Histone Demethylase Activity Research Articles

Regulation of c-Myc expression by the histone demethylase JMJD1A is essential for prostate cancer cell growth and survival.

The histone demethylase JMJD1A, which controls gene expression by epigenetic regulation of H3K9 methylation marks, functions in diverse activities, including spermatogenesis, metabolism, and stem cell self-renewal and differentiation. Here, we found that JMJD1A knockdown in prostate cancer cells antagonizes their proliferation and survival. Profiling array analyses revealed that JMJD1A-dependent genes function in cellular growth, proliferation and survival, and implicated that the c-Myc transcriptional network is de-regulated following JMJD1A inhibition. Biochemical analyses confirmed that JMJD1A enhances c-Myc transcriptional activity by upregulating c-Myc expression levels. Mechanistically, JMJD1A activity promoted recruitment of androgen receptor (AR) to the c-Myc gene enhancer and induced H3K9 demethylation, increasing AR-dependent transcription of c-Myc mRNA. In parallel, we found that JMJD1A regulated c-Myc stability, likely by inhibiting HUWE1, an E3 ubiquitin ligase known to target degradation of several substrates including c-Myc. JMJD1A (wild-type or mutant lacking histone demethylase activity) bound to HUWE1, attenuated HUWE1-dependent ubiquitination and subsequent degradation of c-Myc, increasing c-Myc protein levels. Furthermore, c-Myc knockdown in prostate cancer cells phenocopied effects of JMJD1A knockdown, and c-Myc re-expression in JMJD1A-knockdown cells partially rescued prostate cancer cell growth in vitro and in vivo. c-Myc protein levels were positively correlated with those of JMJD1A in a subset of human prostate cancer specimens. Collectively, our findings identify a critical role for JMJD1A in regulating proliferation and survival of prostate cancer cells by controlling c-Myc expression at transcriptional and post-translational levels.

Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation.

Histone methylation regulates DNA repair. However, the mechanisms that underlie the regulation of histone methylation during this repair remain to be further defined. Here, we show that ionizing radiation (IR) induces DNA-PK-dependent phosphorylation of nuclear fumarase at T236, which leads to an interaction between fumarase and the histone variant H2A.Z at DNA double-strand break (DSB) regions. Locally generated fumarate inhibits KDM2B histone demethylase activity, resulting in enhanced dimethylation of histone H3 K36; in turn, this increases the accumulation of the Ku70-containing DNA-PK at DSB regions for non-homologous end joining (NHEJ) DNA repair and cell survival. These findings reveal a feedback mechanism that underlies DNA-PK regulation by chromatin-associated fumarase and an instrumental function of fumarase in regulating histone H3 methylation and DNA repair.

Abstract 2684: An early innate stress response precedes acquired drug resistance in melanoma

Abstract Acquired drug resistance constitutes a major challenge for effective cancer therapies. The dynamics of early drug resistance leading to permanent resistance are poorly understood. Melanoma cell lines were exposed to molecular targeted inhibitors like BRAF or MEK inhibitors or chemotherapy at sublethal drug concentrations for over 90 days. Alternatively melanoma cells were exposed to hypoxic conditions or low glucose media. Cells surviving drug exposure, hypoxia or nutrient starvation were monitored for the expression of CD271, ALDH activity, differentiation markers, ABCB5, chromatin remodeling, histone demethylases and markers for angiogenesis to characterize cells exposed for a minimum of 12 days. Further gene expression analyses, RPPA analyses and in vivo tumorigenicity were performed in these cells. Drug exposure, hypoxia or nutrient starvation leads to an early innate cell response in melanoma cells resulting in multi-drug resistance, termed induced drug tolerant cells (IDTC). Transition into the IDTC state seems to be an inherent stress reaction for survival towards unfavorable environmental conditions or drug exposure independent of any subpopulation or cancer stem cell. The response comprises chromatin remodeling, activation of signaling cascades, and markers proposed to be stem cell markers with higher angiogenic potential and tumorigenicity. These changes are characterized by a common increase in CD271 expression concomitantly with loss of differentiation markers such as melan-A and tyrosinase, enhanced ALDH activity and upregulation of histone demethylases. Accordingly, IDTCs show a loss of H3K4me3, H3K27me3 and gain of H3K9me3 suggesting activation and repression of differential genes. Drug holidays at the IDTC state allow for reversion into parental cells re-sensitizing them to the drug they were primarily exposed to. However, upon continuous drug exposure IDTCs eventually transform into permanent and irreversible drug resistant cells. Knockdown of CD271 or KDM5B decreases transition into the IDTC state substantially but does not prevent it. Our results suggest a phenotypic shift of parental cells to the induced drug tolerant cell (IDTC) state irrespective of a given subpopulation thus not representing cancer stem cells. Targeting IDTCs would be crucial for sustainable disease management and prevention of acquired drug resistance. Citation Format: Dinoop Ravindran Menon, Suman Das, Clemens Krepler, Adina Vultur, Gao Zhang, Nikolas Haass, Peter H. Soyer, Brian Gabrielli, Rajasekharan Somasundaram, Gerald Hoefler, Meenhard Herlyn, Helmut Schaider. An early innate stress response precedes acquired drug resistance in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2684. doi:10.1158/1538-7445.AM2015-2684

