Histone H3K4 Methyltransferase Complex Research Articles

Role of histone H3K4 methyltransferase in regulating Monascus pigments production by red light-coupled magnetic field.

Light, magnetic field, and methylation affected the growth and secondary metabolism of fungi. The regulation effect of the three factors on the growth and Monascus pigments (MPs) synthesis of Monascus purpureus was investigated in this study. 5-azacytidine (5-AzaC), DNA methylation inhibitor, was used to treat M. purpureus (wild-type, WT). Twenty micromolar 5-AzaC significantly promoted the growth, development, and MPs yield. Moreover, 250 lux red light and red light coupled magnetic field (RLCMF) significantly promoted the biomass. For WT, red light, and RLCMF significantly promoted MPs yield. But compared with red light treatment, only 0.2 mT RLCMF promoted the alcohol-soluble MPs yield. For histone H3K4 methyltransferase complex subunit Ash2 gene knockout strain (ΔAsh2), only 0.2 mT RLCMF significantly promoted water-soluble MPs yield. Yet red light, 1.0 and 0.2 mT RLCMF significantly promoted alcohol-soluble MPs yield. This indicated that methylation affected the MPs biosynthesis. Red light and weaker MF had a synergistic effect on the growth and MPs synthesis of ΔAsh2. This result was further confirmed by the expression of related genes. Therefore, histone H3K4 methyltransferase was involved in the regulation of the growth, development, and MPs synthesis of M. purpureus by the RLCMF.

DPY30 Promotes Proliferation and Cell Cycle Progression of Colorectal Cancer Cells via Mediating H3K4 Trimethylation.

DPY30, a core subunit of the SET1/MLL histone H3K4 methyltransferase complexes, plays an important role in diverse biological functions through the epigenetic regulation of gene transcription, especially in cancer development. However, its involvement in human colorectal carcinoma (CRC) has not been elucidated yet. Here we demonstrated that DPY30 was overexpressed in CRC tissues, and significantly associated with pathological grading, tumor size, TNM stage, and tumor location. Furthermore, DPY30 knockdown remarkably suppressed the CRC cell proliferation through downregulation of PCNA and Ki67 in vitro and in vivo, simultaneously induced cell cycle arrest at S phase by downregulating Cyclin A2. In the mechanistic study, RNA-Seq analysis revealed that enriched gene ontology of cell proliferation and cell growth was significantly affected. And ChIP result indicated that DPY30 knockdown inhibited H3 lysine 4 trimethylation (H3K4me3) and attenuated interactions between H3K4me3 with PCNA, Ki67 and cyclin A2 respectively, which led to the decrease of H3K4me3 establishment on their promoter regions. Taken together, our results demonstrate overexpression of DPY30 promotes CRC cell proliferation and cell cycle progression by facilitating the transcription of PCNA, Ki67 and cyclin A2 via mediating H3K4me3. It suggests that DPY30 may serve as a potential therapeutic molecular target for CRC.

Loss of the Ash2l subunit of histone H3K4 methyltransferase complexes reduces chromatin accessibility at promoters

Changes in gene expression programs are intimately linked to cell fate decisions. Post-translational modifications of core histones contribute to control gene expression. Methylation of lysine 4 of histone H3 (H3K4) correlates with active promoters and gene transcription. This modification is catalyzed by KMT2 methyltransferases, which require interaction with 4 core subunits, WDR5, RBBP5, ASH2L and DPY30, for catalytic activity. Ash2l is necessary for organismal development and for tissue homeostasis. In mouse embryo fibroblasts (MEFs), Ash2l loss results in gene repression, provoking a senescence phenotype. We now find that upon knockout of Ash2l both H3K4 mono- and tri-methylation (H3K4me1 and me3, respectively) were deregulated. In particular, loss of H3K4me3 at promoters correlated with gene repression, especially at CpG island promoters. Ash2l loss resulted in increased loading of histone H3 and reduced chromatin accessibility at promoters, accompanied by an increase of repressing and a decrease of activating histone marks. Moreover, we observed altered binding of CTCF upon Ash2l loss. Lost and gained binding was noticed at promoter-associated and intergenic sites, respectively. Thus, Ash2l loss and reduction of H3K4me3 correlate with altered chromatin accessibility and transcription factor binding. These findings contribute to a more detailed understanding of mechanistic consequences of H3K4me3 loss and associated repression of gene transcription and thus of the observed cellular consequences.

