Intragenic Enhancer Research Articles

Association of MGMT Promoter and Enhancer Methylation with Genetic Variants, Clinical Parameters, and Demographic Characteristics in Glioblastoma.

The response of glioblastoma (GBM) patients to the alkylating agent temozolomide (TMZ) vitally depends on the expression level of the repair protein O6-methylguanine-DNA methyltransferase (MGMT). Since MGMT is strongly regulated by promoter methylation, the methylation status of the MGMT promoter has emerged as a prognostic and predictive biomarker for GBM patients. By determining the methylation levels of the four enhancers located within or close to the MGMT gene, we recently found that enhancer methylation contributes to MGMT regulation. In this study, we investigated if methylation of the four enhancers is associated with SNP rs16906252, TERT promoter mutations C228T and C250T, TERT SNP rs2853669, proliferation index Ki-67, overall survival (OS), age, and sex of the patients. In general, associations with genetic variants, clinical parameters, and demographic characteristics were caused by a complex interplay of multiple CpGs in the MGMT promoter and of multiple CpGs in enhancer regions. The observed associations for intragenic enhancer 4, located in intron 2 of MGMT, differed from associations observed for the three intergenic enhancers. Some findings were restricted to subgroups of samples with either methylated or unmethylated MGMT promoters, underpinning the relevance of the MGMT promoter status in GBMs.

Association between MGMT Enhancer Methylation and MGMT Promoter Methylation, MGMT Protein Expression, and Overall Survival in Glioblastoma.

The repair protein O6-methylguanine-DNA methyltransferase (MGMT) is regulated epigenetically, mainly by the methylation of the MGMT promoter. MGMT promoter methylation status has emerged as a prognostic and predictive biomarker for patients with newly diagnosed glioblastoma (GBM). However, a strong negative correlation between MGMT promoter methylation and MGMT protein expression cannot be applied as a rule for all GBM patients. In order to investigate if the DNA methylation status of MGMT enhancers is associated with MGMT promoter methylation, MGMT expression, and the overall survival (OS) of GBM patients, we established assays based on high-resolution melting analysis and pyrosequencing for one intragenic and three intergenic MGMT enhancers. For CpGs in an enhancer located 560 kb upstream of the MGMT promoter, we found a significant negative correlation between the methylation status and MGMT protein levels of GBM samples expressing MGMT. The methylation status of CpGs in the intragenic enhancer (hs696) was strongly negatively correlated with MGMT promoter methylation and was significantly higher in MGMT-expressing GBM samples than in MGMT-non-expressing GBM samples. Moreover, low methylation of CpGs 01-03 and CpGs 09-13 was associated with the longer OS of the GBM patients. Our findings indicate an association between MGMT enhancer methylation and MGMT promoter methylation, MGMT protein expression, and/or OS.

EVI1 drives leukemogenesis through aberrant ERG activation

AbstractChromosomal rearrangements involving the MDS1 and EVI1 complex locus (MECOM) on chromosome 3q26 define an aggressive subtype of acute myeloid leukemia (AML) that is associated with chemotherapy resistance and dismal prognosis. Established treatment regimens commonly fail in these patients, therefore, there is an urgent need for new therapeutic concepts that will require a better understanding of the molecular and cellular functions of the ecotropic viral integration site 1 (EVI1) oncogene. To characterize gene regulatory functions of EVI1 and associated dependencies in AML, we developed experimentally tractable human and murine disease models, investigated the transcriptional consequences of EVI1 withdrawal in vitro and in vivo, and performed the first genome-wide CRISPR screens in EVI1-dependent AML. By integrating conserved transcriptional targets with genetic dependency data, we identified and characterized the ETS transcription factor ERG as a direct transcriptional target of EVI1 that is aberrantly expressed and selectively required in both human and murine EVI1–driven AML. EVI1 controls the expression of ERG and occupies a conserved intragenic enhancer region in AML cell lines and samples from patients with primary AML. Suppression of ERG induces terminal differentiation of EVI1-driven AML cells, whereas ectopic expression of ERG abrogates their dependence on EVI1, indicating that the major oncogenic functions of EVI1 are mediated through aberrant transcriptional activation of ERG. Interfering with this regulatory axis may provide entry points for the development of rational targeted therapies.

FOXA1 inhibits hypoxia programs through transcriptional repression of HIF1A.

