Suppression of Enhancer Overactivation by a RACK7-Histone Demethylase Complex.

Regulation of enhancer activity is important for controlling gene expression programs. Here, we report that a biochemical complex containing a potential chromatin reader, RACK7, and the histone lysine 4 tri-methyl (H3K4me3)-specific demethylase KDM5C occupies many active enhancers, including almost all super-enhancers. Loss of RACK7 or KDM5C results in overactivation of enhancers, characterized by the deposition of H3K4me3 and H3K27Ac, together with increased transcription of eRNAs and nearby genes. Furthermore, loss of RACK7 or KDM5C leads to de-repression of S100A oncogenes and various cancer-related phenotypes. Our findings reveal a RACK7/KDM5C-regulated, dynamic interchange between histone H3K4me1 and H3K4me3 at active enhancers, representing an additional layer of regulation of enhancer activity. We propose that RACK7/KDM5C functions as an enhancer "brake" to ensure appropriate enhancer activity, which, when compromised, could contribute to tumorigenesis.

Dynamic control of chromatin-associated m6A methylation regulates nascent RNA synthesis.

N6-methyladenosine (m6A) methylation is co-transcriptionally deposited on mRNA, but a possible role of m6A on transcription remains poorly understood. Here, we demonstrate that the METTL3/METTL14/WTAP m6A methyltransferase complex (MTC) is localized to many promoters and enhancers and deposits the m6A modification on nascent transcripts, including pre-mRNAs, promoter upstream transcripts (PROMPTs), and enhancer RNAs. PRO-seq analyses demonstrate that nascent RNAs originating from both promoters and enhancers are significantly decreased in the METTL3-depleted cells. Furthermore, genes targeted by the Integrator complex for premature termination are depleted of METTL3, suggesting a potential antagonistic relationship between METTL3 and Integrator. Consistently, we found the Integrator complex component INTS11 elevated at promoters and enhancers upon loss of MTC or nuclear m6A binders. Taken together, our findings suggest that MTC-mediated m6A modification protects nascent RNAs from Integrator-mediated termination and promotes productive transcription, thus unraveling an unexpected layer of gene regulation imposed by RNA m6A modification.

Crystal structure of TET2-DNA complex: insight into TET-mediated 5mC oxidation.

TET proteins oxidize 5-methylcytosine (5mC) on DNA and play important roles in various biological processes. Mutations of TET2 are frequently observed in myeloid malignance. Here, we present the crystal structure of human TET2 bound to methylated DNA at 2.02 Å resolution. The structure shows that two zinc fingers bring the Cys-rich and DSBH domains together to form a compact catalytic domain. The Cys-rich domain stabilizes the DNA above the DSBH core. TET2 specifically recognizes CpG dinucleotide and shows substrate preference for 5mC in a CpG context. 5mC is inserted into the catalytic cavity with the methyl group orientated to catalytic Fe(II) for reaction. The methyl group is not involved in TET2-DNA contacts so that the catalytic cavity allows TET2 to accommodate 5mC derivatives for further oxidation. Mutations of Fe(II)/NOG-chelating, DNA-interacting, and zinc-chelating residues are frequently observed in human cancers. Our studies provide a structural basis for understanding the mechanisms of TET-mediated 5mC oxidation.

Tet3 CXXC domain and dioxygenase activity cooperatively regulate key genes for Xenopus eye and neural development.

Ten-Eleven Translocation (Tet) family of dioxygenases dynamically regulates DNA methylation and has been implicated in cell lineage differentiation and oncogenesis. Yet their functions and mechanisms of action in gene regulation and embryonic development are largely unknown. Here, we report that Xenopus Tet3 plays an essential role in early eye and neural development by directly regulating a set of key developmental genes. Tet3 is an active 5mC hydroxylase regulating the 5mC/5hmC status at target gene promoters. Biochemical and structural studies further demonstrate that the Tet3 CXXC domain is critical for specific Tet3 targeting. Finally, we show that the enzymatic activity and CXXC domain are both crucial for Tet3's biological function. Together, these findings define Tet3 as a transcription regulator and reveal a molecular mechanism by which the 5mC hydroxylase and DNA binding activities of Tet3 cooperate to control target gene expression and embryonic development.

METTL3 regulates heterochromatin in mouse embryonic stem cells.

