Mark H3K27me3 Research Articles

Abstract B027: The transcription factor HIF2alpha partakes in the differentiation block of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is characterized by epigenetic silencing of differentiation genes and accumulation of aberrant myeloid cells arrested at different stages of myeloid development. The combination of epigenetic and differentiation therapies represents an attractive opportunity for AML patients by promoting chromatin remodeling at differentiation genes and induction of terminal maturation and leukemia debulking. The aim of our work is to define the function of the hypoxia inducible transcription factor HIF2alpha in the pathogenesis of AML. By its genetic inactivation and pharmacological inhibition in AML cell lines and patient-derived xenograft models, we found that HIF2alpha acts as a novel regulator of the AML differentiation block and promotes leukemia progression. Gene expression profiling of HIF2alpha-dependent transcriptome revealed that HIF2alpha mainly promotes the expression of genes involved in transcriptional modulation and epigenetic modifications, whilst inhibiting genes of myeloid maturation and activation. Mechanistically, inhibition of HIF2alpha results in decreased global levels of the heterochromatin marker H3K27me3, which is specifically deposited by the catalytic subunit EZH2 of the Polycomb repressive complex 2. Intriguingly, we found that HIF2alpha and EZH2 cooperate at favoring EZH2-mediated deposition of H3K27me3 at reverse hypoxia responsive elements in the regulatory regions of myeloid maturation genes. Additionally, we demonstrate that HIF2alpha is positively regulated by the pro-differentiation agent all-trans retinoic acid (ATRA), and its inhibition cooperates with ATRA in triggering AML cell differentiation. In conclusion, we provide new mechanistic insights into the role of HIF2alpha in the pathogenesis of AML, by promoting an undifferentiated state via EZH2-mediated epigenetic silencing of myeloid differentiation genes. Importantly, small molecule inhibitors of HIF2alpha have been recently generated and are tested for solid cancers. Therefore, HIF2alpha inhibition may open new therapeutic avenues for AML patients and add therapeutic value to ATRA-based therapies by interrupting HIF2alpha upregulation and promoting an epigenetic state permissive to maturation of leukemic blasts and leukemia exhaustion. Citation Format: Daniela Magliulo, Carolina Caserta, Serena Belluschi, Cristina Fracassi, Kety Giannetti, Raffaella Pini, Ettore Zapparoli, Stefano Beretta, Eleonora Draghi, Marco J. Morelli, Raffaella Di Micco, Bernhard Gentner, Luca Vago, Rosa Bernardi. The transcription factor HIF2alpha partakes in the differentiation block of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B027.

Chromatin dynamics associated with seed desiccation tolerance/sensitivity at early germination in Medicago truncatula.

Desiccation tolerance (DT) has contributed greatly to the adaptation of land plants to severe water-deficient conditions. DT is mostly observed in reproductive parts in flowering plants such as seeds. The seed DT is lost at early post germination stage but is temporally re-inducible in 1mm radicles during the so-called DT window following a PEG treatment before being permanently silenced in 5mm radicles of germinating seeds. The molecular mechanisms that activate/reactivate/silence DT in developing and germinating seeds have not yet been elucidated. Here, we analyzed chromatin dynamics related to re-inducibility of DT before and after the DT window at early germination in Medicago truncatula radicles to determine if DT-associated genes were transcriptionally regulated at the chromatin levels. Comparative transcriptome analysis of these radicles identified 948 genes as DT re-induction-related genes, positively correlated with DT re-induction. ATAC-Seq analyses revealed that the chromatin state of genomic regions containing these genes was clearly modulated by PEG treatment and affected by growth stages with opened chromatin in 1mm radicles with PEG (R1P); intermediate openness in 1mm radicles without PEG (R1); and condensed chromatin in 5mm radicles without PEG (R5). In contrast, we also showed that the 103 genes negatively correlated with the re-induction of DT did not show any transcriptional regulation at the chromatin level. Additionally, ChIP-Seq analyses for repressive marks H2AK119ub and H3K27me3 detected a prominent signal of H3K27me3 on the DT re-induction-related gene sequences at R5 but not in R1 and R1P. Moreover, no clear H2AK119ub marks was observed on the DT re-induction-related gene sequences at both developmental radicle stages, suggesting that silencing of DT process after germination will be mainly due to H3K27me3 marks by the action of the PRC2 complex, without involvement of PRC1 complex. The dynamic of chromatin changes associated with H3K27me3 were also confirmed on seed-specific genes encoding potential DT-related proteins such as LEAs, oleosins and transcriptional factors. However, several transcriptional factors did not show a clear link between their decrease of chromatin openness and H3K27me3 levels, suggesting that their accessibility may also be regulated by additional factors, such as other histone modifications. Finally, in order to make these comprehensive genome-wide analyses of transcript and chromatin dynamics useful to the scientific community working on early germination and DT, we generated a dedicated genome browser containing all these data and publicly available at https://iris.angers.inrae.fr/mtseedepiatlas/jbrowse/?data=Mtruncatula.

ZBTB18 inhibits SREBP-dependent lipid synthesis by halting CTBPs and LSD1 activity in glioblastoma.

