GSK-J4-Mediated Transcriptomic Alterations in Differentiating Embryoid Bodies

Mandal, Chanchal,Kim, Sun Hwa,Kang, Sung Chul,Chai, Jin Choul,Lee, Young Seek,Jung, Kyoung Hwa,Chai, Young Gyu Korean Society for Molecular and Cellular Biology 2017 Molecules and cells Vol.40 No.10

Histone-modifying enzymes are key players in the field of cellular differentiation. Here, we used GSK-J4 to profile important target genes that are responsible for neural differentiation. Embryoid bodies were treated with retinoic acid ($10{\mu}M$) to induce neural differentiation in the presence or absence of GSK-J4. To profile GSKJ4-target genes, we performed RNA sequencing for both normal and demethylase-inhibited cells. A total of 47 and 58 genes were up- and down-regulated, respectively, after GSK-J4 exposure at a log2-fold-change cut-off value of 1.2 (p-value < 0.05). Functional annotations of all of the differentially expressed genes revealed that a significant number of genes were associated with the suppression of cellular proliferation, cell cycle progression and induction of cell death. We also identified an enrichment of potent motifs in selected genes that were differentially expressed. Additionally, we listed upstream transcriptional regulators of all of the differentially expressed genes. Our data indicate that GSK-J4 affects cellular biology by inhibiting cellular proliferation through cell cycle suppression and induction of cell death. These findings will expand the current understanding of the biology of histone-modifying enzymes, thereby promoting further investigations to elucidate the underlying mechanisms.

GSK-J4-Mediated Transcriptomic Alterations in Differentiating Embryoid Bodies

Chanchal Mandal,김선화,강성철,채진철,이영식,정경화,채영규 한국분자세포생물학회 2017 Molecules and cells Vol.40 No.10

Histone-modifying enzymes are key players in the field of cellular differentiation. Here, we used GSK-J4 to profile important target genes that are responsible for neural differentiation. Embryoid bodies were treated with retinoic acid (10 M) to induce neural differentiation in the presence or absence of GSK-J4. To profile GSKJ4-target genes, we performed RNA sequencing for both normal and demethylase-inhibited cells. A total of 47 and 58 genes were up- and down-regulated, respectively, after GSK-J4 exposure at a log2-fold-change cut-off value of 1.2 (p-value < 0.05). Functional annotations of all of the differentially expressed genes revealed that a significant number of genes were associated with the suppression of cellular proliferation, cell cycle progression and induction of cell death. We also identified an enrichment of potent motifs in selected genes that were differentially expressed. Additionally, we listed upstream tran-scriptional regulators of all of the differentially expressed genes. Our data indicate that GSK-J4 affects cellular biology by inhibiting cellular proliferation through cell cycle suppression and induction of cell death. These findings will expand the current understanding of the biology of histone-modifying enzymes, thereby promoting further investigations to elucidate the underlying mechanisms.

MoJMJ1, Encoding a Histone Demethylase Containing JmjC Domain, Is Required for Pathogenic Development of the Rice Blast Fungus, Magnaporthe oryzae

Huh, Aram,Dubey, Akanksha,Kim, Seongbeom,Jeon, Junhyun,Lee, Yong-Hwan The Korean Society of Plant Pathology 2017 Plant Pathology Journal Vol.33 No.2

Histone methylation plays important roles in regulating chromatin dynamics and transcription in eukaryotes. Implication of histone modifications in fungal pathogenesis is, however, beginning to emerge. Here, we report identification and functional analysis of a putative JmjC-domain-containing histone demethylase in Magnaporthe oryzae. Through bioinformatics analysis, we identified seven genes, which encode putative histone demethylases containing JmjC domain. Deletion of one gene, MoJMJ1, belonging to JARID group, resulted in defects in vegetative growth, asexual reproduction, appressorium formation as well as invasive growth in the fungus. Western blot analysis showed that global H3K4me3 level increased in the deletion mutant, compared to wild-type strain, indicating histone demethylase activity of MoJMJ1. Introduction of MoJMJ1 gene into ${\Delta}Mojmj1$ restored defects in pre-penetration developments including appressorium formation, indicating the importance of histone demethylation through MoJMJ1 during infection-specific morphogenesis. However, defects in penetration and invasive growth were not complemented. We discuss such incomplete complementation in detail here. Our work on MoJMJ1 provides insights into H3K4me3-mediated regulation of infection-specific development in the plant pathogenic fungus.