METTL3 (methyltransferase-like 3) mediates the N6-methyladenosine (m6A) methylation of mRNA, which affects the stability of mRNA and its translation into protein1. METTL3 also binds chromatin2-4, but the role of METTL3 and m6A methylation in chromatin is not fully understood. Here we show that METTL3 regulates mouse embryonic stem-cell heterochromatin, the integrity of which is critical for silencing retroviral elements and for mammalian development5. METTL3 predominantly localizes to the intracisternal A particle (IAP)-type family of endogenous retroviruses. Knockout of Mettl3 impairs the deposition of multiple heterochromatin marks onto METTL3-targeted IAPs, and upregulates IAP transcription, suggesting that METTL3 is important for the integrity of IAP heterochromatin. We provide further evidence that RNA transcripts derived from METTL3-bound IAPs are associated with chromatin and are m6A-methylated. These m6A-marked transcripts are bound by the m6A reader YTHDC1, which interacts with METTL3 and in turn promotes the association of METTL3 with chromatin. METTL3 also interacts physically with the histone 3 lysine 9 (H3K9) tri-methyltransferase SETDB1 and its cofactor TRIM28, and is important for their localization to IAPs. Our findings demonstrate that METTL3-catalysed m6A modification of RNA is important for the integrity of IAP heterochromatin in mouse embryonic stem cells, revealing a mechanism of heterochromatin regulation in mammals.

Zc3h13 Regulates Nuclear RNA m6A Methylation and Mouse Embryonic Stem Cell Self-Renewal.

N6-methyladenosine (m6A) is an abundant modification in eukaryotic mRNA, regulating mRNA dynamics by influencing mRNA stability, splicing, export, and translation. However, the precise m6A regulating machinery still remains incompletely understood. Here we demonstrate that ZC3H13, a zinc-finger protein, plays an important role in modulating RNA m6A methylation in the nucleus. We show that knockdown of Zc3h13 in mouse embryonic stem cell significantly decreases global m6A level on mRNA. Upon Zc3h13 knockdown, a great majority of WTAP, Virilizer, and Hakai translocate to the cytoplasm, suggesting that Zc3h13 is required for nuclear localization of the Zc3h13-WTAP-Virilizer-Hakai complex, which is important for RNA m6A methylation. Finally, Zc3h13 depletion, as does WTAP, Virilizer, or Hakai, impairs self-renewal and triggers mESC differentiation. Taken together, our findings demonstrate that Zc3h13 plays a critical role in anchoring WTAP, Virilizer, and Hakai in the nucleus to facilitate m6A methylation and to regulate mESC self-renewal.

Glucose-regulated phosphorylation of TET2 by AMPK reveals a pathway linking diabetes to cancer.

Diabetes is a complex metabolic syndrome that is characterized by prolonged high blood glucose levels and frequently associated with life-threatening complications1,2. Epidemiological studies have suggested that diabetes is also linked to an increased risk of cancer3-5. High glucose levels may be a prevailing factor that contributes to the link between diabetes and cancer, but little is known about the molecular basis of this link and how the high glucose state may drive genetic and/or epigenetic alterations that result in a cancer phenotype. Here we show that hyperglycaemic conditions have an adverse effect on the DNA 5-hydroxymethylome. We identify the tumour suppressor TET2 as a substrate of the AMP-activated kinase (AMPK), which phosphorylates TET2 at serine 99, thereby stabilizing the tumour suppressor. Increased glucose levels impede AMPK-mediated phosphorylation at serine 99, which results in the destabilization of TET2 followed by dysregulation of both 5-hydroxymethylcytosine (5hmC) and the tumour suppressive function of TET2 in vitro and in vivo. Treatment with the anti-diabetic drug metformin protects AMPK-mediated phosphorylation of serine 99, thereby increasing TET2 stability and 5hmC levels. These findings define a novel 'phospho-switch' that regulates TET2 stability and a regulatory pathway that links glucose and AMPK to TET2 and 5hmC, which connects diabetes to cancer. Our data also unravel an epigenetic pathway by which metformin mediates tumour suppression. Thus, this study presents a new model for how a pernicious environment can directly reprogram the epigenome towards an oncogenic state, offering a potential strategy for cancer prevention and treatment.

Targeting the LSD1/KDM1 Family of Lysine Demethylases in Cancer and Other Human Diseases.