Enhanced fatty acid synthesis is a hallmark of tumors, including glioblastoma. SREBF1/2 regulate the expression of enzymes involved in fatty acid and cholesterol synthesis. Yet, little is known about the precise mechanism regulating SREBP gene expression in glioblastoma. Here, we show that a novel interaction between the co-activator/co-repressor CTBP and the tumor suppressor ZBTB18 regulates the expression of SREBP genes. In line with our findings, metabolic assays and glucose tracing analysis confirm the reduction in several phospholipid species upon ZBTB18 expression. Our study identifies CTBP1/2 and LSD1 as co-activators of SREBP genes and indicates that the functional activity of the CTBP-LSD1 complex is altered by ZBTB18. ZBTB18 binding to the SREBP gene promoters is associated with reduced LSD1 demethylase activity of H3K4me2 and H3K9me2 marks. Concomitantly, the interaction between LSD1, CTBP, and ZNF217 is increased, suggesting that ZBTB18 promotes LSD1 scaffolding function. Our results outline a new epigenetic mechanism enrolled by ZBTB18 and its co-factors to regulate fatty acid synthesis that could be targeted to treat glioblastoma patients.

Characterization of the promoter region of the murine Catsper2 gene.

CATSPER2 (Cation channel sperm-associated protein 2) protein, which is part of the calcium CATSPER channel located in the membrane of the flagellar principal piece of the sperm cell, is only expressed in the testis during spermatogenesis. Deletions or mutations in the Catsper2 gene are associated with the deafness-infertility syndrome (DIS) and non-syndromic male infertility. However, the mechanisms by which Catsper2 is regulated are unknown. Here, we report the characterization of the promoter region of murine Catsper2 and the role of CTCF and CREMτ in its transcription. We report that the promoter region has transcriptional activity in both directions, as determined by observing luciferase activity in mouse Sertoli and GC-1 spg transfected cells. WGBS data analysis indicated that a CpG island identified in silico is non-methylated; Chromatin immunoprecipitation (ChIP)-seq data analysis revealed that histone marks H3K4me3 and H3K36me3 are present in the promoter and body of the Catsper2 gene respectively, indicating that Catsper2 is subject to epigenetic regulation. In addition, the murine Catsper2 core promoter was delimited to a region between -54/+189 relative to the transcription start site (TSS), where three CTCF and one CRE binding site were predicted. The functionality of these sites was determined by mutation of the CTCF sites and deletion of the CRE site. Finally, ChIP assays confirmed that CREMτ and CTCF bind to the Catsper2 minimal promoter region. This study represents the first functional analysis of the murine Catsper2 promoter region and the mechanisms that regulate its expression.

CTR9 drives osteochondral lineage differentiation of human mesenchymal stem cells via epigenetic regulation of BMP-2 signaling.

Cell fate determination of human mesenchymal stem/stromal cells (hMSCs) is precisely regulated by lineage-specific transcription factors and epigenetic enzymes. We found that CTR9, a key scaffold subunit of polymerase-associated factor complex (PAFc), selectively regulates hMSC differentiation to osteoblasts and chondrocytes, but not to adipocytes. An in vivo ectopic osteogenesis assay confirmed the essentiality of CTR9 in hMSC-derived bone formation. CTR9 counteracts the activity of Enhancer Of Zeste 2 (EZH2), the epigenetic enzyme that deposits H3K27me3, in hMSCs. Accordingly, CTR9 knockdown (KD) hMSCs gain H3K27me3 mark, and the osteogenic differentiation defects of CTR9 KD hMSCs can be partially rescued by treatment with EZH2 inhibitors. Transcriptome analyses identified bone morphology protein-2 (BMP-2) as a downstream effector of CTR9. BMP-2 secretion, membrane anchorage, and the BMP-SMAD pathway were impaired in CTR9 KD MSCs, and the effects were rescued by BMP-2 supplementation. This study uncovers an epigenetic mechanism engaging the CTR9-H3K27me3-BMP-2 axis to regulate the osteochondral lineage differentiation of hMSCs.

PAX5 Epigenetically Orchestrates CD58 Transcription and Modulates Blinatumomab Response in Acute Lymphoblastic Leukemia

