Epigenetic balance ensures mechanistic control of MLL amplification and rearrangement.

MLL/KMT2A amplifications and translocations are prevalent in infant, adult, and therapy-induced leukemia. However, the molecular contributor(s) to these alterations are unclear. Here, we demonstrate that histone H3 lysine 9 mono- and di-methylation (H3K9me1/2) balance at the MLL/KMT2A locus regulates these amplifications and rearrangements. This balance is controlled by the crosstalk between lysine demethylase KDM3B and methyltransferase G9a/EHMT2. KDM3B depletion increases H3K9me1/2 levels and reduces CTCF occupancy at the MLL/KMT2A locus, in turn promoting amplification and rearrangements. Depleting CTCF is also sufficient to generate these focal alterations. Furthermore, the chemotherapy doxorubicin (Dox), which associates with therapy-induced leukemia and promotes MLL/KMT2A amplifications and rearrangements, suppresses KDM3B and CTCF protein levels. KDM3B and CTCF overexpression rescues Dox-induced MLL/KMT2A alterations. G9a inhibition in human cells or mice also suppresses MLL/KMT2A events accompanying Dox treatment. Therefore, MLL/KMT2A amplifications and rearrangements are controlled by epigenetic regulators that are tractable drug targets, which has clinical implications.

Targeting JMJD1C to selectively disrupt tumor Treg cell fitness enhances antitumor immunity.

Regulatory T (Treg) cells are critical for immune tolerance but also form a barrier to antitumor immunity. As therapeutic strategies involving Treg cell depletion are limited by concurrent autoimmune disorders, identification of intratumoral Treg cell-specific regulatory mechanisms is needed for selective targeting. Epigenetic modulators can be targeted with small compounds, but intratumoral Treg cell-specific epigenetic regulators have been unexplored. Here, we show that JMJD1C, a histone demethylase upregulated by cytokines in the tumor microenvironment, is essential for tumor Treg cell fitness but dispensable for systemic immune homeostasis. JMJD1C deletion enhanced AKT signals in a manner dependent on histone H3 lysine 9 dimethylation (H3K9me2) demethylase and STAT3 signals independently of H3K9me2 demethylase, leading to robust interferon-γ production and tumor Treg cell fragility. We have also developed an oral JMJD1C inhibitor that suppresses tumor growth by targeting intratumoral Treg cells. Overall, this study identifies JMJD1C as an epigenetic hub that can integrate signals to establish tumor Treg cell fitness, and we present a specific JMJD1C inhibitor that can target tumor Treg cells without affecting systemic immune homeostasis.

Jmjd1c demethylates STAT3 to restrain plasma cell differentiation and rheumatoid arthritis.

Appropriate regulation of B cell differentiation into plasma cells is essential for humoral immunity while preventing antibody-mediated autoimmunity; however, the underlying mechanisms, especially those with pathological consequences, remain unclear. Here, we found that the expression of Jmjd1c, a member of JmjC domain histone demethylase, in B cells but not in other immune cells, protected mice from rheumatoid arthritis (RA). In humans with RA, JMJD1C expression levels in B cells were negatively associated with plasma cell frequency and disease severity. Mechanistically, Jmjd1c demethylated STAT3, rather than histone substrate, to restrain plasma cell differentiation. STAT3 Lys140 hypermethylation caused by Jmjd1c deletion inhibited the interaction with phosphatase Ptpn6 and resulted in abnormally sustained STAT3 phosphorylation and activity, which in turn promoted plasma cell generation. Germinal center B cells devoid of Jmjd1c also acquired strikingly increased propensity to differentiate into plasma cells. STAT3 Lys140Arg point mutation completely abrogated the effect caused by Jmjd1c loss. Mice with Jmjd1c overexpression in B cells exhibited opposite phenotypes to Jmjd1c-deficient mice. Overall, our study revealed Jmjd1c as a critical regulator of plasma cell differentiation and RA and also highlighted the importance of demethylation modification for STAT3 in B cells.

An oncogenic JMJD6-DGAT1 axis tunes the epigenetic regulation of lipid droplet formation in clear cell renal cell carcinoma.