LBODP106 Multi-omics Analyses Of MEN1 Missense Mutations Identify Disruption Of Menin-MLL And Menin-JunD Interactions As Critical Requirements For Molecular Pathogenicity

Abstract Loss-of-function mutations of the multiple endocrine neoplasia type 1 (MEN1) gene are causal to the MEN1 endocrine tumor syndrome, but they are also commonly found in sporadic pancreatic neuroendocrine tumors and other types of cancers. The MEN1 gene product, menin, is involved in transcriptional and chromatin regulation, most prominently as an integral component of KMT2A/MLL1 and KMT2B/MLL2 containing COMPASS-like histone H3K4 methyltransferase complexes. In a mutually exclusive fashion, menin also interacts with the JunD subunit of the AP-1 and ATF/CREB transcription factors. After in silico screening of 253 disease-related MEN1 missense mutations, we selected a set of nine menin mutations in surface-exposed residues. The protein interactomes of these mutants were assessed by quantitative mass spectrometry, which indicated that seven of the nine mutants disrupt interactions with both MLL1/2 and JunD complexes. Interestingly, we identified three missense mutations, R52G, E255K and E359K, which predominantly reduce the interaction with MLL1 compared to JunD. This observation was supported by a pronounced loss of binding of the R52G, E255K and E359K mutant proteins at unique MLL1 genomic binding sites with less effect on unique JunD sites. These findings support the general importance of the menin-MLL1 and menin-JunD interactions in MEN1 gene-associated pathogenic conditions. Presentation: No date and time listed

Multi-omics analyses of MEN1 missense mutations identify disruption of menin–MLL and menin–JunD interactions as critical requirements for molecular pathogenicity

BackgroundLoss-of-function mutations of the multiple endocrine neoplasia type 1 (MEN1) gene are causal to the MEN1 tumor syndrome, but they are also commonly found in sporadic pancreatic neuroendocrine tumors and other types of cancers. The MEN1 gene product, menin, is involved in transcriptional and chromatin regulation, most prominently as an integral component of KMT2A/MLL1 and KMT2B/MLL2 containing COMPASS-like histone H3K4 methyltransferase complexes. In a mutually exclusive fashion, menin also interacts with the JunD subunit of the AP-1 and ATF/CREB transcription factors.ResultsHere, we applied and in silico screening approach for 253 disease-related MEN1 missense mutations in order to select a set of nine menin mutations in surface-exposed residues. The protein interactomes of these mutants were assessed by quantitative mass spectrometry, which indicated that seven of the nine mutants disrupt interactions with both MLL1/MLL2 and JunD complexes. Interestingly, we identified three missense mutations, R52G, E255K and E359K, which predominantly reduce the MLL1 and MLL2 interactions when compared with JunD. This observation was supported by a pronounced loss of binding of the R52G, E255K and E359K mutant proteins at unique MLL1 genomic binding sites with less effect on unique JunD sites.ConclusionsOur results underline the effects of MEN1 gene mutations in both familial and sporadic tumors of endocrine origin on the interactions of menin with the MLL1 and MLL2 histone H3K4 methyltransferase complexes and with JunD-containing transcription factors. Menin binding pocket mutants R52G, E255K and E359K have differential effects on MLL1/MLL2 and JunD interactions, which translate into differential genomic binding patterns. Our findings encourage future studies addressing the pathophysiological relevance of the separate MLL1/MLL2- and JunD-dependent functions of menin mutants in MEN1 disease model systems.

Euchromatin factors HULC and Set1C affect heterochromatin organization and mating-type switching in fission yeast Schizosaccharomyces pombe.

Mating-type (P or M) of fission yeast Schizosaccharomyces pombe is determined by the transcriptionally active mat1 cassette and is switched by gene conversion using a donor, either mat2 or mat3, located in an adjacent heterochromatin region (mating-type switching; MTS). In the switching process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the action of two recombination enhancers, SRE2 promoting the use of mat2-P and SRE3 promoting the use of mat3-M, leading to replacement of the content of the expressed mat1 cassette. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1Δ. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant, we found that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in MTS in these mutants suggested that HULC and Set1C inhibit the use of the SRE3 recombination enhancer in M cells, thus favoring SRE2 and mat2-P. Consistent with this, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain.