Intratumoral hypoxia is associated with castration-resistant prostate cancer (CRPC), a lethal disease. FOXA1 is an epithelial transcription factor that is down-regulated in CRPC. We have previously reported that FOXA1 loss induces epithelial-mesenchymal transition (EMT) and cell motility through elevated TGFβ signaling. However, whether FOXA1 directly regulates hypoxia pathways of CRPC tumors has not been previously studied. Here we report that FOXA1 down-regulation induces hypoxia transcriptional programs, and FOXA1 level is negatively correlated with hypoxia markers in clinical prostate cancer (PCa) samples. Mechanistically, FOXA1 directly binds to an intragenic enhancer of HIF1A to inhibit its expression, and HIF1A, in turn, is critical in mediating FOXA1 loss-induced hypoxia gene expression. Further, we identify CCL2, a chemokine ligand that modulates tumor microenvironment and promotes cancer progression, as a crucial target of the FOXA1-HIF1A axis. We found that FOXA1 loss leads to immunosuppressive macrophage infiltration and increased cell invasion, dependent on HIF1A expression. Critically, therapeutic targeting of HIF1A-CCL2 using pharmacological inhibitors abolishes FOXA1 loss-induced macrophage infiltration and PCa cell invasion. In summary, our study reveals an essential role of FOXA1 in controlling the hypoxic tumor microenvironment and establishes the HIF1A-CCL2 axis as one mechanism of FOXA1 loss-induced CRPC progression.

HTLV-1 intragenic viral enhancer influences immortalization phenotype in vitro, but is dispensable for persistence and disease development in animal models.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL) and chronic neurological disease. The disparity between silenced sense transcription versus constitutively active antisense (Hbz) transcription from the integrated provirus is not fully understood. The presence of an internal viral enhancer has recently been discovered in the Tax gene near the 3’ long terminal repeat (LTR) of HTLV-1. In vitro, this enhancer has been shown to bind SRF and ELK-1 host transcription factors, maintain chromatin openness and viral gene transcription, and induce aberrant host gene transcription near viral integration sites. However, the function of the viral enhancer in the context of early HTLV-1 infection events remains unknown. In this study, we generated a mutant Enhancer virus (mEnhancer) and evaluated its effects on HTLV-1-mediated in vitro immortalization, establishment of persistent infection with an in vivo rabbit model, and disease development in a humanized immune system (HIS) mouse model. The mEnhancer virus was able to establish persistent infection in rabbits, and there were no significant differences in proviral load or HTLV-1-specific antibody responses over a 25-week study. However, rabbits infected with the mEnhancer virus had significantly decreased sense and antisense viral gene expression at 12-weeks post-infection. HIS mice infected with wt or mEnhancer virus showed similar disease progression, proviral load, and viral gene expression. While mEnhancer virus was able to sufficiently immortalize primary T-lymphocytes in cell culture, the immortalized cells had an altered phenotype (CD8+ T-cells), decreased proviral load, decreased sense and anti-sense gene expression, and altered cell cycle progression compared to HTLV-1.wt immortalized cells (CD4+ T-cells). These results suggest that the HTLV-1 enhancer element alone does not determine persistence or disease development but plays a pivotal role in regulating viral gene expression.

ATAC-seq on iPSC derived astrocytes to assess chromatin accessibility differences between African and European local ancestry.

Local ancestry (LA) surrounding the APOE4 allele is associated with an increased risk for Alzheimer Disease (AD) in European (EU) LA compared to African (AF) LA. We recently demonstrated that APOE4 has significantly higher expression in astrocytes in brain from EU compared to AF APOE4 LA carriers, but the specific molecular mechanism leading to this difference is not known. We investigated whether evaluating chromatin accessibility and gene expression profiles of inducible pluripotent stem cell (iPSC) derived astrocytes in the LA region in both AF and EU LA could lead us to identify potential regulatory factors affecting chromatin remodeling and expression of APOE. We assayed for Transposase Accessible Chromatin (ATAC-seq), and RNA-seq in three iPSC derived astrocytes from two AF LA and one EU LA individuals with AD. We processed the ATAC-seq data with the ENCODE ATAC-seq pipeline and called peaks using MACS2. An average of 692 peaks were detected in the LA region with no statistical difference between EU and AF LA. Interestingly, an African APOE4 homozygote LA astrocyte line with high APOE expression had an exclusive ATAC peak at the APOE promoter. Amongst the transcription factors (TFs) suggested to bind this differentially accessible peak in APOE are HNF4A and TEAD4. Both TFs had significantly increased expression in this astrocyte line compared to the other lines. Additionally, differential accessibility in this line include peaks in an intragenic enhancer of PVRL2 and the promoter of CBLC, both shown to be bound by HNF4A and both overexpressed in this cell line and one in the promoter of CYP2S1 with TEAD4 binding, which was also overexpressed. This study is one of the first studies to compare chromatin accessibility in AF and EU ancestries. It also represents initial efforts to investigate potential mechanisms and factors, using iPSC-derived cell lines, that could contribute to the differential expression we have previously reported in APOE4 between AF and EU LA brains. Our data suggest that elevated astrocytic expression of APOE4 is associated with rs.