Blinatumomab is a bi-specific molecule that engages CD3+ T cells with CD19-expressing malignant B cells, thus inducing tumor lysis by T-cell immune response. While large frontline ALL trials of blinatumomab are lacking, existing data strongly indicate substantial inter-patient variability in response and toxicities. Complete remission rate ranges from 36 to 66%, depending on the regimen used and patient characteristics. Moreover, the duration of remission varies among trials, with a significant proportion of patients experiencing relapse or progressive disease following blinatumomab treatment, raising concerns about acquired drug resistance. In particular, mechanisms of resistance in CD19+ relapse remain largely unclear. Using genome-wide CRISPR screen in B-ALL cell line in vitro, we comprehensively identified leukemia intrinsic factors of blinatumomab sensitivity. In addition to CD19 deletion the loss of CD58 emerged as a top driver for resistance, plausibly compromising the CD2-CD58-mediated interaction between T cells and B-ALL. Genomic editing of the CD58 gene or blocking with anti-CD58 antibody completely abrogated blinatumomab sensitivity across different B-ALL cell lines. Screening 1,639 transcription factor genes, we then identified PAX5 as the key activator of CD58. Interestingly, primary ALL samples with PAX5 P80R mutation also showed complete loss of CD58 expression, based on RNA-seq of 1,988 B-ALL samples across 20 molecular subtypes. To further validate this, we developed isogenic clones of the B-ALL cell line 697, with different PAX5 diplotypes: wildtype/wildtype (WT/WT), wildtype/frameshift (WT/fs), and P80R/frameshift (P80R/fs). P80R/fs and WT/fs clones exhibited significantly lower CD58 expression as compared to the WT/WT clone, and both were markedly resistant to blinatumomab. PAX5 mutation did not influence CD19 expression in these B-ALL cells. By testing a panel of primary ALL samples in vitro, we found that blast harboring the PAX5 P80R mutation has the lowest CD58 expression and was the most resistant to blinatumomab, which was reversed by ectopic expression of CD58. To delineate the mechanism by which PAX5 mutation affects CD58 suppression in ALL, we comprehensively profiled epigenomic marks within the CD58 locus in 697 cells with WT/fs, P80R/fs, and WT/WT genotypes. Using ChIP-seq, we identified PAX5 binding sites within Intron 1 of the CD58 genes. By contrast, PAX5 binding at this locus was decreased in WT/fs cells and completely eliminated in the P80R/fs clones, suggesting a loss-of-function effect of the P80R mutation. This region was also characterized by H3K27ac and H3K4me3 marks, signifying its enhancer activity, which was also lost in P80R/fs cells. ATAC-seq profiling of these samples showed a similar pattern of PAX5-dependent open chromatin status. CRISPR interference showed that blocking the genomic sequence encompassing this enhancer resulted in significant downregulation of CD58 expression. PAX5 binding was also observed in the CD19 promoter region, but P80R mutation had only modest effects on histone modification and chromatin accessibility at this locus. Finally, we explored the effects of CD58 loss on T cell activation. T cell cocultured with CD58KO Nalm6 clone showed a substantial decrease in activation and proliferation, as compared to that cocultured with parental Nalm6. Defects of activation and proliferation were also observed in T cells cocultured with PAX5 P80R/fs 697 clone as compared to those with parental 697. Using RNA-seq, we identified 111 genes that were highly expressed in T cells cocultured with parental (relative to those with CD58KO Nalm6). Among these hits, there was significant enrichment of genes involved in oxygen metabolisms, immune response, and cytokine signaling pathways, indicating the loss of CD58 abolished blinatumomab-induced T cell activation. ATAC-seq of these samples further confirmed the findings of RNA-seq studies. In conclusion, our study identified the critical role of CD58-mediated interaction between B-ALL and T cells for blinatumomab sensitivity in this type of leukemia, pointing to potential strategies for genomics-guided blinatumomab treatment individualization.

Dysregulation of Stem-Progenitor and Differentiation Programmes through Modulation of Bivalent Chromatin Drives Leukemogenesis in Mutant IDH2 Acute Myeloid Leukaemia

Differentiation arrest is a hallmark in isocitrate dehydrogenase mutant (mIDH) acute myeloid leukaemia (AML). MIDH enzyme produces d-2-hydroxyglutarate, which inhibits α-ketoglutarate-dependent dioxygenases, including TET2 and Jumonji histone demethylases (J-KDM). The underlying leukemogenic mechanism is unclear and may be due to impaired DNA and/ or histone demethylation. Bivalent chromatin, with both K4me3 and K27me3 marks, maintain genes in a 'poised’ state and is important in modulating expression during cell differentiation. We hypothesised that this may be an important mechanism by which mIDH drives differentiation arrest. Using an mIDH2 inhibitor Enasidenib (ENA), we stimulated granulocytic differentiation of IDH2m AML progenitors (L-Prog), and using a multiomic approach, we tracked epigenetic events that reprogramme arrested L-Prog. We previously reported downregulation of stem-progenitor signatures and co-incident expression of cell cycle and granulocytic transcriptional networks during ENA-induced differentiation. We also observed increased chromatin accessibility in ENA-treated cells and hypothesised that regulation of H3K27me3 is critical in creating a more permissive epigenetic landscape (Silveira et al., Blood 2021; 138 (Supplement 1): 3467). To test this hypothesis, we performed enhanced reduced representation methylation sequencing and chromatin immunoprecipitation (ChIPseq) assay in parallel with RNAseq. Compared with controls, ENA stimulated global CpG demethylation (mean LogFC=2.01, range 1.79-2.36). However, promoter CpG islands were only modestly hypomethylated in genes upregulated by ENA. This suggests that mechanisms other than DNA hypermethylation are crucial for IDHm leukemogenesis. Focusing on histone regulation, we studied H3-K4me3, K27me3 and K27ac marks in ENA-treated L-prog from responsive (R) and non-responsive (NR) samples. We annotated gene promoters as monovalent (mono), bivalent (biv) and partial bivalent (part-K27me3 or part-K4me3), depending on relative enrichment of K27me3 and K4me3 marks, and show correlation between gene expression and repressive (mono-K27me3) to active (mono-K4me3) histone states in a continuum. Consistent with the role of bivalent states in modulating gene expression, the most dynamically differentially expressed genes (DEG, LogFC > 0.25) had the greatest proportion of bivalent promoters. Furthermore, ENA-treated R samples have significantly increased (OR range: 1.11 - 2.17, p < 10e-5) bivalent and part-K4me3 marked promoters respectively compared with DMSO, or with ENA-treated NR samples. 67-84% of ENA-upregulated genes where we detected altered chromatin states transitioned from bivalent to more active partial or monovalent K4me3 states. These transitions were 3x more likely to arise from loss of K27me3 than gain K4me3 (p < 10e-16), suggesting specific enhancement of KDM4/6 activity. Conversely, >95% of downregulated genes moved from relatively activated states (mono-K4me3 or part-K4me3) towards bivalency, mainly through gain of K27me3. These genes were highly enriched with polycomb repressor complex 2 (PRC2)/ EZH2 targets (OR range: 4.28-11.03, p < 0.01). The switch from part-H3K27me3 to part-H3K4me3 via bivalency in ENA-upregulated differentiation genes is associated with increased H3K27ac, which in turn is correlated with increased chromatin accessibility measured by single cell (sc)ATACseq. Motif usage analysis across the ENA-induced differentiation trajectory revealed that differentiation genes are dual regulated: repressed by stem-progenitor transcription factors (prog-TFs), e.g. GATA2, FOSL1 and MYC which are active in early differentiation, and then activated by granulocyte-monocyte TFs EGR1, CEBPE, JUNB/FOSB and SPI1 in late differentiation, as prog-TFs themselves are downregulated. Our model (Figure 1) of d-2HG-induced differentiation arrest places modulation of promoter chromatin accessibility and gene expression via KDM4/5 and PRC2-mediated changes to H3K27 methylation centre-stage. For the first time, we show how these chromatin changes drive dynamic transcriptional programmes that initiate and sustain restoration of granulocytic differentiation in primary AML progenitors, and in so doing, identified a novel mechanism of leukemogenesis which may be targetable in other AML subtypes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EZH2 Targeting Induces CD38 Upregulation and Response to Anti-CD38 Antibodies in Multiple Myeloma

