Transcription Factor Motif Enrichment Research Articles

Abstract C056: Characterization of chromatin accessibility patterns in the human pancreas and early pancreatic neoplastic lesions using snATAC-seq

Abstract Study of early pancreas neoplasia in humans had previously been limited by lack of available tissue. Recent work by our group on deceased donor pancreata identified pancreatic intraepithelial neoplasia (PanIN) lesions in non-diseased organs and defined a transcriptomic signature for the microenvironment of these pre-cancerous lesions. Prevalence of PanINs in healthy human tissue was higher than expected and established a novel model to expand understanding of the complex biology of these precursor lesions. Epigenetic changes in chromatin structure and the subsequent effects on gene expression are essential processes in neoplasia that have yet to be described at the single cell level in the human pancreas. We hypothesized that previously defined gene signatures could identify acinar, ductal, and PanIN cells in donor pancreata based on chromatin accessibility profiling by single nuclear transposase-accessible chromatin preparation followed by high-throughput sequencing (snATAC-seq). Comparison between the accessibility patterns, transcription factor motifs, and differences in accessibility versus expression profiles can then be used to expand prior knowledge of early pancreatic neoplasia and identify novel targets for further study and therapy selection. To test this hypothesis, single nuclei from seventeen donor pancreata (twenty total samples) were isolated for snATAC-seq using the 10x Genomics platform. Data analysis was conducted using CellRanger, Seurat, Signac, AUCell, and other programs to identify and label chromatin peaks in individual nuclei corresponding to acinar, ductal, and PanIN origin according to gene signatures identified by scRNA-seq and special transcriptomics on matched donor pancreata. Using this method, cells corresponding to these gene signatures, including PanIN- like cells, were indeed identifiable with differentially accessible regions of the genome then analyzed for transcription factor motif enrichment between these cell types. Motifs enriched in cells identified as PanINs compared to either ductal or acinar cells included several transcription factors implicated in tumorigenesis. These results are similar to previously demonstrated essential factors in pancreatic neoplasia, suggesting this strategy is a relevant method for data analysis and discovery of novel factors implicated in tumorigenesis. Ongoing studies include validation of these findings in vitro using human pancreatic normal epithelial cells, human tumor cell lines, and organoid models derived from human normal and tumor samples. Together, this study demonstrates that PanINs can be identified in human pancreas tissue by snATAC-seq and that differentially expressed motifs can be identified, providing opportunity for further integration with transcriptomic information to elucidate detailed understanding of early neoplasia in the pancreas for future drug development and targeted therapeutics studies. Citation Format: Jamie N Mills, Aaron Dendekker, Joyce K Thompson, Hannah Watkoske, Simone Benitz, Padma Kadiyala, Ahmed Elhossiny, Howard Crawford, Eileen Carpenter, Marina Pasca di Magliano, Filip Bednar. Characterization of chromatin accessibility patterns in the human pancreas and early pancreatic neoplastic lesions using snATAC-seq [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C056.

TGFβ macrophage reprogramming: a new dimension of macrophage plasticity.

The August 2023 article in Science Signaling, "TGF-β uncouples glycolysis and inflammation in macrophages and controls survival during sepsis," challenges the traditional M1/M2 macrophage classification by investigating the impact of transforming growth factor β on macrophage metabolism and function. Despite its conventional anti-inflammatory role, transforming growth factor β-treated macrophages exhibit a distinct phenotype marked by heightened glycolysis, suppressed proinflammatory cytokines, and increased coagulation factor expression. The study identifies phosphofructokinase, liver type as a crucial glycolytic enzyme regulated by transforming growth factor β via the mTOR-c-MYC pathway. Epigenetic changes induced by transforming growth factor β, such as increased Smad3 activation and reduced proinflammatory transcription factor motif enrichment, contribute to the anti-inflammatory profile. The research extends its implications to sepsis, revealing the role of transforming growth factor β in exacerbating coagulation and reducing survival in mouse models. This effect involves upregulation of coagulation factor F13A1, dependent on phosphofructokinase, liver type activity and glycolysis in macrophages. Connections to COVID-19 pathology are drawn, as transforming growth factor β-treated macrophages and SARS-CoV-2 E protein-exposed cells display similar metabolic profiles. Bioinformatic analysis of COVID-19 patient data suggests correlations between myeloid expression of TGFβR1, PFKL, and F13A1 with disease severity. The study challenges the M1/M2 classification, emphasizing the complexity of macrophage responses influenced by transforming growth factor β, proposing transforming growth factor β as a potential therapeutic target for conditions like sepsis and COVID-19 where dysregulated coagulation is significant. Overall, the research provides valuable insights into transforming growth factor β-mediated immunometabolic regulation, paving the way for future investigations and potential therapeutic interventions.