Characterized by intracellular lipid droplet accumulation, clear cell renal cell carcinoma (ccRCC) is resistant to cytotoxic chemotherapy and is a lethal disease. Through an unbiased siRNA screen of 2-oxoglutarate (2-OG)-dependent enzymes, which play a critical role in tumorigenesis, we identified Jumonji domain-containing 6 (JMJD6) as an essential gene for ccRCC tumor development. The downregulation of JMJD6 abolished ccRCC colony formation in vitro and inhibited orthotopic tumor growth in vivo. Integrated ChIP-seq and RNA-seq analyses uncovered diacylglycerol O-acyltransferase 1 (DGAT1) as a critical JMJD6 effector. Mechanistically, JMJD6 interacted with RBM39 and co-occupied DGAT1 gene promoter with H3K4me3 to induce DGAT1 expression. JMJD6 silencing reduced DGAT1, leading to decreased lipid droplet formation and tumorigenesis. The pharmacological inhibition (or depletion) of DGAT1 inhibited lipid droplet formation in vitro and ccRCC tumorigenesis in vivo. Thus, the JMJD6-DGAT1 axis represents a potential new therapeutic target for ccRCC.

NSD1 mediates antagonism between SWI/SNF and polycomb complexes and is required for transcriptional activation upon EZH2 inhibition.

Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.

Long noncoding RNA lncKdm2b is required for ILC3 maintenance by initiation of Zfp292 expression.

Innate lymphoid cells (ILCs) communicate with other hematopoietic and nonhematopoietic cells to regulate immunity, inflammation and tissue homeostasis. How ILC lineages develop and are maintained remains largely unknown. In this study we observed that a divergent long noncoding RNA (lncRNA), lncKdm2b, was expressed at high levels in intestinal group 3 ILCs (ILC3s). LncKdm2b deficiency in the hematopoietic system led to reductions in the number and effector functions of ILC3s. LncKdm2b expression sustained the maintenance of ILC3s by promoting their proliferation through activation of the transcription factor Zfp292. Mechanistically, lncKdm2b recruited the chromatin organizer Satb1 and the nuclear remodeling factor (NURF) complex onto the Zfp292 promoter to initiate its transcription. Deletion of Zfp292 or Bptf also abrogated the maintenance of ILC3s, leading to susceptibility to bacterial infection. Therefore, our findings reveal that lncRNAs may represent an additional layer of regulation of ILC development and function.

Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.

Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.

Taking Me away: the function of phosphorylation on histone lysine demethylases.

Histone lysine demethylases (KDMs) regulate eukaryotic gene transcription by catalysing the removal of methyl groups from histone proteins. These enzymes are intricately regulated by the kinase signalling system in response to internal and external stimuli. Here, we review the mechanisms by which kinase-mediated phosphorylation influence human histone KDM function. These include the changing of histone KDM subcellular localisation or chromatin binding, the altering of protein half-life, changes to histone KDM complex formation that result in histone demethylation, non-histone demethylation or demethylase-independent effects, and effects on histone KDM complex dissociation. We also explore the structural context of phospho-sites on histone KDMs and evaluate how this relates to function.

Histone demethylase KDM2A recruits HCFC1 and E2F1 to orchestrate male germ cell meiotic entry and progression.

In mammals, the transition from mitosis to meiosis facilitates the successful production of gametes. However, the regulatory mechanisms that control meiotic initiation remain unclear, particularly in the context of complex histone modifications. Herein, we show that KDM2A, acting as a lysine demethylase targeting H3K36me3 in male germ cells, plays an essential role in modulating meiotic entry and progression. Conditional deletion of Kdm2a in mouse pre-meiotic germ cells results in complete male sterility, with spermatogenesis ultimately arrested at the zygotene stage of meiosis. KDM2A deficiency disrupts H3K36me2/3 deposition in c-KIT+ germ cells, characterized by a reduction in H3K36me2 but a dramatic increase in H3K36me3. Furthermore, KDM2A recruits the transcription factor E2F1 and its co-factor HCFC1 to the promoters of key genes required for meiosis entry and progression, such as Stra8, Meiosin, Spo11, and Sycp1. Collectively, our study unveils an essential role for KDM2A in mediating H3K36me2/3 deposition and controlling the programmed gene expression necessary for the transition from mitosis to meiosis during spermatogenesis.

