Assay For Transposase-accessible Chromatin With High-throughput Sequencing Research Articles

Platelet CD47 Maintains Circulating Monocyte Immune Homeostasis

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and white blood cells, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of both vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. Circulating monocytes are a part of the first line of host defenses that recognize and clear pathogens in part by releasing inflammatory cytokines, including IL-6, IL-8 and TNFα, that trigger a robust inflammatory reaction. However, this needs to be tightly regulated and in disease conditions like sepsis, dysregulated monocyte responses contribute to a “cytokine storm” that leads to exaggerated inflammation, tissue damage and potentially mortality. The cellular and molecular mechanisms that regulate monocyte immune phenotypes are complex and include roles for platelets. Considering the immune regulatory role of platelets and that thrombocytopenia is associated with worse sepsis outcomes, we hypothesize that resting platelets maintain monocyte immune homeostasis in healthy conditions and thrombocytopenia independently leads to monocyte dysregulation that exacerbates sepsis responses in a manner that is dependent on direct platelet-monocyte CD47 homotypic interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, circulating monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) induced cytokine responses. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming skewed toward glycolysis, consistent with our in vitro and in vivo RNA-Seq results. Enhanced glycolysis was mediated by AKT/mTOR pathway activation and resulted in durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed open chromatin at LPS response genes, and resting platelet interactions with monocytes induced histone H3K9 and H3K27 trimethylation in a CD47 dependent manner. Using mouse models of thrombocytopenia and sepsis, we found that normal platelet numbers limited monocyte immune dysregulation and systemic inflammation. This was dependent on platelet CD47 as only WT platelets, not CD47 -/- platelet transfusion rescued the exaggerated inflammatory responses in thrombocytopenic mice (Fig 1). Both plasma IL-6 and IL-8 and circulating monocyte IL6 and IL8 gene expression from human patients with sepsis and non-septic conditions inversely correlated with patient platelet counts, further suggesting a resting platelet role in maintaining monocyte immune quiescence. Overall, our studies demonstrate that resting platelets maintain monocyte immune homeostasis by limiting TLR responses through regulating monocyte immunometabolic processes that lead to epigenetic changes in monocyte immune response genes (Fig 2). Thrombocytopenia therefore independently leads to immune dysregulation in sepsis, exacerbating systemic inflammatory processes. This is the first demonstration of sterile endogenous cell interactions that regulate innate immune-metabolism and monocyte pathogen responses.

Abstract 163: Thrombocytopenia Independently Leads To Monocyte Immune Dysfunction

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and leukocytes, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of both vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. We have now found that thrombocytopenia (a low platelet count) leads to monocyte dysfunction, independent of the cause of thrombocytopenia, in a manner that is dependent on direct platelet-monocyte CD47 interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) responses, including increased IL-6 production. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming and durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed persistently open chromatin at LPS response genes. Using mouse models of thrombocytopenia and sepsis we found that normal platelet numbers are needed to limit immune dysregulation in sepsis. IL6 and TNF expression in monocytes from human patients with sepsis also inversely correlated with patient platelet counts. Our studies demonstrate that in healthy conditions resting platelets maintain monocyte immune tolerance by regulating monocyte immunometabolic processes that lead to epigenetic changes in TLR-related genes. Platelets therefore function as immune rheostats in both health and disease.

Dose rate dependent reduction in chromatin accessibility at transcriptional start sites long time after exposure to gamma radiation

