H3K4 Methyltransferase Research Articles

H3K36 methyltransferase NSD1 protects against osteoarthritis through regulating chondrocyte differentiation and cartilage homeostasis.

Osteoarthritis (OA) is one of the most common joint diseases, there are no effective disease-modifying drugs, and the pathological mechanisms of OA need further investigation. Here, we show that H3K36 methylations were decreased in senescent chondrocytes and age-related osteoarthritic cartilage. Prrx1-Cre inducible H3.3K36M transgenic mice showed articular cartilage destruction and osteophyte formation. Conditional knockout Nsd1Prrx1-Cre mice, but not Nsd2Prrx1-Cre or Setd2Prrx1-Cre mice, replicated the phenotype of K36M/+; Prrx1-Cre mice. Immunostaining results showed decreased anabolic and increased catabolic activities in Nsd1Prrx1-Cre mice, along with decreased chondrogenic differentiation. Transcriptome and ChIP-seq data revealed that Osr2 was a key factor affected by Nsd1. Intra-articular delivery of Osr2 adenovirus effectively improved the homeostasis of articular cartilage in Nsd1Prrx1-Cre mice. In human osteoarthritic cartilages, both mRNA and protein levels of NSD1 and OSR2 were decreased. Our results indicate that NSD1-induced H3K36 methylations and OSR2 expression play important roles in articular cartilage homeostasis and OA. Targeting H3K36 methylation and OSR2 would be a novel strategy for OA treatment.

Chaetocin-mediated SUV39H1 inhibition targets stemness and oncogenic networks of diffuse midline gliomas and synergizes with ONC201.

Diffuse intrinsic pontine gliomas (DIPG/DMG) are devastating pediatric brain tumors with extraordinarily limited treatment options and uniformly fatal prognosis. Histone H3K27M mutation is a common recurrent alteration in DIPG and disrupts epigenetic regulation. We hypothesize that genome-wide H3K27M-induced epigenetic dysregulation makes tumors vulnerable to epigenetic targeting. We performed a screen of compounds targeting epigenetic enzymes to identify potential inhibitors for the growth of patient-derived DIPG cells. We further carried out transcriptomic and genomic landscape profiling including RNA-seq and CUT&RUN-seq as well as shRNA-mediated knockdown to assess the effects of chaetocin and SUV39H1, a target of chaetocin, on DIPG growth. High-throughput small-molecule screening identified an epigenetic compound chaetocin as a potent blocker of DIPG cell growth. Chaetocin treatment selectively decreased proliferation and increased apoptosis of DIPG cells and significantly extended survival in DIPG xenograft models, while restoring H3K27me3 levels. Moreover, the loss of H3K9 methyltransferase SUV39H1 inhibited DIPG cell growth. Transcriptomic and epigenomic profiling indicated that SUV39H1 loss or inhibition led to the downregulation of stemness and oncogenic networks including growth factor receptor signaling and stemness-related programs; however, D2 dopamine receptor (DRD2) signaling adaptively underwent compensatory upregulation conferring resistance. Consistently, a combination of chaetocin treatment with a DRD2 antagonist ONC201 synergistically increased the antitumor efficacy. Our studies reveal a therapeutic vulnerability of DIPG cells through targeting the SUV39H1-H3K9me3 pathway and compensatory signaling loops for treating this devastating disease. Combining SUV39H1-targeting chaetocin with other agents such as ONC201 may offer a new strategy for effective DIPG treatment.

Histone lysine methyltransferases MpDot1 and MpSet9 are involved in the production of lovastatin and MonAzPs by histone crosstalk modification

Increasing data suggested that histone methylation modification plays an important role in regulating biosynthesis of secondary metabolites (SMs). Monascus spp. have been applied to produce hypolipidemic drug lovastatin (also called monacolin K, MK) and edible Monascus-type azaphilone pigments (MonAzPs). However, little is known about how histone methylation regulates MK and MonAzPs. In this study, we constructed H3K9 methyltransferase deletion strain ΔMpDot1 and H4K20 methyltransferase deletion strain ΔMpSet9 using Monascus pilosus MS-1 as the parent. The result showed that deletion of MpDot1 reduced the production of MK and MonAzPs, and deletion of MpSet9 increased MonAzPs production. Real-time quantitative PCR (RT-qPCR) showed inactivation of mpdot1 and mpset9 disturbed the expression of genes responsible for the biosynthesis of MK and MonAzPs. Western blot suggested that deletion of MpDot1 reduced H3K79me and H4K16ac, and deletion of MpSet9 decreased H4K20me3 and increased H4pan acetylation. Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) showed ΔMpDot1 strain and ΔMpSet9 strain reduced the enrichment of H3K79me2 and H4K20me3 in the promoter regions of key genes for MK and MonAzPs biosynthesis, respectively. These results suggested that MpDot1 and MpSet9 affected the synthesis of SMs by regulating gene transcription and histone crosstalk, providing alternative approach for regulation of lovastatin and MonAzPs.