Epithelial Transcription Factor FOXA1 Regulates Prostate Cancer Immune Response

Background: While localized prostate cancer (PCa) can be mitigated by surgery and radiation, metastatic PCa remains a challenge to treat. Androgen deprivation therapies and androgen receptor (AR) pathway inhibitors are mainstay treatments for advanced PCa. Yet, resistance often develops leading to castration-resistant prostate cancer (CRPC). Forkhead Box A1 (FOXA1) is a pioneer transcription factor that plays pivotal roles in regulating AR activity and promoting epithelial differentiation. Studies have shown that FOXA1 is frequently downregulated in CRPC tumors. Congruently, FOXA1 loss is reported to induce aberrant AR signaling, epithelial-mesenchymal transition, and PCa de-differentiation. However, the role of FOXA1 in regulating PCa immune response, an area of much interest recently, has not been reported. CRPC has shown poor response to immune checkpoint inhibitors, due to its immunosuppressive nature. A better understanding of the tumor intrinsic mechanisms regulating PCa tumor immunity will inform the design of better targeted immunotherapeutic approaches. Methods: We performed RNA-seq, ChIP-seq, qPCR, western blot, and ELISA analyses to evaluate how FOXA1 regulates inflammatory response genes. We utilized an in vitro macrophage infiltration transwell assay, in which M2-like macrophages were added to the upper chamber and PCa cells were plated in the lower chamber, to examine how perturbations to PCa cells affect macrophage migration. Finally, we performed bioinformatic analyses of patient datasets to confirm the clinical relevance of FOXA1 repression of inflammatory genes in PCa. Results: Through integration of RNA-seq and ChIP-seq data, we uncovered a novel function of FOXA1 in suppressing inflammatory response pathways. In accordance, patient data analyses revealed that inflammatory response genes were upregulated in FOXA1-low PCa tumors. Mechanistically, we showed that FOXA1 proteins bound an intragenic enhancer of Hypoxia-inducible factor 1-alpha (HIF1A) gene to directly repress its expression, such that FOXA1 loss induced HIF1A upregulation. We further showed that Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) became upregulated upon FOXA1 depletion in a HIF1A-dependent manner. This led to infiltration by immunosuppressive, tumor promoting M2-like macrophages. Inhibiting this HIF1A-CCL2 axis with a HIF1A inhibitor or CCL2 neutralizing antibody blocked macrophage infiltration. Future studies using immunocompetent mouse models are needed to confirm the effect of FOXA1 on macrophage infiltration in vivo and evaluate the preclinical potential of targeting the FOXA1-HIF1A-CCL2 axis in CRPC. Conclusion: This study proposes a novel role for FOXA1 loss in promoting macrophage infiltration via the HIF1A-CCL2 axis. Moreover, our findings suggest that targeting this axis may be a promising approach for the treatment of FOXA1-low CRPC tumors.

Epigenetics of Muscle- and Brain-Specific Expression of KLHL Family Genes.

