Distinct Regulatory Programs Research Articles

Abstract A007: Pancreatic cancer comprises co-existing transcriptional states regulated by distinct master regulator programs

Abstract Despite extensive efforts, reproducible assessment of pancreatic ductal adenocarcinoma (PDA) heterogeneity and plasticity at the single cell level remains elusive. Systematic, network-based analysis of single cell RNA-seq profiles showed that most PDA tumors comprise three coexisting lineages whose aberrant transcriptional state is mechanistically controlled by distinct regulatory programs. These lineages were characterized by the aberrant activation of either gastrointestinal lineage markers (GLS), transcriptional effectors of morphogen pathways (MOS) and acinar to ductal metaplasia markers (ALS). Each lineage was characterized by cells in two different cell states determined by the differential activation of MEK signaling (M+/M-) and high cellular plasticity. These states were confirmed in multiple cohorts, cell lines, PDX models and harmonized with bulk profile analyses. Master regulators (MRs) of GLS and MOS state were predictive of patient’s survival in bulk profiles. Cross-state plasticity was confirmed by lineage tracing assays, while pooled CRISPR/Cas9 assays confirmed the essentiality of identified MR proteins. Finally, mechanistic MR-mediated cell state control was confirmed by MR expression-mediated reprogramming of MOS cells to a GLS state. Our work provided a mechanistic model of pancreatic cancer heterogeneity and testable hypothesis to target cell state-specific pancreatic cancer dependencies. Citation Format: Pasquale Laise, Mikko Turunen, Hans Carlo Maurer, Alvaro Curiel Garcia, Ela Elyada, Bernhard Schmierer, Lorenzo Tomassoni, Jeremy Worley, Mariano J. Alvarez, Jordan Kesner, Xiangtian Tan, Somnath Tagore, Ester Calvo Fernandez, Kelly Wong, Alexander L. E. Wang, Sabrina Ge, Alina C. Iuga, Aaron T. Griffin, Winston Wong, Gulam A. Manji, Faiyaz Notta, David A. Tuveson, Kenneth P. P. Olive, Andrea Califano. Pancreatic cancer comprises co-existing transcriptional states regulated by distinct master regulator programs [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A007.

Whole-genome profiling of DNA methylation and hydroxymethylation identifies distinct regulatory programs among innate lymphocytes.

Innate lymphocytes encompass a diverse array of phenotypic identities with specialized functions. DNA methylation and hydroxymethylation are essential for epigenetic fidelity and fate commitment. The landscapes of these modifications are unknown in innate lymphocytes. Here, we characterized the whole-genome distribution of methyl-CpG and 5-hydroxymethylcytosine in mouse ILC3, ILC2, and NK cells. We identified differentially methylated and hydroxymethylated DNA regions between ILC-NK subsets and correlated them with transcriptional signatures. We associated lineage-determining transcription factors with demethylation and demonstrated unique patterns of DNA methylation/hydroxymethylation in relationship to open chromatin regions, histone modifications, and transcription factor binding sites. We further discovered a novel association between hydroxymethylation and NK cell super-enhancers. Using mice lacking DNA hydroxymethylase TET2, we showed its requirement for optimal production of hallmark cytokines by ILC3 and IL-17A by inflammatory ILC2. These findings provide a powerful resource for studying innate lymphocyte epigenetic regulation and decode the regulatory logic governing their identity.

Enhanced pro-apoptosis gene signature following the activation of TAp63\u03b1 in oocytes upon \u03b3 irradiation

Specialized surveillance mechanisms are essential to maintain the genetic integrity of germ cells, which are not only the source of all somatic cells but also of the germ cells of the next generation. DNA damage and chromosomal aberrations are, therefore, not only detrimental for the individual but affect the entire species. In oocytes, the surveillance of the structural integrity of the DNA is maintained by the p53 family member TAp63α. The TAp63α protein is highly expressed in a closed and inactive state and gets activated to the open conformation upon the detection of DNA damage, in particular DNA double-strand breaks. To understand the cellular response to DNA damage that leads to the TAp63α triggered oocyte death we have investigated the RNA transcriptome of oocytes following irradiation at different time points. The analysis shows enhanced expression of pro-apoptotic and typical p53 target genes such as CDKn1a or Mdm2, concomitant with the activation of TAp63α. While DNA repair genes are not upregulated, inflammation-related genes become transcribed when apoptosis is initiated by activation of STAT transcription factors. Furthermore, comparison with the transcriptional profile of the ΔNp63α isoform from other studies shows only a minimal overlap, suggesting distinct regulatory programs of different p63 isoforms.