ABSTRACT Ionizing radiation (IR) impact cellular and molecular processes that require chromatin remodelling relevant for cellular integrity. However, the cellular implications of ionizing radiation (IR) delivered per time unit (dose rate) are still debated. This study investigates whether the dose rate is relevant for inflicting changes to the epigenome, represented by chromatin accessibility, or whether it is the total dose that is decisive. CBA/CaOlaHsd mice were whole-body exposed to either chronic low dose rate (2.5 mGy/h for 54 d) or the higher dose rates (10 mGy/h for 14 d and 100 mGy/h for 30 h) of gamma radiation (60Co, total dose: 3 Gy). Chromatin accessibility was analysed in liver tissue samples using Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq), both one day after and over three months post-radiation (>100 d). The results show that the dose rate contributes to radiation-induced epigenomic changes in the liver at both sampling timepoints. Interestingly, chronic low dose rate exposure to a high total dose (3 Gy) did not inflict long-term changes to the epigenome. In contrast to the acute high dose rate given to the same total dose, reduced accessibility at transcriptional start sites (TSS) was identified in genes relevant for the DNA damage response and transcriptional activity. Our findings link dose rate to essential biological mechanisms that could be relevant for understanding long-term changes after ionizing radiation exposure. However, future studies are needed to comprehend the biological consequence of these findings.

Lymphocyte crosstalk is required for monocyte-intrinsic trained immunity to Plasmodium falciparum.

Plasmodium falciparum (P. falciparum) induces trained innate immune responses in vitro, where initial stimulation of adherent PBMCs with P. falciparum–infected RBCs (iRBCs) results in hyperresponsiveness to subsequent ligation of TLR2. This response correlates with the presence of T and B lymphocytes in adherent PBMCs, suggesting that innate immune training is partially due to adaptive immunity. We found that T cell–depleted PBMCs and purified monocytes alone did not elicit hyperproduction of IL-6 and TNF-α under training conditions. Analysis of P. falciparum–trained PBMCs showed that DCs did not develop under control conditions, and IL-6 and TNF-α were primarily produced by monocytes and DCs. Transwell experiments isolating purified monocytes from either PBMCs or purified CD4+ T cells, but allowing diffusion of secreted proteins, enabled monocytes trained with iRBCs to hyperproduce IL-6 and TNF-α after TLR restimulation. Purified monocytes stimulated with IFN-γ hyperproduced IL-6 and TNF-α, whereas blockade of IFN-γ in P. falciparum–trained PBMCs inhibited trained responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from patients with malaria showed persistently open chromatin at genes that appeared to be trained in vitro. Together, these findings indicate that the trained immune response of monocytes to P. falciparum is not completely cell intrinsic but depends on soluble signals from lymphocytes.

Sites of active gene regulation in the prenatal frontal cortex and their role in neuropsychiatric disorders.

Common genetic variation appears to largely influence risk for neuropsychiatric disorders through effects on gene regulation. It is therefore possible to shed light on the biology of these conditions by testing for enrichment of associated genetic variation within regulatory genomic regions operating in specific tissues or cell types. Here, we have used the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) to map open chromatin (an index of active regulatory genomic regions) in bulk tissue, NeuN+ and NeuN- nuclei from the prenatal human frontal cortex, and tested enrichment of single-nucleotide polymorphism (SNP) heritability for five neuropsychiatric disorders (autism spectrum disorder, attention deficit hyperactivity disorder [ADHD], bipolar disorder, major depressive disorder, and schizophrenia) within these regions. We observed significant enrichment of SNP heritability for ADHD, major depressive disorder, and schizophrenia within open chromatin regions (OCRs) mapped in bulk fetal frontal cortex, and for all five tested neuropsychiatric conditions when we restricted these sites to those overlapping histone modifications indicative of enhancers (H3K4me1) or promoters (H3K4me3) in fetal brain. SNP heritability for neuropsychiatric disorders was significantly enriched in OCRs identified in fetal frontal cortex NeuN- as well as NeuN+ nuclei overlapping fetal brain H3K4me1 or H3K4me3 sites. We additionally demonstrate the utility of our mapped OCRs for prioritizing potentially functional SNPs at genome-wide significant risk loci for neuropsychiatric disorders. Our data provide evidence for an early neurodevelopmental component to a range of neuropsychiatric conditions and highlight an important role for regulatory genomic regions active within both NeuN+ and NeuN- cells of the prenatal brain.