KLHL and the related KBTBD genes encode components of the Cullin-E3 ubiquitin ligase complex and typically target tissue-specific proteins for degradation, thereby affecting differentiation, homeostasis, metabolism, cell signaling, and the oxidative stress response. Despite their importance in cell function and disease (especially, KLHL40, KLHL41, KBTBD13, KEAP1, and ENC1), previous studies of epigenetic factors that affect transcription were predominantly limited to promoter DNA methylation. Using diverse tissue and cell culture whole-genome profiles, we examined 17 KLHL or KBTBD genes preferentially expressed in skeletal muscle or brain to identify tissue-specific enhancer and promoter chromatin, open chromatin (DNaseI hypersensitivity), and DNA hypomethylation. Sixteen of the 17 genes displayed muscle- or brain-specific enhancer chromatin in their gene bodies, and most exhibited specific intergenic enhancer chromatin as well. Seven genes were embedded in super-enhancers (particularly strong, tissue-specific clusters of enhancers). The enhancer chromatin regions typically displayed foci of DNA hypomethylation at peaks of open chromatin. In addition, we found evidence for an intragenic enhancer in one gene upregulating expression of its neighboring gene, specifically for KLHL40/HHATL and KLHL38/FBXO32 gene pairs. Many KLHL/KBTBD genes had tissue-specific promoter chromatin at their 5′ ends, but surprisingly, two (KBTBD11 and KLHL31) had constitutively unmethylated promoter chromatin in their 3′ exons that overlaps a retrotransposed KLHL gene. Our findings demonstrate the importance of expanding epigenetic analyses beyond the 5′ ends of genes in studies of normal and abnormal gene regulation.

JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-mediated Transgene Expression in the Lung and Liver of Mice.

Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats. A putative intragenic enhancer, termed JE, localized to the 3′ end of the JSRV env gene, has been previously described. Herein we provide further evidence that the JE functions as a transcriptional enhancer, as it was able to enhance gene expression when placed in either forward or reverse orientation when combined with a heterologous chicken beta actin promoter. We then generated novel composite promoters designed to improve transgene expression from adeno-associated virus (AAV) gene therapy vectors. A hybrid promoter consisting of the shortest JE sequence examined (JE71), the U3 region of the JSRV long terminal repeat (LTR), and the chicken beta actin promoter, demonstrated robust expression in vitro and in vivo, when in the context of AAV vectors. AAV-mediated transgene expression in vivo from the hybrid promoter was marginally lower than that observed for AAV vectors encoding the strong CAG promoter, but greatly reduced in the heart, making this promoter/enhancer combination attractive for non-cardiac applications, particularly respiratory tract or liver directed therapies. Replacement of the murine leukemia virus intron present in the original vector construct with a modified SV40 intron reduced the promoter/enhancer/intron cassette size to 719 bp, leaving an additional ~4 kb of coding capacity when packaged within an AAV vector. Taken together, we have developed a novel, compact promoter that is capable of directing high level transgene expression from AAV vectors in both the liver and lung with diminished transgene expression in the heart.

LacZ-reporter mapping of Dlx5/6 expression and genoarchitectural analysis of the postnatal mouse prethalamus.

We present here a thorough and complete analysis of mouse P0‐P140 prethalamic histogenetic subdivisions and corresponding nuclear derivatives, in the context of local tract landmarks. The study used as fundamental material brains from a transgenic mouse line that expresses LacZ under the control of an intragenic enhancer of Dlx5 and Dlx6 (Dlx5/6‐LacZ). Subtle shadings of LacZ signal, jointly with pan‐DLX immunoreaction, and several other ancillary protein or RNA markers, including Calb2 and Nkx2.2 ISH (for the prethalamic eminence, and derivatives of the rostral zona limitans shell domain, respectively) were mapped across the prethalamus. The resulting model of the prethalamic region postulates tetrapartite rostrocaudal and dorsoventral subdivisions, as well as a tripartite radial stratification, each cell population showing a characteristic molecular profile. Some novel nuclei are proposed, and some instances of potential tangential cell migration were noted.

AP-1 Signaling by Fra-1 Directly Regulates HMGA1 Oncogene Transcription in Triple-Negative Breast Cancers.