Chromatin Accessibility Analysis Reveals Epigenetic Evolution Is a Common Mechanism of Relapse in Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is associated with a poor prognosis and high rates of relapse despite aggressive treatments including high dose chemotherapy. To understand the clonal dynamics and genetic evolution of relapsed AML, we analyzed a cohort of 142 previously published genotyped and paired diagnosis-relapse AML samples. 40% of queried cases exhibited no major changes in somatic mutations upon relapse, and genetically stable clonal structure correlated with increased relapse probability. Thus, we hypothesized epigenetic reprogramming plays a role in these cases and AML relapse in general. Here, we examine the epigenetic landscape of relapsed AML and characterize the cis and trans regulatory elements that correlate with AML relapse in the presence or absence of genetic evolution.Methods: We identified 27 viable cryopreserved paired diagnosis and relapse samples from the same patient treated at Stanford with high-dose chemotherapy. Leukemic blasts and (when possible) leukemia stem cell (LSC) enriched populations were FACS purified and prepared for genotyping with a myeloid malignancy targeted sequencing panel as well as ATAC-seq for chromatin accessibility profiling. Single-cell ATAC-seq was further performed on select samples to investigate regulatory reprogramming in cell subpopulations. We then performed integrative analysis to uncover the interplay between genetic lesions, epigenetic regulatory programs, and gene accessibility in relapsed AML.Results: Genotyping analysis of these AML specimens revealed that 40% of samples exhibited no changes in AML-related genetic alterations upon relapse (hereby referred to as “stable” samples). Chromatin accessibility analysis revealed these stable samples had a distinct epigenetic signature, modulating similar gene accessibility programs and sharing enhancer loci that become accessible across all stable relapse samples. These sequences included genes involved in chromatin organization and compaction, as well as those involved in transcriptional control of hematopoietic differentiation and myeloid cell maturation. We also observed several regulatory signatures present at relapse specific for AML subtypes, including NPM1/FLT3 double mutant AML and those with mutations in transcription factors such as CEBPA or RUNX1.We then performed single-cell ATAC-seq on genetically stable samples to further characterize the sub-clonal epigenetic dynamics between the diagnosis and relapse cells. Several samples exhibited regulatory heterogeneity in multiple cell subpopulations that changed significantly at relapse. One subpopulation of interest was characterized by increased GATA and RUNX family transcription factor motif accessibility at relapse, indicating a shift toward a less differentiated progenitor cell phenotype. In addition, we identified a subpopulation of cells at diagnosis that were epigenetically similar to the major epigenetic states present at relapse, indicating that selection for specific epigenetic subclones may occur in AML patients during therapy in the absence of additional genetic lesions.Finally, given the critical role of LSCs in AML pathogenesis and their possible role as a reservoir for AML relapse, we analyzed LSC-enriched subpopulations in a subset of our cohort. ATAC-seq analysis indicated these cells shared several LSC-specific epigenetic features between samples, are distinguished from leukemia blasts by distinct regulatory programs, and undergo epigenetic remodeling between initial diagnosis and relapse. Gene accessibility analysis also revealed a shared LSC gene expression signature that also shifted at relapse. These data indicate a specific, distinct epigenomic signature for LSC enriched cell populations, and that epigenetic evolution at relapse occurs intracellularly, rather than reflecting heterogeneity in cellular subpopulations upon AML relapse.Conclusion: This study reveals that epigenetic remodeling in the absence of genetic evolution is a mechanism through which AML relapse occurs. We show that chromatin reorganization of genes and regulatory sequences occurs in these AML cells, leading to a permissive cell state that might be resistant to conventional treatment. Ongoing work includes dissecting the subclonal structure of these AML cells and identifying the relationship between gene regulatory networks that contribute to relapse. DisclosuresEdiriwickrema: Nanosive SAS: Patents & Royalties. Majeti: BeyondSpring Inc.: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc..