Epigenetic targets and drug discovery Part 2: Histone demethylation and DNA methylation.

Chromatin structure is dynamically modulated by various chromatin modifications, such as histone/DNA methylation and demethylation. We have reviewed histone methyltransferases and methyllysine binders in terms of small molecule screening and drug discovery in the first part of this review series. In this part, we will summarize recent progress in chemical probe and drug discovery of histone demethylases and DNA methyltransferases. Histone demethylation and DNA methylation have attracted a lot of attention regarding their biology and disease implications. Correspondingly, many small molecule compounds have been designed to modulate the activity of histone demethylases and DNA methyltransferases, and some of them have been developed into therapeutic drugs or put into clinical trials.

Histone Demethylase LSD2 Acts as an E3 Ubiquitin Ligase and Inhibits Cancer Cell Growth through Promoting Proteasomal Degradation of OGT

Histone demethylases play important roles in various biological processes in a manner dependent on their demethylase activities. However, little is known about their demethylase-independent activities. Here, we report that LSD2, a well-known histone H3K4me1/me2 demethylase, possesses an unexpected E3 ubiquitin ligase activity. LSD2 directly ubiquitylates and promotes proteasome-dependent degradation of O-GlcNAc transferase (OGT), and inhibits A549 lung cancer cell growth in a manner dependent on its E3 ligase activity, but not demethylase activity. The depletion of LSD2 stabilizes OGT and promotescolony formation of 293T cells. LSD2 regulates distinct groups of target genes through histone demethylase and E3 ligase activities, respectively. Such regulation suggests a mechanism through which LSD2 suppresses tumorigenesis by promoting the degradation of OGT and other substrates yet to be discovered. Our study reveals an antigrowth function of LSD2 dependent on its E3 ligase activity and establishes a connection between histone demethylase and ubiquitin-dependent pathway.

Cadmium induces histone H3 lysine methylation by inhibiting histone demethylase activity.

Cadmium is an established human lung carcinogen with weak mutagenicity. However, the mechanisms underlying cadmium-induced carcinogenesis remain obscure. It has been suggested that epigenetic mechanisms may play a role in cadmium-induced carcinogenesis. In this study, we investigated the effects of cadmium on histone methylation and histone demethylases, and the role of histone methylation in transformation of immortalized normal human bronchial epithelial (BEAS-2B) cells. Exposure to 0.625, 1.25, 2.5, and 5.0 μM of cadmium for 6, 24, and 48 h increased global trimethylated histone H3 on lysine 4 (H3K4me3) and dimethylated histone H3 on lysine 9 (H3K9me2) in BEAS-2B cells compared with untreated cells, and most of these changes remained after the removal of cadmium (P < .05 or P < .01 for most modifications). Meanwhile, cadmium inhibited the activities of histone H3 on lysine 4 (H3K4) and histone H3 on lysine 9 (H3K9) demethylases which were detected by histone demethylation assay. However, there was no significant change in the protein levels of the H3K4 demethylase lysine-specific demethylase 5A (KDM5A) and the H3K9 demethylase lysine-specific demethylase 3A (KDM3A). Interestingly, during transformation of BEAS-2B cells by 20 weeks of exposure to 2.0 μM cadmium as assessed by anchorage-independent growth in soft agar, global H3K4me3, and H3K9me2 were significantly increased at 4 weeks (P < .05 or P < .01), whereas no significant change was observed at 8, 12, 16, and 20 weeks compared with control. Our study suggests that cadmium increases global H3K4me3 and H3K9me2 by inhibiting the activities of histone demethylases, and aberrant histone methylation that occurs early (48 h) and at 4 weeks is associated with cadmium-induced transformation of BEAS-2B cells at the early stage.