The architectural chromatin protein HMGA1 and the transcription factor Fra-1 are both overexpressed in aggressive triple-negative breast cancers (TNBC), where they both favor epithelial-to-mesenchymal transition, invasion, and metastasis. We therefore explored the possibility that Fra-1 might be involved in enhanced transcription of the HMGA1 gene in TNBCs by exploiting cancer transcriptome datasets and resorting to functional studies combining RNA interference, mRNA and transcriptional run-on assays, chromatin immunoprecipitation, and chromosome conformation capture approaches in TNBC model cell lines. Our bioinformatic analysis indicated that Fra-1 and HMGA1 expressions positively correlate in primary samples of patients with TNBC. Our functional studies showed that Fra-1 regulates HMGA1 mRNA expression at the transcriptional level via binding to enhancer elements located in the last two introns of the gene. Although Fra-1 binding is required for p300/CBP recruitment at the enhancer domain, this recruitment did not appear essential for Fra-1-stimulated HMGA1 gene expression. Strikingly, Fra-1 binding is required for efficient recruitment of RNA Polymerase II at the HMGA1 promoter. This is permitted owing to chromatin interactions bringing about the intragenic Fra-1-binding enhancers and the gene promoter region. Fra-1 is, however, not instrumental for chromatin loop formation at the HMGA1 locus but rather exerts its transcriptional activity by exploiting chromatin interactions preexisting to its binding. IMPLICATIONS: We demonstrate that Fra-1 bound to an intragenic enhancer region is required for RNA Pol II recruitement at the HMGA1 promoter. Thereby, we provide novel insights into the mechanisms whereby Fra-1 exerts its prooncogenic transcriptional actions in the TNBC pathologic context.

PF153 MLL‐AF4 CAUSES ABERRANT UPREGULATION OF PROM1 (CD133) IN ACUTE LYMPHOBLASTIC LEUKEMIA BY CONTROLLING ENHANCER‐PROMOTER INTERACTIONS

Background:MLL rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukaemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL‐AF4 fusion protein. MLL‐AF4 is thought to promote leukaemogenesis by activating key target genes, mainly by altering the epigenetic profile of the cell through recruitment of DOT1L and increasing histone H3 lysine 79 methylation (H3K79me2/3). One of these key genes is PROM1 which encodes the cell surface protein CD133 (Prominin‐1; Mak2016). CD133 has previously been shown to be expressed on ALL blasts and is of potential therapeutic interest. However, the mechanism by which MLL‐AF4 dysregulates PROM1 expression and the impact of PROM1 expression on leukaemia cell survival remains unclear.Aims:To determine the mechanism by which MLL‐AF4 causes aberrant expression of PROM1 in leukaemia.Methods:We characterised normal fetal haematopoietic stem and progenitor cells (HSPC), blasts from patient leukaemia samples, and SEM cells (a MLL‐AF4+ cell line) by immunophenotyping and transcriptome analysis. ChIP‐Seq and Capture C (a high resolution chromosome conformation capture technique) was used to elucidate enhancer‐promoter structure at the PROM1 locus.Results:We find that the MLL‐AF4 complex aberrantly upregulates PROM1 transcription by controlling enhancer promoter‐interactions.Immunophenotyping and transcriptome analysis: Immunophenotypic analysis of normal human fetal bone marrow HSPC confirmed that CD133 expression is confined to the stem cell compartment and is downregulated through B cell commitment. In contrast, MLLr patient blasts and SEM cells showed high levels of surface CD133 expression. Analysis of RNA‐Sequencing data showed that PROM1 gene expression in normal fetal B progenitors, patient blasts and SEM cells reflected the pattern of surface CD133 expressionFunctional assays: Upon inhibition of MLL‐AF4 binding by a DOT1L inhibitor, CD133 expression is downregulated in SEM cells and nascent RNA‐Seq shows down‐regulation of PROM1 at the transcriptional level. Direct knock down of PROM1 in SEM cells confers slower growth rates/less colony formation and increased cell death.ChIP‐Seq and Capture‐C: ChIP‐sequencing identified MLL‐AF4 and associated complex components binding to the PROM1 promoter and within the gene body at putative enhancer elements. Capture C profiles identified intragenic enhancer elements within the PROM1 gene body and within the nearby TAPT1 gene. Inhibition of MLL‐AF4 complex activity upon treatment with DOT1L inhibitors causing loss of H34K79me2/3 disrupted gene expression as well as enhancer‐promoter interactions.Summary/Conclusion:Together these results indicate that MLL‐AF4 directly up‐regulates PROM1 expression in ALL cells and promotes their proliferation and survival. Therefore, CD133 expression should not only be a useful marker for diagnosis and residual disease monitoring but might also be exploited as a therapeutic target in MLL re‐arranged ALL.

The BAF and PRC2 Complex Subunits Dpf2 and Eed Antagonistically Converge on Tbx3 to Control ESC Differentiation.