GATA1 and GATA2 Sense/Antisense Transcriptional Switching in Early Hematopoietic Development

The GATA1 and GATA2 genes play essential roles in hematopoietic development. GATA1 is the master regulator of erythropoiesis and GATA2 is required for the expansion and survival of hematopoietic progenitor and stem cells, as well as the development of mast cells. A switch in GATA expression occurs during erythropoiesis, with silencing of the GATA2 gene and increased expression of GATA1. A GATA2 antisense transcript ( GATA2-AS1) has been associated with silencing of GATA2 expression. We have identified a novel GATA1 antisense promoter located in the first intron that opposes the GATA1 promoter and generates a spliced antisense transcript complementary to the SUV39H1 H3K9 methyltransferase gene. The competition between opposing sense and antisense promotersin the GATA1 and GATA2 genes may play a central role in the switching of GATA expression that occurs during hematopoiesis. To address this hypothesis, gene expression analysis of cells expressing sense versus antisense transcripts from the GATA1 or GATA2 genes was performed using 5'scRNAseq data from in vitro differentiated CD34 cells. Cells expressing only sense or only antisense GATA1 transcriptswere readily detected and comprised the majority (90%) of cells with GATA1 transcripts. Differential gene expression analysis of cells with GATA1 sense-only versus cells with GATA1 antisense-only showed association of sense transcription with erythrocyte genes, whereas antisense expression was associated with a mast cell phenotype and upregulation of GATA2 transcript levels. Conversely, GATA2 antisense expressing cells had increased GATA1 transcription and an erythrocyte phenotype, and GATA2 sense expressing cells demonstrated mast cell-specific gene expression. Single cell RNAseq analysis of HL60 cells induced to differentiate in vitro identified significant changes in GATA2 gene expression. Induction of granulocyte differentiation in HL60 cells treated with DMSO resulted in increased GATA2 antisense transcription, whereas PMA induction of monocyte differentiation was associated with increased GATA2 sense transcription. Taken together these results indicate a central role for sense/antisense promoter switching in hematopoiesis.

Uncoupling the distinct functions of HP1 proteins during heterochromatin establishment and maintenance

H3K9 methylation (H3K9me) marks transcriptionally silent genomic regions called heterochromatin. HP1 proteins are required to establish and maintain heterochromatin. HP1 proteins bind to H3K9me, recruit factors that promote heterochromatin formation, and oligomerize to form phase-separated condensates. We do not understand how these different HP1 properties are involved in establishing and maintaining transcriptional silencing. Here, we demonstrate that the S.pombe HP1 homolog, Swi6, can be completely bypassed to establish silencing at ectopic and endogenous loci when an H3K4 methyltransferase, Set1, and an H3K14 acetyltransferase, Mst2, are deleted. Deleting Set1 and Mst2 enhances Clr4 enzymatic activity, leading to higher H3K9me levels and spreading. In contrast, Swi6 and its capacity to oligomerize were indispensable during epigenetic maintenance. Our results demonstrate the role of HP1 proteins in regulating histone modification crosstalk during establishment and identify a genetically separable function in maintaining epigenetic memory.

G-protein coupled receptor 5C (GPRC5C) is required for osteoblast differentiation and responds to EZH2 inhibition and multiple osteogenic signals.