Epigenetic regulation of the expression of Il12 and Il23 and autoimmune inflammation by the deubiquitinase Trabid.

The proinflammatory cytokines interleukin 12 (IL-12) and IL-23 connect innate responses and adaptive immune responses and are also involved in autoimmune and inflammatory diseases. Here we describe an epigenetic mechanism for regulation of the genes encoding IL-12 (Il12a and Il12b; collectively called 'Il12' here) and IL-23 (Il23a and Il12b; collectively called 'Il23' here) involving the deubiquitinase Trabid. Deletion of Zranb1 (which encodes Trabid) in dendritic cells inhibited induction of the expression of Il12 and Il23 by Toll-like receptors (TLRs), which impaired the differentiation of inflammatory T cells and protected mice from autoimmune inflammation. Trabid facilitated TLR-induced histone modifications at the promoters of Il12 and Il23, which involved deubiqutination and stabilization of the histone demethylase Jmjd2d. Our findings highlight an epigenetic mechanism for the regulation of Il12 and Il23 and establish Trabid as an innate immunological regulator of inflammatory T cell responses.

KDM4A lysine demethylase induces site-specific copy gain and rereplication of regions amplified in tumors.

Acquired chromosomal instability and copy number alterations are hallmarks of cancer. Enzymes capable of promoting site-specific copy number changes have yet to be identified. Here, we demonstrate that H3K9/36me3 lysine demethylase KDM4A/JMJD2A overexpression leads to localized copy gain of 1q12, 1q21, and Xq13.1 without global chromosome instability. KDM4A-amplified tumors have increased copy gains for these same regions. 1q12h copy gain occurs within a single cell cycle, requires S phase, and is not stable but is regenerated each cell division. Sites with increased copy number are rereplicated and have increased KDM4A, MCM, and DNA polymerase occupancy. Suv39h1/KMT1A or HP1γ overexpression suppresses the copy gain, whereas H3K9/K36 methylation interference promotes gain. Our results demonstrate that overexpression of a chromatin modifier results in site-specific copy gains. This begins to establish how copy number changes could originate during tumorigenesis and demonstrates that transient overexpression of specific chromatin modulators could promote these events.

Lineage commitment of dermal fibroblast progenitors is controlled by Kdm6b-mediated chromatin demethylation.

Dermal Fibroblast Progenitors (DFPs) differentiate into distinct fibroblast lineages during skin development. However, the epigenetic mechanisms that regulate DFP differentiation are not known. Our objective was to use multimodal single-cell approaches, epigenetic assays, and allografting techniques to define a DFP state and the mechanism that governs its differentiation potential. Our initial results indicated that the overall transcription profile of DFPs is repressed by H3K27me3 and has inaccessible chromatin at lineage-specific genes. Surprisingly, the repressive chromatin profile of DFPs renders them unable to reform the skin in allograft assays despite their multipotent potential. We hypothesized that chromatin derepression was modulated by the H3K27me3 demethylase, Kdm6b/Jmjd3. Dermal fibroblast-specific deletion of Kdm6b/Jmjd3 in mice resulted in adipocyte compartment ablation and inhibition of mature dermal papilla functions, confirmed by additional single-cell RNA-seq, ChIP-seq, and allografting assays. We conclude that DFPs are functionally derepressed during murine skin development by Kdm6b/Jmjd3. Our studies therefore reveal a multimodal understanding of how DFPs differentiate into distinct fibroblast lineages and provide a novel publicly available multiomics search tool.

The H3K4 demethylase JMJ1 is required for proper timing of flowering in Brachypodium distachyon.

Flowering is a key developmental transition in the plant life cycle. In temperate climates, flowering often occurs in response to the perception of seasonal cues such as changes in day-length and temperature. However, the mechanisms that have evolved to control the timing of flowering in temperate grasses are not fully understood. We identified a Brachypodium distachyon mutant whose flowering is delayed under inductive long-day conditions due to a mutation in the JMJ1 gene, which encodes a Jumonji domain-containing protein. JMJ1 is a histone demethylase that mainly demethylates H3K4me2 and H3K4me3 in vitro and in vivo. Analysis of the genome-wide distribution of H3K4me1, H3K4me2, and H3K4me3 in wild-type plants by chromatin immunoprecipitation and sequencing combined with RNA sequencing revealed that H3K4m1 and H3K4me3 are positively associated with gene transcript levels, whereas H3K4me2 is negatively correlated with transcript levels. Furthermore, JMJ1 directly binds to the chromatin of the flowering regulator genes VRN1 and ID1 and affects their transcription by modifying their H3K4me2 and H3K4me3 levels. Genetic analyses indicated that JMJ1 promotes flowering by activating VRN1 expression. Our study reveals a role for JMJ1-mediated chromatin modification in the proper timing of flowering in B. distachyon.

Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs.

Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo.

KDM3A Senses Oxygen Availability to Regulate PGC-1α-Mediated Mitochondrial Biogenesis.

Hypoxia, which occurs during tumor growth, triggers complex adaptive responses in which peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) plays a critical role in mitochondrial biogenesis and oxidative metabolism. However, how PGC-1α is regulated in response to oxygen availability remains unclear. We demonstrated that lysine demethylase 3A (KDM3A) binds to PGC-1α and demethylates monomethylated lysine (K) 224 of PGC-1α under normoxic conditions. Hypoxic stimulation inhibits KDM3A, which has a high KM of oxygen for its activity, and enhances PGC-1α K224 monomethylation. This modification decreases PGC-1α's activity required for NRF1- and NRF2-dependent transcriptional regulation of TFAM, TFB1M, and TFB2M, resulting in reduced mitochondrial biogenesis. Expression of PGC-1α K224R mutant significantly increases mitochondrial biogenesis, reactive oxygen species (ROS) production, and tumor cell apoptosis under hypoxia and inhibits brain tumor growth in mice. This study revealed that PGC-1α monomethylation, which is dependent on oxygen availability-regulated KDM3A, plays a critical role in the regulation of mitochondrial biogenesis.

Acetylation of PHF5A Modulates Stress Responses and Colorectal Carcinogenesis through Alternative Splicing-Mediated Upregulation of KDM3A.

Alternative pre-mRNA-splicing-induced post-transcriptional gene expression regulation is one of the pathways for tumors maintaining proliferation rates accompanying the malignant phenotype under stress. Here, we uncover a list of hyperacetylated proteins in the context of acutely reduced Acetyl-CoA levels under nutrient starvation. PHF5A, a component of U2 snRNPs, can be acetylated at lysine 29 in response to multiple cellular stresses, which is dependent on p300. PHF5A acetylation strengthens the interaction among U2 snRNPs and affects global pre-mRNA splicing pattern and extensive gene expression. PHF5A hyperacetylation-induced alternative splicing stabilizes KDM3A mRNA and promotes its protein expression. Pathologically, PHF5A K29 hyperacetylation and KDM3A upregulation axis are correlated with poor prognosis of colon cancer. Our findings uncover a mechanism of an anti-stress pathway through which acetylation on PHF5A promotes the cancer cells' capacity for stress resistance and consequently contributes to colon carcinogenesis.

An HSF1-JMJD6-HSP feedback circuit promotes cell adaptation to proteotoxic stress.

Heat Shock Factor 1 (HSF1) is best known as the master transcriptional regulator of the heat-shock response (HSR), a conserved adaptive mechanism critical for protein homeostasis (proteostasis). Combining a genome-wide RNAi library with an HSR reporter, we identified Jumonji domain-containing protein 6 (JMJD6) as an essential mediator of HSF1 activity. In follow-up studies, we found that JMJD6 is itself a noncanonical transcriptional target of HSF1 which acts as a critical regulator of proteostasis. In a positive feedback circuit, HSF1 binds and promotes JMJD6 expression, which in turn reduces heat shock protein 70 (HSP70) R469 monomethylation to disrupt HSP70-HSF1 repressive complexes resulting in enhanced HSF1 activation. Thus, JMJD6 is intricately wired into the proteostasis network where it plays a critical role in cellular adaptation to proteotoxic stress.

Dynamic evolution of the heterochromatin sensing histone demethylase IBM1.