Multi-Omic Analysis Identifies Epigenetic Evolution in Relapsed Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is associated with a poor prognosis even with aggressive treatments including high dose chemotherapy. While most patients enter clinical remission, these remissions are often short-lived leading to chemotherapy-resistant relapsed disease that accounts for the majority of deaths. We undertook a meta-analysis of published datasets consisting of 142 genotyped paired diagnosis-relapse AML samples to understand the genetic evolution of AML between the two disease states. This analysis determined that a plurality of cases exhibited the same mutations at diagnosis and relapse, and that genetically stable clones were associated with an increased probability of relapse. The finding that many cases exhibited no clonal genetic evolution upon relapse, yet exhibited chemotherapy resistance, lead us to hypothesize that epigenetic evolution plays a significant role in AML relapse. Here, our objective was to investigate the epigenetic evolution and cis and trans regulatory elements that correlate with AML relapse. Methods: We identified 27 paired diagnosis and relapse specimens from patients treated at Stanford with high dose chemotherapy regimens. Leukemic blasts, and in some cases leukemia stem cell (LSC)-enriched fractions, were purified by FACS. Cells were then analyzed through a multi-omic platform including genotyping with a myeloid malignancy targeted panel, RNA-seq, and ATAC-seq to obtain a molecular and chromatin accessibility profile of each sample. The resulting data set was analyzed to investigate epigenetic evolution in relapsed AML. Results: Genotyping analysis of banked AML specimens identified a similar pattern of genetic evolution as our meta-analysis, with several samples exhibiting the same mutations at diagnosis and relapse. We used an epigenetic matrix of chromatin accessibility data obtained from purified cell populations within the hematopoietic hierarchy and implemented this with the CIBERSORT algorithm to map the regulatory programs active in diagnosis and relapsed AML blasts. This analysis revealed a general trend of epigenetic states associated with more primitive cells (such as hematopoietic stem and progenitor cells) active at relapse, as opposed to more differentiated myeloid cell programs active at diagnosis. Focusing further on samples with no genetic changes between the two disease states, we observed several samples with substantial epigenetic evolution at relapse, with AML blasts shifting from a more differentiated myeloid cell profile to that of stem and progenitor cells. These changes were associated with a loss of accessibility in PU.1 and CEBPα transcription factor motifs, with a corresponding increase in GATA and RUNX motifs, suggesting epigenetic remodeling contributes to relapse even in the absence of genetic changes. We have additionally identified various categories of relapse samples in our cohort that share similar epigenetic profiles relating to genotype; NPM1 and FLT3 double mutant samples, for example, shared active chromatin accessibility features. Given the key importance of LSCs in AML pathogenesis and their potential role in chemotherapy resistance, we further undertook an analysis of cellular subpopulations enriched for these cells in a subset of our sample cohort. ATAC-seq analysis of CD34+CD38- cell fractions revealed these cells share many epigenetic features between samples, yet also have distinct regulatory programs from those active in leukemia non-stem cells and exhibit similar epigenetic reprogramming between diagnosis and relapse. This analysis further indicates that epigenetic evolution at relapse occurs at the single cell level, rather than reflecting selection of cellular subpopulations at relapse. Ongoing work involves identifying the specific regulatory programs upregulated in relapse samples, and LSCs specifically, to understand how these programs contribute to relapse at the gene regulatory level. Conclusion: Our results indicate a substantial role for epigenetic evolution in AML, with the activation of more primitive stem and progenitor programs upon relapse. We have also identified epigenetic classifications for several relapse samples that correspond to genotype and characterized the regulatory programs associated with relapse. We hope this work will permit a deeper understanding of the evolutionary factors that guide AML relapse. Disclosures Majeti: Zenshine Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Coherus BioSciences: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Research Funding; BeyondSpring Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Stanford University: Patents & Royalties: pending patent application on CD93 CAR ; Forty-Seven Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences, Inc.: Patents & Royalties: inventor on patents related to CD47 cancer immunotherapy.

Distinct Regulatory Programs Control the Latent Regenerative Potential of Dermal Fibroblasts during Wound Healing.