Lysine-specific demethylase 1 (LSD1) was the first histone demethylase discovered and the founding member of the flavin-dependent lysine demethylase family (KDM1). The human KDM1 family includes KDM1A and KDM1B, which primarily catalyze demethylation of histone H3K4me1/2. The KDM1 family is involved in epigenetic gene regulation and plays important roles in various biological and disease pathogenesis processes, including cell differentiation, embryonic development, hormone signaling, and carcinogenesis. Malfunction of many epigenetic regulators results in complex human diseases, including cancers. Regulators such as KDM1 have become potential therapeutic targets because of the reversibility of epigenetic control of genome function. Indeed, several classes of KDM1-selective small molecule inhibitors have been developed, some of which are currently in clinical trials to treat various cancers. In this chapter, we review the discovery, biochemical, and molecular mechanisms, atomic structure, genetics, biology, and pathology of the KDM1 family of lysine demethylases. Focusing on cancer, we also provide a comprehensive summary of recently developed KDM1 inhibitors and related preclinical and clinical studies to provide a better understanding of the mechanisms of action and applications of these KDM1-specific inhibitors in therapeutic treatment.

TET2 promotes anti-tumor immunity by governing G-MDSCs and CD8+ T-cell numbers.

The host immune response is a fundamental mechanism for attenuating cancer progression. Here we report a role for the DNA demethylase and tumor suppressor TET2 in host anti-tumor immunity. Deletion of Tet2 in mice elevates IL-6 levels upon tumor challenge. Elevated IL-6 stimulates immunosuppressive granulocytic myeloid-derived suppressor cells (G-MDSCs), which in turn reduce CD8+ T cells upon tumor challenge. Consequently, systematic knockout of Tet2 in mice leads to accelerated syngeneic tumor growth, which is constrained by anti-PD-1 blockade. Removal of G-MDSCs by the anti-mouse Ly6g antibodies restores CD8+ T-cell numbers in Tet2-/- mice and reboots their anti-tumor activity. Importantly, anti-IL-6 antibody treatment blocks the expansion of G-MDSCs and inhibits syngeneic tumor growth. Collectively, these findings reveal a TET2-mediated IL-6/G-MDSCs/CD8+ T-cell immune response cascade that safeguards host adaptive anti-tumor immunity, offering a cell non-autonomous mechanism of TET2 for tumor suppression.

BS69/ZMYND11 reads and connects histone H3.3 lysine 36 trimethylation-decorated chromatin to regulated pre-mRNA processing.

BS69 (also called ZMYND11) contains tandemly arranged PHD, BROMO, and PWWP domains, which are chromatin recognition modalities. Here, we show that BS69 selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3) via its chromatin-binding domains. We further identify BS69 association with RNA splicing regulators, including the U5 snRNP components of the spliceosome, such as EFTUD2. Remarkably, RNA sequencing shows that BS69 mainly regulates intron retention (IR), which is the least understood RNA alternative splicing event in mammalian cells. Biochemical and genetic experiments demonstrate that BS69 promotes IR by antagonizing EFTUD2 through physical interactions. We further show that regulation of IR by BS69 also depends on its binding to H3K36me3-decorated chromatin. Taken together, our study identifies an H3.3K36me3-specific reader and a regulator of IR and reveals that BS69 connects histone H3.3K36me3 to regulated RNA splicing, providing significant, important insights into chromatin regulation of pre-mRNA processing.

Nono deficiency compromises TET1 chromatin association and impedes neuronal differentiation of mouse embryonic stem cells.

NONO is a DNA/RNA-binding protein, which plays a critical regulatory role during cell stage transitions of mouse embryonic stem cells (mESCs). However, its function in neuronal lineage commitment and the molecular mechanisms of its action in such processes are largely unknown. Here we report that NONO plays a key role during neuronal differentiation of mESCs. Nono deletion impedes neuronal lineage commitment largely due to a failure of up-regulation of specific genes critical for neuronal differentiation. Many of the NONO regulated genes are also DNA demethylase TET1 targeted genes. Importantly, re-introducing wild type NONO to the Nono KO cells, not only restores the normal expression of the majority of NONO/TET1 coregulated genes but also rescues the defective neuronal differentiation of Nono-deficient mESCs. Mechanistically, our data shows that NONO directly interacts with TET1 via its DNA binding domain and recruits TET1 to genomic loci to regulate 5-hydroxymethylcytosine levels. Nono deletion leads to a significant dissociation of TET1 from chromatin and dysregulation of DNA hydroxymethylation of neuronal genes. Taken together, our findings reveal a key role and an epigenetic mechanism of action of NONO in regulation of TET1-targeted neuronal genes, offering new functional and mechanistic understanding of NONO in stem cell functions, lineage commitment and specification.