Menin directs regionalized decidual transformation through epigenetically setting PTX3 to balance FGF and BMP signaling

During decidualization in rodents, uterine stroma undergoes extensive reprograming into distinct cells, forming the discrete regions defined as the primary decidual zone (PDZ), the secondary decidual zone (SDZ) and the layer of undifferentiated stromal cells respectively. Here we show that uterine deletion of Men1, a member of the histone H3K4 methyltransferase complex, disrupts the terminal differentiation of stroma, resulting in chaotic decidualization and pregnancy failure. Genome-wide epigenetic profile reveals that Men1 binding in chromatin recapitulates H3K4me3 distribution. Further transcriptomic investigation demonstrates that Men1 directly regulates the expression of PTX3, an extra-cellular trap for FGF2 in decidual cells. Decreased Ptx3 upon Men1 ablation leads to aberrant activation of ERK1/2 in the SDZ due to the unrestrained FGF2 signal emanated from undifferentiated stromal cells, which blunt BMP2 induction and decidualization. In brief, our study provides genetic and molecular mechanisms for epigenetic rewiring mediated decidual regionalization by Men1 and sheds new light on pregnancy maintenance.

COMPASS functions as a module of the INO80 chromatin remodeling complex to mediate histone H3K4 methylation in Arabidopsis.

In the INO80 chromatin remodeling complex, all of the accessory subunits are assembled on the following three domains of INO80: N-terminal domain (NTD), HSA domain, and ATPase domain. Although the ATPase and HSA domains and their interacting accessory subunits are known to be responsible for chromatin remodeling, it is largely unknown how the accessory subunits that interact with the INO80 NTD regulate chromatin status. Here, we identify both conserved and nonconserved accessory subunits that interact with the three domains in the INO80 complex in Arabidopsis thaliana. While the accessory subunits that interact with all the three INO80 domains can mediate transcriptional repression, the INO80 NTD and the accessory subunits interact with it can contribute to transcriptional activation even when the ATPase domain is absent, suggesting that INO80 has an ATPase-independent role. A subclass of the COMPASS histone H3K4 methyltransferase complexes interact with the INO80 NTD in the INO80 complex and function together with the other accessory subunits that interact with the INO80 NTD, thereby facilitating H3K4 trimethylation and transcriptional activation. This study suggests that the opposite effects of the INO80 complex on transcription are required for the balance between vegetative growth and flowering under diverse environmental conditions.

Myocardin-Related Transcription Factor A Mediates LPS-Induced iNOS Transactivation.

Macrophage-dependent inflammation plays a critical role in atherogenesis. Inducible nitric oxide synthase (iNOS) is one of key pro-inflammatory mediators produced in macrophages and its levels can be upregulated by lipopolysaccharide (LPS). The epigenetic mechanism whereby LPS induces iNOS transcription is incompletely understood. We show here myocardin-related transcription factor A (MRTF-A) potentiated iNOS promoter activity in macrophages. There was a decrease in LPS-induced iNOS expression in several cell models due to the lack of MRTF-A. LPS treatment promoted nuclear accumulation of MRTF-A and its interaction with NF-κB/p65 on the iNOS promoter. The absence of MRTF-A prevented the accumulation of active histone marks on the iNOS promoter in response to LPS treatment. Mechanistically, MRTF-A recruited ASH2, a key component of the mammalian histone H3K4 methyltransferase complex, to the iNOS promoter. Silencing of ASH2 attenuated iNOS expression following LPS treatment. Together, our data highlight a role for MRTF-A-dependent recruitment of H3K4 methyltransferase in iNOS induction and as such provide a novel target in the intervention of atherosclerosis.

Lack of the Histone Methyltransferase Gene Ash2 Results in the Loss of Citrinin Production in Monascus purpureus

Absent, small, or homeotic discs 2 (Ash2), a histone H3K4 methyltransferase complex, has been implicated in the control of hyphal development and secondary metabolism in many kinds of filamentous fungi. We constructed an Ash2 deletion mutant (ΔAsh2) by using an Agrobacterium-mediated gene knockout method to investigate the function of the Ash2 gene in the mold Monascus purpureus. Lack of the Ash2 gene resulted in the formation of a lower colony phenotype with fluffy aerial hyphae that autolyzed as the colony grew on potato dextrose agar at 30°C. The production of pigments and the number of conidia were significantly lower in the ΔAsh2 than in the wild type. Citrinin production by the ΔAsh2 was not detected during 15 days of fermentation. Relative expression levels of secondary metabolite regulatory genes PigR and CTNR, secondary metabolite synthesizing genes PKSPT and CTN, key genes of mitogen-activated protein kinase pathway Spk1 and its downstream gene mam2, the conidium development control gene BrlA, and global regulatory genes LaeA and VeA were detected by the quantitative real-time PCR. These results indicate that the Ash2 gene is involved in conidial germination, pigment production, and citrinin production and plays a key role in development and secondary metabolism in M. purpureus.