SummaryBAF complexes are composed of different subunits with varying functional and developmental roles, although many subunits have not been examined in depth. Here we show that the Baf45 subunit Dpf2 maintains pluripotency and ESC differentiation potential. Dpf2 co-occupies enhancers with Oct4, Sox2, p300, and the BAF subunit Brg1, and deleting Dpf2 perturbs ESC self-renewal, induces repression of Tbx3, and impairs mesendodermal differentiation without dramatically altering Brg1 localization. Mesendodermal differentiation can be rescued by restoring Tbx3 expression, whose distal enhancer is positively regulated by Dpf2-dependent H3K27ac maintenance and recruitment of pluripotency TFs and Brg1. In contrast, the PRC2 subunit Eed binds an intragenic Tbx3 enhancer to oppose Dpf2-dependent Tbx3 expression and mesendodermal differentiation. The PRC2 subunit Ezh2 likewise opposes Dpf2-dependent differentiation through a distinct mechanism involving Nanog repression. Together, these findings delineate distinct mechanistic roles for specific BAF and PRC2 subunits during ESC differentiation.

1031 - Reactivation of fetal hemoglobin for therapy: From GWAS to the clinic

The major hemoglobin disorders, b-thalassemia and sickle cell disease (SCD), are among the most common severe Mendelian disorders worldwide. Although the molecular bases of these diseases have been known for decades, treatment has been largely supportive, except for curative bone marrow transplantation. Classical family and genetic studies long ago established the beneficial impact of elevated fetal hemoglobin on the clinical phenotypes, and stimulated investigation into approaches for reactivation of silenced fetal hemoglobin as a therapeutic strategy. Despite elucidation of the principal regulators of erythroid gene transcription more than 2 decades ago, the basis for repression of fetal hemoglobin (HbF) during late gestation and after birth was elusive. Genome-wide association studies of HbF levels in 2007/8 revealed candidate loci, including the zinc-finger transcription factor BCL11A. Subsequently, combined surrogate genetics in cells, human genetics, and CRISPR/Cas9 saturating mutagenesis have established BCL11A as a principal repressor of HbF expression and identified discrete DNA sequences within an intragenic erythroid enhancer that are favorable for gene editing therapy. Through unbiased screening of potential BCL11A binding sequences we recently identified a novel recognition motif present in embryonic and fetal, but not adult, globin genes and showed that this site is mutated in rare individuals with classical hereditary persistence of hemoglobin, an entitly described 3 decades ago. Translation of basic findings to the clinic, through genetic or pharmacological means, is under intense investigation. Summary of the current state of these efforts will be discussed. Studies of BCL11A have greatly advanced a mechanistic understanding of the switch from fetal to adult hemoglobin, and illustrate the power of integrative genetic, biochemical, and hematologic approaches in providing opportunities for transformative management of the major hemoglobin disorders.

Abstract 5324: Intragenic DNA-hypomethylation promotes overexpression of ITGB7 in MF subgroup of multiple myeloma