ATRT-07. DEFINING LOST AND GAINED TRANSCRIPTIONAL REGULATORY NETWORKS IN ATYPICAL TERATOID RHABDOID TUMOR

Atypical teratoid rhabdoid tumor (ATRT) is a central nervous system cancer of infancy and early childhood that may occur anywhere along the neuraxis and is associated with a high rate of mortality. While contemporary multimodal therapeutic approaches have significantly improved overall survival, targeted therapy remains elusive, and treatment is often associated with significant morbidity. ATRT is unique in its genomic stability, with the only recurrent genetic abnormality being bi-allelic loss of the SMARCB1 gene, which encodes a core subunit of the BAF chromatin remodeling complex. The epigenetic mechanisms by which SMARCB1 loss leads to tumorigenesis are not yet well-defined and addressing this gap in understanding is necessary for creating efficacious, targeted therapeutics. To better understand the epigenetic features gained and lost in ATRT, we re-expressed SMARCB1 in a library of patient-derived and established ATRT cell lines of multiple molecular subtypes. SMARCB1 restoration significantly reduced or eliminated the proliferative and clonogenic capacity of each cell line. We performed assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) and RNA sequencing (RNA-Seq) to define putative transcriptional regulatory networks that are gained and lost in ATRT. SMARCB1 restoration was associated with global changes in chromatin openness consistent with the creation of new regulatory elements throughout the genome, and these were associated with induction of a diverse developmental transcriptional signature. Motif enrichment analysis of regions with increased accessibility defined a small but consistent number of centrally enriched transcription factor motifs across cell lines indicative of putative pioneer factors whose functions may be lost in ATRT. Pertinent chromatin immunoprecipitation with sequencing (ChIP-Seq) data will be discussed in the context of lost and gained transcriptional regulatory networks in ATRT and normal cellular development.

Abstract 13435: Characterization of Open Chromatin in Human Mitral Valves to Identify Regulatory Mechanisms at Mitral Valve Prolapse Genetic Loci

Introduction: Mitral valve prolapse (MVP) is a common valve disease that leads to mitral insufficiency, heart failure, and sudden death. The identification of genetic risk loci provided insight into its genetic architecture, although the causal variants and target genes need to be fully characterized. Hypothesis: Functional genomic annotation, including chromatin accessibility profiles of human mitral valves, allows the characterization of variants and target genes at MVP loci. Methods: We determined open chromatin regions using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) from surgically removed human valves and primary cultures of fibroblasts. We analyzed the spatial conformation of MVP-associated loci using circular chromatin conformation capture (4C-Seq) with candidate causal variants as viewpoints. Candidate causal variants were followed-up using reporter assays and in silico analyses. Results: We mapped the open chromatin from 11 human mitral valves and 3 primary cell cultures. We found that mitral valve-specific ATAC-Seq peaks were enriched near genes involved in extracellular matrix organization, chondrocyte differentiation, and connective tissue development. We also found that MVP-associated variants (p<10 -5 ) observed in the MVP GWAS were significantly enriched (p<0.05) in mitral valve open chromatin regions, but not in heart tissue from ENCODE. We found that rs6723013 on Chr2 belongs to a valve-specific enhancer at the IGFBP5/TNS1 locus and is the most likely causal variant at this locus. We also identified several plausible causal SNPs and, by using 4C-Seq, potential target genes ( HIC1 and DPH1 ) at the SMG6/SRR locus. Conclusions: Here we describe unprecedented genome-wide open chromatin profiles from human mitral valves that indicate specific gene regulation profiles, compared to the heart. We also report in vitro functional evidence for potential causal variants and target genes at MVP risk loci involving established and new biological mechanisms relevant to mitral valve disease.

Tn5 Transposase Applied in Genomics Research.

The development of high-throughput sequencing (next-generation sequencing technology (NGS)) and the continuous increase in experimental throughput require the upstream sample processing steps of NGS to be as simple as possible to improve the efficiency of the entire NGS process. The transposition system has fast “cut and paste” and “copy and paste” functions, and has been innovatively applied to the NGS field. For example, the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) uses high-throughput sequencing to detect chromatin regions accessible by Tn5 transposase. Linear Amplification via Transposon Insertion (LIANTI) uses Tn5 transposase for linear amplification, haploid typing, and structural variation detection. Not only is it efficient and simple, it effectively shortens the time for NGS sample library construction, realizes large-scale and rapid sequencing, improves sequencing resolution, and can be flexibly modified for more technological innovation.