Multiple myeloma (MM) is the second most common hematological malignancy characterized by the accumulation of tumor plasma cells within the bone marrow. MM is characterized by high molecular and clinical heterogeneity. During the last 10 years new therapeutic classes including targeted immunotherapies significantly improved the overall survival of MM patients. However, drug resistance and relapse remain major challenges in MM. In MM, marked reduction of CD38 expression via exocytosis, endocytosis, degradation of the antigen-antibody complex and trogocytosis has been associated with resistance to CD38 targeted immunotherapies. Our group previously described a link and a negative correlation between CD38 and EZH2 (Enhancer of Zeste homolog 2) expression during normal B to plasma cell differentiation in association with a transcriptional control of CD38 expression involving the polycomb PRC2 complex (Herviou et al. Leukemia. 2019). Here we identified a significant negative correlation between CD38 expression and EZH2 expression in 2 independent cohorts of MM patients (p<0.05; Montpellier cohort n=198; Compass cohort n=631). Thus, we hypothesized that PRC2 targeting using EZH2 inhibitor could result in CD38 re-expression to overcome daratumumab resistance. We first characterized the membrane CD38 expression by calculating the Antibody Binding Capacity in our unique panel of 40 Human Myeloma Cell lines (HMCL) representative of MM heterogeneity. We observed a heterogeneous expression of the membrane CD38 expression on MM cell lines with cell lines demonstrating no membrane expression and cell lines demonstrating high membrane expression. Next, we selected 3 HMCLs with low (JJN3), intermediate (XG2), and high (XG7) CD38 expression, and treated these cell lines with sublethal concentration of EZH2 inhibitor (EPZ-6438) for 9 days. CD38 membrane expression was characterized at Day 3, Day 6 and Day 9. Interestingly, EZP-6438 treatment induced upregulation of cell surface CD38 expression on all the HMCLs tested with the highest effect measured at day 9. Similar re-expression pattern was observed by western blot at these timing in the JJN3 CD38low cell line. Remarkably, ChIPseq analyses underlined a significant association of H3K27me3 mark at CD38 promoter in CD38neg and CD38low cell lines compared to CD38highcells. In addition, we investigated the efficiency of CD38 monoclonal antibodies (Daratumumab and Isatuximab) lysis through ADCC (Antibody dependent cellular cytotoxicity) assay after 9 days of EPZ-6438 pretreatment. Using JJN3 CD38low HMCL, EPZ-6438 pretreatment significantly improved the ADCC lysis induced by low doses of Daratumumab and Isatuximab treatment, in presence of purified Natural Killer (NK) from healthy donor, together with CD38 re-expression (p<0.05; n=3). Finally, we searched to validate these results using primary MM cells from patients at relapse after Daratumumab treatment. EPZ-6438 pretreatment resulted in a significant CD38 membrane expression upregulation with a 2 fold difference compared to the control condition together with improved ADCC toxicity of anti-CD38 antibodies in vitro. Altogether we demonstrated that treatment of MM cells with EZH2 inhibitor leads to significant upregulation of membrane CD38 expression in cell lines and primary MM cells from patients. CD38 re-expression was linked to an improvement of Daratumumab and Isatuximab ADCC efficiency. Thus, EZH2 targeting may be of therapeutic interest to overcome resistance to anti-CD38 targeted immunotherapies in Multiple Myeloma.

Functional Characterization of Cooperating MGA Deletions in RUNX1::RUNX1T1 Acute Myeloid Leukemia