Osteoblast differentiation is epigenetically suppressed by the H3K27 methyltransferase EZH2, and induced by the morphogen BMP2 and transcription factor RUNX2. These factors also regulate distinct G protein coupled receptors (GPRCs; e.g., PTH1R, GPR30/GPER1). Because GPRCs transduce many physiological stimuli, we examined whether BMP2 or EZH2 inhibition (i.e., GSK126) regulates other GPRC genes in osteoblasts. RNA-seq screening of >400 mouse GPRC-related genes showed that many GPRCs are downregulated during osteogenic differentiation. The orphan receptor GPRC5C, along with a small subset of other GPRCs, is induced by BMP2 or GSK126 during Vitamin C dependent osteoblast differentiation, but not by all-trans retinoic acid. ChIP-seq analysis revealed that GSK126 reduces H3K27me3 levels at the GPRC5C gene locus in differentiating MC3T3-E1 osteoblasts, consistent with enhanced GPRC5C mRNA expression. Loss of function analyses revealed that shRNA-mediated depletion of GPRC5C decreases expression of bone markers (e.g., BGLAP and IBSP) and mineral deposition in response to BMP2 or GSK126. GPRC5C mRNA was found to be reduced in the osteopenic bones of KLF10 null mice which have compromised BMP2 signaling. GPRC5C mRNA is induced by the bone-anabolic activity of 17β-estradiol in trabecular but not cortical bone following ovariectomy. Collectively, these findings suggest that GPRC5C protein is a key node in a pro-osteogenic axis that is normally suppressed by EZH2-mediated H3K27me3 marks and induced during osteoblast differentiation by GSK126, BMP2, and/or 17β-estradiol. Because GPRC5C protein is an understudied orphan receptor required for osteoblast differentiation, identification of ligands that induce GPRC5C signaling may support therapeutic strategies to mitigate bone-related disorders.

The nuclear cytokine IL-37a controls lethal cytokine storms primarily via IL-1R8-independent transcriptional upregulation of PPARγ.

Cytokine storms are crucial in the development of various inflammatory diseases, including sepsis and autoimmune disorders. The immunosuppressive cytokine INTERLEUKIN (IL)-37 consists of five isoforms (IL-37a-e). We identified IL-37a as a nuclear cytokine for the first time. Compared to IL-37b, IL-37a demonstrated greater efficacy in protecting against Toll-like receptor-induced cytokine hypersecretion and lethal endotoxic shock. The full-length (FL) form of IL-37a and the N-terminal fragment, which is processed by elastase, could translocate into cell nuclei through a distinctive nuclear localization sequence (NLS)/importin nuclear transport pathway. These forms exerted their regulatory effects independent of the IL-1R8 receptor by transcriptionally upregulating the nuclear receptor peroxisome proliferator-activated receptor (PPARγ). This process involved the recruitment of the H3K4 methyltransferase complex WDR5/MLL4/C/EBPβ and H3K4me1/2 to the enhancer/promoter of Pparg. The receptor-independent regulatory pathway of the nuclear IL-37a-PPARγ axis and receptor-dependent signaling by secreted IL-37a maintain homeostasis and are potential therapeutic targets for various inflammatory diseases, including sepsis.

SETD2 mutations do not contribute to clonal fitness in response to chemotherapy in childhood B cell acute lymphoblastic leukemia

Mutations in genes encoding epigenetic regulators are commonly observed at relapse in B cell acute lymphoblastic leukemia (B-ALL). Loss-of-function mutations in SETD2, an H3K36 methyltransferase, have been observed in B-ALL and other cancers. Previous studies on mutated SETD2 in solid tumors and acute myelogenous leukemia support a role in promoting resistance to DNA damaging agents. We did not observe chemoresistance, an impaired DNA damage response, nor increased mutation frequency in response to thiopurines using CRISPR-mediated knockout in wild-type B-ALL cell lines. Likewise, restoration of SETD2 in cell lines with hemizygous mutations did not increase sensitivity. SETD2 mutations affected the chromatin landscape and transcriptional output that was unique to each cell line. Collectively our data does not support a role for SETD2 mutations in driving clonal evolution and relapse in B-ALL, which is consistent with the lack of enrichment of SETD2 mutations at relapse in most studies.

G9a and Sirtuin6 epigenetically modulate host cholesterol accumulation to facilitate mycobacterial survival.