Oxidative stress alters global histone modification and DNA methylation

The JmjC domain-containing histone demethylases can remove histone lysine methylation and thereby regulate gene expression. The JmjC domain uses iron Fe(II) and α-ketoglutarate (αKG) as cofactors in an oxidative demethylation reaction via hydroxymethyl lysine. We hypothesize that reactive oxygen species will oxidize Fe(II) to Fe(III), thereby attenuating the activity of JmjC domain-containing histone demethylases. To minimize secondary responses from cells, extremely short periods of oxidative stress (3h) were used to investigate this question. Cells that were exposed to hydrogen peroxide (H2O2) for 3h exhibited increases in several histone methylation marks including H3K4me3 and decreases of histone acetylation marks including H3K9ac and H4K8ac; preincubation with ascorbate attenuated these changes. The oxidative stress level was measured by generation of 2′,7′-dichlorofluorescein, GSH/GSSG ratio, and protein carbonyl content. A cell-free system indicated that H2O2 inhibited histone demethylase activity where increased Fe(II) rescued this inhibition. TET protein showed a decreased activity under oxidative stress. Cells exposed to a low-dose and long-term (3 weeks) oxidative stress also showed increased global levels of H3K4me3 and H3K27me3. However, these global methylation changes did not persist after washout. The cells exposed to short-term oxidative stress also appeared to have higher activity of class I/II histone deacetylase (HDAC) but not class III HDAC. In conclusion, we have found that oxidative stress transiently alters the epigenetic program process through modulating the activity of enzymes responsible for demethylation and deacetylation of histones.

Pharmacological Inhibitors of LSD1 Promote Differentiation of Myeloid Leukemia Cells through a Mechanism Independent of Histone Demethylation

Lysine Specific Demethylase 1 (LSD1 or KDM1A) is one of a number of epigenetic regulators which have recently emerged as candidate therapeutic targets in acute myeloid leukaemia (AML). It is a flavin adenine dinucleotide (FAD) dependent homolog of the amine oxidase family with an ability to demethylate monomethyl or dimethyl lysine 4 (K4) of histone H3, in addition to other substrates. Pharmacological inhibitors of LSD1 such as the tranylcypromine derivatives have already commenced evaluation in early phase clinical trials. While it has been widely assumed that these compounds promote differentiation of AML cells through inhibition of the demethylase activity of LSD1, the precise mechanisms by which LSD1 inhibitors function has not yet been determined.If changes in histone methylation are a central and critical mediator of the effects of LSD1 inhibitors in promoting AML cell differentiation, it would be expected that global changes in transcription would be tightly linked temporally to changes in histone methylation following drug treatment of cells. Through RNA sequencing and ChIP sequencing experiments performed in human THP1 AML cells treated for 24 hours with a potent and specific tranylcypromine-derivative LSD1 inhibitor (ORY86, trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine), we have established that wholescale up regulation of a myeloid differentiation transcription programme occurs in the absence of any significant genome-wide changes in mono- and di-methyl H3K4 and H3K9 (which are key enzymatic targets of LSD1). Thus LSD1 inhibitor-induced up regulation of myeloid differentiation gene expression is not downstream of changes in histone methylation.We further demonstrated that non-enzymatic functions of LSD1 are essential in AML cells by expressing either wild-type (WT) or catalytically inactive LSD1 (K661A) in LSD1 knockdown (KD) THP1 cells. While LSD1 KD cells exhibit myeloid differentiation and loss of clonogenic potential, both the WT and mutant versions of LSD1 were able to rescue the in vitro clonogenic potential of KD cells to an equivalent extent. Thus the histone demethylase activity of LSD1 is not required to sustain AML blasts in an undifferentiated state.Comparison of the transcriptional consequences of LSD1 inhibition with the transcriptional consequences of transcription factor knockdown in THP1 AML cells using GSEA revealed that pharmacological inhibition of LSD1 mimics depletion of GFI1. Immunoprecipitation experiments confirmed the previously described physical association of GFI1 with LSD1. Critically, the physical interactions of LSD1 with GFI1 was reversed by pharmacological inhibition of LSD1 with ORY86. Furthermore, in ChIP sequencing experiments drug treatment led to dissociation of LSD1 from promoters and enhancers. By contrast, there was no disruption of the endogenous level interaction of LSD1 with RCOR1, HDAC1 and HDAC2 (i.e. the CoREST complex) following drug treatment.To determine whether the inhibitor-induced separation of LSD1 from GFI1 is required for induction of myeloid differentiation by ORY86, we performed experiments using a GFI1-LSD1 fusion construct expressed in THP1 cells under the control of a doxycycline-regulated promoter. This construct tethers LSD1 directly to the transcription factor and circumvents any drug induced physical separation. THP1 cells expressing GFI1-LSD1 were drug resistant (as determined by immunophenotyping and clonogenic potential), in contrast to control cells expressing GFI1, LSD1 or an empty vector in the same inducible system. Thus, drug-induced physical separation of GFI1 from LSD1 is required for THP1 AML cells to undergo differentiation.Our data support a model whereby the physical association of LSD1 with transcription factors such as GFI1 is essential to maintain the differentiation block in AML. Unexpectedly, tranylcypromine-derivative inhibitors target this novel scaffolding function of LSD1, rather than its histone demethylase activity, to promote differentiation of AML cells. DisclosuresLynch:Astra Zeneca: Employment. Ciceri:Oryzon Genomics: Employment. Somervaille:Oryzon Genomics: Research Funding; Imago Biosciences: Consultancy.