Regeneration after acute kidney injury requires PTIP-mediated epigenetic modifications.

A terminally differentiated cellular phenotype is thought to be maintained, at least in part, by both active and repressive histone marks. However, it is unclear whether regenerating cells after injury need to replicate such epigenetic marks to recover. To test whether renal epithelial cell regeneration is dependent on histone H3K4 methylation, we generated a mouse model that deleted the Paxip1 gene in mature renal proximal tubules. Paxip1 encodes PTIP, an essential protein in the Mll3/4 histone H3K4 methyltransferase complex. Mice with PTIP deletions in the adult kidney proximal tubules were viable and fertile. Upon acute kidney injury, such mice failed to regenerate damaged tubules, leading to scarring and interstitial fibrosis. The inability to repair damage was likely due to a failure to reenter mitosis and reactivate regulatory genes such as Sox9. PTIP deletion reduced histone H3K4 methylation in uninjured adult kidneys but did not significantly affect function or the expression of epithelial specific markers. Strikingly, cell lineage tracing revealed that surviving PTIP mutant cells could alter their phenotype and lose epithelial markers. These data demonstrate that PTIP and associated MLL3/4-mediated histone methylation are needed for regenerating proximal tubules and to maintain or reestablish the cellular epithelial phenotype.

WDR5 inhibition halts metastasis dissemination by repressing the mesenchymal phenotype of breast cancer cells

BackgroundDevelopment of metastases and drug resistance are still a challenge for a successful systemic treatment in breast cancer (BC) patients. One of the mechanisms that confer metastatic properties to the cell relies in the epithelial-to-mesenchymal transition (EMT). Moreover, both EMT and metastasis are partly modulated through epigenetic mechanisms, by repression or induction of specific related genes.MethodsWe applied shRNAs and drug targeting approaches in BC cell lines and metastatic patient-derived xenograft (PDX) models to inhibit WDR5, the core subunit of histone H3 K4 methyltransferase complexes, and evaluate its role in metastasis regulation.ResultWe report that WDR5 is crucial in regulating tumorigenesis and metastasis spreading during BC progression. In particular, WDR5 loss reduces the metastatic properties of the cells by reverting the mesenchymal phenotype of triple negative- and luminal B-derived cells, thus inducing an epithelial trait. We also suggest that this regulation is mediated by TGFβ1, implying a prominent role of WDR5 in driving EMT through TGFβ1 activation. Moreover, such EMT reversion can be induced by drug targeting of WDR5 as well, leading to BC cell sensitization to chemotherapy and enhancement of paclitaxel-dependent effects.ConclusionsWe suggest that WDR5 inhibition could be a promising pharmacologic approach to reduce cell migration, revert EMT, and block metastasis formation in BC, thus overcoming resistance to standard treatments.

Structural basis for histone H3K4me3 recognition by the N-terminal domain of the PHD finger protein Spp1.

Saccharomyces cerevisiae Spp1, a plant homeodomain (PHD) finger containing protein, is a critical subunit of the histone H3K4 methyltransferase complex of proteins associated with Set1 (COMPASS). The chromatin binding affinity of the PHD finger of Spp1 has been proposed to modulate COMPASS activity. During meiosis, Spp1 plays another role in promoting programmed double-strand break (DSB) formation by binding H3K4me3 via its PHD finger and interacting with a DSB protein, Mer2. However, how the Spp1 PHD finger performs site-specific readout of H3K4me3 is still not fully understood. In the present study, we determined the crystal structure of the highly conserved Spp1 N-terminal domain (Sc_Spp1NTD) in complex with the H3K4me3 peptide. The structure shows that Sc_Spp1NTD comprises a PHD finger responsible for methylated H3K4 recognition and a C3H-type zinc finger necessary to ensure the overall structural stability. Our isothermal titration calorimetry results show that binding of H3K4me3 to Sc_Spp1NTD is mildly inhibited by H3R2 methylation, weakened by H3T6 phosphorylation, and abrogated by H3T3 phosphorylation. This histone modification cross-talk, which is conserved in the Saccharomyces pombe and mammalian orthologs of Sc_Spp1 in vitro, can be rationalized structurally and might contribute to the roles of Spp1 in COMPASS activity regulation and meiotic recombination.

LncRNA Gm10451 regulates PTIP to facilitate iPSCs-derived β-like cell differentiation by targeting miR-338-3p as a ceRNA

iPSCs-derived insulin-producing cell transplantation is a promising strategy for diabetes therapy. Although there have been many protocols of mature, glucose-responsive β cells induced in vitro over the past few years, many underlying problems remain to be resolved. As a crucial regulator, long noncoding RNAs (lncRNAs) participate in numerous biological processes, including the maintenance of pluripotency, and stem cell differentiation. In this study, we identified a novel lncRNA Gm10451 as a functional regulator for β-like cell differentiation. Localized to the cytoplasm, Gm10451 regulates histone H3K4 methyltransferase complex PTIP to facilitate Insulin+/Nkx6.1+ β-like cell differentiation by targeting miR-338-3p as a competing endogenous RNA (ceRNA). miR-338-3p has also been shown to suppress Nkx6.1+ early-stage β-like cell differentiation by targeting PTIP. Following transplantation into streptozotocin (STZ)-mice, Gm10451 loss in β-like cells prevented the expression of mature β-cell makers, such as Insulin, Nkx6.1, and Mafa. Accordingly, hyperglycemia in the mice was not resolved. Taken together, this study provides an efficient epigenetic target for generating more mature and functional iPSCs-derived β-like cells. We anticipate that pancreatic organoids, which are generated from human stem cells, biological materials, and epigenetic modifications, can be used in the future as a novel diabetes treatment option.

Hoxblinc Is Aberrantly Expressed in Acute Myeloid Leukemia and Functions As a Potent Oncogenic Long Non-Coding RNA in Leukemogenesis

Aberrant expression of long non-coding RNAs (lncRNAs) might contribute to the development and progression of leukemia. However, functional studies on the actual role of lncRNAs during the development of leukemia remain scarce, and very few lncRNAs have been shown to be involved in leukemogenesis. HoxBlinc is an anterior HoxB gene-associated intergenic lncRNA. It is a cis-acting lncRNA and functions as an epigenetic regulator to coordinate anterior HoxB gene expression. Giving the dysregulation of HOXA/B genes is a dominant mechanism of leukemic transformation, HoxBlinc might be an oncogenic lncRNA of leukemia.To determine whether HOXBLINC lncRNA is aberrantly expressed in human AML samples, we performed RT-qPCR on bone marrow mononuclear cells (BMMNCs) from a cohort of 73 AML patients. A dramatic up-regulation of HOXBLINC was observed in over 60% of the patients. When TCGA-AML datasets of a cohort of 179 AML patients were analyzed for their HOXBLINC expression, a significant portion of these AML patients had high levels of HOXBLINC expression. Interestingly, AML patients with high HOXBLINC expression (the top thirty percentile of patients) had a significantly shortened survival as compared to patients with low HOXBLINC expression (the bottom thirty percentile).To investigate the impact of HoxBlinc overexpression on normal hematopoiesis and the pathogenesis of hematological malignancies in vivo, we generated a HoxBlinc transgenic(Tg) mouse model. Within 1 year of age, 67% of the HoxBlincTg mice (10 of 15) died or were sacrificed because of a moribund condition due to AML.We then assessed whether overexpression of HoxBlinc affects the pools of HSC/HPCs by flow cytometric analysis on the BM cells of young WT and HoxBlincTg mice (8-10 weeks of age). HoxBlincTg BM had a dramatically greater number of LT-HSC, ST-HSC, MPP cells, and a significantly higher percentage of GMP, but a lower percentage of MEP/CMP cell populations as compared to WT group. To determine the effect of HoxBlinc overexpression on the function of HSC/HPCs, we performed paired-daughter cell assay, replating assay and liquid culture on sorted LT-HSC, LSK or LK cells from young WT and HoxBlincTg mice, the results indicate that transgenic expression of HoxBlinc enhances HSC self-renewal and impairs HSC/HPC differentiation.To assess whether HoxBlinc overexpression-mediated changes in HSC/HPC function are cell-autonomous, we performed competitive transplantation assays to examine the repopulating capacity of HoxBlincTg BM cells. When the donor cell chimerism was analyzed kinetically in the PB of recipient mice, the CD45.2 cell population remained ~50% in mice receiving WT BM cells, whereas the CD45.2 chimerism in the recipients transplanted with HoxBlincTg BM cells steadily increased. Interestingly, mice receiving HoxBlincTg BM cells developed AML at 2-6 months after transplantation.Previous data reported that HoxBlinc can recruit the Setd1a/Mll1 histone H3K4 methyltransferase complex to mediate formation of the active topologically associated domain (TAD) in the anterior HoxB locus for transcription of the anterior HoxB genes. In this study, LSK or LK cells sorted from young WT and HoxBlincTg mice were analyzed by RNA-seq, ATAC-seq, H3K4me3 CHIP-seq and 4C analysis. Mechanistically, HoxBlinc overexpression alters HoxB locus chromatin three-dimensional organization to enhance enhancer/promoter chromatin accessibility and coordinate the expression of not only HoxB1-5 but also HoxA9, Runx1, Meis1 and so on, which are critical genes for HSC regulation and/or leukemogenesis.Our study provides novel insights into the HSC regulation by lncRNAs and identifies HOXBLINC, which coordinates to maintain an oncogenic transcription program for leukemic transformation, as a potent oncogenic lncRNA in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

DPY30 functions in glucose homeostasis via integrating activated histone epigenetic modifications.

Glucose homeostasis is a key event during many physiological and pathological processes. Histone modifications have emerged as vital factors influencing this process. DPY30, a core subunit of SET1/MLL family histone H3K4 methyltransferase complexes, has been reported to be amplified in cancers. However, the role of DPY30 in glucose homeostasis remains unclear. Here we reported that DPY30 regulated H3K4me3 recruitment to control the expression of Hif1α and its targeted glycolytic genes. Specifically, DPY30 promoted H3K9Ac recruitment via inhibiting SIRT6 occupancy on these gene promoters. Finally, we observed significant upregulation of DPY30 mRNA expression in hepatocellular carcinoma samples from datasets. Taken together, our results reveal a critical role of DPY30 in glucose homeostasis and might offer new therapeutic and diagnostic opportunities for cancers.

Structure and Conformational Dynamics of a COMPASS Histone H3K4 Methyltransferase Complex

The methylation of histone 3 lysine 4 (H3K4) is carried out by an evolutionarily conserved family of methyltransferases referred to as complex of proteins associated with Set1 (COMPASS). The activity of the catalytic SET domain (su(var)3-9, enhancer-of-zeste, and trithorax) is endowed through forming a complex with a set of core proteins that are widely shared from yeast to humans. We obtained cryo-electron microscopy (cryo-EM) maps of the yeast Set1/COMPASS core complex at overall 4.0- to 4.4-Å resolution, providing insights into its structural organization and conformational dynamics. The Cps50 C-terminal tail weaves within the complex to provide a central scaffold for assembly. The SET domain, snugly positioned at the junction of the Y-shaped complex, is extensively contacted by Cps60 (Bre2), Cps50 (Swd1), and Cps30 (Swd3). The mobile SET-I motif of the SET domain is engaged by Cps30, explaining its key role in COMPASS catalytic activity toward higher H3K4 methylation states.

Abstract 057: Adenosine Kinase Epigenetically Regulates Abdominal Aortic Aneurysm Formation

Objectives: Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality worldwide; however, the molecular mechanisms involved in AAA formation are not well understood. The nucleoside adenosine plays important roles in modulating vascular homeostasis. In this study, we sought to determine whether adenosine kinase (ADK), the adenosine-metabolizing enzyme, modulates AAA formation via control of intracellular adenosine level and investigate the involvement of ADK-mediated epigenetic mechanism in this aortic disease. Methods and results: ADK expression and activity were increased in murine AAA models, including calcium chloride-stimulated AAA and angiotensin II-induced AAA. Using a loss-of-function genetic mouse model, we demonstrated that genetic ADK knockout mice led to increased Intracellular adenosine level in macrophages and vascular cells and developed significantly smaller aortic expansion when subjected to AAA inductions. Notably, knockout of ADK alleviated arterial inflammation and diminished infiltration of macrophages, as well as suppressed arterial extracellular matrix degradation, which was associated with down-regulation of CXCL12, MMP2/9 and adhesion molecule expression. Mechanistically, elevation of intracellular adenosine by genetic inactivation of ADK or exogenous adenosine reduces activation of the transmethylation pathway via inactivation of SAH hydrolase (SAHH) and histone H3K4 methyltransferase (HMT), thus down-regulating expression and signaling of mitogen-activated protein kinases in activated macrophages and vascular cells with pro-inflammatory stimuli. Blocking of the H3K4 transmethylation pathway by knockdown of WDR5, a core subunit of HMT complex, abrogated the beneficial effects of ADK inactivation. Conclusions: Our findings reveal a previously unrecognized role of ADK mediated epigenetic pathways in the pathogenesis of AAA and provides a therapeutic target for the prevention of AAA.

Twenty years of menin: emerging opportunities for restoration of transcriptional regulation in MEN1.

Since the discovery of the multiple endocrine neoplasia type 1 (MEN1) gene in 1997, elucidation of the molecular function of its protein product, menin, has been a challenge. Biochemical, proteomics, genetics and genomics approaches have identified various potential roles, which converge on gene expression regulation. The most consistent findings show that menin connects transcription factors and chromatin-modifying enzymes, in particular, the histone H3K4 methyltransferase complexes MLL1 and MLL2. Chromatin immunoprecipitation combined with next-generation sequencing has enabled studying genome-wide dynamics of chromatin binding by menin. We propose that menin regulates cell type-specific transcriptional programs by linking chromatin regulatory complexes to specific transcription factors. In this fashion, the MEN1 gene is a tumor suppressor gene in the endocrine tissues that are affected in MEN1. Recent studies have hinted at possibilities to pharmacologically restore the epigenetic changes caused by loss of menin function as therapeutic strategies for MEN1, for example, by inhibition of histone demethylases. The current lack of appropriate cellular model systems for MEN1-associated tumors is a limitation for compound testing, which needs to be addressed in the near future. In this review, we look back at the past twenty years of research on menin and the mechanism of disease of MEN1. In addition, we discuss how the current understanding of the molecular function of menin offers future directions to develop novel treatments for MEN1-associated endocrine tumors.

Abstract 12514: Elevated Intracellular Adenosine Epigenetically Regulates Endothelial Inflammation (Best of Basic Science Abstract)

Background and Objectives: Adenosine is a potent modulator of endothelial inflammation. However, the molecular mechanisms by which adenosine regulates endothelial inflammation has not been fully understood. This study aims to determine the effect of elevated intracellular adenosine on endothelial inflammation and investigate the involvement of epigenetic mechanism in this effect. Methods and Results: Adenosine but not adenosine receptor agonists alleviated the TNF-α-induced expression of endothelial adhesion molecules (E-selectin, ICAM-1 and VCAM-1). This effect was abrogated by nucleoside transporter inhibitor NBMPR but not by adenosine receptor antagonists. Knockdown of adenosine metabolic enzyme adenosine kinase (ADK) by shRNA or inhibition of ADK by inhibitors augmented intracellular adenosine and also attenuated TNF-α-induced expression of adhesion molecules. This effect was also not affected by adenosine receptor antagonists. Mechanistically, adenosine treatment or ADK knockdown deactivated the SAH hydrolase (SAHH) and histone H3K4 methyltransferase (HMT) and lowered H3K4 di- and tri- methylation levels. Erasing these histone marks by inhibition of SAHH and depletion of WDR5, a core subunit of HMT complex blocked the anti-inflammatory effects of adenosine and ADK knockdown. Co-IP assay showed that ADK can bind with SAHH and WDR5 to methylate H3K4 in a spatially effective manner. With the intravital microscopy we observed the compromised rolling and adhesion of leukocytes in postcapillary venues of endothelial specific ADK deficient mice. Furthermore, atheroprotection in Apoe-/- mice on a Western diet and neuroprotection in acute cerebral ischemic mice were conferred in endothelial specific ADK deficient mice. Conclusion: Our study provides evidence that intracellular adenosine attenuates endothelial inflammation through epigenetic pathways and ADK is a promising therapeutic target to treat inflammatory vascular diseases.