Max-gene associated (MGA) is a dual-specificity transcription factor that is recurrently mutated in 2-5% of all cancers and commonly seen in lung adenocarcinoma, endometrial carcinoma, and colorectal cancer. Our group has previously described heterozygous loss-of-function mutations in 9% of RUNX1::RUNX1T1 pediatric AMLs. Similar loss-of-function mutations are seen in other hematopoietic malignancies, including chronic lymphocytic leukemia (CLL), natural killer/T-cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), and T-cell acute lymphoblastic leukemia (T-ALL). MGA is a component of the non-canonical polycomb repressive complex (ncPRC1.6) containing an amino-terminal T-box domain and a carboxy-terminal Myc-like basic helix-loop-helix (bHLH) domain which are used to regulate MAX-network and T-box family targets. In hematological malignancies such as RUNX1::RUNX1T1 AMLs, MGA mutations disrupt or remove the Myc-like bHLH domain. The role of MGA in normal hematopoietic regulation and leukemic development are understudied. This is likely due to the lack of in vivo MGA deficiency models which are embryonic lethal. We hypothesize that the loss of MGA leads to disruption of the ncPRC1.6 complex, causing a loss in global transcriptional repression of many pathways, including Myc and E2F target genes, leading to enhanced hematopoietic cell growth and self-renewal, as well as shortening the latency of AML driven by RUNX1::RUNXT1. To test this hypothesis, we designed a multidisciplinary approach to characterize the hematopoietic function of MGA. IP-Westerns of overexpressed flag-tagged MGA and its subsequent variants in K562 cells revealed that MGA truncations disrupt interactions with components of the ncPRC1.6 complex. Because these truncations lead to the loss of MGA's molecular function, we next generated CRISPR/Cas9 to knockout MGA in Molm13 cells. Using RNA-Seq and CUT&RUN to assess the genomic localization of MGA, we determined that MGA binds to an average of 85,119 peaks and 17.7% of those sites are at transcription start sites, including cell cycle genes such as CCND2. Deletion of MGA led to the removal of repressive H3K27me3 marks and an increase in MYC binding as well as active histone marks such as H3K4me3 and H3K27Ac at the transcription start site of CCND2. This led to an increase in CCND2 expression (logFC 1.56, FDR 1.56E-12) which is supported by in vitro EdU assays showing a 10.43% increase in cells cycling into S-phase. To further define the hematopoietic function of MGA, we designed a conditional knockout Mga mouse model with LoxP sites flanking exon 3, leading to deletion of Mga in hematopoietic stem and progenitor cells (HSPCs) when crossed with Vav1-cre. Transcriptome profiling analysis of HSPCs isolated from heterozygous (Mga+/) and homozygous (Mga-/-) mice reveals that Mga-/- mice, but not Mga+/-, leads to up-regulation of cell cycle (FDR 4.2E-06), Myc (FDR 6.3E-10), and E2f (FDR 2.9E-17) signaling pathways. Ex vivo serial CFU replating and cell proliferation assays support this, showing that Mga-/- leads to increased stem cell self-renewal and cell growth. However, bone marrow and spleen analysis of both Mga+/- and Mga-/- mice reveal no changes in immunophenotype when compared to WT controls. Expression of RUNX1::RUNX1T1 isoform 9a (AML-ETO9a) in Mga+/- or Mga-/- HSPCs reveals a significant increase (69% and 74% respectively, p<0.01) in leukemic stem cell self-renewal, when compared to WT controls expressing AML-ETO9a at week 4. When transplanted in mice, Mga+/- and Mga-/- HSPCs expressing AML-ETO9a led to a rapid expansion of blasts (GFP+, lin-, cKit+) found in the peripheral blood as well as a shortened latency compared to wild-type controls expressing AML-ETO9a (Figure 1). These studies demonstrated MGA as a regulator of hematopoietic cell proliferation (Figure 2), and how MGA deficiency cooperates with oncogenes to create more aggressive leukemia. Because MGA mutations occur in many tumor types, these data also provide functional insights into ncPRC1.6 activity in leukemia and other tumor types with recurrent mutations in MGA or other components of this complex. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EPCO-44. HOXD13 GENE PLAYS A ROLE AS A PUTATIVE MASTER EPIGENETIC REGULATOR IN GLIOMA RECURRENCE AND PROGRESSION

Abstract BACKGROUND Adult diffuse glioma is a heterogenous brain tumor group that has malignant potential and recurring properties. Glioma CpG island methylator phenotype (G-CIMP) associated with chromatin remodeling encompasses two different methylation degrees: G-CIMP-high and G-CIMP-low that have high levels of DNA methylation and loss of DNA methylation, respectively. G-CIMP-high confers a more favorable prognosis and better predictive values by altering transcriptional dynamics and the gene expression. METHODS We have performed Chromatin Immunoprecipitation Sequencing (ChIP-Seq) on DNA extracted from fresh frozen primary tumor tissues (G-CIMP-high and G-CIMP-low). We have processed the sequencing data by using FastQC (v.:0.11.5), bwa-mem (v.:0.7.15) and picard tool (v.:2.7.1). The ChIP-seq peaks and the differentially bound peaks were obtained using MACS2 (v.:2.1.1) and DiffBind (v.:3.4.11), respectively. We have also integrated transcriptomic, epigenomic and genomic data. RESULTS Initial results showed that the differentially bound H3K27ac and H3K4me3 peak gains and losses are located mainly in intronic and intergenic regions. The integration between epigenetic and transcriptomic data revealed that the promoter of the HOXD13 gene upregulated in GCIMP-low presents an active regulatory region and is enriched by H3K27ac and H3K4me3 sites. We were able to identify that GCIMP-low, in addition to presenting more downregulated genes than GCIMP-high, also has greater affinity to the H3K27ac peaks. In GCIMP-low, we identified that regions enriched by H3K27ac are highly associated with the presence H3K4me3 marks. CONCLUSION We mapped the enrichment of histone modifications in the TSS of differentially expressed genes in G-CIMP-high and G-CIMP-low identifying both the susceptibility of GIMP-low to interact with H3K27ac and the fact that the progression of GCIMP-high to G-CIMP-low can be explained by the association between H3K27ac gain and gene downregulation. Also, we predicted the HOXD13 gene as a putative master epigenetic regulator of the G-CIMP demethylation process and, consequently, of tumor recurrence and progression, over time.