Publisher Correction: N6-Methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation.

In the version of this article originally published, the references were incorrectly re-ordered during production. The hyphen in "N6-methyladenosine" in the title was also superscript. The errors have been corrected in the HTML and PDF versions of the paper.

N6-Methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation.

N6-Methyladenosine (m6A) RNA modification is present in messenger RNAs (mRNA), ribosomal RNAs (rRNA), and spliceosomal RNAs (snRNA) in humans. Although mRNA m6A modifications have been extensively studied and shown to play critical roles in many cellular processes, the identity of m6A methyltransferases for rRNAs and the function of rRNA m6A modifications are unknown. Here we report a new m6A methyltransferase, ZCCHC4, which primarily methylates human 28S rRNA and also interacts with a subset of mRNAs. ZCCHC4 knockout eliminates m6A4220 modification in 28S rRNA, reduces global translation, and inhibits cell proliferation. We also find that ZCCHC4 protein is overexpressed in hepatocellular carcinoma tumors, and ZCCHC4 knockout significantly reduces tumor size in a xenograft mouse model. Our results highlight the functional significance of an rRNA m6A modification in translation and in tumor biology.

LSD2/KDM1B and its cofactor NPAC/GLYR1 endow a structural and molecular model for regulation of H3K4 demethylation.

Dynamic regulation of histone methylation represents a fundamental epigenetic mechanism underlying eukaryotic gene regulation, yet little is known about how the catalytic activities of histone demethylases are regulated. Here, we identify and characterize NPAC/GLYR1 as an LSD2/KDM1b-specific cofactor that stimulates H3K4me1 and H3K4me2 demethylation. We determine the crystal structures of LSD2 alone and LSD2 in complex with the NPAC linker region in the absence or presence of histone H3 peptide, at resolutions of 2.9, 2.0, and 2.25 Å, respectively. These crystal structures and further biochemical characterization define a dodecapeptide of NPAC (residues 214-225) as the minimal functional unit for its cofactor activity and provide structural determinants and a molecular mechanism underlying the intrinsic cofactor activity of NPAC in stimulating LSD2-catalyzed H3K4 demethylation. Thus, these findings establish a model for how a cofactor directly regulates histone demethylation and will have a significant impact on our understanding of catalytic-activity-based epigenetic regulation.

Kdm1a promotes SCLC progression by transcriptionally silencing the tumor suppressor Rest.

Small cell lung carcinoma (SCLC) is one of the deadliest cancer types, with a 5-year survival rate less than 10%. Kdm1a/Lsd1 has recently been implicated as a potential therapeutic target for SCLC. However, the underlying molecular mechanism by which Kdm1a promotes the oncogenesis of SCLC has not been fully understood. Kdm1a is significantly elevated in most human SCLC specimens, whereas Rest, a tumor suppressor and neuronal repressive transcriptional factor, is typically inactivated. Knock-out of Kdm1a (Kdm1a-KO) in mouse SCLC cell lines resulted in the suppression of cell growth and soft agar colony formation. RNA-Seq analysis of the Kdm1a-KO cells revealed significant repression of a program of neuroendocrine signature genes, and conversely, a significant upregulation of a network of genes capable of inhibiting tumor cell growth. Rest was identified among the top 10 upregulated genes in Kdm1a-KO cells. The treatment of the SCLC cells with Kdm1a demethylase inhibitors resulted in a dramatic up-regulation of Rest similar to the extent of that in Kdm1a-KO cells. Importantly, accompanying the restored expression of the SCLC signature genes, knock-out of Rest in Kdm1a-KO cells rescued the restricted cell growth and soft agar colony formation. Taken together, these novel findings show that Kdm1a is a key transcriptional repressor of Rest, and that suppression of SCLC progression by the targeted inhibition of Kdm1a depends on the reactivation of Rest, suggesting a new strategy for effective SCLC treatment by targeting the Kdm1a/Rest molecular pathway.

PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression.

Histone methylation occurs on both lysine and arginine residues, and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD finger (PHD(UHRF1)), an important regulator of DNA CpG methylation, as a histone H3 unmodified arginine 2 (H3R2) recognition modality. This conclusion is based on binding studies and cocrystal structures of PHD(UHRF1) bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1's ability to repress its direct target gene expression is dependent on PHD(UHRF1) binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function.

Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells.

DNA methylation at the 5 position of cytosine (5mC) in the mammalian genome is a key epigenetic event critical for various cellular processes. The ten-eleven translocation (Tet) family of 5mC-hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), offers a way for dynamic regulation of DNA methylation. Here we report that Tet1 binds to unmodified C or 5mC- or 5hmC-modified CpG-rich DNA through its CXXC domain. Genome-wide mapping of Tet1 and 5hmC reveals mechanisms by which Tet1 controls 5hmC and 5mC levels in mouse embryonic stem cells (mESCs). We also uncover a comprehensive gene network influenced by Tet1. Collectively, our data suggest that Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting 5mC to 5hmC through hydroxylase activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 targets, ultimately contributing to mESC differentiation and the onset of embryonic development.

ERα is a negative regulator of PD-L1 gene transcription in breast cancer.

The programmed death-ligand 1 (PD-L1) expression by tumors results in potent antitumor immune suppression through binding to programmed death-1 (PD-1) on T cells and subsequent inhibition of T cells activity. Although recent pathological studies have shown that PD-L1 is actively expressed in certain ERα-negative breast cancer, little is known about whether ER signaling regulates PD-L1 gene expression. Here, we investigated the relationship between ERα and PD-L1 in breast cancer. Analysis of Comprehensive Cell Line Encyclopedia (CCLE) data showed that the average mRNA level of PD-L1 in ERα-positive breast cancer cell lines was significantly lower than that in ERα-negative breast cancer cell lines. E2 treatment inhibited PD-L1 mRNA expression in hormone-depleted ERα-positive MCF7 cells. Moreover, ectopic expression of ERα in triple-negative MDA-MB-231 cells reduced PD-L1 mRNA and protein expression. Consistently, analysis of The Cancer Genome Atlas (TCGA) data revealed an inverse correlation between ERα and PD-L1 expression in ERα-positive breast cancer. Taken together, our results identify ERα as a negative regulator of PD-L1 gene transcription in breast cancer cells, suggesting that ERα loss-of-function may facilitate the immune evasion of breast cancer cells via up-regulation of PD-L1.

Human LSD2/KDM1b/AOF1 regulates gene transcription by modulating intragenic H3K4me2 methylation.

Dynamic histone H3K4 methylation is an important epigenetic component of transcriptional regulation. However, most of our current understanding of this histone mark is confined to the regulation of transcriptional initiation. We now show that human LSD2/KDM1b/AOF1, the human homolog of LSD1, is an H3K4me1/2 demethylase that specifically regulates histone H3K4 methylation within intragenic regions of its target genes. Genome-wide mapping reveals that LSD2 associates predominantly with the gene bodies of actively transcribed genes, but is markedly absent from promoters. Depletion of endogenous LSD2 results in an increase of H3K4me2 as well as a decrease of H3K9me2 at LSD2-binding sites and a consequent dysregulation of target gene transcription. Furthermore, characterization of the LSD2 complex reveals that LSD2 forms active complexes with euchromatic histone methyltransferases G9a and NSD3 as well as cellular factors involved in transcription elongation. These data provide a possible molecular mechanism linking LSD2 to transcriptional regulation after initiation.

A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands.

Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs' role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT), but not catalytic inactive mutant (Mut), TET2 in low-TET-expressing cells results in an increase in the level of 5hmC with accompanying DNA demethylation at a subset of CGIs. Most importantly, this alteration is sufficient in making de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of essential developmental gene promoters, which are located within CGIs and are previously silenced due to DNA methylation. On the other hand, deletion of Tet1 and Tet2 in mouse embryonic stem (ES) cells results in an apparent loss of H3K27me3 at bivalent domains, which are associated with a particular set of key developmental gene promoters. Collectively, this study demonstrates the critical role of TET proteins in regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at CGIs associated with developmental genes.