A stress-induced early innate response causes multidrug tolerance in melanoma.

Acquired drug resistance constitutes a major challenge for effective cancer therapies with melanoma being no exception. The dynamics leading to permanent resistance are poorly understood but are important to design better treatments. Here we show that drug exposure, hypoxia or nutrient starvation leads to an early innate cell response in melanoma cells resulting in multidrug resistance, termed induced drug-tolerant cells (IDTCs). Transition into the IDTC state seems to be an inherent stress reaction for survival toward unfavorable environmental conditions or drug exposure. The response comprises chromatin remodeling, activation of signaling cascades and markers implicated in cancer stemness with higher angiogenic potential and tumorigenicity. These changes are characterized by a common increase in CD271 expression concomitantly with loss of differentiation markers such as melan-A and tyrosinase, enhanced aldehyde dehydrogenase (ALDH) activity and upregulation of histone demethylases. Accordingly, IDTCs show a loss of H3K4me3, H3K27me3 and gain of H3K9me3 suggesting activation and repression of differential genes. Drug holidays at the IDTC state allow for reversion into parental cells re-sensitizing them to the drug they were primarily exposed to. However, upon continuous drug exposure IDTCs eventually transform into permanent and irreversible drug-resistant cells. Knockdown of CD271 or KDM5B decreases transition into the IDTC state substantially but does not prevent it. Targeting IDTCs would be crucial for sustainable disease management and prevention of acquired drug resistance.

Restoration of desmosomal junction protein expression and inhibition of H3K9-specific histone demethylase activity by cytostatic proline-rich polypeptide-1 leads to suppression of tumorigenic potential in human chondrosarcoma cells.

Disruption of cell-cell junctions and the concomitant loss of polarity, downregulation of tumor-suppressive adherens junctions and desmosomes represent hallmark phenotypes for several different cancer cells. Moreover, a variety of evidence supports the argument that these two common phenotypes of cancer cells directly contribute to tumorigenesis. In this study, we aimed to determine the status of intercellular junction proteins expression in JJ012 human malignant chondrosarcoma cells and investigate the effect of the antitumorigenic cytokine, proline-rich polypeptide-1 (PRP-1) on their expression. The cell junction pathway array data indicated downregulation of desmosomal proteins, such as desmoglein (1,428-fold), desmoplakin (620-fold) and plakoglobin (442-fold). The tight junction proteins claudin 11 and E-cadherin were also downregulated (399- and 52-fold, respectively). Among the upregulated proteins were the characteristic for tumors gap junction β-5 protein (connexin 31.1) and the pro-inflammatory pathway protein intercellular adhesion molecule (upregulated 129- and 43-fold, respectively). We demonstrated that PRP-1 restored the expression of the abovementioned downregulated in chondrosarcoma desmosomal proteins. PRP-1 inhibited H3K9-specific histone demethylase activity in chondrosarcoma cells in a dose-dependent manner (0.5 µg/ml PRP, 63%; 1 µg/ml PRP, 74%; and 10 µg/ml PRP, 91% inhibition). Members of the H3K9 family were shown to transcriptionally repress tumor suppressor genes and contribute to cancer progression. Our experimental data indicated that PRP-1 restores tumor suppressor desmosomal protein expression in JJ012 human chondrosarcoma cells and inhibits H3K9 demethylase activity, contributing to the suppression of tumorigenic potential in chondrosarcoma cells.