Serine-threonine kinase ROCK2 regulates germinal center B cell positioning and cholesterol biosynthesis.

Germinal center (GC) responses require B cells to respond to a dynamic set of intercellular and microenvironmental signals that instruct B cell positioning, differentiation, and metabolic reprogramming. RHO-associated coiled-coil-containing protein kinase 2 (ROCK2), a serine-threonine kinase that can be therapeutically targeted by ROCK inhibitors or statins, is a key downstream effector of RHOA GTPases. Although RHOA-mediated pathways are emerging as critical regulators of GC responses, the role of ROCK2 in B cells is unknown. Here, we found that ROCK2 was activated in response to key T cell signals like CD40 and IL-21 and that it regulated GC formation and maintenance. RNA-Seq analyses revealed that ROCK2 controlled a unique transcriptional program in GC B cells that promoted optimal GC polarization and cholesterol biosynthesis. ROCK2 regulated this program by restraining AKT activation and subsequently enhancing FOXO1 activity. ATAC-Seq (assay for transposase-accessible chromatin with high-throughput sequencing) and biochemical analyses revealed that the effects of ROCK2 on cholesterol biosynthesis were instead mediated via a novel mechanism. ROCK2 directly phosphorylated interferon regulatory factor 8 (IRF8), a crucial mediator of GC responses, and promoted its interaction with sterol regulatory element-binding transcription factor 2 (SREBP2) at key regulatory regions controlling the expression of cholesterol biosynthetic enzymes, resulting in optimal recruitment of SREBP2 at these sites. These findings thus uncover ROCK2 as a multifaceted and therapeutically targetable regulator of GC responses.

SA97 - CHANGES OF OPEN CHROMATIN REGIONS REVEAL STAGE-SPECIFIC TRANSCRIPTIONAL NETWORK DYNAMICS IN HUMAN IPSC-DERIVED NEURONS

Background The application of human induced Pluripotent Stem Cells (iPSCs)-differentiated neurons has served as a promising model for gaining insights into the molecular and cellular mechanisms of genetic risk for mental disorders. Multiple factors determine the fate and trajectory of neuronal cell differentiation, of which transcriptional regulation plays a major role. Chromatin accessibility (openness) to Transcription Factors (TF) strongly influences gene transcription and cell differentiation. However, the dynamic changes of open chromatin and the TF networks during neuronal differentiation from iPSCs are poorly understood. Methods Here, we performed a global mapping of Open Chromatin Regions (OCRs) using the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) at different cell stages of glutamatergic neuronal differentiation, examined the correlation of changes of open chromatin and mRNA abundances (assayed by RNA-Seq), and constructed the neuronal stage-specific TF networks through a genome-wide inference of TF-binding footprints in OCRs. Results We found that OCRs were highly dynamic during neuronal differentiation, and more importantly that OCR accessibility at core promoter regions was positively correlated with mRNA abundances at their respective cell stages. Furthermore, we found that the dynamic changes of OCRs during neuronal differentiation were accompanied by the binding events of cell stage-specific TFs. As expected, binding footprints of OCT4 and NANOG, genes that encode pluripotent stem cell-specific markers, are most enriched in iPSCs. Intriguingly, binding footprints of NEUROD1 and NEUROG2, the two TFs that have been reported to rapidly induce high efficient conversion of iPSCs to excitatory neurons, are among those most enriched in the relatively mature neurons. Discussion Further TF network analysis based on the inferred TF binding footprints in OCRs successfully identified core TF networks and their master regulators for different neuronal stages. Interestingly, both NEUROD1 and NEUROG2 are in the same core TF network specific to more mature neurons, suggesting their pivotal role in epigenetic control of neuronal differentiation and maturation.