Abstract 11486: Hormone-Independent Sex Dimorphisms in Cardiac Reparative Functions of Bone Marrow Stem Cells: Underlying Epigenetic Insights

Introduction: This work was designed to identify the hormone-independent mechanisms that govern the superiority of female endothelial progenitor cells (F-EPC) for cardiac repair post myocardial infarction (MI). A direct comparison of therapeutic efficacy of gender-specific stem cells or estrogen-independent mechanisms of gender-specific dimorphism in the reparative properties of EPCs has not been established. Hypothesis: We hypothesized that epigenetic mechanisms contribute to the sex-specific functional dimorphisms of Sca-1+/CD31+ EPCs in regulating cardiac reparative functions post-MI. Methods: Male (M), female (F), and ovariectomized (OVX) mice derived Sca-1+/CD31+ EPCs were subjected through a series of cytokine quantifications and epigenetic CUT&amp;Tag screening, followed by assessment of their therapeutic function in vitro and in vivo . Results: F-EPC and OVX EPC secrete high levels of proangiogenic factors and low amounts of pro-inflammatory cytokines compared to M-EPC. Further evaluation of the inflammasome showed that the M-EPC secreted the most elevated amounts of CCL3, and other inflammatory chemokines compared to F-EPC and OVX EPC. The secretome of F-EPC and OVX EPC was found to be substantially proangiogenic in vitro than the male EPCs. Post-MI injection of F-EPC and OVX EPC resulted in remarkable preservation of cardiac structure and functions. In contrast, post-MI injection of M-EPC resulted in an inflamed myocardium. Epigenetic sequencing of the sex differential EPCs for the H3K9me3 mark showed high methylation in M-EPC compared to F-EPC and OVX EPC. The inhibition of histone methyltransferase, G9a using BIX-01294 upregulated the secretion of inflammatory factors in all EPCs. The conditioned medium from all EPCs with high levels of CCL3 inhibited angiogenesis in vitro . Neutralizing CCL3 in the same medium restored in vitro angiogenesis. Conclusion: EPCs from both fertile and OVX female mice retain better cardiac reparative activity than their male counterparts irrespective of sex hormone influence. Mechanistically, we show that this gender-based functional dimorphism of EPCs is mediated, in part, by differential epigenetic regulation of chemokine CCL3.

37. Epigenetic reprogramming of brain development pathways during non-small cell lung cancer metastasis to brain

Non-small cell lung cancer (NSCLC) is one of the most common and deadliest cancers, with roughly half of all patients initially presenting with both primary and metastatic disease. While the major events in the metastatic cascade have been identified, a mechanistic understanding of how NSCLC routinely, successfully colonizes the brain is largely unknown. To better understand this process, we profiled a combination of genomic, transcriptomic, and methylomic landscapes of 45 paired NSCLC primary and brain metastasis samples. 75 genes displayed recurrent metastasis enriched variants, largely implicated in focal adhesion and extracellular matrix receptor interactions. Variant allele frequencies over a wide range of epigenetic regulators displayed an increase in metastases, suggesting that epigenetic misregulation may be selected for and possibly contribute to NSCLC metastasis to brain. Consistent with these observations, we observed widespread changes in DNA methylation throughout disease progression, many found within brain-specific active enhancers and correlated with increased nearby gene expression. The greatest recurrent methylation changes during metastatic progression occurred over a subset of DNA methylation valleys (DMVs) enriched for H3K9me3 and bivalent marks H3K27me3 and H3K4me1 in normal lung. Mapping EZH2, the catalytic subunit of polycomb repressive complex 2 (PCR2), binding locations in H1299, a lymph node-derived lung cancer cell line, revealed a pervasive loss of EZH2 binding within DMVs accompanied by an increase in DNA methylation, exemplifying epigenetic switching. The vast majority of these DMR-associated DMVs harbor developmental genes, suggesting that altered epigenetic regulation of developmentally important genes may confer a selective advantage during metastatic progression.

Pathological Nuclear Hallmarks in Dentate Granule Cells of Alzheimer's Patients: A Biphasic Regulation of Neurogenesis.

The dentate gyrus (DG) of the human hippocampus is a complex and dynamic structure harboring mature and immature granular neurons in diverse proliferative states. While most mammals show persistent neurogenesis through adulthood, human neurogenesis is still under debate. We found nuclear alterations in granular cells in autopsied human brains, detected by immunohistochemistry. These alterations differ from those reported in pyramidal neurons of the hippocampal circuit. Aging and early AD chromatin were clearly differentiated by the increased epigenetic markers H3K9me3 (heterochromatin suppressive mark) and H3K4me3 (transcriptional euchromatin mark). At early AD stages, lamin B2 was redistributed to the nucleoplasm, indicating cell-cycle reactivation, probably induced by hippocampal nuclear pathology. At intermediate and late AD stages, higher lamin B2 immunopositivity in the perinucleus suggests fewer immature neurons, less neurogenesis, and fewer adaptation resources to environmental factors. In addition, senile samples showed increased nuclear Tau interacting with aged chromatin, likely favoring DNA repair and maintaining genomic stability. However, at late AD stages, the progressive disappearance of phosphorylated Tau forms in the nucleus, increased chromatin disorganization, and increased nuclear autophagy support a model of biphasic neurogenesis in AD. Therefore, designing therapies to alleviate the neuronal nuclear pathology might be the only pathway to a true rejuvenation of brain circuits.