Cholesterol derived from the host milieu forms a critical factor for mycobacterial pathogenesis. However, the molecular circuitry co-opted by Mycobacterium tuberculosis (Mtb) to accumulate cholesterol in host cells remains obscure. Here, we report that the coordinated action of WNT-responsive histone modifiers G9a (H3K9 methyltransferase) and SIRT6 (H3K9 deacetylase) orchestrate cholesterol build-up in in vitro and in vivo mouse models of Mtb infection. Mechanistically, G9a, along with SREBP2, drives the expression of cholesterol biosynthesis and uptake genes; while SIRT6 along with G9a represses the genes involved in cholesterol efflux. The accumulated cholesterol in Mtb infected macrophages promotes the expression of antioxidant genes leading to reduced oxidative stress, thereby supporting Mtb survival. In corroboration, loss-of-function of G9a in vitro and pharmacological inhibition in vivo; or utilization of BMDMs derived from Sirt6-/- mice or in vivo infection in haplo-insufficient Sirt6-/+ mice; hampered host cholesterol accumulation and restricted Mtb burden. These findings shed light on the novel roles of G9a and SIRT6 during Mtb infection and highlight the previously unknown contribution of host cholesterol in potentiating anti-oxidative responses for aiding Mtb survival.

Hypoxia stabilizes SETDB1 to maintain genome stability.

Von Hippel-Lindau (VHL) is a tumor suppressor that functions as the substrate recognition subunit of the CRL2VHL E3 complex. While substrates of VHL have been identified, its tumor suppressive role remains to be fully understood. For further determination of VHL substrates, we analyzed the physical interactome of VHL and identified the histone H3K9 methyltransferase SETBD1 as a novel target. SETDB1 undergoes oxygen-dependent hydroxylation by prolyl hydroxylase domain proteins and the CRL2VHL complex recognizes hydroxylated SETDB1 for ubiquitin-mediated degradation. Under hypoxic conditions, SETDB1 accumulates by escaping CRL2VHL activity. Loss of SETDB1 in hypoxia compared with that in normoxia escalates the production of transposable element-derived double-stranded RNAs, thereby hyperactivating the immune-inflammatory response. In addition, strong derepression of TEs in hypoxic cells lacking SETDB1 triggers DNA damage-induced death. Our collective results support a molecular mechanism of oxygen-dependent SETDB1 degradation by the CRL2VHL E3 complex and reveal a role of SETDB1 in genome stability under hypoxia.

Natural products modulating epigenetic mechanisms by affecting histone methylation/demethylation: targeting cancer cells.

Many natural products can exert anticancer or chemopreventive activity by interfering with the cellular epigenetic machinery. While a plethora of studies indicate the relevance of affecting DNA methylation and histone acetylation, the influence on the mechanisms related to histone methylation is often overlooked. This may be associated with the lagging evidence that changes in the action of histone methylation writers and erasers and subsequent alterations in the profile of histone methylation are causally related with carcinogenesis. However, recent animal studies have shown that targeting histone methylation/demethylation affects the course of experimentally induced carcinogenesis. Existing data suggest that numerous natural compounds from different chemical groups, including green tea polyphenols and other flavonoids, curcuminoids, stilbene derivatives, phenolic acids, isothiocyanates, alkaloids and terpenes, can affect the expression and activity of crucial enzymes involved in the methylation and demethylation of histone lysine and arginine residues. These activities have been associated with the modulation of cancer-related gene expression and functional changes, including reduced cell proliferation and migration, and enhanced apoptosis in various cancer models. Most studies focused on the modulation of the expression and/or activity of two proteins - EZH2 (a H3K27 methyltransferase) and LSD1 (a H3K4/9 demethylase), or the effects on the global levels of histone methylation marks by the phytochemicals, but data regarding other histone methyltransferases or demethylases are scarce. While the field still remains relatively unexplored, this review aims to explore the impact of natural products on the enzymes related to histone methylation/demethylation, showing their relevance to carcinogenesis and cancer progression.

Prolonged Inhibition of the MEK1/2-ERK Signaling Axis Primes Interleukin-1 Beta Expression through Histone 3 Lysine 9 Demethylation in Murine Macrophages.