ARID1A Is Critical for Maintaining Normal Hematopoiesis in Mice

ARID1A is a key component of ATP-dependent SWI/SNF complex involved in chromatin remodeling. Chromatin remodeling mediated by SWI/SNF complex is crucial for gene expression and affects a broad range of biological processes including hematopoietic development. ARID1A is frequently mutated across several solid tumors as well as hematopoietic malignancies, including Burkitt's lymphoma, diffuse large B-cell lymphoma and acute promyelocytic leukemia. Nevertheless, function of ARID1A in adult hematopoiesis and implications of its deficiency in development and progression of hematopoietic diseases has not been explored. In this study, we used a murine model of ARID1A deficiency to establish its essential function in maintaining normal hematopoietic development.Germline loss of Arid1a is embryonic lethal; therefore, we generated mice with deletion of Arid1a specifically in the hematopoietic compartment using Vav-iCre and Mx1-Cre transgenic mice. Arid1afl/fl;Vav-iCre+ mice occurred at a lower than expected frequency, suggesting some perinatal mortality. For the Mx1-Cre model, Arid1a exon 9 was excised by administrating poly(I:C) to adult mice and hematopoiesis was evaluated using flow cytometry. An increase in both percentage and absolute number of long-term hematopoietic stem cells (LTHSCs) defined as Lin-Sca1+Kit+CD34-FLT3- or Lin-Sca1+Kit+CD48-CD150+ occurred in the bone marrow using both models of Arid1a deficiency. RNA-sequencing of sorted LTHSCs from Arid1a KO bone marrow revealed dysregulated expression of several genes involved in cell cycle, G2/M checkpoint and related pathways. In vivo BrdU incorporation assays showed a substantially lower proportion of quiescent hematopoietic stem cells in Arid1a deficient bone marrow. To assess the reconstitution ability of ARID1A deficient HSCs, sorted KO or WT LTHSCs were transplanted into irradiated congenic recipient mice in competitive repopulation assays. Proportion of donor-derived cells in recipients transplanted with KO cells was strikingly lower compared to wild-type cells, suggesting poor reconstitution ability of Arid1a KO LTHSCs.Also, differentiation of both myeloid and lymphoid lineages was impaired in Arid1a KO mice compared to WT controls. To investigate the mechanism of perturbed differentiation of the myeloid and erythroid lineages, RNA-Seq was performed on sorted CMPs, GMPs and MEPs from WT and Arid1a KO BM. Our analysis showed significant decrease in expression of several transcription factors (Runx1, Gata2, Cebpa), which play a crucial role in lineage differentiation.To determine how Arid1a deficiency alters chromatin accessibility in myeloid precursors, Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-Seq) was performed on sorted Lin-Kit+ BM cells from both Arid1a KO and WT mice. A global reduction in open chromatin in Arid1a KO cells was noted compared to WT cells. A substantial overlap occurred between down regulated genes (RNA-seq) and reduced chromatin accessibility in Arid1a KO myeloid progenitors. Motifs for PU.1, RUNX1, GATA and CEBPA were significantly enriched in loci with reduced ATAC-seq signals in Arid1a KO cells. Our findings demonstrate an indispensable function of Arid1a in hematopoietic development and underline the importance of precise chromatin dynamics maintained by ARID1A-containing SWI/SNF complex in hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Multiomic Insights into Novel Treg Subset