DNA Methylation-Based Classification of Hairy Cell Leukemia and Splenic B Cell Lymphoma

Improved classification of rare lymphoid neoplasms would be aided by a deeper understanding of their underlying molecular features and is important for diagnosis, prognosis and therapy. Tumor entities classified within the WHO category of splenic B cell lymphomas and leukemias often exhibit heterogenous, transecting features, and include hairy cell leukemia (HCL), splenic diffuse red pulp lymphoma (SDRPL), splenic marginal zone lymphoma (SMZL), and the newly described WHO entity, splenic B cell lymphoma/leukemia with prominent nucleoli (SBLPN); the latter including patients formerly classified as HCL-variant (HCL-V). Genome-wide epigenetic information provides a tumor cell fingerprint combining cell-of-origin and tumor-specific events. Here we used DNA methylation to perform an unbiased molecular subclassification and to explore novel biological aspects of these patients. Samples from patients with a pathological diagnosis of HCL, HCL-V, SDRPL and SMZL (made prior to the 5 th WHO revision and ICC classifications) were obtained from 19 institutions across 9 countries, totaling 367 patients. Cells were FACS-purified where necessary and DNA was analyzed by 450/850K Illumina DNA methylation arrays. Genetic mutations were assessed by whole-exome or targeted sequencing, IGHV-D-J sequences by Sanger sequencing, and copy number alterations (CNAs) by Illumina arrays. The 1000 most variable CpG methylation sites were used for k-means clustering. Recursive feature elimination/random forest algorithms were used to develop a classifier for DNA methylation-based subgroups with 98% accuracy. Unsupervised clustering of 197 patients diagnosed with HCL, HCL-V or SDRPL revealed 5 distinct DNA methylation (M) subgroups ( Figure 1). Subgroup assignment was stable throughout longitudinal sampling (including pre/post-treatment) and consistent between splenic, bone marrow and PBMC derived cells. A subgroup with universally clonal BRAF-V600E mutations and majority diagnosed as HCL was termed the M-HCL subgroup ( Table 1). Four other groups termed M-SBLPN1-4 contained all HCL-V and SDRPL diagnosed samples and were devoid of BRAF-V600E mutations. M-SBLPN1 comprised MAP2K1 mutations (91%) and was enriched for CREBBP, ARIDIA and TERT-promoter mutations. These patients displayed an HCL-like immunophenotype (64.3% CD25+) with 1/3 diagnosed as HCL. M-SBLPN2 exhibited the highest prevalence of TP53 mutations and concomitant genomic instability. Patients in M-SBLPN1,2 were enriched in unmutated IGHV4-34 rearrangements. M-SBLPN3,4 subgroups displayed an immunophenotype more dissimilar to HCL, mutated IGHV genes, and enrichment of IGLL5, SYK and BIRC3 mutations. M-SBLPN4 contained the most SDRPL samples, suggesting it may represent the SDRPL entity retained by the WHO. We next uncovered that 29/170 SMZL patients displayed DNA methylation patterns mapping to M-SBLPN2-4. These patients were phenotypically and molecularly similar to SBLPN (70% displaying villous morphology and depleted in IGHV1-2*04, NOTCH2, KLF2 mutations), likely representing SMZL patients suggested for reassignment to SBLPN in the updated WHO classification. To elucidate molecular pathways governing the biology of M-SBLPN subgroups, transcription factor motif enrichment analysis in hypomethylated genomic regions revealed selective activation of AP-1 in M-HCL along with ETS in M-SBLPN1,2. Both transcription factors are downstream of MAPK signaling, consistent with activating BRAF and MAP2K1 mutations in these subgroups. However, we observed strong ETS enrichment in the absence of MAP2K1 in M-SBLPN along with mutual exclusivity of MAP2K1 and TP53 mutations, suggesting TP53 mutations are driving ETS activation. Although lymphoid neoplasms rarely exhibit TERT promoter mutations, 83% of M-SBLPN1 patients showed the c.-124C>T mutation commonly observed in other cancers producing an ETS binding site and ectopic TERT activation. ETS activation and gain of an ETS site by mutation implies oncogenesis involves aberrant TERT activation in this subgroup. In summary, we have developed a DNA methylation-based classifier that resolves 4 SBLPN subgroups with distinct molecular features, and reclassifies a subset of SMZL and HCL patients, adding further information to the updated WHO/ICC entities. We reveal distinct biological pathways operating in M-SBLPN subgroups that may aid targeted therapy approaches.