The Histone Demethylase PHF2 Promotes Fat Cell Differentiation as an Epigenetic Activator of Both C/EBPα and C/EBPδ

이경화,주욱일,송정엽,전양숙 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.10

Histone modifications on major transcription factor target genes are one of the major regulatory mechanisms controlling adipogenesis. Plant homeodomain finger 2 (PHF2) is a Jumonji domain-containing protein and is known to demethylate the histone H3K9, a repressive gene marker. To better understand the function of PHF2 in adipocyte differentiation, we constructed stable PHF2 knock-down cells by using the mouse pre-adipocyte cell line 3T3-L1. When induced with adipogenic media, PHF2 knock-down cells showed reduced lipid accumulation compared to control cells. Differential expression using a cDNA microarray revealed significant reduction of metabolic pathway genes in the PHF2 knock-down cell line after differentiation. The reduced expression of major transcription factors and adipokines was confirmed with reverse transcription- quantitative polymerase chain reaction and Western blotting. We further performed co-immunoprecipitation analysis of PHF2 with four major adipogenic transcription factors, and we found that CCATT/enhancer binding protein (C/EBP) and C/EBP physically interact with PHF2. In addition, PHF2 binding to target gene promoters was confirmed with a chromatin immunoprecipitation experiment. Finally, histone H3K9 methylation markers on the PHF2-binding sequences were increased in PHF2 knock-down cells after differentiation. Together, these results demonstrate that PHF2 histone demethylase controls adipogenic gene expression during differentiation.

Sumoylation of the histone demethylase KDM4A is required for binding to tumor suppressor p53 in HCT116 colon cancer cell lines

유승은,박수형,장연규 한국통합생물학회 2018 Animal cells and systems Vol.22 No.1

The histone demethylase lysine-specific demethylase 4A (KDM4A/Jmjd2A) has diverse functions, including involvement in gene regulation and cell cycle, and plays an oncogenic role in cancer cells. The modulation of KDM4A through post-translational modifications remains unclear. Here, we show that small ubiquitin-like modifier (SUMO) 1-mediated modification of KDM4A was required for interaction with tumor suppressor p53. Our data revealed that KDM4A is mainly sumoylated at lysine residue 471. However, the SUMO modification resulted in little change in subcellular localization, demethylase activity, or protein stability of KMD4A. Intriguingly, coimmunoprecipitation data revealed that sumoylation-defective mutants of KDM4A had a lower binding ability with p53 compared to that of wild-type KDM4A, suggesting a positive role for sumoylation in the interaction between KDM4A and p53. Together, these data suggest that KDM4A is post-translationally modified by SUMO, and this sumoylation may be a novel regulatory switch for controlling the interplay between KDM4A and p53.

Sumoylation of the histone demethylase KDM4A is required for binding to tumor suppressor p53 in HCT116 colon cancer cell lines

Yu, Seung Eun,Park, Su Hyung,Jang, Yeun Kyu The Korean Society for Integrative Biology 2018 Animal cells and systems Vol.22 No.1

The histone demethylase lysine-specific demethylase 4A (KDM4A/Jmjd2A) has diverse functions, including involvement in gene regulation and cell cycle, and plays an oncogenic role in cancer cells. The modulation of KDM4A through post-translational modifications remains unclear. Here, we show that small ubiquitin-like modifier (SUMO) 1-mediated modification of KDM4A was required for interaction with tumor suppressor p53. Our data revealed that KDM4A is mainly sumoylated at lysine residue 471. However, the SUMO modification resulted in little change in subcellular localization, demethylase activity, or protein stability of KMD4A. Intriguingly, co-immunoprecipitation data revealed that sumoylation-defective mutants of KDM4A had a lower binding ability with p53 compared to that of wild-type KDM4A, suggesting a positive role for sumoylation in the interaction between KDM4A and p53. Together, these data suggest that KDM4A is post-translationally modified by SUMO, and this sumoylation may be a novel regulatory switch for controlling the interplay between KDM4A and p53.

The Histone Demethylase PHF2 Promotes Fat Cell Differentiation as an Epigenetic Activator of Both C/EBPα and C/EBPδ

Lee, Kyoung-Hwa,Ju, Uk-Il,Song, Jung-Yup,Chun, Yang-Sook Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.10

Histone modifications on major transcription factor target genes are one of the major regulatory mechanisms controlling adipogenesis. Plant homeodomain finger 2 (PHF2) is a Jumonji domain-containing protein and is known to demethylate the histone H3K9, a repressive gene marker. To better understand the function of PHF2 in adipocyte differentiation, we constructed stable PHF2 knock-down cells by using the mouse pre-adipocyte cell line 3T3-L1. When induced with adipogenic media, PHF2 knock-down cells showed reduced lipid accumulation compared to control cells. Differential expression using a cDNA microarray revealed significant reduction of metabolic pathway genes in the PHF2 knock-down cell line after differentiation. The reduced expression of major transcription factors and adipokines was confirmed with reverse transcription- quantitative polymerase chain reaction and Western blotting. We further performed co-immunoprecipitation analysis of PHF2 with four major adipogenic transcription factors, and we found that CCATT/enhancer binding protein (C/EBP)${\alpha}$ and C/EBP${\delta}$ physically interact with PHF2. In addition, PHF2 binding to target gene promoters was confirmed with a chromatin immunoprecipitation experiment. Finally, histone H3K9 methylation markers on the PHF2-binding sequences were increased in PHF2 knock-down cells after differentiation. Together, these results demonstrate that PHF2 histone demethylase controls adipogenic gene expression during differentiation.

Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial <i>N</i>-Demethylase Complex

Kim, Jun Hoe,Kim, Bong Heon,Brooks, Shelby,Kang, Seung Yeon,Summers, Ryan M.,Song, Hyun Kyu Elsevier 2019 Journal of molecular biology Vol.431 No.19

<P><B>Abstract</B></P> <P>Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as <I>Pseudomonas putida</I> CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential <I>N</I>-demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial <I>N</I>-demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial <I>N</I> <SUB>1</SUB>- and <I>N</I> <SUB>3</SUB>-specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper <I>N</I>-demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during <I>N</I>-demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Crystal structures of bacterial N-demethylase A and B were determined in apo and substrate-bound states. </LI> <LI> NdmAB forms a unique heterocomplex with 3:3 stoichiometry. </LI> <LI> Structure of the binary complex between NdmA and a ferredoxin domain of NdmD was determined. </LI> <LI> The structural and biochemical results provide a framework for engineering of caffeine degradation enzymes and thus increase the reaction products, methylxanthines, which are high-value biochemicals. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Expression of a Viral Histone H4 Suppresses Expressions of lysine-specific Demethylase and Serine Proteinase Inhibitor to Inhibit Host Growth and Development

Sunil Kumar,Yonggyun Kim 한국응용곤충학회 2014 한국응용곤충학회 학술대회논문집 Vol.2014 No.10

Cotesia plutellae, an endoparasitoids braconid wasp, possesses a polydnaviruses (PDVs) called Cotesia plutellae bracovirus (CpBV) that encodes viral histone H4 (= CpBV-H4). This viral histone H4 shares high sequence homology (82.5%) with host`s H4 of P.xylostella, except an extended N-terminal tail consisting of 38 amino acid residues with nine lysines. Its extended N-terminal tail has been postulated to play a crucial role in suppressing host immunity, growth and development-associated genes, presumably through an epigenetic control mechanism. A suppression subtractive hybridization (SSH) analysis was analyzed in transcriptome by short-read sequencing technology and provided several target and non-target genes of a viral histone H4. In this study, we analyzed the effect CpBV-H4 on the expression of two target genes: Lysine-specific demethylase (KDM) and Serine proteinase inhibitor (Serpins). Transient expression of CpBV-H4 into non parasitized P. xylostella was performed by microinjection of a recombinant expression vector, and showed the expression up to 70 h. Under this transient expression condition, we analyzed the effect of CpBV-H4 on the expression of target genes by RT-PCR at different time points. Interestingly, the CpBV-H4 significantly inhibited the expression of these target genes, while the truncated CpBV-H4 deleting the N-terminal tail did not show this inhibitory effect. This study also showed that the viral histone H4 suppresses expressions of lysine-specific demethylase and serine proteinase inhibitor (Serpin2) to inhibit host growth and development.

Possible Role of Lysine Demethylase 2A in the Pathophysiology of Psoriasis

( Dong Ha Kim ),( Mi-ra Choi ),( Jae Kyung Lee ),( Dong-kyun Hong ),( Kyung Eun Jung ),( Chong Won Choi ),( Young Lee ),( Chang-deok Kim ),( Young-joon Seo ),( Jeung-hoon Lee ) 대한피부과학회 2020 Annals of Dermatology Vol.32 No.6

Background: Psoriasis is a common chronic inflammatory skin disease. The development of psoriasis is dependent on many intercellular events such as innate immunity and T cell-mediated inflammation. Furthermore, genetic factors are strongly implicated in the pathophysiology of psoriasis. Although a variety of susceptible genes are identified, it is likely that many important genes remain undisclosed. Objective: The aim of this study is to investigate the possible role of lysine demethylase 2A (KDM2A) in the pathophysiology of psoriasis. Methods: We examined the expression of KDM2A using a well established imiquimod-induced psoriasiform dermatitis model. Results: Immunohistochemistry analysis showed that expression of KDM2A was increased in imiquimod-induced psoriasiform dermatitis. Consistent with this result, KDM2A level was markedly increased in the epidermis of psoriatic patient. When keratinocytes were stimulated with TLR3 agonist poly(I:C), KDM2A was increased at both the mRNA and protein levels. Poly(I:C) increased the expression of psoriasis-related cytokines including tumor necrosis factor-α, interleukin-8, and CCL20, and KDM2A inhibitor daminozide enhanced the poly(I:C)-induced cytokine expression. Finally, topical co-application of imiquimod and daminozide exacerbated the imiquimod-induced psoriasiform dermatitis. Conclusion: Together, these results suggest that KDM2A is increased to negatively regulate the inflammatory reaction of epidermal keratinocytes in psoriasis. (Ann Dermatol 32(6) 481∼486, 2020)