Using epigenetic reprogramming through Somatic Cell Nuclear Transfer, we developed two regulatory T cell (Treg) models, T138 (Ku M., et al., PNAS 2016) and T143 (Ku M., et al., JACI 2018). While both Treg models were thymically derived, T143 displayed characteristics of agonist-selection (aTreg), which had been postulated for all Treg cells. However, T138 was quite distinct with a higher degree of interaction between TCR beta-chain and MHC-II rather than TCR and agonist. We refer to as T138 as natural-occurring Treg cell (nTreg). Strikingly, we identified FoxP3-negative T cells expressing the same TCR as nTreg and aTreg. Especially, FoxP3-negative T cells isolated from T138 were heavily poised in their ability to differentiate into FoxP3-expressing cells rather than Th1-differentation.In order to further identify new transcription factors that mediated this epigenetically poised state in FoxP3-negative T138 (pre-nTreg) cells and to identify accessible promoters and enhancers that are associated with the pro-FoxP3 poised state, we performed ATAC-Seq (assay for transposase-accessible chromatin with high-throughput sequencing). We identified several loci that are more accessible in pre-nTreg cells than in wild-type (WT) cells. Motif analysis revealed enrichment of several lineage-determining transcription factor binding motifs, such as Atf3. Strikingly, transcriptomic analysis demonstrated that Atf3 is specifically expressed in T138 and not in WT. Interestingly; it has previously been shown that the FoxP3-locus contains an Atf-binding site, suggesting an important link of pre-nTreg transcriptional programs to its development into nTreg.Since ATAC-Seq signals show similar enrichment at both active and poised (bivalently-marked by both H3K4me2 and H3K27me3 marks) promoters, we performed H3K4me2 and H3K27me3 chromatin-immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) to further identify poised promoters, which gear e.g. pre-nTreg towards a FoxP3+ cellular fate.In order to identify unique phosphorylation sites during activation of pre-nTreg and nTreg cells, we analyzed the phospho-proteome. We identified several differential phosphorylated peptides. Particularly, we found about 100 peptides uniquely phosphorylated in FoxP3- pre-nTreg cells. Together with the transcriptional analysis this clearly demonstrates the unique characteristics of this novel pre-nTreg population.Several mechanisms had been shown to mediate immune regulatory function of Treg cells. Strikingly, we identified Ctla4 and Lag3 to be differentially expressed in T138, T143 and the Treg pool in WT mice. This is of particular interest since, e.g. CTLA4 is currently being explored for immunotherapy. Differential expression of Ctla4 provides evidence for selective modifications of a Treg subset through anti-CTLA4 treatment. Our data supports the concept that anti-CTLA4 treatment might function through targeting of a tumor-infiltrating Treg subset rather than check point inhibition.Taken together our multiomics and functional data on Treg cells clearly demonstrated the existence of distinct Treg subsets, which has major implications in better understanding disease mechanisms and in devising new future therapies. DisclosuresSabouri-Ghomi:Fate Therapeutics: Employment.

Simultaneous Profiling of DNA Accessibility and Gene Expression Dynamics with ATAC-Seq and RNA-Seq.

This chapter describes sequencing-based methods for profiling dynamic changes in DNA accessibility and gene expression in Saccharomyces cerevisiae. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) is a powerful technique for identifying nucleosome-free regions of the genome. Combining ATAC-Seq with RNA Sequencing (RNA-Seq) is a rapid approach for studying the relationship between genome structure and changes in global patterns of gene expression from a single experiment. A laboratory protocol is presented for these methods as well as examples of typical results and visualizations.

Mapping of the Multiple Myeloma Transcriptional Core Regulatory Circuitry Reveals TCF3 As a Novel Dependency and an Oncogenic Collaborator of MYC