Systematic benchmarking of single-cell ATAC-sequencing protocols

Single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) has emerged as a powerful tool for dissecting regulatory landscapes and cellular heterogeneity. However, an exploration of systemic biases among scATAC-seq technologies has remained absent. In this study, we benchmark the performance of eight scATAC-seq methods across 47 experiments using human peripheral blood mononuclear cells (PBMCs) as a reference sample and develop PUMATAC, a universal preprocessing pipeline, to handle the various sequencing data formats. Our analyses reveal significant differences in sequencing library complexity and tagmentation specificity, which impact cell-type annotation, genotype demultiplexing, peak calling, differential region accessibility and transcription factor motif enrichment. Our findings underscore the importance of sample extraction, method selection, data processing and total cost of experiments, offering valuable guidance for future research. Finally, our data and analysis pipeline encompasses 169,000 PBMC scATAC-seq profiles and a best practices code repository for scATAC-seq data analysis, which are freely available to extend this benchmarking effort to future protocols.

Abstract A062: Alternative promoter usage is linked to transcriptional and epigenetic alterations during prostate cancer progression

Abstract Background: Prostate tumor progression is driven by transcription factors (TFs), most prominently the Androgen Receptor (AR), that drive proliferation and therapy resistance. Many genes have several promoters that can be bound by distinct TFs. It is unclear what role alternative promoter selection plays in tumor progression. We hypothesized that driver TFs target novel gene promoters during prostate tumor progression, and that understanding how this targeting occurs would reveal novel aspects of prostate tumor biology. Methods: We performed ultra-deep RNA sequencing on 104 metastatic Castration Resistant Prostate Cancer (mCRPC) biopsies, generating > 450M reads/sample (10X the typical depth), in samples with matched whole genome DNA and methylation sequencing. We combined these data with published deep transcriptomes in normal prostate and localized prostate cancer for a total of 274 samples. Promoter activity was evaluated in all 274 biopsies using the proActiv tool. Differential promoter activity levels were integrated with transcription factor motif enrichment, chromatin immunoprecipitation sequencing, and previously published somatic and methylation sequencing data in the mCRPC samples. Results: Using normal prostate tissue as a baseline, we identified 463 and 3,237 APs with differential activity in localized prostate cancer and mCRPC, respectively. Increased alternative promoter (AP) usage was associated with upregulation of prostate tumor driver genes. Elevated androgen signaling in both localized and mCRPC was correlated with increased AP use. In localized prostate tumors, APs preferentially switch to promoters harboring binding sites for the AR pioneer factor FOXA1. In mCPRC, APs were further enriched for binding of MYC, E2F1, and HIF1A. Furthermore, AP usage reflects transcriptional dysregulation related to lineage plasticity in response to therapy. APs in neuroendocrine prostate cancer (NEPC) were bound by neuroendocrine TFs such as ASCL1 and HAND2. While canonical promoters are usually unmethylated, methylation levels at APs were strongly linked to AP activity. Conclusions: Our data show that transcription initiated at APs is a key contributor to increased gene activity in prostate tumors. APs that are upregulated in prostate tumors are linked to transcription factors activated by recurrent somatic alterations and lineage plasticity. Methylation at APs is an important and underappreciated driver of promoter choice in mCRPC. Understanding how somatic alterations and epigenetic reprogramming of TFs affect APs is essential to understanding how these processes impact therapy resistance. Citation Format: Meng Zhang, Martin Sjöström, Raunak Shrestha, Arian Lundberg, Thaidy Moreno-Rodriguez, Adam Foye, Ha X. Dang, Joshi J. Alumkal, Rahul Aggarwal, Eric J. Small, Christopher A. Maher, Felix Y. Feng, David Quigley. Alternative promoter usage is linked to transcriptional and epigenetic alterations during prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A062.