Abstract Integrin-β7 is part of an extensive family of glycoproteins and is overexpressed in the MF subgroups in Multiple Myeloma (MM), contributing to drug resistance and poor survival. ITGB7 promotes adhesion and proliferation of myeloma cells in bone-marrow stroma by supplementing growth factors like VEGF. Here, we investigate the possible epigenetic mechanism of ITGB7 overexpression in the MF subgroup, comprising of t(14;16) and t(14;20) myeloma. Newly diagnosed MM (NDMM) patient bone marrow aspirates underwent CD138 cell selection to enrich tumor cells to >98%. Samples consisted of those with a t(14;16) (n=17), t(14;20) (n=7), t(4;14) (n=9), t(11;14) (n=10) and hyperdiploidy (n=19, separated into D1 (n=12) or D2 (n=7 subgroups). NDMM patient samples were compared to plasma cells isolated from age-matched healthy donors (n=4), to determine differential changes in epitranscripts. We performed unbiased genome-wide reduced representation bisulfite sequencing (RRBS) to identify differentially methylated regions (DMRs) in CpG islands, which were validated by Infinium MethylationEPIC arrays (Illumina) in conjunction with gene expression array data (U133 Plus 2.0, Affymetrix) to determine the epitranscriptomic profile in all samples. RRBS was performed using 75 bp reads to a minimum of 20 M reads per sample. Data from MM cells at the Blueprint consortium were also used for annotation of epigenetic marks. Using the RRBS data we identified 26 hypomethylated, overexpressed genes in the MF group samples, which were not present in the other subgroups. In comparison, only 1 gene, FUT7, was hypermethylated and down-regulated in the MF cluster. Most interestingly, we identified ITGB7 amongst the hypomethylated, overexpressed genes. With an in-depth analysis we identified 4 significant (p<0.05) DMRs across intragenic regions of ITGB7, within a 2.5 kb region. Mean methylation across the DMRs reduced from 58% to 22% (p<0.01) in t(14;16) and 39% (p=0.02) in t(14;20) with concomitant overexpression of ITGB7 in the t(14;16), 19.5-fold increase, and the t(14;20), 23.4-fold increase, subgroups. These 4 DMRs belong to CpG islands, annotated to be the part of possible intragenic enhancer site with promoter like activity, and enriched for open chromatin structures. The DMRs also align with the hotspot of H3K4me1 marks and a putative binding site of activating transcription factors such as AP1/2 or Sp1. This suggests that the identified DMRs could be within the enhancer that regulates ITGB7 overexpression through DNA-hypomethylation. ITGB7 is a known oncogenic factor in high-risk MM, contributing to cell adhesion, migration and homing. Here we show using combined DNA methylation and expression data that ITGB7 is regulated through hypomethylation of the an enhancer region in the MF subgroup in MM. Citation Format: Samrat Roy Choudhury, Cody Ashby, Ruslana Tytarenko, Yan Wang, Purvi H. Patel, Aneta Mikulasova, Michael Bauer, Shayu Deshpande, Faith E. Davies, Gareth J. Morgan, Brian A. Walker. Intragenic DNA-hypomethylation promotes overexpression of ITGB7 in MF subgroup of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5324.

Hdac3 regulates lymphovenous and lymphatic valve formation.

Lymphedema, the most common lymphatic anomaly, involves defective lymphatic valve development; yet the epigenetic modifiers underlying lymphatic valve morphogenesis remain elusive. Here, we showed that during mouse development, the histone-modifying enzyme histone deacetylase 3 (Hdac3) regulates the formation of both lymphovenous valves, which maintain the separation of the blood and lymphatic vascular systems, and the lymphatic valves. Endothelium-specific ablation of Hdac3 in mice led to blood-filled lymphatic vessels, edema, defective lymphovenous valve morphogenesis, improper lymphatic drainage, defective lymphatic valve maturation, and complete lethality. Hdac3-deficient lymphovenous valves and lymphatic vessels exhibited reduced expression of the transcription factor Gata2 and its target genes. In response to oscillatory shear stress, the transcription factors Tal1, Gata2, and Ets1/2 physically interacted with and recruited Hdac3 to the evolutionarily conserved E-box–GATA–ETS composite element of a Gata2 intragenic enhancer. In turn, Hdac3 recruited histone acetyltransferase Ep300 to form an enhanceosome complex that promoted Gata2 expression. Together, these results identify Hdac3 as a key epigenetic modifier that maintains blood-lymph separation and integrates both extrinsic forces and intrinsic cues to regulate lymphatic valve development.

Intragenic Enhancers Attenuate Host Gene Expression

Eukaryotic gene transcription is regulated at many steps, including RNA polymerase II (Pol II) recruitment, transcription initiation, promoter-proximal Pol II pause release, and transcription termination; however, mechanisms regulating transcription during productive elongation remain poorly understood. Enhancers, which activate gene transcription, themselves undergo Pol II-mediated transcription, but our understanding of enhancer transcription and enhancer RNAs (eRNAs) remains incomplete. Here we show that transcription at intragenic enhancers interferes with and attenuates host gene transcription during productive elongation. While the extent of attenuation correlates positively with nascent eRNA expression, the act of intragenic enhancer transcription alone, but not eRNAs, explains the attenuation. Through CRISPR/Cas9-mediated deletions, we demonstrate a physiological role for intragenic enhancer-mediated transcription attenuation in cell fate determination. We propose that intragenic enhancers not only enhance transcription of one or more genes from a distance but also fine-tune transcription of their host gene through transcription interference, facilitating differential utilization of the same regulatory element for disparate functions.

