Hong Wen

ZMYND11 links histone H3.3K36me3 to transcription elongation and tumour suppression

Recognition of modified histones by ‘reader’ proteins plays a critical role in the regulation of chromatin. H3K36 trimethylation (H3K36me3) is deposited onto the nucleosomes in the transcribed regions after RNA polymerase II elongation. In yeast, this mark in turn recruits epigenetic regulators to reset the chromatin to a relatively repressive state, thus suppressing cryptic transcription. However, much less is known about the role of H3K36me3 in transcription regulation in mammals. This is further complicated by the transcription-coupled incorporation of the histone variant H3.3 in gene bodies. Here we show that the candidate tumour suppressor ZMYND11 specifically recognizes H3K36me3 on H3.3 (H3.3K36me3) and regulates RNA polymerase II elongation. Structural studies show that in addition to the trimethyl-lysine binding by an aromatic cage within the PWWP domain, the H3.3-dependent recognition is mediated by the encapsulation of the H3.3-specific ‘Ser 31’ residue in a composite pocket formed by the tandem bromo–PWWP domains of ZMYND11. Chromatin immunoprecipitation followed by sequencing shows a genome-wide co-localization of ZMYND11 with H3K36me3 and H3.3 in gene bodies, and its occupancy requires the pre-deposition of H3.3K36me3. Although ZMYND11 is associated with highly expressed genes, it functions as an unconventional transcription co-repressor by modulating RNA polymerase II at the elongation stage. ZMYND11 is critical for the repression of a transcriptional program that is essential for tumour cell growth; low expression levels of ZMYND11 in breast cancer patients correlate with worse prognosis. Consistently, overexpression of ZMYND11 suppresses cancer cell growth in vitro and tumour formation in mice. Together, this study identifies ZMYND11 as an H3.3-specific reader of H3K36me3 that links the histone-variant-mediated transcription elongation control to tumour suppression.

Recognition of Histone H3K4 Trimethylation by the Plant Homeodomain of PHF2 Modulates Histone Demethylation

Distinct lysine methylation marks on histones create dynamic signatures deciphered by the “effector” modules, although the underlying mechanisms remain unclear. We identified the plant homeodomain- and Jumonji C domain-containing protein PHF2 as a novel histone H3K9 demethylase. We show in biochemical and crystallographic analyses that PHF2 recognizes histone H3K4 trimethylation through its plant homeodomain finger and that this interaction is essential for PHF2 occupancy and H3K9 demethylation at rDNA promoters. Our study provides molecular insights into the mechanism by which distinct effector domains within a protein cooperatively modulate the “cross-talk” of histone modifications.

Regulation of estrogen receptor α by histone methyltransferase SMYD2-mediated protein methylation

Significance Histone-modifying enzymes play an important role in regulating chromatin-associated processes such as transcription. In addition to modifying histones, these enzymes control gene expression through modifying nonhistone proteins, including transcription factors. In this study, we show that SET and MYND domain containing 2 (SMYD2), a histone H3K4 and H3K36 methyltransferase, directly methylates estrogen receptor alpha (ERα) protein at lysine 266 and represses ERα transactivation activity. Upon estrogen activation, this repressive mark is relieved by the histone H3K4 demethylase lysine-specific demethylase 1, followed by p300/cAMP response element-binding protein–binding protein (CBP)-mediated protein acetylation. Our study suggests that the cross-talk of distinct posttranslational modifications in the hinge region of ERα plays an important role in fine tuning the functions of ERα at chromatin in hormone response. Estrogen receptor alpha (ERα) is a ligand-activated transcription factor. Upon estrogen stimulation, ERα recruits a number of coregulators, including both coactivators and corepressors, to the estrogen response elements, modulating gene activation or repression. Most coregulator complexes contain histone-modifying enzymes to control ERα target gene expression in an epigenetic manner. In addition to histones, these epigenetic modifiers can modify nonhistone proteins including ERα, thereby constituting another layer of transcriptional regulation. Here we show that SET and MYND domain containing 2 (SMYD2), a histone H3K4 and H3K36 methyltransferase, directly methylates ERα protein at lysine 266 (K266) both in vitro and in cells. In breast cancer MCF7 cells, SMYD2 attenuates the chromatin recruitment of ERα to prevent ERα target gene activation under an estrogen-depleted condition. Importantly, the SMYD2-mediated repression of ERα target gene expression is mediated by the methylation of ERα at K266 in the nucleus, but not the methylation of histone H3K4. Upon estrogen stimulation, ERα–K266 methylation is diminished, thereby enabling p300/cAMP response element-binding protein–binding protein to acetylate ERα at K266, which is known to promote ERα transactivation activity. Our study identifies a previously undescribed inhibitory methylation event on ERα. Our data suggest that the dynamic cross-talk between SMYD2-mediated ERα protein methylation and p300/cAMP response element-binding protein–binding protein-dependent ERα acetylation plays an important role in fine-tuning the functions of ERα at chromatin and the estrogen-induced gene expression profiles.

ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia

Cancer cells are characterized by aberrant epigenetic landscapes and often exploit chromatin machinery to activate oncogenic gene expression programs. Recognition of modified histones by ‘reader’ proteins constitutes a key mechanism underlying these processes; therefore, targeting such pathways holds clinical promise, as exemplified by the development of bromodomain and extra-terminal (BET) inhibitors. We recently identified the YEATS domain as an acetyl-lysine-binding module, but its functional importance in human cancer remains unknown. Here we show that the YEATS domain-containing protein ENL, but not its paralogue AF9, is required for disease maintenance in acute myeloid leukaemia. CRISPR–Cas9-mediated depletion of ENL led to anti-leukaemic effects, including increased terminal myeloid differentiation and suppression of leukaemia growth in vitro and in vivo. Biochemical and crystal structural studies and chromatin-immunoprecipitation followed by sequencing analyses revealed that ENL binds to acetylated histone H3, and co-localizes with H3K27ac and H3K9ac on the promoters of actively transcribed genes that are essential for leukaemia. Disrupting the interaction between the YEATS domain and histone acetylation via structure-based mutagenesis reduced the recruitment of RNA polymerase II to ENL-target genes, leading to the suppression of oncogenic gene expression programs. Notably, disrupting the functionality of ENL further sensitized leukaemia cells to BET inhibitors. Together, our data identify ENL as a histone acetylation reader that regulates oncogenic transcriptional programs in acute myeloid leukaemia, and suggest that displacement of ENL from chromatin may be a promising epigenetic therapy, alone or in combination with BET inhibitors, for aggressive leukaemia.

G9a-mediated methylation of ERα links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression

The euchromatin histone methyltransferase 2 (also known as G9a) methylates histone H3K9 to repress gene expression, but it also acts as a coactivator for some nuclear receptors. The molecular mechanisms underlying this activation remain elusive. Here we show that G9a functions as a coactivator of the endogenous oestrogen receptor α (ERα) in breast cancer cells in a histone methylation-independent manner. G9a dimethylates ERα at K235 both in vitro and in cells. Dimethylation of ERαK235 is recognized by the Tudor domain of PHF20, which recruits the MOF histone acetyltransferase (HAT) complex to ERα target gene promoters to deposit histone H4K16 acetylation promoting active transcription. Together, our data suggest the molecular mechanism by which G9a functions as an ERα coactivator. Along with the PHF20/MOF complex, G9a links the crosstalk between ERα methylation and histone acetylation that governs the epigenetic regulation of hormonal gene expression.

ZMYND11: an H3.3-specific reader of H3K36me3.

