (Tregs) by enhancing the activity of a DNA demethylase ten-eleven-translocation (Tet). However, it is not clear whether vitamin C affects other helper T cell lineages like T helper type 17 (Th17) cells which are related with Tregs. Here, we show that the expression of interleukin-17A (IL17) increases with the treatment of vitamin C but not with other antioxidants. Interestingly, the upregulation of IL17 was not accompanied by DNA demethylation in Il17 promoter and was independent of Tet enzymes. Rather, vitamin C reduced the trimethylation of histone H3 lysine 9 (H3K9me3) in the regulatory elements of the Il17 locus, and the effects of vitamin C were abrogated by knockdown of jumonji-C domain-containing protein 2 (jmjd2). These results suggest that vitamin C can affect the expression of IL17 by modulating the histone demethylase activity.

• 한국식품영양과학회지
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• 제46권2호
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• pp.169-176
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• 2017

본 연구에서는 쓴메밀 새싹 추출물(TBS)을 대상으로 histone acetyltransferase(HAT) 활성 저해능을 평가하고 oleic acid와 palmitic acid(OPA)를 이용하여 HepG2 세포에서 비알코올성 지방간을 유도하여 그 효과를 검토하였다. HeLa 세포의 nuclear extract(NE)를 HAT의 source로 하여 in vitro에서 TBS에 의한 HAT 활성 저해능을 평가한 결과 추출물의 처리에 의하여 HAT 활성이 억제됨을 관찰할 수 있었다. 또한, 대표적인 HAT 단백질인 p300과 CBP를 이용하여 동일한 방식으로 HAT 억제능을 평가한 결과 TBS 처리에 의하여 두 단백질 모두 활성이 감소하였으며, 특히 TBS는 p300의 활성을 특이적으로 저해함을 확인할 수 있었다. 그뿐만 아니라 HepG2 세포에 $400{\mu}M$의 oleic acid 및 $100{\mu}M$의 palmitic acid와 함께 $200{\mu}g/mL$, $500{\mu}g/mL$의 TBS를 처리한 후 NE를 이용하여 세포 내 HAT 활성을 측정한 결과 역시 추출물 처리에 의하여 세포 내 HAT 활성이 저해되어 있음이 관찰되었다. TBS에 의한 HAT 활성의 억제는 세포 내 다양한 단백질들의 아세틸화 저해와 지질축적에 의하여 아세틸화 변형을 일으키는 것으로 알려진 histone H3K9, H4K8의 아세틸화 및 H3K36의 아세틸화를 감소시켰으며, 세포 내 지질합성과 관련된 대표적 유전자인 SREBP1c, ACLY, FAS의 전사 활성 역시 저해함을 관찰하였다. 이와 같은 변화를 통하여 OPA에 의하여 HepG2 세포 내에 축적되었던 지질은 TBS의 처리에 의하여 효과적으로 감소하였으며 이때 처리된 OPA와 소재에 의한 세포 내 독성은 관찰되지 않았다. 그러므로 이러한 결과는 TBS에 의한 HAT 활성의 저해가 히스톤 단백질의 아세틸활 변형을 억제하고 이를 통하여 지방 합성 관련 유전자들의 전사 활성을 감소시켜 결과적으로 세포 내 지질축적을 방지하는 것으로 생각되며, TBS은 비알코올성 지방간질환의 예방에 좋은 천연물 소재로 활용될 수 있을 것이라 여겨진다.

• Biomolecules & Therapeutics
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• 제15권2호
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• pp.78-82
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• 2007

The proto-oncogene protein DEK is a nuclear binding phosphoprotein that has been associated with various human diseases including leukemia. Histone acetylation is an important post-translational modification which plays important role in transcriptional regulation. Auto-acetylation of histone acetyltransferase PCAF results in increment of its HAT activity and facilitation of its nuclear localization. In this study, we report that DEK inhibits PCAF auto-acetylation through direct interaction. The C-terminal acidic domains of DEK are responsible for the interaction with PCAF. Using confocal microscopy, we have shown that nuclear localization of PCAF is severely inhibited by DEK. Taken together, our results suggest that DEK may be involved in various cellular signal transduction pathways accommodated by PCAF through the regulation of PCAF auto-acetylation.