Injury-induced hair growth response is promoted by tissue macrophages via insulin-like growth factor 1

Abstract Hair follicles (HFs) produce hair to mediate the first line of defense in mammals. HFs undergo cyclical production of hair (anagen), and while this process is unsynchronized during adulthood, external cues, including shaving, induce synchronized anagen and bolster the surface barrier. Herein, we show that macrophages were crucial in promoting anagen. Shaving triggered the accumulation of macrophages to the secondary hair germs (SHG), which underwent proliferation as early as Day3 post-induction. Constitutive ablation of macrophages markedly delayed anagen, and targeted ablation of macrophages at Day 3, but not at Day 0 or Day 6 post-shaving, impaired SHG activation with delayed anagen. Anagen entry was dependent on Folr2 +tissue macrophages, but not on CCR2 +monocyte-derived macrophages. Single-cell RNA-sequencing and cell-cell communication analyses revealed that Folr2 +macrophages communicated with the SHGs via Igf1 (encoding insulin-like growth factor 1 or IGF1). Ablation of macrophages and deletion of IGF1 from macrophages decreased AKT signaling in SHG and led to delayed anagen, both which were rescued via injection of recombinant IGF1 in vivo. Single-cell ATAC-sequencing revealed that the lack of IGF1-from macrophages resulted in alteration of transcription factor motif enrichment and decreased chromatin accessibility in various genes in SHGs, demonstrating that macrophages primed the SHGs to enter anagen by promoting IGF1R signaling. These results highlight the importance of immune-tissue crosstalk in facilitating hair growth and provide impetus for strategies to promote hair growth.

A pituitary gene network linking vgll3 to regulators of sexual maturation in male Atlantic salmon

Age at maturity is a key life history trait and a significant contributor to life history strategy variation. The maturation process is influenced by genetic and environmental factors, but specific causes of variation in maturation timing remain elusive. In many species, the increase in the regulatory gonadotropin-releasing hormone 1 (GnRH1) marks the onset of puberty. Atlantic salmon, however, lacks the gnrh1 gene, suggesting gnrh3 and/or other regulatory factors are involved in the maturation process. Earlier research in Atlantic salmon has found a strong association between alternative alleles of vgll3 and maturation timing. Recently we reported strong induction of gonadotropin genes (fshb and lhb) in the pituitary of Atlantic salmon homozygous for the early maturation allele (E) of vgll3. The induction of gonadotropins was accompanied by increased expression of their direct upstream regulators, c-jun and sf1 (nr5a1b) but the regulatory connection between vgll3 and these regulators has never been investigated in any organism. In this study, we investigated the potential regulatory connection between vgll3 genotypes and these regulators through a stepwise approach of identifying a gene regulatory network (GRN) containing c-jun and sf1, and transcription factor motif enrichment analysis. We found a GRN containing c-jun with predicted upstream regulators, e2f1, egr1, foxj1 and klf4, to be differentially expressed in the pituitary. Finally, we suggest a vgll3 and Hippo pathway -dependent model for transcriptional regulation of c-jun and sf1 in the pituitary, which may have broader implications across vertebrates.

3D genome alterations in T cells associated with disease activity of systemic lupus erythematosus

ObjectivesThree-dimensional (3D) genome alterations can dysregulate gene expression by rewiring physical interactions within chromosomes in a tissue-specific or cell-specific manner and lead to diseases. We aimed to elucidate the 3D...

Single‐cell multi‐omics analysis identifies dynamic regulation of SREBF1 in Alzheimer’s disease

The gene-regulatory landscape of the brain is highly dynamic in health and disease, coordinating a menagerie of biological processes across distinct cell-types. Understanding these regulatory programs requires a holistic experimental and analytical approach, especially in understanding neurodegenerative disorders such as Alzheimer's Disease (AD).In this study, we used single-nucleus ATAC-seq (snATAC-seq) to profile the epigenome in all major cell types of the human brain in AD, and we single-nucleus RNA-seq (snRNA-seq) in the same biological samples, together painting a more complete portrait of the changes brought on by disease. We constructed an oligodendrocyte trajectory using 58,221 single-nuclei chromatin profiles and 36,773 single-nuclei transcriptomes. We developed a new analysis approach, scWGCNA, a method for interrogation of cell population-specific co-expression networks.We investigated the dynamics of transcription factor (TF) binding motifs throughout the oligodendrocyte trajectory, and evaluated chromatin accessibility, TF motif enrichment, gene expression, and cis-regulatory elements to identify SREBF1, a master regulator of cholesterol biosynthesis and myelination, downregulated in late-stage AD in oligodendrocytes. We extend our analysis of SREBF1 in oligodendrocytes by examining its downstream regulatory targets in the context of gene co-expression networks. Notably, we found several oligodendrocyte modules significantly enriched for targets of SREBF1, indicating the importance of SREBF1 in regulating gene expression in these modules. Using a multi-scale dataset of bulk-tissue RNA-seq, high-throughput proteomics, and SREBF1 ChIP-seq data (ENCODE), we defined a protein-protein interaction (PPI) network of SREBF1 target genes. Additionally, we found module eigengene expression of SREBF1 targets downregulated in early- and late-pathology AD cases at the level of proteins and RNA, corroborated by downregulation of SREBF1 activity in snATAC-seq data.Altogether, our data and analyses highlight key biological role of SREBF1 in AD biology. While the causative molecular mechanisms of sporadic AD remain unknown, our work is an important stepping stone in fully unraveling the nature of gene regulation in AD, especially in regards to genomic loci with well-described heritable disease risk. Additional work is needed to spatially resolve the complexity of single-cell gene expression and epigenomics in AD and neurodegeneration, in general.