Dermal fibroblasts exhibit considerable heterogeneity during homeostasis and in response to injury. Defining lineage origins of reparative fibroblasts and regulatory programs that drive fibrosis or, conversely, promote regeneration will be essential for improving healing outcomes. Using complementary fate-mapping approaches, we show that hair follicle mesenchymal progenitors make limited contributions to wound repair. In contrast, extrafollicular progenitors marked by the quiescence-associated factor Hic1 generated the bulk of reparative fibroblasts and exhibited functional divergence, mediating regeneration in the center of the wound neodermis and scar formation in the periphery. Single-cell RNA-seq revealed unique transcriptional, regulatory, and epithelial-mesenchymal crosstalk signatures that enabled mesenchymal competence for regeneration. Integration with scATAC-seq highlighted changes in chromatin accessibility within regeneration-associated loci. Finally, pharmacological modulation of RUNX1 and retinoic acid signaling or genetic deletion of Hic1 within wound-activated fibroblasts was sufficient to modulate healing outcomes, suggesting that reparative fibroblasts have latent but modifiable regenerative capacity.

Allele-specific expression changes dynamically during T cell activation in HLA and other autoimmune loci.

Genetic studies have revealed that autoimmune susceptibility variants are over-represented in memory CD4+ T cell regulatory elements1–3. Understanding how genetic variation affects gene expression in different T cell physiological states is essential for deciphering genetic mechanisms of autoimmunity4,5. Here we characterized the dynamics of genetic regulatory effects at eight time points during memory CD4+ T cell activation with high depth RNA-seq in healthy individuals. We discovered widespread dynamic allele-specific expression across the genome, where the balance of alleles changes over time. These genes were four-fold enriched within autoimmune loci. We found pervasive dynamic regulatory effects within six HLA genes. HLA-DQB1 alleles had one of three distinct transcriptional regulatory programs. Using CRISPR/Cas9 genomic editing we demonstrated that a promoter variant is causal for T cell-specific control of HLA-DQB1 expression. Our study shows that genetic variation in cis regulatory elements affects gene expression in a lymphocyte activation status-dependent manner contributing to the inter-individual complexity of immune responses.

Morphological and stage-specific transcriptome analyses reveal distinct regulatory programs underlying yam (Dioscorea alata L.) bulbil growth.

In yam (Dioscorea spp) species, bulbils at leaf axils are the most striking species-specific axillary structure and exhibit important ecological niches. Genetic regulation underlying bulbil growth remains largely unclear so far. Here, we characterize yam (Dioscorea alata L.) bulbil development using histological analysis, and perform full transcriptional profiling on key developmental stages together with phytohormone analyses. Using the stage-specific scoring algorithm, we have identified 3451 stage-specifically expressed genes that exhibit a tight link between major transcriptional changes and stages. Co-expressed gene clusters revealed an obvious over-representation of genes associated with cell division and expansion at the initiation stage of bulbils (T1). Transcriptional changes of hormone-related genes highly coincided with hormone levels, indicating that bulbil initiation and growth are coordinately controlled by multiple phytohormones. In particular, localized auxin is transiently required to trigger bulbil initiation, and be further depleted or exported from bulbils to promote growth by up-regulation of genes involved in auxinconjugation and efflux. The sharp increase in supply of sucrose and an enhanced trehalose-6-phophate pathway at T1 were observed, suggesting that sucrose probably functions as a key signal and promotes bulbil initiation. Analysis of the expression of transcription factors (TFs) predicated 149 TFs as stage-specifically expressed; several T1-specific TFs (from Aux/IAA, E2F, MYB, and bHLH families) have been shown to play key roles in triggering bulbil formation. Together, our work provides a crucial angle for in-depth understanding of the molecular programs underlying yam’s unique bulbil development processes. Stage-specific gene sets can be queried to obtain key candidates regulating bulbil growth, serving as valuable resources for further functional research.

CIC-DUX4 oncoprotein drives sarcoma metastasis and tumorigenesis via distinct regulatory programs.

Transcription factor fusion genes create oncoproteins that drive oncogenesis and represent challenging therapeutic targets. Understanding the molecular targets by which such fusion oncoproteins promote malignancy offers an approach to develop rational treatment strategies to improve clinical outcomes. Capicua-double homeobox 4 (CIC-DUX4) is a transcription factor fusion oncoprotein that defines certain undifferentiated round cell sarcomas with high metastatic propensity and poor clinical outcomes. The molecular targets regulated by the CIC-DUX4 oncoprotein that promote this aggressive malignancy remain largely unknown. We demonstrated that increased expression of ETS variant 4 (ETV4) and cyclin E1 (CCNE1) occurs via neomorphic, direct effects of CIC-DUX4 and drives tumor metastasis and survival, respectively. We uncovered a molecular dependence on the CCNE-CDK2 cell cycle complex that renders CIC-DUX4-expressing tumors sensitive to inhibition of the CCNE-CDK2 complex, suggesting a therapeutic strategy for CIC-DUX4-expressing tumors. Our findings highlight a paradigm of functional diversification of transcriptional repertoires controlled by a genetically aberrant transcriptional regulator, with therapeutic implications.