S1-1 - Glutathione and cellular redox control in epigenetic regulation

Epigenetics is defined as the mitotically/meiotically heritable changes in gene expression that are not due to changes in the primary DNA sequence. Over recent years, growing evidence has suggested a link between redox metabolism and the control of epigenetic mechanisms. The effect of the redox control, oxidative stress, and glutathione (GSH) on the epigenetic mechanisms occur at different levels affecting DNA methylation, miRNAs expression, and histone post-translational modifications (PTMs). Furthermore, a number of redox PTMs are being described, so enriching the histone code. Pioneer works showed how oxidized GSH inhibits the activity of S-adenosyl methionine synthetase, MAT1A, a key enzyme involved in the synthesis of S-adenosyl methionine (SAM), which is used by DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs). Alteration in NAD /NADH ratio affects the activity of class III histone deacetylases (HDACs) and poly-ADP ribosyltransferases (PARPs). Furthermore, the iron redox state of the catalytic center of key enzymes influences the activity of HDACs and the activity of Tet methylcytosine dioxygenases (DNA demetylases) and JmjC histone demethylases. In this communication, we will show the intricate mechanisms that participate in the redox control of the epigenetic mechanisms. We specially focus our work in the characterization of new PTMs in histones, such as histone carbonylation and glutathionylation. Demonstrating how GSH influences the epigenetic mechanisms beyond a mere regulation of SAM levels. The mechanisms described in this communication place GSH and redox control in the landscape of the epigenetic regulation. The results shown underscore the relevant role that oxidative stress and GSH play as key factors in epigenetics, opening a new window for understating the underlying mechanisms that control cell differentiation, proliferation, development, and disease.

Abstract 5161: The development of therapeutic inhibitors of the KDM5 histone demethylases

Abstract Histone methyl marks contribute to the regulation of chromatin architecture and of DNA-based processes such as replication, repair and gene transcription. There is increasing evidence for alterations in histone methylation patterns in cancer and other human diseases. Importantly, aberrant methylation can contribute to deregulated gene expression and thereby to excessive proliferation, survival and metastasis of cancer cells. Histone methyl transferases and demethylases establish a dynamic equilibrium of histone modifications, and the expression and activity of several members of these enzyme families are deregulated in human cancer. The jumonji C-domain histone H3K4me3/2 demethylase KDM5B/PLU1/JARID1B as well as its close relative KDM5A/JARID1A are potential oncogenes. Both proteins are overexpressed in human tumors and the gene encoding KDM5B is e.g. amplified in invasive breast cancer cells. Results obtained with RNA interference and targeted deletions in mice have provided additional support for a critical role of KDM5 demethylases in cancer cells. EpiTherapeutics is developing novel cancer drugs targeting the enzymatic activity of histone demethylases. Here we present our recent progress in obtaining selective, potent, and cell-permeable inhibitors of the KDM5 demethylases. Biochemical and cellular activities, pharmacological data, and in vivo antitumor activity will be discussed. Citation Format: Heidi R. Hudlebusch, Bo Heinemann, Jesper M. Nielsen, Daniela Kleine-Kohlbrecher, Camilla Hauge, Thomas Boesen, Marc Labelle, Lars-Ole Gerlach, Peter Staller, Peter Birk. The development of therapeutic inhibitors of the KDM5 histone demethylases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5161. doi:10.1158/1538-7445.AM2014-5161

The histone demethylase activity of Rph1 is not essential for its role in the transcriptional response to nutrient signaling.