ZMYND11 (also known as BS69), a candidate tumor suppressor that interacts with adenovirus E1A protein, contains a tandem “reader” modules of histone modifications, including a plant homeodomain (PHD) zinc finger, a bromodomain and a PWWP domain. Recently, we found that this tandem PHD-bromo-PWWP domains of ZMYND11 recognize histone H3K36 trimethylation (H3K36me3) and importantly, this recognition is specific for the histone variant H3.3. The PWWP domain is predominantly responsible for the readout of the trimethyl moiety, whereas a second composite pocket at the junction of the bromodomain, PWWP, and an embedded zinc finger motif recognizes the “Serine 31-Threonine 32 (S31T32)” segment, thus accounting for the H3.3 specificity. The PWWP domain belongs to the “Royal Family” of protein domains that also include Tudor, chromo, and MBT domains. It was initially identified as a DNA-binding domain, whereas recently it has been shown that the PWWP domain, like other “Royal Family” members, also possesses methyl-lysine recognition activity, especially for methylation on histone H3K36. Notably, ZMYND11 distinguishes itself from these PWWP proteins in that it is a histone variant H3.3-specific reader of K36me3. The PWWP domain adopts a 5-bladed β-barrel fold with an extended C-terminal α-helix (α P ). Histone H3K36me3 is recognized by a conserved aromatic cage formed at the center of the PWWP β-barrel. The ZMYND11 bromodomain adopts a typical 4 α-helical bundle fold but lacks canonical key residues for acetyllysine recognition. One unique feature of ZMYND11’s reader module is the newly identified “C2CH”-type zinc finger motif embedded between bromo and PWWP. It stabilizes the V-shaped compact fold of the bromo-PWWP cassette and forms a composite pocket for the H3.3S31 recognition. Moreover, the surface of ZMYND11 bromo-PWWP is electrostatically patchy, implying a co-recognition mechanism of histone H3K36me3 and DNA in nucleosomal context. In addition, although the structure of H3K36me3-bound ZMYND11 tandem PHD-bromo-PWWP domains has yet to be determined, our biochemical studies demonstrated that the PHD finger also facilitates ZMYND11 association with H3K36me3 both in vitro and in vivo. It remains an interesting topic for future studies to explore the exact functions of the PHD finger and the bromodomain of ZMYND11 and their contributions to the combinatorial readout of H3.3K36me3 at the nucleosomal level in concert with the PWWP domain. H3.3 is a conserved histone H3 variant that is structurally close to the canonical H3. It differs from canonical H3.2 and H3.1 at only 4 or 5 amino acids, respectively, with 3 or 4 located in the core histone fold regions and only one residue (S31) located in the N-terminal unstructured tail. The “A87...I89G90” residues located in the histone-fold domain account for particular properties of histone H3.3, such as nucleosome stability and chaperone recognitions. Although H3.3S31 is phosphorylated during mitosis, the role of this modification or S31 itself in transcriptional regulation is currently unknown. To our knowledge, our study is the first to define a critical role of H3.3 S31 in substrate recognition. Substitution of H3.3 S31 with Alanine (A31) present in canonical H3.1 severely impacted the binding of ZMYND11 to the H3K36me3 peptide and histones in vitro and in cells, respectively, suggesting that the H3.3-specific S31 plays an indispensable role in ZMYND11’s combinatorial recognition of H3.3K36me3. Our ChIP-seq analysis revealed that in accordance with previous reports, H3.3 is enriched in the transcribed regions in addition to regulatory elements such as promoters and enhancers, and gene bodyenriched H3.3 correlates with the distribution of H3K36me3. The combination of H3K36me3 and H3.3 establishes a unique epigenetic state that defines the genomic distribution of ZMYND11, offering a spatiotemporal control of gene expression for both normal and neoplastic growth. Although ZMYND11 is associated with genes with high H3K36me3 and expression levels, knockdown of ZMYND11 caused only a moderate change in gene expression. We propose that ZMYND11, rather than working as an essential “on/off switch”, mainly functions to “fine-tune” gene expression. A similar “fine-tuning” mechanism has also been proposed for the actions of histone demethylases in transcriptional

YEATS2 links histone acetylation to tumorigenesis of non-small cell lung cancer

Recognition of modified histones by “reader” proteins constitutes a key mechanism regulating diverse chromatin-associated processes important for normal and neoplastic development. We recently identified the YEATS domain as a novel acetyllysine-binding module; however, the functional importance of YEATS domain-containing proteins in human cancer remains largely unknown. Here, we show that the YEATS2 gene is highly amplified in human non-small cell lung cancer (NSCLC) and is required for cancer cell growth and survival. YEATS2 binds to acetylated histone H3 via its YEATS domain. The YEATS2-containing ATAC complex co-localizes with H3K27 acetylation (H3K27ac) on the promoters of actively transcribed genes. Depletion of YEATS2 or disruption of the interaction between its YEATS domain and acetylated histones reduces the ATAC complex-dependent promoter H3K9ac levels and deactivates the expression of essential genes. Taken together, our study identifies YEATS2 as a histone H3K27ac reader that regulates a transcriptional program essential for NSCLC tumorigenesis.Histone modification recognition is an important mechanism for gene expression regulation in cancer. Here, the authors identify YEATS2 as a histone H3K27ac reader, regulating a transcriptional program essential for tumorigenesis in human non-small cell lung cancer.