Macrophages undergo different cellular states upon activation that can be hyporesponsive (tolerated) or hyperresponsive (primed or trained) to subsequent stimuli. Epigenetic modifications are known to play key roles in determining these cellular states. However, little is known about the role of signaling pathways that lead to these epigenetic modifications. Here, we examined the effects of various inhibitors targeting key signaling pathways induced by lipopolysaccharide (LPS) on tolerance and priming in murine macrophages. We found that a prolonged inhibition (>18 h) of the mitogen-activated protein kinase (MEK)1/2-extracellular signal-regulated kinase (ERK)1/2 signaling axis reversed tolerance and primed cells in expressing interleukin (IL)-1β and other inflammatory cytokines such as IL-6, tumor necrosis factor (TNF)α, and CXCL10. The ectopic expression of catalytically active and inactive MEK1 mutants suppressed and enhanced IL-1β expression, respectively. A transcriptomic analysis showed that cells primed by the MEK1/2 inhibitor U0126 expressed higher levels of gene sets associated with immune responses and cytokine/chemokine production, but expressed lower levels of genes with cell cycle progression, chromosome organization, and heterochromatin formation than non-primed cells. Of interest, the mRNA expressions of the histone 3 lysine 9 (H3K9) methyltransferase Suv39h1 and the H3K9 methylation reader Cbx5 were substantially suppressed, whereas the H3K9 demethylase Kdm7a was enhanced, suggesting a role of the MEK1/2-ERK signaling axis in H3K9 demethylation. The H3K9 trimethylation levels in the genomic regions of IL-1β, TNFα, and CXCL10 were decreased by U0126. Also, the H3K9 methyltransferase inhibitor BIX01294 mimicked the U0126 training effects and the overexpression of chromobox homolog (CBX)5 prevented the U0126 training effects in both RAW264.7 cells and bone-marrow-derived macrophages. Collectively, these data suggest that the prolonged inhibition of the MEK1/2-ERK signaling axis reverses tolerance and primed macrophages likely through decreasing the H3K9 methylation levels.

H3K9 and H4K20 methyltransferases are directly involved in the heterochromatinization of the paternal chromosomes in male Planococcus citri embryos.

Using a new method for bulk preparation of early stage embryos, we have investigated the role played by putative Planococcus citri H3K9 and H4K20 histone methyl transferases (HMTases) in regulating heterochromatinization of the imprinted paternal chromosomal set in male embryos. We found that H3K9 and H420 HMTases are required for heterochromatinization of the paternal chromosomes. We present evidence that both HMTases maintain the paternal "imprint" during the cleavage divisions when both parental chromosome sets are euchromatic. A testable model that accommodates our findings is proposed.

A functional crosstalk between the H3K9 methylation writers and their reader HP1 in safeguarding embryonic stem cell identity.

Histone H3 lysine 9 (H3K9) methylation, as a hallmark of heterochromatin, has a central role in cell lineage and fate determination. Although evidence of a cooperation between H3K9 methylation writers and their readers has started to emerge, their actual interplay remains elusive. Here, we show that loss of H3K9 methylation readers, the Hp1 family, causes reduced expression of H3K9 methyltransferases, and that this subsequently leads to the exit of embryonic stem cells (ESCs) from pluripotency and a reciprocal gain of lineage-specific characteristics. Importantly, the phenotypes of Hp1-null ESCs can be rescued by ectopic expression of Setdb1, Nanog, and Oct4. Furthermore, Setdb1 ablation results in loss of ESC identity, which is accompanied by a reduction in the expression of Hp1 genes. Together, our data support a model in which the safeguarding of ESC identity involves the cooperation between the H3K9 methylation writers and their readers.

SET Domain Group 703 Regulates Planthopper Resistance by Suppressing the Expression of Defense-Related Genes.

Plant defense responses against insect pests are intricately regulated by highly complex regulatory networks. Post-translational modifications (PTMs) of histones modulate the expression of genes involved in various biological processes. However, the role of PTMs in conferring insect resistance remains unclear. Through the screening of a T-DNA insertion activation-tagged mutant collection in rice, we identified the mutant planthopper susceptible 1 (phs1), which exhibits heightened expression of SET domain group 703 (SDG703). This overexpression is associated with increased susceptibility to the small brown planthopper (SBPH), an economically significant insect pest affecting rice crops. SDG703 is constitutively expressed in multiple tissues and shows substantial upregulation in response to SBPH feeding. SDG703 demonstrates the activity of histone H3K9 methyltransferase. Transcriptomic analysis revealed the downregulation of genes involved in effector-triggered immunity (ETI) and pattern-triggered immunity (PTI) in plants overexpressing SDG703. Among the downregulated genes, the overexpression of SDG703 in plants resulted in a higher level of histone H3K9 methylation compared to control plants. Collectively, these findings indicate that SDG703 suppresses the expression of defense-related genes through the promotion of histone methylation, consequently leading to reduced resistance against SBPH. The defense-related genes regulated by histone methylation present valuable targets for developing effective pest management strategies in future studies. Furthermore, our study provides novel insight into the epigenetic regulation involved in plant-insect resistance.