Rph1 and Gis1 are two related yeast zinc finger proteins that function as downstream effectors in the Ras/PKA, TOR and Sch9 nutrient signaling pathways. Both proteins also contain JmjC histone demethylase domains, but only Rph1 is known to be an active enzyme, demethylating lysine 36 of histone H3. We have studied to what extent the demethylase activity of Rph1 contributes to its role in nutrient signaling by performing gene expression microarray experiments on a yeast strain containing a catalytically inactive allele of RPH1. We find that the enzymatic activity of Rph1 is not essential for its role in growth phase dependent gene regulation. However, the ability of Rph1 to both activate and repress transcription is partially impaired in the active site mutant, indicating that the demethylase activity may enhance its function in vivo. Consistent with this, we find that the Rph1 mutation and a deletion of the histone H3 methylase Set2 affect the same target genes in opposite directions. Genes that are differentially expressed in the Rph1 mutant are also enriched for binding of Rpd3, a downstream effector in silencing, to their promoters. The expression of some subtelomeric genes and genes involved in sporulation and meiosis are also affected by the mutation, suggesting a role for Rph1-dependent demethylation in regulating these genes. A small set of genes are more strongly affected by the active site mutation, indicating a more pronounced role for the demethylase activity in their regulation by Rph1.

The KDM5 family of histone demethylases as targets in oncology drug discovery.

There is growing evidence for a causal role of the KDM5 family of histone demethylases in human cancer. In particular, KDM5A (JARID1A/RBP2) and KDM5B (JARID1B/PLU1) contribute to cancer cell proliferation, reduce the expression of tumor suppressor genes, promote the development of drug tolerance and maintain tumor-initiating cells. KDM5 enzymes remove tri- and di-methylations of lysine 4 of histone H3 - modifications that occur at the start site of transcription in actively transcribed genes. However, the importance of the histone demethylase activity of KDM5 proteins for cancer cells has not been resolved so far. The currently available approaches suppress or remove the targeted proteins and thereby affect their putative functions as structural components and recruitment factors for other chromatin-associated proteins. Therefore, the development of specific enzymatic inhibitors for KDM5 will promote our understanding of the biological role of their catalytic activity and yield potential novel anticancer therapeutics.

The epigenetic promotion of osteogenic differentiation of human adipose-derived stem cells by the genetic and chemical blockade of histone demethylase LSD1

Human adipose-derived stem cells (hASCs) are a highly attractive source in bone tissue engineering. It has become increasingly clear that chromatin regulators play an important role in cell fate determination. However, how osteogenic differentiation of hASCs is controlled by epigenetic mechanisms is not fully understood. Here we use genetic tools and chemical inhibitors to modify the epigenetic program of hASCs and identify lysine-specific demethylase 1 (LSD1), a histone demethylase that specifically catalyzes demethylation of di- and mono- methyl histone H3 lysine 4 (H3K4me2/1), as a key regulator in osteogenic differentiation of hASCs. Specifically, we demonstrated that genetic depletion of LSD1 with lentiviral strategy for gene knockdown promoted osteogenic differentiation of hASCs by cell studies and xenograft assays. At the molecular level, we found that LSD1 regulates osteogenesis-associated genes expression through its histone demethylase activity. Significantly, we demonstrated LSD1 demethylase inhibitors could efficiently block its catalytic activity and epigenetically boost osteogenic differentiation of hASCs. Altogether, our study defined the functional and biological roles of LSD1 and extensively explored the effects of its enzymatic activity in osteogenic differentiation of hASCs. A better understanding of how LSD1 influences on osteogenesis associated epigenetic events will provide new insights into the modulation of hASCs based cell therapy and improve the development of bone tissue engineering with epigenetic intervention.

The Jumonji gene family in Crassostrea gigas suggests evolutionary conservation of Jmj-C histone demethylases orthologues in the oyster gametogenesis and development

Jumonji (Jmj) proteins are histone demethylases, which control the identity of stem cells. Jmj genes were characterized from plants to mammals where they have been implicated in the epigenetic regulation of development. Despite the Pacific oyster Crassostrea gigas representing one of the most important aquaculture resources worldwide, the molecular mechanisms governing the embryogenesis and reproduction of this lophotrochozoan species remain poorly understood. However, annotations in the C. gigas EST library suggested the presence of putative Jumonji genes, raising the question of the conservation of this family of histone demethylases in the oyster.Using Primer walking, 5′-RACE PCR and in silico analyses, we characterized nine Jumonji orthologues in the oyster, called Cg-Jmj, bearing conserved domains critical for putative histone demethylase activity. Phylogenic analyses revealed that oyster Jumonji cluster into two distinct groups: ‘single-domain Jmj’ and ‘multi-domain Jmj’, and define 8 subgroups corresponding to each cognate orthologues in metazoans. RT-qPCR investigations showed specific regulations of Cg-Jmj mRNAs during the early development and along the reproduction cycle. Furthermore, in situ and in toto hybridizations indicate that oyster Jumonji genes are transcribed mostly within the gonad in adult oysters whereas they display a ubiquitous expression during embryonic and larval development.Our study demonstrates the presence of nine Jumonji orthologues in the oyster C. gigas. Their domain conservation and their expression profile suggest an implication during reproduction and development, questioning about the epigenetic regulation by histone methylation in lophotrochozoans.