Genome-wide chromatin accessibility analysis unveils open chromatin convergent evolution during polyploidization in cotton

Allopolyploidization, resulting in divergent genomes in the same cell, is believed to trigger a "genome shock", leading to broad genetic and epigenetic changes. However, little is understood about chromatin and gene-expression dynamics as underlying driving forces during allopolyploidization. Here, we examined the genome-wide DNase I-hypersensitive site (DHS) and its variations in domesticated allotetraploid cotton (Gossypium hirsutum and Gossypium barbadense, AADD) and its extant AA (Gossypium arboreum) and DD (Gossypium raimondii) progenitors. We observed distinct DHS distributions between G. arboreum and G. raimondii. In contrast, the DHSs of the two subgenomes of G. hirsutum and G. barbadense showed a convergent distribution. This convergent distribution of DHS was also present in the wild allotetraploids Gossypium darwinii and G. hirsutum var. yucatanense, but absent from a resynthesized hybrid of G. arboreum and G. raimondii, suggesting that it may be a common feature in polyploids, and not a consequence of domestication after polyploidization. We revealed that putative cis-regulatory elements (CREs) derived from polyploidization-related DHSs were dominated by several families, including Dof, ERF48, and BPC1. Strikingly, 56.6% of polyploidization-related DHSs were derived from transposable elements (TEs). Moreover, we observed positive correlations between DHS accessibility and the histone marks H3K4me3, H3K27me3, H3K36me3, H3K27ac, and H3K9ac, indicating that coordinated interplay among histone modifications, TEs, and CREs drives the DHS landscape dynamics under polyploidization. Collectively, these findings advance our understanding of the regulatory architecture in plants and underscore the complexity of regulome evolution during polyploidization.

The H3K9me2-binding protein AGDP3 limits DNA methylation and transcriptional gene silencing in Arabidopsis.

DNA methylation, a conserved epigenetic mark, is critical for tuning temporal and spatial gene expression. The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci. However, how ROS1 is recruited to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor that is required to prevent gene silencing and DNA hypermethylation. AGDP3 binds to H3K9me2 marks in its target DNA via its AGD12 cassette. Analysis of the crystal structure of the AGD12 cassette of AGDP3 in complex with an H3K9me2 peptide revealed that dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. A histidine residue located in the methyllysine binding aromatic cage provides AGDP3 with pH-dependent H3K9me2 binding capacity. Our results uncover a molecular mechanism for the regulation of DNA demethylation by the gene silencing mark H3K9me2.

PpyABF3 recruits the COMPASS-like complex to regulate bud dormancy maintenance via integrating ABA signaling and GA catabolism.

Bud dormancy is essential for perennial trees that survive the cold winters and to flower on time in the following spring. Histone modifications have been reported to be involved in the control of the dormancy cycle and DAM/SVPs are considered targets. However, how the histone modification marks are added to the specific gene loci during bud dormancy cycle is still unknown. Using yeast-two hybrid library screening and co-immunoprecipitation assays, we found that PpyABF3, a key protein regulating bud dormancy, recruits Complex of Proteins Associated with Set1-like complex via interacting with PpyWDR5a, which increases the H3K4me3 deposition at DAM4 locus. Chromatin immunoprecipitation-quantitative polymerase chain reaction showed that PpyGA2OX1 was downstream gene of PpyABF3 and it was also activated by H3K4me3 deposition. Silencing of GA2OX1 in pear calli and pear buds resulted in a similar phenotype with silencing of ABF3. Furthermore, overexpression of PpyWDR5a increased H3K4me3 levels at DAM4 and GA2OX1 loci and inhibited the growth of pear calli, whereas silencing of PpyWDR5a in pear buds resulted in a higher bud-break percentage. Our findings provide new insights into how H3K4me3 marks are added to dormancy-related genes in perennial woody plants and reveal a novel mechanism by which ABF3 integrates abscisic acid signaling and gibberellic acid catabolism during bud dormancy maintenance.

Upregulation of programmed cell death 1 by interferon gamma and its biological functions in human monocytes

Programmed cell death 1 (PD-1) is a co-inhibitory checkpoint receptor expressed in various immune cells, especially in activated T cells. Engagement of PD-1 with its ligand leads to the exhausted T cells and impaired antitumor immunity. To date, PD-1 expression and its roles have been widely reported in T cells but not well defined in innate immune cells including monocytes. In this study, expression of PD-1 was investigated in human monocytes. Here we observed that among cytokines tested, IFN-γ significantly upregulated the PD-1 expression in both THP-1 cell line and human primary monocytes in a dose- and time-dependent manner. This effect was reduced by PI3K inhibitor, suggesting that the involvement of PI3K/AKT pathway. Furthermore, enrichment of active histone mark H3K4me3 in the Pdcd1 promotor was also observed in IFN-γ-induced THP-1, indicating that epigenetic regulation also plays a role in IFN-γ-induced PD-1 expression. To investigate the biological functions of PD-1, Pdcd1 was deleted in THP-1 cell line by CRISPR/Cas9 system and the phagocytic ability was investigated. The results showed that the PD-1 deficiency in THP-1 cell line resulted in significantly poor phagocytic potency against carboxylated-modified latex beads. Moreover, the PD-1 deficiency or blocking PD-1/PD-L1 interaction by immune checkpoint inhibitor resulted in an impaired induction of IL-4-induced CD163 expression in THP-1 cell line. Taken together, these results highlighted the importance of PD-1 expression in some of key monocyte functions.