The ZZ-type zinc finger of ZZZ3 modulates the ATAC complex-mediated histone acetylation and gene activation.

Recognition of histones by epigenetic readers is a fundamental mechanism for the regulation of chromatin and transcription. Most reader modules target specific post-translational modifications on histones. Here, we report the identification of a reader of histone H3, the ZZ-type zinc finger (ZZ) domain of ZZZ3, a subunit of the Ada-two-A-containing (ATAC) histone acetyltransferase complex. The solution NMR structure of the ZZ in complex with the H3 peptide reveals a unique binding mechanism involving caging of the N-terminal Alanine 1 of histone H3 in an acidic cavity of the ZZ domain, indicating a specific recognition of H3 versus other histones. Depletion of ZZZ3 or disruption of the ZZ-H3 interaction dampens ATAC-dependent promoter histone H3K9 acetylation and target gene expression. Overall, our study identifies the ZZ domain of ZZZ3 as a histone H3 reader that is required for the ATAC complex-mediated maintenance of histone acetylation and gene activation.Histones are recognized by epigenetic readers, which play essential roles in regulation of chromatin and transcription. Here the authors provide evidence that the ZZ-type zinc finger domain of ZZZ3 functions as a reader of histone H3, which is required for the ATAC complex-mediated maintenance of histone acetylation and gene activation.

CBP/p300: intramolecular and intermolecular regulations

BackgroundCREB binding protein (CBP) and its close paralogue p300 are transcriptional coactivators with intrinsic acetyltransferase activity. Both CBP/p300 play critical roles in development and diseases. The enzymatic and biological functions of CBP/p300 are tightly regulated by themselves and by external factors. However, a comprehensive up-to-date review of the intramolecular and intermolecular regulations is lacking.ObjectiveTo summarize the molecular mechanisms regulating CBP/p300s functions.MethodsA systematic literature search was conducted using the PubMed (https://doi.org/www.ncbi.nlm.nih.gov/pubmed/) for literatures published during 1985–2018. Keywords “CBP regulation” or “p300 regulation” were used for the search.ResultsThe functions of CBP/p300, especially their acetyltransferase activity and chromatin association, are regulated both intramolecularly by their autoinhibitory loop (AIL), bromodomain, and PHD-RING region and intermolecularly by their interacting partners. The intramolecular mechanisms equip CBP/p300 with the capability of self-regulation while the intermolecular mechanisms allow them to respond to various cell signaling pathways.ConclusionInvestigations into those regulation mechanisms are crucial to our understanding of CBP/p300s role in development and pathogenesis. Pharmacological interventions targeting these regulatory mechanisms have therapeutic potentials.

Gas41 links histone acetylation to H2A.Z deposition and maintenance of embryonic stem cell identity

The histone variant H2A.Z is essential for maintaining embryonic stem cell (ESC) identity in part by keeping developmental genes in a poised bivalent state. However, how H2A.Z is deposited into the bivalent domains remains unknown. In mammals, two chromatin remodeling complexes, Tip60/p400 and SRCAP, exchange the canonical histone H2A for H2A.Z in the chromatin. Here we show that Glioma Amplified Sequence 41 (Gas41), a shared subunit of the two H2A.Z-depositing complexes, functions as a reader of histone lysine acetylation and recruits Tip60/p400 and SRCAP to deposit H2A.Z into specific chromatin regions including bivalent domains. The YEATS domain of Gas41 bound to acetylated histone H3K27 and H3K14 both in vitro and in cells. The crystal structure of the Gas41 YEATS domain in complex with the H3K27ac peptide revealed that, similar to the AF9 and ENL YEATS domains, Gas41 YEATS forms a serine-lined aromatic cage for acetyllysine recognition. Consistently, mutations in the aromatic residues of the Gas41 YEATS domain abrogated the interaction. In mouse ESCs, knockdown of Gas41 led to flattened morphology of ESC colonies, as the result of derepression of differentiation genes. Importantly, the abnormal morphology was rescued by expressing wild-type Gas41, but not the YEATS domain mutated counterpart that does not recognize histone acetylation. Mechanically, we found that Gas41 depletion led to reduction of H2A.Z levels and a concomitant reduction of H3K27me3 levels on bivalent domains. Together, our study reveals an essential role of the Gas41 YEATS domain in linking histone acetylation to H2A.Z deposition and maintenance of ESC identity.