Comprehensive analysis of the longan transcriptome reveals distinct regulatory programs during the floral transition

BackgroundLongan (Dimocarpus longan Lour.) is an important fruit tree in the subtropical regions of Southeast Asia and Australia. Among the factors affecting D. longan fruit yield, the difficulty and instability of blossoming is one of the most challenging issues. Perpetual flowering (PF) is a crucial trait for fruit trees and is directly linked to production potential. Therefore, studying the molecular regulatory mechanism of longan PF traits is crucial for understanding and solving problems related to flowering. In this study, comparative transcriptome analysis was performed using two longan cultivars that display opposite flowering phenotypes during floral induction.ResultsWe obtained 853.72 M clean reads comprising 125.08 Gb. After comparing these data with the longan genome, 27,266 known genes and 1913 new genes were detected. Significant differences in gene expression were observed between the two genotypes, with 6150 and 6202 differentially expressed genes (DEGs) for ‘SJ’ and ‘SX’, respectively. The transcriptional landscape of floral transition at the early stage was very different in these two longan genotypes with respect to key hormones, circadian rhythm, sugar metabolism, and transcription factors. Almost all flowering-related DEGs identified are involved in photoperiod and circadian clock pathways, such as CONSTANS-like (COL), two-component response regulator-like (APRRs), gigantea (GI), and early flowering (EFL). In addition, the leafy (LFY) gene, which is the central floral meristem identity gene, may inhibit PF formation in ‘SJ’.ConclusionThis study provides a platform for understanding the molecular mechanisms responsible for changes between PF and seasonal flowering (SF) longan genotypes and may benefit studies on PF trait mechanisms of evergreen fruit trees.

Breaking Away: The Role of Homeostatic Drive in Perpetuating Depression.

We propose that the complexity of regulatory interactions modulating brain neurochemistry and behavior is such that multiple stable responses may be supported, and that some of these alternate regulatory programs may play a role in perpetuating persistent psychological dysfunction. To explore this, we constructed a model network representing major neurotransmission and behavioral mechanisms reported in literature as discrete logic circuits. Connectivity and information flow through this biobehavioral circuitry supported two distinct and stable regulatory programs. One such program perpetuated a depressive state with a characteristic neurochemical signature including low serotonin. Further analysis suggested that small irregularities in glutamate levels may render this pathology more directly accessible. Computer simulations mimicking selective serotonin reuptake inhibitor (SSRI) therapy in the presence of everyday stressors predicted recidivism rates similar to those reported clinically and highlighted the potentially significant benefit of concurrent behavioral stress management therapy.

Comprehensive transcriptome analysis reveals distinct regulatory programs during vernalization and floral bud development of orchardgrass (Dactylis glomerata L.)

BackgroundVernalization and the transition from vegetative to reproductive growth involve multiple pathways, vital for controlling floral organ formation and flowering time. However, little transcription information is available about the mechanisms behind environmental adaption and growth regulation. Here, we used high-throughput sequencing to analyze the comprehensive transcriptome of Dactylis glomerata L. during six different growth periods.ResultsDuring vernalization, 4689 differentially expressed genes (DEGs) significantly increased in abundance, while 3841 decreased. Furthermore, 12,967 DEGs were identified during booting stage and flowering stage, including 7750 up-regulated and 5219 down-regulated DEGs. Pathway analysis indicated that transcripts related to circadian rhythm, photoperiod, photosynthesis, flavonoid biosynthesis, starch, and sucrose metabolism changed significantly at different stages. Coexpression and weighted correlation network analysis (WGCNA) analysis linked different stages to transcriptional changes and provided evidence of inner relation modules associated with signal transduction, stress responses, cell division, and hormonal transport.ConclusionsWe found enrichment in transcription factors (TFs) related to WRKY, NAC, AP2/EREBP, AUX/IAA, MADS-BOX, ABI3/VP1, bHLH, and the CCAAT family during vernalization and floral bud development. TFs expression patterns revealed intricate temporal variations, suggesting relatively separate regulatory programs of TF modules. Further study will unlock insights into the ability of the circadian rhythm and photoperiod to regulate vernalization and flowering time in perennial grass.