Heterochromatin is critical for maintaining genome stability, especially in flowering plants, where it relies on a feedback loop involving the H3K9 methyltransferase, KRYPTONITE (KYP), and the DNA methyltransferase CHROMOMETHYLASE3 (CMT3). The H3K9 demethylase INCREASED IN BONSAI METHYLATION 1 (IBM1) counteracts the detrimental consequences of KYP-CMT3 activity in transcribed genes. IBM1 expression in Arabidopsis is uniquely regulated by methylation of the 7th intron, allowing it to monitor global H3K9me2 levels. We show the methylated intron is prevalent across flowering plants and its underlying sequence exhibits dynamic evolution. We also find extensive genetic and expression variations in KYP, CMT3, and IBM1 across flowering plants. We identify Arabidopsis accessions resembling weak ibm1 mutants and Brassicaceae species with reduced IBM1 expression or deletions. Evolution towards reduced IBM1 activity in some flowering plants could explain the frequent natural occurrence of diminished or lost CMT3 activity and loss of gene body DNA methylation, as cmt3 mutants in A. thaliana mitigate the deleterious effects of IBM1.

DNA methylation near MAD1L1, KDM2B, and SOCS3 mediates the effect of socioeconomic status on elevated body mass index in African American adults.

Obesity and poverty disproportionally affect African American persons. Epigenetic mechanisms could partially explain the association between socioeconomic disadvantage and body mass index (BMI). We examined the extent to which epigenetic mechanisms mediate the effect of socioeconomic status (SES) on BMI. Using data from African American adults from the Atherosclerosis Risk in Communities (ARIC) Study (n = 2664, mean age = 57 years), education, income, and occupation were used to create a composite SES score at visit 1 (1987-1989). We conducted two methylation-wide association analyses to identify associations between SES (visit 1), BMI and cytosine-phosphate-guanine (CpG) sites measured at a subsequent visit (1990-1995). We then utilized structural equation modeling (SEM) to test whether identified sites mediated the association between earlier SES and BMI in sex-stratified models adjusted for demographic and risk factor covariates. Independent replication and meta-analyses were conducted using the Jackson Heart Study (JHS, n = 874, mean age 51 years, 2000-2004). Three CpG sites near MAD1L1, KDM2B, and SOCS3 (cg05095590, cg1370865, and cg18181703) were suggestively associated (P-value < 1.3×10-5) in ARIC and at array-wide significance (P-value < 1.3×10-7) in a combined meta-analysis of ARIC with JHS. SEM of these three sites revealed significant indirect effects in females (P-value < 5.8×10-3), each mediating 7%-20% of the total effect of SES on BMI. Nominally significant indirect effects were observed for two sites near MAD1L1 and KDM2B in males (P-value < 3.4×10-2), mediating -17 and -22% of the SES-BMI effect. These results provide further evidence that epigenetic modifications may be a potential pathway through which SES may "get under the skin" and contribute to downstream health disparities.

Identification of a molecular network regulated by multiple ASD high risk genes.

Genetic sequencing has identified high-confidence ASD risk genes with loss-of-function mutations. How the haploinsufficiency of distinct ASD risk genes causes ASD remains to be elucidated. In this study, we examined the role of four top-ranking ASD risk genes, ADNP, KDM6B, CHD2, and MED13, in gene expression regulation. ChIP-seq analysis reveals that gene targets with the binding of these ASD risk genes at promoters are enriched in RNA processing and DNA repair. Many of these targets are found in ASD gene database (SFARI), and are involved in transcription regulation and chromatin remodeling. Common gene targets of these ASD risk genes include a network of high confidence ASD genes associated with gene expression regulation, such as CTNNB1 and SMARCA4. We further directly examined the transcriptional impact of the deficiency of these ASD risk genes. Our mRNA profiling with qPCR assays in cells with the knockdown of Adnp, Kdm6b, Chd2 or Med13 has revealed an intricate pattern of their cross-regulation, as well as their influence on the expression of other ASD genes. In addition, some synaptic genes, such as Snap25 and Nrxn1, are strongly regulated by deficiency of the four ASD risk genes, which could be through the direct binding at promoters or indirectly through the targets like Ctnnb1 or Smarca4. The identification of convergent and divergent gene targets that are regulated by multiple ASD risk genes will help to understand the molecular mechanisms underlying common and unique phenotypes associated with haploinsufficiency of ASD-associated genes.