A novel approach applying a chemical biology strategy in phenotypic screening reveals pathway-selective regulators of histone 3 K27 tri-methylation

Epigenetic regulation by histone methylation is crucial for proper programming of the genome during development. Homeostasis of histone methylation is balanced by the activities of histone methyltransferases and demethylases. Although these methyltransferases and demethylases represent logical targets for potential drug discovery, the activities of methyltransferases and demethylases regulated in response to a complex biological stimulus are also important and not yet clear. To manipulate and study histone methylation in biological systems, we screened a Biologically Diverse Compound Set (BDCS) utilizing a phenotypic assay system that directly measures the Histone 3 K27 tri-methylation (H3K27me3) level in cells. The BDCS is a unique set of target-annotated chemical probes, containing a total of 5853 compounds targeting 736 unique proteins with multiple maximally selective compounds for each target. A number of targets, with multiple hits against each target, were identified in the screen. This gave us confidence that these targets and pathways may be relevant, and included the identification of non-methyltransferase/demethylase targets as potential upstream regulators of H3K27me3. Our study suggests that a systematically designed chemical probe library can serve as a powerful drug discovery tool when combined with phenotypic screening. Follow-up studies using these findings may reveal novel therapeutically useful pathways and targets of H3K27me3 regulation.

Hairless is a histone H3K9 demethylase

The hairless (HR) protein contains a Jumonji C (JmjC) domain that is conserved among a family of proteins with histone demethylase (HDM) activity. To test whether HR possesses HDM activity, we performed a series of in vitro demethylation assays, which demonstrated that HR can demethylate monomethylated or dimethylated histone H3 lysine 9 (H3K9me1 or me2). Moreover, ectopic expression of wild-type HR, but not JmjC-mutant HR, led to pronounced demethylation of H3K9 in cultured human HeLa cells. We also show that two missense mutations in HR, which we and others described in patients with atrichia with papular lesions, abolished the demethylase activity of HR, demonstrating the role of HR demethylase activity in human disease. By ChIP-Seq analysis, we identified multiple new HR target genes, many of which play important roles in epidermal development, neural function, and transcriptional regulation, consistent with the predicted biological functions of HR. Our findings demonstrate for the first time that HR is a H3K9 demethylase that regulates epidermal homeostasis via direct control of its target genes.

Brd4 and JMJD6-Associated Anti-Pause Enhancers in Regulation of Transcriptional Pause Release

Distal enhancers characterized by the H3K4me(1) mark play critical roles in developmental and transcriptional programs. However, potential roles of specific distal regulatory elements in regulating RNA polymerase II (Pol II) promoter-proximal pause release remain poorly investigated. Here, we report that a unique cohort of jumonji C-domain-containing protein 6 (JMJD6) and bromodomain-containing protein 4 (Brd4) cobound distal enhancers, termed anti-pause enhancers (A-PEs), regulate promoter-proximal pause release of a large subset of transcription units via long-range interactions. Brd4-dependent JMJD6 recruitment on A-PEs mediates erasure of H4R3me(2(s)), which is directly read by 7SK snRNA, and decapping/demethylation of 7SK snRNA, ensuring the dismissal of the 7SK snRNA/HEXIM inhibitory complex. The interactions of both JMJD6 and Brd4 with the P-TEFb complex permit its activation and pause release of regulated coding genes. The functions of JMJD6/ Brd4-associated dual histone and RNA demethylase activity on anti-pause enhancers have intriguing implications for these proteins in development, homeostasis, and disease.

Erratum: A small molecule modulates Jumonji histone demethylase activity and selectively inhibits cancer growth

Nature Communications 4: Article number: 2035 (2013); Published: 24 June 2013; Updated: 22 October 2013. This Article contains errors in Figs 4 and 6 that were introduced during the production process. In Fig. 4d, the lane labels ‘HA-JMJD2B mut’ and ‘HA-JMJD2B’ on the western blot were inadvertentlyswitched.