Multiple Myeloma (MM) is a complex plasma cell malignancy driven by numerous genetic and epigenetic alterations that are acquired over time. The events controlling and modifying transcriptomic changes that drive MM cell growth and progression remains undefined. To reveal the epigenetic circuitry governing myeloma cells, we performed a comprehensive analysis integrating data obtained from Multiplexed Indexed T7 Chromatin IP (Mint-ChIP), Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq), and RNA-seq in 10 primary MM cells as well as 3 MM cell lines to identify genome-wide the master transcription factors (TFs), the enhancer elements they occupy, and the genes they regulate. Using these data, we have identified myeloma-specific core regulatory circuitry which includes several well-established regulators of MM such as IKZF, E2F, MYC and IRF family of genes. For example, our data show elevated MYC at numerous tissue specific enhancers in myeloma cells, including those that regulate lineage specifying transcription factors such as IRF4 and TCF3 (aka E2A). When translocated to the immunoglobulin enhancer, MYC in turn is regulated by these lineage transcription factors thus integrating MYC into the interconnected transcriptional core regulatory circuitry of MM (Figure 1a,b).We propose that this oncogenic “re-wiring” accounts for the observed addiction of MM cells to lineage factors such as IRF4 and in this work, we implicate the B-cell factor TCF3 as a novel multiple myeloma dependency. Using myeloma cell lines and primary samples, we observed elevated enhancer activity at TCF3 in primary CD138+ cells from myeloma patients compared to normal plasma cells (NPCs) (Figure 1c). As a result, TCF3 expression is significantly upregulated in our large cohort of MM patients (n=370) compared to normal bone marrow plasma cells (n=18). As MYC proteins can only bind pre-established and acetylated regions of active chromatin, we hypothesize that enhancer specifying lineage transcription factors such as TCF3 may cooperate with MYC to alter tissue specific gene expression programs. We show that TCF3 is regulated by a large proximal enhancer that is bound by MYC, and is highly sensitive to chemical perturbation of enhancer co-activators such as BRD4. As a helix-loop-helix transcription factor that similar to MYC binds short (CANNTG) E-box sequences, we computationally predict co-occupancy of MYC and TCF3 at ~80% of all enhancers that form the multiple myeloma transcriptional core regulatory circuitry. To evaluate the functional role of TCF3 in myeloma cells, we established TCF3 knock down myeloma cell lines and followed the cell growth over time. Stable knockdown of TCF3 preferentially blocks proliferation of IgH MYC translocated cell lines (such as MM1.S cells) versus non-translocated lines (such as U266 cells). Finally, high expression of TCF3 correlates with poor clinical outcome in myeloma patients. Together these data suggest TCF3 acts as an oncogenic collaborator with deregulated MYC and implicates transcriptional control of lineage as a dependency in multiple myeloma.Figure 1: Transcriptional core regulatory circuitry of multiple myeloma: A) ChIP-Seq tracks of IRF4, MYC, BRD4, and H3K27ac occupancy at the IRF4, IgH enhancer, and TCF3 loci respectively. B) Schematic of transcription factor to enhancer connectivity of the partial multiple myeloma transcriptional core regulatory circuitry highlighting interactions between IRF4, MYC, and TCF3 (computationally predicted based on TCF3 motif data). C) ChIP-Seq tracks of H3K27ac occupancy at the TCF3 locus in patient multiple myeloma (top, n=3) or normal plasma cells (bottom, n=2). [Display omitted] DisclosuresBradner:Acetylon: Other: Scientific Founder; Novartis: Employment. Anderson:Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees.

The Repression of Atoh1 by Neurogenin1 during Inner Ear Development.

Atonal homolog 1 (Atoh1) and Neurogenin1 (Neurog1) are basic Helix-Loop-Helix (bHLH) transcription factors crucial for the generation of hair cells (HCs) and neurons in the inner ear. Both genes are induced early in development, but the expression of Atoh1 is counteracted by Neurog1. As a result, HC development is prevented during neurogenesis. This work aimed at understanding the molecular basis of this interaction. Atoh1 regulation depends on a 3’Atoh1-enhancer that is the site for Atoh1 autoregulation. Reporter assays on chick embryos and P19 cells show that Neurog1 hampers the autoactivation of Atoh1, the effect being cell autonomous and independent on Notch activity. Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) analysis shows that the region B of the 3’Atoh1-enhancer is accessible during development and sufficient for both activation and repression. Neurog1 requires the regions flanking the class A E-box to show its repressor effect, however, it does not require binding to DNA for Atoh1 repression. This depends on the dimerization domains Helix-1 and Helix-2 and the reduction of Atoh1 protein levels. The results point towards the acceleration of Atoh1 mRNA degradation as the potential mechanism for the reduction of Atoh1 levels. Such a mechanism dissociates the prevention of Atoh1 expression in neurosensory progenitors from the unfolding of the neurogenic program.