Understanding Tissue-Specific Gene Regulation

SummaryAlthough all human tissues carry out common processes, tissues are distinguished by gene expression patterns, implying that distinct regulatory programs control tissue specificity. In this study, we investigate gene expression and regulation across 38 tissues profiled in the Genotype-Tissue Expression project. We find that network edges (transcription factor to target gene connections) have higher tissue specificity than network nodes (genes) and that regulating nodes (transcription factors) are less likely to be expressed in a tissue-specific manner as compared to their targets (genes). Gene set enrichment analysis of network targeting also indicates that the regulation of tissue-specific function is largely independent of transcription factor expression. In addition, tissue-specific genes are not highly targeted in their corresponding tissue network. However, they do assume bottleneck positions due to variability in transcription factor targeting and the influence of non-canonical regulatory interactions. These results suggest that tissue specificity is driven by context-dependent regulatory paths, providing transcriptional control of tissue-specific processes.

Gene-regulatory interactions in embryonic stem cells represent cell-type specific gene regulatory programs.

Pluripotency, the ability of embryonic stem cells to differentiate into specialized cell types, is determined by ESC-specific gene regulators such as transcription factors and chromatin modification factors. It is not well understood how ESCs are poised for differentiation, however, and methods are needed for prognosis of the molecular changes in the differentiation of ESCs into specific organs. We describe a new approach to infer cell-type specific gene regulatory programs based on gene regulatory interactions in ESCs. Our method infers the molecular logic of gene regulatory mechanisms by mapping the position-specific combinatory patterns of numerous regulators in ESCs into cell-type specific gene regulations. We validate the proposed approach by recapitulating the RNA-seq and microarray data of neuronal progenitor cells, adult liver cells, and ESCs from the integrated patterns of diverse gene regulators in ESCs. We find that the collective functions of diverse gene regulators in ESCs represent distinct gene regulatory programs in specialized cell types. Our new approach expands our understanding of the differential gene regulatory information in developments encoded in regulatory networks of ESCs.

Genome-Wide Binding of Posterior HOXA/D Transcription Factors Reveals Subgrouping and Association with CTCF

Homeotic genes code for key transcription factors (HOX-TFs) that pattern the animal body plan. During embryonic development, Hox genes are expressed in overlapping patterns and function in a partially redundant manner. In vitro biochemical screens probing the HOX-TF sequence specificity revealed largely overlapping sequence preferences, indicating that co-factors might modulate the biological function of HOX-TFs. However, due to their overlapping expression pattern, high protein homology, and insufficiently specific antibodies, little is known about their genome-wide binding preferences. In order to overcome this problem, we virally expressed tagged versions of limb-expressed posterior HOX genes (HOXA9-13, and HOXD9-13) in primary chicken mesenchymal limb progenitor cells (micromass). We determined the effect of each HOX-TF on cellular differentiation (chondrogenesis) and gene expression and found that groups of HOX-TFs induce distinct regulatory programs. We used ChIP-seq to determine their individual genome-wide binding profiles and identified between 12,721 and 28,572 binding sites for each of the nine HOX-TFs. Principal Component Analysis (PCA) of binding profiles revealed that the HOX-TFs are clustered in two subgroups (Group 1: HOXA/D9, HOXA/D10, HOXD12, and HOXA13 and Group 2: HOXA/D11 and HOXD13), which are characterized by differences in their sequence specificity and by the presence of cofactor motifs. Specifically, we identified CTCF binding sites in Group 1, indicating that this subgroup of HOX-proteins cooperates with CTCF. We confirmed this interaction by an independent biological assay (Proximity Ligation Assay) and demonstrated that CTCF is a novel HOX cofactor that specifically associates with Group 1 HOX-TFs, pointing towards a possible interplay between HOX-TFs and chromatin architecture.