• 식물조직배양학회지
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• 제27권5호
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• pp.359-366
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• 2000

Epigenetics represents a chromatin-mediated transcriptional repression which plays a control role in both animal and plant development. A number of different mechanisms have been described to be involved in the formation of chromatin structure: especially DNA methylation, nucleosomal histone modification, DNA replication timing, and binding of chromatin remodelling proteins. Epigenetic phenomena include genomic imprinting, dosage compensation of X-chromosome linked genes, mutual allelic interactions, paramutation, transvection, silencing of invasive DNA sequences, etc. They are often unstable and inherited in a non-Mendelian way. A number of epigenetic defects has been preferentially described in floral development. Here, epigenetic phenomena in model angiosperm plants and their corresponding mechanisms are reviewed.

• 생명과학회지
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• 제25권6호
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• pp.703-708
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• 2015

Hox 유전자는 호메오도메인을 포함한 전사인자로써, 발생 과정 중 전후축을 따라 몸의 형태 형성을 조절하는 역할을 한다. 레티노산(RA)은 발생 과정에서 필수적인 형태형성인자이며 세포의 특성을 결정하는데 중요한 조절자이다. 특히, RA는 생쥐나 인간으로부터 만들어진 배아암종(EC)세포에서 Hox 유전자의 발현을 조절한다고 밝혀져 있다. 또한 RA에 의한 세포 분화와 유전자 조절 과정에 히스톤 변이가 중요한 역할을 하는 것으로 보고되어 있다. 히스톤 변이가 RA에 의해 유도되는 Hox 유전자의 발현에 특이적인 역할을 할 것으로 유추되기 때문에, 이 연구의 목적은 F9 생쥐배아 기형암종세포에서 RA에 의해 유도되는 Hoxc 유전자의 순차적인 발현이 히스톤 변이에 의해 일어나는 것인지를 조사하는 것이다. Hox 유전자의 발현 양상과 히스톤 변이는 semi-quantitative RT-PCR, RNA-sequencing과 chromatin immuno-precipitation (ChIP)-PCR 기법을 이용하여 관찰하였다. RA 처리 후(0일(D0), 1일(D1), 3일(D3)), Hoxc4 유전자의 발현(D1)은 Hoxc5부터 –c10 유전자(D3)보다 먼저 시작되었다. Hox가 발현하지 않는 D0 샘플은 전사 억제 마커인 H3K27me3이 모든 Hoxc 좌위에 강하게 표지 되어 있었으나 D1과 D3 샘플에서는 모든 좌위의 H3K27me3 표지가 확연히 줄어들어 있었다. 전사 발현 마커인 H3K4me3가 Hoxc 유전자의 순차적인 발현과 더 연관성이 있는 것으로 보이는데 D1에서 Hoxc4 발현과 함께 H3K4me3이 표지 되어 있었고, D3에서는 Hoxc 유전자 발현과 함께 모든 좌위에서 H3K4me3 마커가 존재했기 때문이다. 모든 결과를 종합해 보았을 때 F9 세포에서 RA에 의해 유도된 Hoxc 유전자의 순차적인 발현은 Hoxc 좌위에서 H3K27me3가 사라지고, H3K4me3가 표지 되는 히스톤 메틸화의 변이에 의해 결정되는 것으로 사료된다.

• 한국발생생물학회지:발생과생식
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• 제17권1호
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• pp.9-16
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• 2013

Coactivator-associated arginine methyltransferase 1 (CARM1) is included in the protein arginine methyltransferase (PRMT) family, which methylates histone arginine residues through posttranslational modification. It has been proposed that CARM1 may up-regulate the expression of pluripotency-related genes through the alteration of the chromatin structure. Mouse embryonic stem cells (mESCs) are pluripotent and have the ability to self-renew. The cells are mainly used to study the genetic function of novel genes, because the cells facilitate the transmission of the manipulated genes into target mice. Since the up-regulated methylation levels of histone arginine residue lead to the maintenance of pluripotency in embryos and stem cells, it may be suggested that CARM1 overexpressing mESCs elevate the expression of pluripotency-related genes in reconstituted embryos for transgenic mice and may resist the differentiation into trophectoderm (TE). We constructed a fusion protein by connecting CARM1 and 7X-arginine (R7). As a cell-penetrating peptide (CPP), can translocate CARM1 protein into mESCs. CPP-CARM1 protein was detected in the nuclei of the mESCs after a treatment of 24 hours. Accordingly, the expression of pluripotency-related genes was up-regulated in CPP-CARM1-treated mESCs. In addition, CPP-CARM1-treated mESC-derived embryoid bodies (EBs) showed an elevated expression of pluripotency-related genes and delayed spontaneous differentiation. This result suggests that the treatment of recombinant CPP-CARM1 protein elevates the expression of pluripotency-related genes of mESCs by epigenetic modification, and this protein-delivery system could be used to modify embryonic fate in reconstituted embryos with mESCs.