Identification and characterization of a FOXA2-regulated transcriptional enhancer at a type 2 diabetes intronic locus that controls GCKR expression in liver cells

BackgroundGenome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes (T2D). However, the underlying biological mechanisms for many of these associations remain unknown. GWAS signals close to the glucokinase regulatory protein gene (GCKR) have been reported for lipid and glucose metabolism traits and the risk of T2D. We investigated the regulatory function of an intronic locus at GCKR represented by the lead single nucleotide polymorphism (SNP) rs780094.MethodsWe used ENCODE project histone modification and transcription factor binding data to determine the regulatory features of a GCKR intronic locus formed by the high linkage disequilibrium rs780094(C/T), rs780095(G/A), and rs780096(G/C) SNPs. Characterization of the transcriptional activity of this region was assessed by luciferase reporter assays in HepG2 cells and mouse primary hepatocytes. ChIP-qPCR was used to determine the levels of haplotype specific transcription factor binding and histone marks. A CRISPR-dCas9 transcriptional activator system and qPCR were used to activate the locus and measure GCKR expression, respectively. Differential haplotype expression was measured from human liver biopsies.ResultsThe ENCODE data suggest the existence of a liver-specific intragenic enhancer at the locus represented by s780094. We observed that FOXA2 increased the transcriptional activity of this region in a haplotype specific way (CGG > TAC; rs780094, rs780095, and rs780096). In addition, the CGG haplotype showed higher binding to FOXA2 and higher levels of the H3K27Ac histone mark. The epigenetic activation of this locus increased the expression of endogenous GCKR in HepG2 cells, confirming that GCKR is the direct target gene of the enhancer. Finally, we confirmed that the CGG haplotype exhibits higher levels of transcription in human liver.ConclusionsOur results demonstrate the existence of a liver-specific FOXA2-regulated transcriptional enhancer at an intronic T2D locus represented by rs780094, rs780095, and rs780096 SNPs that increases GCKR expression. Differential haplotype regulation suggests the existence of cis regulatory effects that may contribute to the associated traits at this locus.

Contraction of intestinal effector T cells by retinoic acid-induced purinergic receptor P2X7

Abstract Vitamin A is a key regulator of immunity and immune tolerance. Here we report that the vitamin A metabolite retinoic acid regulates the population size of intestinal effector T cells by inducing purinergic receptor P2X7 expression. RA induces P2X7 expression by direct binding to an intragenic enhancer of the P2rx7 gene in CD4+T cells, rendering them highly susceptible to apoptosis triggered by P2X7 activation in response to extracellular ATP and NAD. Utilizing P2X7-null mice, we found that this tolerogenic mechanism is critical for reining in the expansion of Th1 and Th17 effector T cell populations in the intestine where the concentrations of extracellular ATP/NAD and retinoic acid are high. We demonstrated that P2X7 activation can ameliorate colitis by inducing the apoptosis of inflammatory effector T cells. We conclude that the RA-P2X7 axis is a novel immune homeostatic mechanism by which the size of potentially inflammatory effector T cell populations is tightly controlled to prevent inflammatory diseases.

Contraction of intestinal effector T cells by retinoic acid-induced purinergic receptor P2X7.

The intestinal environment harbors a large number of activated T cells, which are potentially inflammatory. To prevent inflammatory responses, intestinal T cells are controlled by various tolerogenic mechanisms, including T cell apoptosis. We investigated the expression mechanism and function of the purinergic receptor P2X7 in contraction of intestinal CD4+ effector T cells. We found that P2X7 up-regulation on CD4+ effector T cells is induced by retinoic acid through retinoic acid receptor α (RARα) binding to an intragenic enhancer region of the P2rx7 gene. P2X7 is highly expressed by most intestinal αβ and γδ T cells, including Th1 and Th17 cells. The intestinal effector T cells are effectively deleted by P2X7 activation-dependent apoptosis. Moreover, P2X7 activation suppressed T cell-induced colitis in Rag1−/− mice. The data from vitamin A-deficient and P2rx7−/− mice indicate that the retinoic acid-P2X7 pathway is important in preventing aberrant build-up of activated T cells. We conclude that retinoic acid controls intestinal effector T cell populations by inducing P2X7 expression. These findings have important ramifications in preventing inflammatory diseases in the intestine.