Massively Parallel Single Cell RNA-Seq of Primary Lymphomas Reveals Distinct Cellular Lineages and Diverse, Intratumoral Transcriptional States

Follicular lymphoma (FL) is a generally incurable B-cell malignancy which has the potential to transform into highly aggressive lymphomas. Genomic studies indicate it is often a small subpopulation rather than the dominant population in the FL that gives rise to the more aggressive subtype.To resolve the underlying transcriptional networks of follicular B-cell lymphomas at single molecule and cell resolution, we leveraged droplet-based barcoding technology for highly parallel single cell RNA-Seq. We analyzed the transcriptomes from tens of thousands of cells derived from five primary FL tumors (average > 5,000 cells/sample). Simultaneously, we conducted multi-dimensional flow cell sorting to validate our characterizing of cellular lineages and critical expressed proteins. For each tumor, we identified multiple cellular subpopulations, matching known hematopoietic lineages. Despite some common features, such as MYC and BCLoverexpression, distinct transcriptional patterns and regulatory programs were evident among the different tumors. Within each tumor, subpopulations of malignant cell transcripts were compared to matched normal B-cells (Figure 1).Transcriptome analysis of >13,500 B-cells across 5 samples reveals sample-specific and joint clusters of B-cells. Each dot represents the t-SNE projection of a single cell’s transcriptome profile. Single cells are colored according to sample origin (control PBMC samples 1 & 2; follicular lymphoma samples 1, 2 & 3). Normal B-cells within follicular lymphoma samples cluster together with PBMC derived B-cells. Malignant B-cells cluster according to sample origin, except for the cycling tumor cells (upper left cell cluster).Malignant B-cells were characterized by expression of restricted immunoglobulin light chain type (either kappa or lambda), BCL2 expression, and CD20 expression. We show evidence for the coexistence of small, malignant subpopulations alongside the dominant FL population in the majority of tumors. These smaller subpopulations harbored several transcriptional changes that were absent from the dominant population, including upregulation of MHC class II expression, downregulation of β-2-microglobulin, and exclusive expression of chemokines (CCL22 and CCL17), that would have resulted in alteration in antigen presentation and of the T cell immune system. In addition, we characterize the transcriptomes of the infiltrating immune cell populations that are characteristic of this disease. Our findings provide an unprecedented resolution of distinct immune lineages as seen by transcriptionally characterized cellular diversity. [Display omitted] DisclosuresLevy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

First trimester infection with Listeria monocytogenes is characterized by decidual and placental inflammation in pregnant cynomolgus macaques

First trimester infection with Listeria monocytogenes is characterized by decidual and placental inflammation in pregnant cynomolgus macaques

Distinct gene regulatory programs define the inhibitory effects of liver X receptors and PPARG on cancer cell proliferation.

BackgroundThe liver X receptors (LXRs, NR1H2 and NR1H3) and peroxisome proliferator-activated receptor gamma (PPARG, NR1C3) nuclear receptor transcription factors (TFs) are master regulators of energy homeostasis. Intriguingly, recent studies suggest that these metabolic regulators also impact tumor cell proliferation. However, a comprehensive temporal molecular characterization of the LXR and PPARG gene regulatory responses in tumor cells is still lacking.MethodsTo better define the underlying molecular processes governing the genetic control of cellular growth in response to extracellular metabolic signals, we performed a comprehensive, genome-wide characterization of the temporal regulatory cascades mediated by LXR and PPARG signaling in HT29 colorectal cancer cells. For this analysis, we applied a multi-tiered approach that incorporated cellular phenotypic assays, gene expression profiles, chromatin state dynamics, and nuclear receptor binding patterns.ResultsOur results illustrate that the activation of both nuclear receptors inhibited cell proliferation and further decreased glutathione levels, consistent with increased cellular oxidative stress. Despite a common metabolic reprogramming, the gene regulatory network programs initiated by these nuclear receptors were widely distinct. PPARG generated a rapid and short-term response while maintaining a gene activator role. By contrast, LXR signaling was prolonged, with initial, predominantly activating functions that transitioned to repressive gene regulatory activities at late time points.ConclusionsThrough the use of a multi-tiered strategy that integrated various genomic datasets, our data illustrate that distinct gene regulatory programs elicit common phenotypic effects, highlighting the complexity of the genome. These results further provide a detailed molecular map of metabolic reprogramming in cancer cells through LXR and PPARG activation. As ligand-inducible TFs, these nuclear receptors can potentially serve as attractive therapeutic targets for the treatment of various cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0328-6) contains supplementary material, which is available to authorized users.

O-004 Analysis of Chromatin and Transcriptional Profiles in Crohnʼs Disease Reveals Molecular Subclasses and Highlights Functional Regulatory Regions Implicated in Disease

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract, characterized by an inappropriate immune response to the enteric microbiota. Chromatin based assays have successfully identified cell type and disease-specific regions of DNA accessibility, and have been linked to differential transcription of target genes, but this approach has yet to be explored in CD. In this study we performed formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) to identify nucleosome-depleted regions associated with regulatory elements, and RNA-seq to quantify expression, to characterize disease-relevant changes in chromatin and establish links to putative target genes.Mucosal Biopsies for 21 CD and 12 non-IBD individuals were taken from macroscopically uninflamed portions of the ascending colon at time of surgery. We performed FAIRE-seq and RNA-seq on each tissue sample. Reads were aligned to personalized genomes constructed with genotypes assayed on the Illumina Immunochip platform. A genome-wide scan for differential regions and genes was performed using 2 sided t-tests on normalized read counts in 300bp regions (FAIRE-seq) and RPKM (RNA-seq).RNA-seq data analysis across all samples identified 2 distinct classes of patients associated with epithelial cell lipid transport (class I, n = 10, CD; n = 1, non-IBD) and maintenance of cellular and luminal pH (class II, n = 11, CD, n = 11, non-IBD). To unravel potential gene regulatory mechanisms we analyzed chromatin profiles using FAIRE-seq and histone annotations using tissue specific ChIP-seq data sets. Class I patients showed increased accessibility at distal sites marked for enhancer activity in small intestine, and reduced accessibility at sites marked as enhancers in colon, suggesting 2 distinct regulatory programs driving the expression of differential genes. This pattern was reversed in class II samples. In an analysis restricting to class II, which was characterized by normal colon enhancer activity, we found regulatory regions specific to CD and non-IBD, respectively, as well as 51 and 507 genes upregulated in the respective cohorts. Top ontologies of genes upregulated in CD included host response to bacteria and immune response. Differential regulatory genes were enriched near TSSs of differentially expressed genes, implying putative causal effect. In an integrative analysis, we identified 72 regulatory regions that associate with both disease status and expression of nearby genes. A retrospective analysis of patients in class I and II revealed that the majority of patients (10/11) in class I required post-operative anti-TNF therapy for disease recurrence compared to only 1/11 CD patients in the class II group.We used chromatin status as a novel marker of disease in Crohn's. We identified a subset of CD patients that are characterized at the chromatin level in the colon by enrichment of enhancer marks specific to small intestine, and loss of enhancer activity specific to colon. Among individuals with enhancer activity indicative of normal colon, we integrated data from well-described transcriptional markers with novel chromatin variables to identify molecular associations and interactions that affect disease predisposition. Despite small patient numbers we were able to use chromatin profiles to identify patients that required post-operative management with anti-TNF agents.

Comprehensive Tissue-Specific Transcriptome Analysis Reveals Distinct Regulatory Programs during Early Tomato Fruit Development.

Fruit formation and early development involve a range of physiological and morphological transformations of the various constituent tissues of the ovary. These developmental changes vary considerably according to tissue type, but molecular analyses at an organ-wide level inevitably obscure many tissue-specific phenomena. We used laser-capture microdissection coupled to high-throughput RNA sequencing to analyze the transcriptome of ovaries and fruit tissues of the wild tomato species Solanum pimpinellifolium. This laser-capture microdissection-high-throughput RNA sequencing approach allowed quantitative global profiling of gene expression at previously unobtainable levels of spatial resolution, revealing numerous contrasting transcriptome profiles and uncovering rare and cell type-specific transcripts. Coexpressed gene clusters linked specific tissues and stages to major transcriptional changes underlying the ovary-to-fruit transition and provided evidence of regulatory modules related to cell division, photosynthesis, and auxin transport in internal fruit tissues, together with parallel specialization of the pericarp transcriptome in stress responses and secondary metabolism. Analysis of transcription factor expression and regulatory motifs indicated putative gene regulatory modules that may regulate the development of different tissues and hormonal processes. Major alterations in the expression of hormone metabolic and signaling components illustrate the complex hormonal control underpinning fruit formation, with intricate spatiotemporal variations suggesting separate regulatory programs.