ECOA-7. Conserved cell-lineage controlled chromatin accessibility in human and mouse glioblastoma stem cells predicts functionally distinct subgroups

Abstract There is ample support for developmental control of glioblastoma stem cells (GSCs), and a deeper knowledge of their epigenetic regulation could be central to more efficient glioblastoma (GBM) therapies. For this purpose, we analyzed the chromatin-accessibility landscape of nine mouse GSC cultures of defined cell of origin and 60 patient-derived GSC cultures by assay for transposase-accessible chromatin using sequencing (ATAC-seq). This uncovered an epigenetic variability of both mouse and human GSC cultures that differed from transcriptome clusters. Both mouse and human chromatin accessibility-guided clusters were predominantly determined by distal regulatory elements, displayed unique sets of transcription factor motif enrichment, and exhibited different functional and drug-response properties. Cross-species analysis of distal regulatory element regions in accessible chromatin of mouse and human cultures revealed conserved epigenetic regulation of GSCs.

Abstract LB206: Simultaneous single cell profiling of open chromatin and gene expression in B cell lymphoma highlights tumor-specific regulatory networks

Abstract Profiling multiple analytes from a single cell enables additional dimensions to characterize tumor biology. Simultaneous measurement of epigenetic information and gene expression in single cells provides a clearer readout of the links between regulatory elements and target genes, allowing a more mechanistic interpretation of cell clusters and better cell type distinctions based on a cell's history and its future potential. Here, we describe simultaneous measurement of gene expression and chromatin accessibility from thousands of single nuclei using the Chromium Single Cell Multiome ATAC + Gene Expression. We leverage a microfluidic platform to generate paired barcoded gene expression and ATAC-seq libraries, linking transcriptomic and open chromatin information at the single cell level. Using this method, we jointly profiled gene expression and chromatin accessibility from over 10,000 immune cells from healthy human PBMCs and over 14,000 cells from a human B cell lymphoma sample. In healthy immune cells, we leveraged chromatin information to better parse distinct cell states within a gene expression-defined cell type, showing that the addition of ATAC profiling to gene expression improves resolution of cell type specification. In B cell lymphoma, we used known markers of malignancy to segregate tumor B cells from normal B cells. We constructed regulatory networks in both cell states using the observed transcription factor motif enrichment in open chromatin regions combined with gene expression from the same cells. Comparing regulatory networks in healthy vs. tumor B cells highlighted the presence of unique regulatory networks in tumor B cells. Notably, the PAX5 tumor regulator was observed only in tumor B cells, and its impacts on the regulation of many downstream genes was characterized. Building this regulatory network analysis was only made possible through the joint profiling of open chromatin and gene expression at the single cell level. Our data demonstrate the power of simultaneous profiling of both epigenomic and transcriptomic data in characterizing cells in cancer state and extracting functional information from tumor samples. Citation Format: Shamoni Maheshwari, Li Wang, Vijay Gopalan, Joe Shuga, Sharmila Chatterjee, Kamila Belhocine. Simultaneous single cell profiling of open chromatin and gene expression in B cell lymphoma highlights tumor-specific regulatory networks [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 LB206.

Single-cell chromatin accessibility maps reveal regulatory programs driving early mouse organogenesis.

During mouse embryonic development, pluripotent cells rapidly divide and diversify, yet the regulatory programs that define the cell repertoire for each organ remain ill-defined. To delineate comprehensive chromatin landscapes during early organogenesis, we mapped chromatin accessibility in 19,453 single nuclei from mouse embryos at 8.25 days post-fertilisation. Identification of cell type-specific regions of open chromatin pinpointed two TAL1-bound endothelial enhancers, which we validated using transgenic mouse assays. Integrated gene expression and transcription factor motif enrichment analyses highlighted cell type-specific transcriptional regulators. Subsequent in vivo experiments in zebrafish revealed a role for the ETS factor FEV in endothelial identity downstream of ETV2 (Etsrp in zebrafish). Concerted in vivo validation experiments in mouse and zebrafish thus illustrate how single-cell open chromatin maps, representative of a mammalian embryo, provide access to the regulatory blueprint for mammalian organogenesis.