Single-cell epigenome analysis reveals age-associated decay of heterochromatin domains in excitatory neurons in the mouse brain

Loss of heterochromatin has been implicated as a cause of pre-mature aging and age-associated decline in organ functions in mammals; however, the specific cell types and gene loci affected by this type of epigenetic change have remained unclear. To address this knowledge gap, we probed chromatin accessibility at single-cell resolution in the brains, hearts, skeletal muscles, and bone marrows from young, middle-aged, and old mice, and assessed age-associated changes at 353,126 candidate cis-regulatory elements (cCREs) across 32 major cell types. Unexpectedly, we detected increased chromatin accessibility within specific heterochromatin domains in old mouse excitatory neurons. The gain of chromatin accessibility at these genomic loci was accompanied by the cell-type-specific loss of heterochromatin and activation of LINE1 elements. Immunostaining further confirmed the loss of the heterochromatin mark H3K9me3 in the excitatory neurons but not in inhibitory neurons or glial cells. Our results reveal the cell-type-specific changes in chromatin landscapes in old mice and shed light on the scope of heterochromatin loss in mammalian aging.

The Drosophila ZAD zinc finger protein Kipferl guides Rhino to piRNA clusters.

RNA interference systems depend on the synthesis of small RNA precursors whose sequences define the target spectrum of these silencing pathways. The Drosophila Heterochromatin Protein 1 (HP1) variant Rhino permits transcription of PIWI-interacting RNA (piRNA) precursors within transposon-rich heterochromatic loci in germline cells. Current models propose that Rhino's specific chromatin occupancy at piRNA source loci is determined by histone marks and maternally inherited piRNAs, but also imply the existence of other, undiscovered specificity cues. Here, we identify a member of the diverse family of zinc finger associated domain (ZAD)-C2H2 zinc finger proteins, Kipferl, as critical Rhino cofactor in ovaries. By binding to guanosine-rich DNA motifs and interacting with the Rhino chromodomain, Kipferl recruits Rhino to specific loci and stabilizes it on chromatin. In kipferl mutant flies, Rhino is lost from most of its target chromatin loci and instead accumulates on pericentromeric Satellite arrays, resulting in decreased levels of transposon targeting piRNAs and impaired fertility. Our findings reveal that DNA sequence, in addition to the H3K9me3 mark, determines the identity of piRNA source loci and provide insight into how Rhino might be caught in the crossfire of genetic conflicts.

A chromatin mark by SETD7 regulates myocardial inflammation in obesity-related heart failure with preserved ejection fraction

Abstract Background Obesity is a major risk factor for heart failure with preserved ejection fraction (HFpEF). Post-translational modification of histones by chromatin modifying enzymes (CMEs) are emerging as pivotal regulators of gene transcription in cardiovascular disease. Purpose To investigate the role of chromatin remodelling in obese HFpEF (obHFpEF). Methods Gene expression profiling of CMEs (PCR array) was performed in left ventricular (LV) myocardial specimens from obHFpEF patients and age-matched control donors (n=8/group). Among myocardial CMEs, the methyltransferase SETD7 showed the highest variation in gene expression. Hence, we investigated the role of SETD7 and its chromatin mark H3K4me1 in a murine model of obHFpEF. Mice with cardiomyocyte-specific deletion of SETD7 (c-SETD7−/−) and control littermates (SETD7fl/fl) were subjected to high fat diet feeding and L-NAME treatment for 15 weeks to induce obHFpEF. Echocardiography and Treadmill exhaustion test were performed. ChIP-Seq datasets were employed to determine the biological pathways regulated by SETD7, whereas chromatin immunoprecipitation assays (ChIP) were performed to investigate SETD7/H3k4me1 enrichment on target gene promoters. SETD7 gain- and loss-of-function experiments were performed in cultured neonatal rat ventricular myocytes (NRVMs) exposed to palmitic acid (200μM) for 48h. Selective inhibition of SETD7 by (R)-PFI-2 was performed in skinned cardiomyocytes isolated from left ventricular specimens of obHFpEF patients. Passive stiffness, a main feature of HFpEF, was assessed before and after (R)-PFI-2 treatment. Results CMEs profiling showed SETD7 as the top-ranking transcript (fold change, 7.36, P&amp;lt;0.01) in myocardial specimens from obHFpEF patients as compared to controls. ChIP-Seq in CMs showed a strong enrichment of SETD7 and H3k4me1 on the promoter of NF-kB p65 gene, a master regulator of inflammation. SETD7 and H3k4me1 were upregulated in HFpEF vs. control mouse hearts, showed enrichment on NF-kB p65 promoter and were associated with IL-1β and IL-6 upregulation. In HFpEF mice, cardiomyocyte-specific deletion of SETD7 protected against LV hypertrophy, diastolic dysfunction (assessed by E/E' ratio) and lung congestion while improving exercise tolerance. At the molecular level, SETD7 deletion blunted H3K4me1 enrichment on p65 promoter thus preventing the upregulation of inflammatory genes and myocardial apoptosis. In cultured CMs exposed to PA, SETD7 inhibition by (R)-PFI-2 prevented H3k4me1-driven p65 upregulation, whereas SETD7 overexpression mimicked HFpEF features. Moreover, knockdown of NF-kB p65 prevented IL-1β/IL-6 transcription in SETD7-overexpressing CMs. Of clinical relevance, (R)-PFI-2 reduced passive stiffness in skinned CMs isolated from obHFpEF patients. Conclusions Our results unveil a new epigenetic mechanism underpinning inflammation in obHFpEF. Targeting SETD7 may represent a novel therapeutic approach to prevent HFpEF in obesity. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation