Enhanced Chromatin Accessibility Research Articles

The High Mobility Group A1 Chromatin Regulator Drives Immune Evasion during MPN Progression By Repressing Genes Involved in Antigen Presentation and Immune Attack

Introduction: Myeloproliferative Neoplasms (MPN) are blood diseases caused by mutations in hematopoietic stem cells (HSCs) which lead to clonal expansion and overproduction of myeloid lineages. Individuals with MPN are at increased risk for transformation to bone marrow fibrosis (myelofibrosis, MF) and acute myeloid leukemia (AML), both of which are associated with poor clinical outcomes. However, targetable mechanisms underlying progression remain elusive. The High Mobility Group A1 (HMGA1) gene encodes chromatin regulators which are enriched in stem cells and aberrantly overexpressed in aggressive tumors (Xian et al Nature Commun 2017;8:15008, Resar et al Cancer Res 2018;78:1890). Transgenic mice misexpressing Hmga1 in lymphoid cells develop lethal leukemia by dysregulating gene networks associated with aberrant proliferation and inflammation (Hillion et al Cancer Res 2008;68:10121, Schuldenfrei et al BMC Genomics, 2011;12:549). We discovered that HMGA1 is overexpressed in MPN with progression and required for leukemic transformation in preclinical models (Resar et al Blood 2018;132 Suppl 1:102). We therefore sought to: 1) test the hypothesis that HMGA1 drives MPN progression by dysregulating gene networks involved in immune evasion, and, 2) identify mediators of immune escape that could be disrupted in therapy.Methods: To elucidate transcriptional networks regulated by HMGA1 during MPN progression to AML, we integrated multi-omics sequencing (seq) analyses, including RNAseq, chromatin immunoprecipitation seq (ChIPseq), and ATACseq in AML cell lines from JAK2 V617Fmutant MPN after leukemic transformation (DAMI, SET-2) + HMGA1 depletion. HMGA1 gene expression was inactivated using CRISPR/Cas9 or short hairpin RNA (shRNA)-mediated gene silencing. Gene set enrichment analysis was used to dissect molecular mechanisms underlying immune invasion by HMGA1. To validate results in human MPN, RNAseq was performed in peripheral blood mononuclear cells (PBMCs) from matched MF patients who transformed to AML. To reconstruct the immune cell composition of primary MPN samples, we applied xCell, a robust computational method that converts gene expression profiles to immune cell types. Transcriptional networks were validated at the level of mRNA and protein via quantitative RT-PCR and flow cytometry. To identify drugs to disrupt HMGA1 immune evasion networks, we applied the Broad Institute Connectivity Map (CMAP) and cytotoxicity assays.Results: Integration of RNAseq, ChIPseq, and ATACseq in MPN AML cells (DAMI, SET-2) revealed that HMGA1 represses genes involved in immune activation (inflammatory response, TNFa signaling, NF-κB networks) and antigen presentation [Interferon gamma (IFNγ) response networks], including genes encoding the major histocompatibility complex (MHC) class I and II antigens. Inhibiting HMGA1 results in up-regulation of MHC class I and II antigen genes, with greatest induction of HLA-DRA (the alpha paralog for HLA Class II antigens). Similarly, HMGA1 depletion increases cell surface expression of HLA-DRA antigens. Strikingly, RNAseq from MPN patients with MF after transformation to AML reveal that HMGA1 is up-regulated in MPN AML concurrent with repression in gene networks of immune activation and antigen presentation. Immune cell transcriptomes are also depleted in MPN cells after leukemic transformation. To determine if immune evasion by HMGA1 could be modulated in therapy, we applied CMAP which identified histone deacetylase inhibitors (HDACi) as drugs targeting the HMGA1 transcriptome. The HDACi, entinostat, is cytotoxic and synergizes with the JAK inhibitor, ruxolitinib, in MPN AML cell lines. Further, entinostat induces expression of MHC class II genes and antigens. Moreover, HMGA1 depletion enhances sensitivity of MPN AML cells to entinostat.Conclusions: We discovered an epigenetic program whereby HMGA1 drives immune evasion during MPN progression by binding to chromatin and enhancing chromatin accessibility to activate transcriptional networks that repress antigen presentation and immune attack. Most importantly, HMGA1 immune evasion networks are dysregulated in human MPN and can be targeted by HDACi therapy. Together, our studies reveal a new paradigm whereby HMGA1 down-regulates MHC antigens during MPN progression, suggesting that targeting HMGA1 networks could activate an immune attack and prevent MPN progression. [Display omitted] DisclosuresRampal: Novartis: Consultancy; Pharmaessentia: Consultancy; Kartos: Consultancy; Blueprint: Consultancy; Disc Medicine: Consultancy; BMS/Celgene: Consultancy; Stemline: Consultancy, Research Funding; Constellation: Research Funding; Jazz Pharmaceuticals: Consultancy; Sierra Oncology: Consultancy; Abbvie: Consultancy; CTI: Consultancy; Incyte: Consultancy, Research Funding; Memorial Sloan Kettering: Current Employment.

Enhanced chromatin accessibility contributes to X chromosome dosage compensation in mammals

BackgroundPrecise gene dosage of the X chromosomes is critical for normal development and cellular function. In mice, XX female somatic cells show transcriptional X chromosome upregulation of their single active X chromosome, while the other X chromosome is inactive. Moreover, the inactive X chromosome is reactivated during development in the inner cell mass and in germ cells through X chromosome reactivation, which can be studied in vitro by reprogramming of somatic cells to pluripotency. How chromatin processes and gene regulatory networks evolved to regulate X chromosome dosage in the somatic state and during X chromosome reactivation remains unclear.ResultsUsing genome-wide approaches, allele-specific ATAC-seq and single-cell RNA-seq, in female embryonic fibroblasts and during reprogramming to pluripotency, we show that chromatin accessibility on the upregulated mammalian active X chromosome is increased compared to autosomes. We further show that increased accessibility on the active X chromosome is erased by reprogramming, accompanied by erasure of transcriptional X chromosome upregulation and the loss of increased transcriptional burst frequency. In addition, we characterize gene regulatory networks during reprogramming and X chromosome reactivation, revealing changes in regulatory states. Our data show that ZFP42/REX1, a pluripotency-associated gene that evolved specifically in placental mammals, targets multiple X-linked genes, suggesting an evolutionary link between ZFP42/REX1, X chromosome reactivation, and pluripotency.ConclusionsOur data reveal the existence of intrinsic compensatory mechanisms that involve modulation of chromatin accessibility to counteract X-to-Autosome gene dosage imbalances caused by evolutionary or in vitro X chromosome loss and X chromosome inactivation in mammalian cells.

The transcription factor reservoir and chromatin landscape in activated plasmacytoid dendritic cells

BackgroundTranscription factors (TFs) control gene expression by direct binding to regulatory regions of target genes but also by impacting chromatin landscapes and modulating DNA accessibility for other TFs. In recent years several TFs have been defined that control cell fate decisions and effector functions in the immune system. Plasmacytoid dendritic cells (pDCs) are an immune cell type with the unique capacity to produce high amounts of type I interferons quickly in response to contact with viral components. Hereby, this cell type is involved in anti-infectious immune responses but also in the development of inflammatory and autoimmune diseases. To date, the global TF reservoir in pDCs early after activation remains to be fully characterized.ResultsTo fill this gap, we have performed a comprehensive analysis in naïve versus TLR9-activated murine pDCs in a time course study covering early timepoints after stimulation (2 h, 6 h, 12 h) integrating gene expression (RNA-Seq) and chromatin landscape (ATAC-Seq) studies. To unravel the biological processes underlying the changes in TF expression on a global scale gene ontology (GO) analyses were performed. We found that 70% of all genes annotated as TFs in the mouse genome (1014 out of 1636) are expressed in pDCs for at least one stimulation time point and are covering a wide range of TF classes defined by their specific DNA binding mechanisms. GO analysis revealed involvement of TLR9-induced TFs in epigenetic modulation, NFκB and JAK-STAT signaling, and protein production in the endoplasmic reticulum. pDC activation predominantly “turned on” the chromatin regions associated with TF genes. Our in silico analyses pointed at the AP-1 family of TFs as less noticed but possibly important players in these cells after activation. AP-1 family members exhibit (1) increased gene expression, (2) enhanced chromatin accessibility in their promoter region, and (3) a TF DNA binding motif that is globally enriched in genomic regions that were found more accessible in pDCs after TLR9 activation.ConclusionsIn this study we define the complete set of TLR9-regulated TFs in pDCs. Further, this study identifies the AP-1 family of TFs as potentially important but so far less well characterized regulators of pDC function.

Chromatin-based, in cis and in trans regulatory rewiring underpins distinct oncogenic transcriptomes in multiple myeloma

Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Distinct myeloma transcriptome profiles are primarily driven by myeloma initiating events (MIE) and converge into a mutually exclusive overexpression of the CCND1 and CCND2 oncogenes. Here, with reference to their normal counterparts, we find that myeloma PC enhanced chromatin accessibility combined with paired transcriptome profiling can classify MIE-defined genetic subgroups. Across and within different MM genetic subgroups, we ascribe regulation of genes and pathways critical for myeloma biology to unique or shared, developmentally activated or de novo formed candidate enhancers. Such enhancers co-opt recruitment of existing transcription factors, which although not transcriptionally deregulated per se, organise aberrant gene regulatory networks that help identify myeloma cell dependencies with prognostic impact. Finally, we identify and validate the critical super-enhancer that regulates ectopic expression of CCND2 in a subset of patients with MM and in chronic lymphocytic leukemia.

Notch signaling via Hey1 and Id2b regulates Müller glia's regenerative response to retinal injury.

Zebrafish Müller glia (MG) respond to retinal injury by suppressing Notch signaling and producing progenitors for retinal repair. A certain threshold of injury-derived signal must be exceeded in order to engage MG in a regenerative response (MG's injury-response threshold). Pan-retinal Notch inhibition expands the zone of injury-responsive MG at the site of focal injury, suggesting that Notch signaling regulates MG's injury-response threshold. We found that Notch signaling enhanced chromatin accessibility and gene expression at a subset of regeneration-associated genes in the uninjured retina. Two Notch effector genes, hey1 and id2b, were identified that reflect bifurcation of the Notch signaling pathway, and differentially regulate MG's injury-response threshold and proliferation of MG-derived progenitors. Furthermore, Notch signaling component gene repression in the injured retina suggests a role for Dll4, Dlb, and Notch3 in regulating Notch signaling in MG and epistasis experiments confirm that the Dll4/Dlb-Notch3-Hey1/Id2b signaling pathway regulates MG's injury-response threshold and proliferation.

CLAMP and Zelda function together to promote Drosophila zygotic genome activation

During the essential and conserved process of zygotic genome activation (ZGA), chromatin accessibility must increase to promote transcription. Drosophila is a well-established model for defining mechanisms that drive ZGA. Zelda (ZLD) is a key pioneer transcription factor (TF) that promotes ZGA in the Drosophila embryo. However, many genomic loci that contain GA-rich motifs become accessible during ZGA independent of ZLD. Therefore, we hypothesized that other early TFs that function with ZLD have not yet been identified, especially those that are capable of binding to GA-rich motifs such as chromatin-linked adaptor for male-specific lethal (MSL) proteins (CLAMP). Here, we demonstrate that Drosophila embryonic development requires maternal CLAMP to (1) activate zygotic transcription; (2) increase chromatin accessibility at promoters of specific genes that often encode other essential TFs; and (3) enhance chromatin accessibility and facilitate ZLD occupancy at a subset of key embryonic promoters. Thus, CLAMP functions as a pioneer factor that plays a targeted yet essential role in ZGA.

Abstract 1406: FOXC1 induces cisplatin resistance through enhancer activation in bladder cancer

Abstract Introduction: We previously reported that an aggressive subpopulation of highly tumorigenic, drug-resistant bladder cancer cells can arise from the bulk tumor cells without mutational events, and that this phenotypic plasticity is driven at least in part by epigenetic mechanisms. In the current work, we analyzed the chromatin accessibility of enhancers to identify transcription factors that contribute to this transition to a drug-resistant state. Materials and methods: We used Hoechst nuclear staining and fluorescence activated cell sorting to isolate the Hoechst-low drug resistant side population (SP) cells from the less drug resistant Hoechst-high non-side population (NSP) cells in bladder cancer cell lines. We performed RNA sequencing and AcceSssIble assay to profile RNA expression, global DNA methylation and chromatin accessibility in SP and NSP cells. Putative enhancer regions were identified using H3K27ac ChIPseq data in bladder cancer cell lines, which are publicly available. We next performed transcription factor motif analysis on accessible enhancers near genes overexpressed in SP cells relative to NSP cells. The differentially expressed candidate target transcription factor by RNA-seq was validated using qRT-PCR. Functional validation was done using CRISPR knock out and drug resistance assays. Results: A higher percentage of enhancers were differentially accessible between SP and NSP cells compared with the promoters. We identified 746 enhancers that are accessible only in SP cells and 465 enhancers only in NSP cells. Transcription factor motif analysis showed that FOX family motif was enriched in accessible enhancers near SP-overexpressed genes. Among FOX family transcription factors, FOXC1 was the only overexpressed transcription factor, and it was also the most significantly overexpressed gene in SP cells. FOXC1 was consistently overexpressed in bladder tumor tissues relative to normal bladder tissues in three independent studies in BaseSpace Correlation Engine. CRISPR knock-out of FOXC1 in multiple bladder cancer cell lines resulted in decreased cisplatin resistance. Conclusion: We found that FOXC1 is overexpressed and its motif is enriched on the accessible enhancers when bladder cancer cells shift to a drug-resistant tumorigenic phenotype. Conversely, FOXC1 knock-out attenuates cisplatin resistance in bladder cancer cells. These observations suggest that differential expression and enhancer binding of FOXC1 promotes the previously observed, mutation-independent shift towards cisplatin resistance in bladder cancer. Citation Format: Yi-Tsung Lu, Tong Xu, Tien-Chan Hsieh, Maheen Iqbal, Zhifei Luo, Peggy J. Farnham, Suhn K. Rhie, Amir Goldkorn. FOXC1 induces cisplatin resistance through enhancer activation in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1406.

High throughput screening identifies SOX2 as a super pioneer factor that inhibits DNA methylation maintenance at its binding sites

Binding of mammalian transcription factors (TFs) to regulatory regions is hindered by chromatin compaction and DNA methylation of their binding sites. Nevertheless, pioneer transcription factors (PFs), a distinct class of TFs, have the ability to access nucleosomal DNA, leading to nucleosome remodelling and enhanced chromatin accessibility. Whether PFs can bind to methylated sites and induce DNA demethylation is largely unknown. Using a highly parallelized approach to investigate PF ability to bind methylated DNA and induce DNA demethylation, we show that the interdependence between DNA methylation and TF binding is more complex than previously thought, even within a select group of TFs displaying pioneering activity; while some PFs do not affect the methylation status of their binding sites, we identified PFs that can protect DNA from methylation and others that can induce DNA demethylation at methylated binding sites. We call the latter super pioneer transcription factors (SPFs), as they are seemingly able to overcome several types of repressive epigenetic marks. Finally, while most SPFs induce TET-dependent active DNA demethylation, SOX2 binding leads to passive demethylation, an activity enhanced by the co-binding of OCT4. This finding suggests that SPFs could interfere with epigenetic memory during DNA replication.

NFAT5 Amplifies Antipathogen Responses by Enhancing Chromatin Accessibility, H3K27 Demethylation, and Transcription Factor Recruitment.

The ability of innate immune cells to respond to pathogen-associated molecular patterns across a wide range of intensities is fundamental to limit the spreading of infections. Studies on transcription responses to pathogen-activated TLRs have often used relatively high TLR ligand concentrations, and less is known about their regulation under mild stimulatory conditions. We had shown that the transcription factor NFAT5 facilitates expression of antipathogen genes under TLR stimulation conditions corresponding to low pathogen loads. In this study, we analyze how NFAT5 optimizes TLR-activated responses in mouse macrophages. We show that NFAT5 was required for effective recruitment of central effectors p65/NF-κB and c-Fos to specific proinflammatory target genes, such as Nos2, Il6, and Tnf in primary macrophages responding to low doses of the TLR4 ligand LPS. By contrast, NFAT5 was not required for p65/NF-κB recruitment in response to high LPS doses. Using the transposase-accessible chromatin with high-throughput sequencing assay, we show that NFAT5 facilitated chromatin accessibility mainly at promoter regions of multiple TLR4-responsive genes. Analysis of various histone marks that regulate gene expression in response to pathogens identified H3K27me3 demethylation as an early NFAT5-dependent mechanism that facilitates p65 recruitment to promoters of various TLR4-induced genes. Altogether, these results advance our understanding about specific mechanisms that optimize antipathogen responses to limit infections.

Low Replicative Stress Triggers Cell-Type Specific Inheritable Advanced Replication Timing.

DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains.

PRDM16 regulates a temporal transcriptional program to promote progression of cortical neural progenitors.

ABSTRACTRadial glia (RG) in the neocortex sequentially generate distinct subtypes of projection neurons, accounting for the diversity and complex assembly of cortical neural circuits. Mechanisms that drive the rapid and precise temporal progression of RG are beginning to be elucidated. Here, we reveal that the RG-specific transcriptional regulator PRDM16 promotes the transition of early to late phase of neurogenesis in the mouse neocortex. Loss of Prdm16 delays the timely progression of RG, leading to defective cortical laminar organization. Our genomic analyses demonstrate that PRDM16 regulates a subset of genes that are dynamically expressed between early and late neurogenesis. We show that PRDM16 suppresses target gene expression through limiting chromatin accessibility of permissive enhancers. We further confirm that crucial target genes regulated by PRDM16 are neuronal specification genes, cell cycle regulators and molecules required for neuronal migration. These findings provide evidence to support the finding that neural progenitors temporally shift the gene expression program to achieve neural cell diversity.

Decitabine Induces Gene Derepression on Monosomic Chromosomes: In Vitro and In Vivo Effects in Adverse-Risk Cytogenetics AML.

Hypomethylating agents (HMA) have become the backbone of nonintensive acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) treatment, also by virtue of their activity in patients with adverse genetics, for example, monosomal karyotypes, often with losses on chromosome 7, 5, or 17. No comparable activity is observed with cytarabine, a cytidine analogue without DNA-hypomethylating properties. As evidence exists for compounding hypermethylation and gene silencing of hemizygous tumor suppressor genes (TSG), we thus hypothesized that this effect may preferentially be reversed by the HMAs decitabine and azacitidine. An unbiased RNA-sequencing approach was developed to interrogate decitabine-induced transcriptome changes in AML cell lines with or without a deletion of chromosomes 7q, 5q or 17p. HMA treatment preferentially upregulated several hemizygous TSG in this genomic region, significantly derepressing endogenous retrovirus (ERV)3-1, with promoter demethylation, enhanced chromatin accessibility, and increased H3K4me3 levels. Decitabine globally reactivated multiple transposable elements, with activation of the dsRNA sensor RIG-I and interferon regulatory factor (IRF)7. Induction of ERV3-1 and RIG-I mRNA was also observed during decitabine treatment in vivo in serially sorted peripheral blood AML blasts. In patient-derived monosomal karyotype AML murine xenografts, decitabine treatment resulted in superior survival rates compared with cytarabine. Collectively, these data demonstrate preferential gene derepression and ERV reactivation in AML with chromosomal deletions, providing a mechanistic explanation that supports the clinical observation of superiority of HMA over cytarabine in this difficult-to-treat patient group. SIGNIFICANCE: These findings unravel the molecular mechanism underlying the intriguing clinical activity of HMAs in AML/MDS patients with chromosome 7 deletions and other monosomal karyotypes.See related commentary by O'Hagan et al., p. 813.

Recent Advances in Lupus B Cell Biology: PI3K, IFNγ, and Chromatin.

In the autoimmune disease Systemic Lupus Erythematosus (SLE), autoantibodies are formed that promote inflammation and tissue damage. There has been significant interest in understanding the B cell derangements involved in SLE pathogenesis. The past few years have been particularly fruitful in three domains: the role of PI3K signaling in loss of B cell tolerance, the role of IFNγ signaling in the development of autoimmunity, and the characterization of changes in chromatin accessibility in SLE B cells. The PI3K pathway coordinates various downstream signaling molecules involved in B cell development and activation. It is governed by the phosphatases PTEN and SHIP-1. Murine models lacking either of these phosphatases in B cells develop autoimmune disease and exhibit defects in B cell tolerance. Limited studies of human SLE B cells demonstrate reduced expression of PTEN or increased signaling events downstream of PI3K in some patients. IFNγ has long been known to be elevated in both SLE patients and mouse models of lupus. New data suggests that IFNγR expression on B cells is required to develop autoreactive germinal centers (GC) and autoantibodies in murine lupus. Furthermore, IFNγ promotes increased transcription of BCL6, IL-6 and T-bet in B cells, which also promote GC and autoantibody formation. IFNγ also induces epigenetic changes in human B cells. SLE B cells demonstrate significant epigenetic reprogramming, including enhanced chromatin accessibility at transcription factor motifs involved in B cell activation and plasma cell (PC) differentiation as well as alterations in DNA methylation and histone modifications. Histone deacetylase inhibitors limit disease development in murine lupus models, at least in part via their ability to prevent B cell class switching and differentiation into plasma cells. This review will discuss relevant discoveries of the past several years pertaining to these areas of SLE B cell biology.

The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness

Abstract Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a low passage primary AML culture model arranged in a hierarchy with functionally defined stem cells in the CD34+CD38-fraction. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. HK2 is the first and rate-limiting enzyme in glycolysis and phosphorylates glucose. In contrast, other metabolic enzymes including phosphofructokinase, fumarase, pyruvate kinase 2, glucose phosphate isomerase, enolase1, citrate synthase, aconitase 2, and succinate dehydrogenase were not detected in the nucleus of these cells. We also detected HK2, but not these other metabolic enzymes, in the nucleus of OCI-AML2, U937, NB4 and TEX leukemia as well as 8 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed selective over-expression of HK2 in the nucleus of these cells without increasing levels in the cytoplasm or mitochondria. Over-expression of nuclear HK2 increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Over expression of HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down total endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation after treatment with retinoic, and decreased the percentage of CD34+CD38- 8227 stem cells without changing basal proliferation. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2 and identified proteins related to chromatin organization and regulation. Therefore, we examined the impact of nuclear HK2 on chromatin accessibility using ATAC-seq. Over expression of nuclear HK2 enhanced chromatin accessibility, whereas the selective knockdown of nuclear HK2 compacted chromatin. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .

Nuclear Heparanase Regulates Chromatin Remodeling, Gene Expression and PTEN Tumor Suppressor Function.

Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate and has been shown in various cancers to promote metastasis, angiogenesis, osteolysis, and chemoresistance. Although heparanase is thought to act predominantly extracellularly or within the cytoplasm, it is also present in the nucleus, where it may function in regulating gene transcription. Using myeloma cell lines, we report here that heparanase enhances chromatin accessibility and confirm a previous report that it also upregulates the acetylation of histones. Employing the Multiple Myeloma Research Foundation CoMMpass database, we demonstrate that patients expressing high levels of heparanase display elevated expression of proteins involved in chromatin remodeling and several oncogenic factors compared to patients expressing low levels of heparanase. These signatures were consistent with the known function of heparanase in driving tumor progression. Chromatin opening and downstream target genes were abrogated by inhibition of heparanase. Enhanced levels of heparanase in myeloma cells led to a dramatic increase in phosphorylation of PTEN, an event known to stabilize PTEN, leading to its inactivity and loss of tumor suppressor function. Collectively, this study demonstrates that heparanase promotes chromatin opening and transcriptional activity, some of which likely is through its impact on diminishing PTEN tumor suppressor activity.

Extrachromosomal DNA is associated with oncogene amplification and poor outcome across multiple cancers.

Extrachromosomal DNA (ecDNA) amplification promotes intratumoral genetic heterogeneity and accelerated tumor evolution1–3, but its frequency and clinical impact are unclear. Here we show, using computational analysis of whole-genome sequencing data from 3,212 cancer patients, that ecDNA amplification frequently occurs in most cancer types, but not in blood or normal tissue. Oncogenes were highly enriched on amplified ecDNA and the most common recurrent oncogene amplifications arise on ecDNA. EcDNA amplifications resulted in higher levels of oncogene transcription compared to copy number matched linear DNA, coupled with enhanced chromatin accessibility and more frequently resulted in transcript fusions. Patients whose cancers carry ecDNAs have significantly shorter survival, even when controlled for tissue type, than do patients whose cancers are not driven by ecDNA-based oncogene amplification. The results presented here demonstrate that ecDNA-based oncogene amplification is common in cancer, is different from chromosomal amplification and drives poor outcome for patients across many cancer types.

Collapse of the hepatic gene regulatory network in the absence of FoxA factors.

The FoxA transcription factors are critical for liver development through their pioneering activity, which initiates a highly complex regulatory network thought to become progressively resistant to the loss of any individual hepatic transcription factor via mutual redundancy. To investigate the dispensability of FoxA factors for maintaining this regulatory network, we ablated all FoxA genes in the adult mouse liver. Remarkably, loss of FoxA caused rapid and massive reduction in the expression of critical liver genes. Activity of these genes was reduced back to the low levels of the fetal prehepatic endoderm stage, leading to necrosis and lethality within days. Mechanistically, we found FoxA proteins to be required for maintaining enhancer activity, chromatin accessibility, nucleosome positioning, and binding of HNF4α. Thus, the FoxA factors act continuously, guarding hepatic enhancer activity throughout adult life.

Inhibition of Glycolysis in Pathogenic TH17 Cells through Targeting a miR -21-Peli1-c-Rel Pathway Prevents Autoimmunity.

It is well known that some pathogenic cells have enhanced glycolysis; the regulatory network leading to increased glycolysis are not well characterized. In this study, we show that CNS-infiltrated pathogenic TH17 cells from diseased mice specifically upregulate glycolytic pathway genes compared with homeostatic intestinal TH17 cells. Bioenergetic assay and metabolomics analyses indicate that in vitro-derived pathogenic TH17 cells are highly glycolytic compared with nonpathogenic TH17 cells. Chromatin landscape analyses demonstrate TH17 cells in vivo that show distinct chromatin states, and pathogenic TH17 cells show enhanced chromatin accessibility at glycolytic genes with NF-κB binding sites. Mechanistic studies reveal that miR-21 targets the E3 ubiquitin ligase Peli1-c-Rel pathway to promote glucose metabolism of pathogenic TH17 cells. Therapeutic targeting c-Rel-mediated glycolysis in pathogenic TH17 cells represses autoimmune diseases. These findings extend our understanding of the regulation TH17 cell glycolysis in vivo and provide insights for future therapeutic intervention to TH17 cell-mediated autoimmune diseases.

Valproic Acid Significantly Improves CRISPR/Cas9-Mediated Gene Editing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has emerged as a powerful technology, with the potential to generate transgenic animals. Particularly, efficient and precise genetic editing with CRISPR/Cas9 offers immense prospects in various biotechnological applications. Here, we report that the histone deacetylase inhibitor valproic acid (VPA) significantly increases the efficiency of CRISPR/Cas9-mediated gene editing in mouse embryonic stem cells and embryos. This effect may be caused through globally enhanced chromatin accessibility, as indicate by histone hyperacetylation. Taken together, our results suggest that VPA can be used to increase the efficacy of CRISPR/Cas9 in generating transgenic systems.

Epigenetic Changes During Human Thyroid Cell Differentiation.

Objective: It has been demonstrated that the transcription factors TAZ (transcriptional coactivator with PDZ-binding motif), paired box gene 8 (PAX8), and NK2 homeobox 1 (NKX2-1) are coexpressed in the nucleus of thyroid cells. Furthermore, TAZ is known to enhance the transcriptional activity of PAX8 and NKX2-1 as well as the key thyroid-specific gene, thyroglobulin (TG), suggesting a critical role for TAZ in the control of thyroid cell speciation. We previously reported that the small molecule ethacridine, identified as a TAZ activator, was able to induce thyroid-specific transcription in endodermal cells differentiated from human embryonic stem (hES) cells using activin A. Since transcription factors are epigenetically regulated in cell differentiation, we investigated the epigenetic changes in the promoter regions of these key transcription factors during in vitro differentiation of hES cells into thyrocytes. Methods: We initially profiled chromatin accessibility using the technique of Assay for Transposase Accessible Chromatin sequencing (ATAC-seq), and then examined DNA methylation and histone acetylation in the promoter regions of the three selected thyroid transcription factors and the thyroid-specific genes during hES cell differentiation. Results: ATAC-seq analysis showed enriched chromatin accessibility of TAZ, NKX2-1, and PAX8 after exposure to activin A and ethacridine. There were no methylation changes found in the NKX2-1, PAX8, and TAZ promoters by bisulfite sequencing. In contrast, acetylation of histone H4, specifically acetylation of lysine 16, was observed in each of the promoters when measured by chromatin immunoprecipitation polymerase chain reaction assays, which correlated with the activity and expression of NKX2-1 and PAX8 as well as sodium/iodide symporter, thyroid stimulating hormone receptor, and TG genes. Conclusions: These results indicate that ethacridine treatment of activin A-derived endodermal hES cells leads to enhanced chromatin accessibility, which, in turn, allows histone H4 acetylation in the regulation of active genes for speciation of thyroid follicular cells from hES cells.