Abstract A026: Overactivation of histone deacetylases and EZH2 in Wilms tumorigenesis

Abstract Wilms tumor (WT) is the most common childhood kidney cancer. Altered epigenetics is central to oncogenesis in many pediatric cancers. The critical contribution of epigenetic dysregulation to pediatric tumors provides a compelling rationale for the therapeutic potential of epigenetic drugs. Histone deacetylases (HDACs, especially HDAC1/2) and Enhancer of Zeste Homolog 2 (EZH2), a histone H3K27 methyltransferase, have been demonstrated to play a critical role in self-renewal and differentiation of mouse nephron progenitor cells (NPCs). WT was considered as the arrest of NPC differentiation at a pre-epithelialized cell state. Altered expression and mutations of HDACs and EZH2 have been linked to many human cancers, including WT. Thus, they are among the most promising therapeutic targets for cancer treatment. The treatment of Panobinostat, an FDA-approved pan-HDAC inhibitor, leaded to apoptosis and growth suppression of WT cells. EZH2 is highly expressed in WT and has been demonstrated to be associated with WT progression. We reasoned that WT results from unrestrained proliferation of progenitor cells due to overactive HDAC1, HDAC2, and EZH2. To prove it, we collected 5 human WT specimens. Immunostaining demonstrated dramatically higher level of HDAC1, HDAC2, SIX1, and SIX2 in the tumor tissues compared with adjacent normal tissues, strongly suggesting a critical role for the overactive HDAC1/2 in the SIX1/2 activation to promote cell proliferation in WT. SIX1 and SIX2 are associated with the high-risk blastemal subtype and with the presence of undifferentiated blastema in WT. In addition, knockdown of HDAC1/2 by siRNAs or CRISPR gene editing resulted in downregulation of SIX1/2 in human embryonic kidney 293 cells. Our study in mouse model also revealed that deletion of Hdac1/2 resulted in deletion of progenitor pool and downregulation of key progenitor genes. We also demonstrated that Ezh2 is the dominant H3K27 methyltransferase in Six2+ NPCs and required for NPC proliferation. Based on these findings, we reason that SIX1/2 upregulation by HDAC1/2 and EZH2 overactivation may play an important role in Wilms tumorigenesis. Concomitant inhibition of HDACs and EZH2 has proven to be highly synergistic and very potent for the treatment of many types of human cancers. Promisingly, combined treatment benzamide-based HDAC1/2-selective inhibitor Merck60 and Tazemetostat (an FDA-approved EZH2 inhibitor) synergistically suppressed cell growth in a wide range of dose combinations in G401 kidney cancer cells. In summary, our studies demonstrated the overactivation of HDAC1/2 in WT, positive regulation of SIX1/SIX2 by HDAC1/2 during tumorigenesis, and synergic effect of HDAC1/2 and EZH2 inhibition in suppressing tumor cell proliferation. Thus, targeting HDAC1/2 and EZH2 may provide a promising therapeutic approach to treat WT and other pediatric kidney cancer with low toxicity and low side effects. Furthermore, availability of epigenetic drugs will facilitate the translation of this research into effective therapies. Citation Format: Hongbing Liu. Overactivation of histone deacetylases and EZH2 in Wilms tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A026.

SETD1A function in leukemia is mediated through interaction with mitotic regulators BuGZ/BUB3.

The H3K4 methyltransferase SETD1A plays a crucial role in leukemia cell survival through its noncatalytic FLOS domain-mediated recruitment of cyclin K and regulation of DNA damage response genes. In this study, we identify a functional nuclear localization signal in and interaction partners of the FLOS domain. Our screen for FLOS domain-binding partners reveals that the SETD1A FLOS domain binds mitosis-associated proteins BuGZ/BUB3. Inhibition of both cyclin K and BuGZ/BUB3-binding motifs in SETD1A shows synergistic antileukemic effects. BuGZ/BUB3 localize to SETD1A-bound promoter-TSS regions and SETD1A-negative H3K4me1-positive enhancer regions adjacent to SETD1A target genes. The GLEBS motif and intrinsically disordered region of BuGZ are required forboth SETD1A-binding and leukemia cell proliferation. Cell-cycle-specific SETD1A restoration assays indicate that SETD1A expression at the G1/S phase of the cell cycle promotes both the expression of DNA damage response genes and cell cycle progression in leukemia cells.