The LRF/ZBTB7A Transcription Factor Is a BCL11A-Independent Repressor of Fetal Hemoglobin

Induction of fetal-type hemoglobin (HbF: α2γ2) is a promising means to treat hemoglobinopathies; however, precisely how HbF expression is silenced in adult erythroid cells is not fully understood. Such knowledge is essential to develop mechanism-based, targeted approaches to reactivate HbF production. Here, we show that Leukemia/lymphoma Related Factor (LRF), encoded by the ZBTB7A gene, is a novel and potent repressor of HbF production.To assess the effects of LRF loss on the mouse erythroid transcriptome, we performed RNA-Seq analysis using splenic erythroblasts from control and LRF conditional knockout (Zbtb7aF/F Mx1-Cre+) mice. LRF-deficient adult erythroblasts showed significant induction of Hbb-bh1, but not Hbb-y. The results were validated at the protein levels via isoelectric focusing of peripheral blood (PB) hemolysates and MALDI-TOFMS analysis. LRF loss also reactivated human fetal globin expression in vivo in LRF conditional KO mice harboring the human β-globin gene cluster as a yeast artificial chromosome transgene (β-YAC).To determine whether LRF loss could induce HbF in human erythroid cells, we employed human CD34+ hematopoietic stem and progenitor (HSPC)-derived primary erythroblasts and determined γ-globin expression levels upon shRNA-mediated LRF knockdown (KD). HbF levels in LRF KD cells (49-70%) were much greater than those seen in parental or scrambled-shRNA transduced cells. We next employed a novel human immortalized erythroid line (HUDEP-2). This line possesses an advantage over lines currently used for globin switching studies because it expresses predominantly adult hemoglobin (HbA: α2β2), with very low background HbF expression. Using CRISPR/cas9 gene modification, we knocked out ZBTB7A in HUDEP-2 cells and performed RNA-Seq analysis. As expected, γ-globin (HBG1 and HBG2) transcripts, but not those of embryonic ε-globin (HBE1), were markedly induced in ZBTB7A KO (ZBTB7AΔ/Δ) HUDEP-2 cells. ZBTB7AΔ/Δ cells exhibited HbF levels greater than 60%, while that of parental cells was less than 3%. Notably, the HbF reactivation occurred without changes in levels of transcripts encoding known HbF repressors, including BCL11A, the principal known switching factor.We next performed chromatin-immunoprecipitation and sequencing (ChIP-Seq) with an anti-LRF antibody using HSPC-derived proerythroblasts and HUDEP-2 cells. The most enriched motif identified in either was concordant with that previously identified in vitro using CAST analysis (Maeda et. al. Nature 2005), confirming antibody specificity. Supporting a direct role of LRF at the β-globin cluster, we observed several significant enrichment of LRF-ChIP binding signals at adult (HBB), fetal (HBG1) globin loci and the upstream hypersensitivity (HS) sites within the locus control region (LCR). ATAC-Seq (for assay for transposase-accessible chromatin with high-throughput sequencing) analysis revealed strong chromatin accessibility at the γ-globin locus in ZBTB7AΔ/Δ cells. Strikingly, differential enrichment of ATAC-signals in ZBTB7AΔ/Δ cells was evident only at the γ-locus. Thus, while LRF binds to the HBB locus and HS sites as well as to the HBG1 locus, LRF depletion specifically opens chromatin at the γ-globin locus.Finally, to determine whether LRF and BCL11A suppress γ-globin expression via distinct mechanisms, we established LRF/BCL11A double knockout HUDEP-2 cells. Strikingly, HUDEP-2 lines lacking both LRF and BCL11A exhibited almost a complete switch in expression from adult- to fetal-type globin, suggesting that these two factors cumulatively represent the near entirety of γ-globin repressive activity in adult erythroid cells. Our findings reveal a novel molecular mechanism regulating γ-globin silencing and may open a new window for therapeutic targeting in the treatment of hemoglobinopathies. DisclosuresBauer:Biogen: Research Funding; Editas Medicine: Consultancy. Orkin:Editas Inc.: Consultancy.