H3K36 Di-Methylation Marks, Mediated by Ash1 in Complex with Caf1-55 and MRG15, Are Required during Drosophila Heart Development.

Methyltransferases regulate transcriptome dynamics during development and aging, as well as in disease. Various methyltransferases have been linked to heart disease, through disrupted expression and activity, and genetic variants associated with congenital heart disease. However, in vivo functional data for many of the methyltransferases in the context of the heart are limited. Here, we used the Drosophila model system to investigate different histone 3 lysine 36 (H3K36) methyltransferases for their role in heart development. The data show that Drosophila Ash1 is the functional homolog of human ASH1L in the heart. Both Ash1 and Set2 H3K36 methyltransferases are required for heart structure and function during development. Furthermore, Ash1-mediated H3K36 methylation (H3K36me2) is essential for healthy heart function, which depends on both Ash1-complex components, Caf1-55 and MRG15, together. These findings provide in vivo functional data for Ash1 and its complex, and Set2, in the context of H3K36 methylation in the heart, and support a role for their mammalian homologs, ASH1L with RBBP4 and MORF4L1, and SETD2, during heart development and disease.

Loss of PHF8 induces a viral mimicry response by activating endogenous retrotransposons

Immunotherapy has become established as major treatment modality for multiple types of solid tumors, including colorectal cancer. Identifying novel immunotherapeutic targets to enhance anti-tumor immunity and sensitize current immune checkpoint blockade (ICB) in colorectal cancer is needed. Here we report the histone demethylase PHD finger protein 8 (PHF8, KDM7B), a Jumonji C domain-containing protein that erases repressive histone methyl marks, as an essential mediator of immune escape. Ablation the function of PHF8 abrogates tumor growth, activates anti-tumor immune memory, and augments sensitivity to ICB therapy in mouse models of colorectal cancer. Strikingly, tumor PHF8 deletion stimulates a viral mimicry response in colorectal cancer cells, where the depletion of key components of endogenous nucleic acid sensing diminishes PHF8 loss-meditated antiviral immune responses and anti-tumor effects in vivo. Mechanistically, PHF8 inhibition elicits H3K9me3-dependent retrotransposon activation by promoting proteasomal degradation of the H3K9 methyltransferase SETDB1 in a demethylase-independent manner. Moreover, PHF8 expression is anti-correlated with canonical immune signatures and antiviral immune responses in human colorectal adenocarcinoma. Overall, our study establishes PHF8 as an epigenetic checkpoint, and targeting PHF8 is a promising viral mimicry-inducing approach to enhance intrinsic anti-tumor immunity or to conquer immune resistance.

Parallels and contrasts between the cnidarian and bilaterian maternal-to-zygotic transition are revealed in Hydractinia embryos.

Embryogenesis requires coordinated gene regulatory activities early on that establish the trajectory of subsequent development, during a period called the maternal-to-zygotic transition (MZT). The MZT comprises transcriptional activation of the embryonic genome and post-transcriptional regulation of egg-inherited maternal mRNA. Investigation into the MZT in animals has focused almost exclusively on bilaterians, which include all classical models such as flies, worms, sea urchin, and vertebrates, thus limiting our capacity to understand the gene regulatory paradigms uniting the MZT across all animals. Here, we elucidate the MZT of a non-bilaterian, the cnidarian Hydractinia symbiolongicarpus. Using parallel poly(A)-selected and non poly(A)-dependent RNA-seq approaches, we find that the Hydractinia MZT is composed of regulatory activities similar to many bilaterians, including cytoplasmic readenylation of maternally contributed mRNA, delayed genome activation, and separate phases of maternal mRNA deadenylation and degradation that likely depend on both maternally and zygotically encoded clearance factors, including microRNAs. But we also observe massive upregulation of histone genes and an expanded repertoire of predicted H4K20 methyltransferases, aspects thus far particular to the Hydractinia MZT and potentially underlying a novel mode of early embryonic chromatin regulation. Thus, similar regulatory strategies with taxon-specific elaboration underlie the MZT in both bilaterian and non-bilaterian embryos, providing insight into how an essential developmental transition may have arisen in ancestral animals.