C-Maf regulates the plasticity of group 3 innate lymphoid cells by restraining the type 1 program.

CCR6- group 3 innate lymphoid cells (ILC3s) are mediators of intestinal immunity and barrier function that possess the capacity to acquire type 1 effector features and fully convert into ILC1s. The molecular mechanisms governing such plasticity are undefined. Here, we identified c-Maf as an essential regulator of ILC3 homeostasis and plasticity that limits physiological ILC1 conversion. Phenotypic analysis of effector status in Maf-deficient CCR6- ILC3s, coupled with evaluation of global changes in transcriptome, chromatin accessibility, and transcription factor motif enrichment, revealed that c-Maf enforces ILC3 identity. c-Maf promoted ILC3 accessibility and supported RORγt activity and expression of type 3 effector genes. Conversely, c-Maf antagonized type 1 programming, largely through restraint of T-bet expression and function. Mapping of the dynamic changes in chromatin landscape accompanying CCR6- ILC3 development and ILC1 conversion solidified c-Maf as a gatekeeper of type 1 regulatory transformation and a controller of ILC3 fate.

MEK inhibition remodels the active chromatin landscape and induces SOX10 genomic recruitment in BRAF(V600E) mutant melanoma cells

BackgroundThe MAPK/ERK signaling pathway is an essential regulator of numerous cell processes that are crucial for normal development as well as cancer progression. While much is known regarding MAPK/ERK signal conveyance from the cell membrane to the nucleus, the transcriptional and epigenetic mechanisms that govern gene expression downstream of MAPK signaling are not fully elucidated.ResultsThis study employed an integrated epigenome analysis approach to interrogate the effects of MAPK/ERK pathway inhibition on the global transcriptome, the active chromatin landscape, and protein–DNA interactions in 501mel melanoma cells. Treatment of these cells with the small-molecule MEK inhibitor AZD6244 induces hyperpigmentation, widespread gene expression changes including alteration of genes linked to pigmentation, and extensive epigenomic reprogramming of transcriptionally distinct regulatory regions associated with the active chromatin mark H3K27ac. Regulatory regions with differentially acetylated H3K27ac regions following AZD6244 treatment are enriched in transcription factor binding motifs of ETV/ETS and ATF family members as well as the lineage-determining factors MITF and SOX10. H3K27ac-dense enhancer clusters known as super-enhancers show similar transcription factor motif enrichment, and furthermore, these super-enhancers are associated with genes encoding MITF, SOX10, and ETV/ETS proteins. Along with genome-wide resetting of the active enhancer landscape, MEK inhibition also results in widespread SOX10 recruitment throughout the genome, including increased SOX10 binding density at H3K27ac-marked enhancers. Importantly, these MEK inhibitor-responsive enhancers marked by H3K27ac and occupied by SOX10 are located near melanocyte lineage-specific and pigmentation genes and overlap numerous human SNPs associated with pigmentation and melanoma phenotypes, highlighting the variants located within these regions for prioritization in future studies.ConclusionsThese results reveal the epigenetic reprogramming underlying the re-activation of melanocyte pigmentation and developmental transcriptional programs in 501mel cells in response to MEK inhibition and suggest extensive involvement of a MEK-SOX10 axis in the regulation of these processes. The dynamic chromatin changes identified here provide a rich genomic resource for further analyses of the molecular mechanisms governing the MAPK pathway in pigmentation- and melanocyte-associated diseases.

Alveolar macrophages generate a noncanonical NRF2-driven transcriptional response to Mycobacterium tuberculosis in vivo.

Alveolar macrophages (AMs) are the first cells to be infected during Mycobacterium tuberculosis (M.tb.) infection. Thus, the AM response to infection is the first of many steps leading to initiation of the adaptive immune response required for efficient control of infection. A hallmark of M.tb. infection is the slow initiation of the adaptive response, yet the mechanisms responsible for this are largely unknown. To study the initial AM response to infection, we developed a system to identify, sort, and analyze M.tb.-infected AMs from the lung within the first 10 days of infection. In contrast to what has been previously described using in vitro systems, M.tb.-infected AMs up-regulate a cell-protective antioxidant transcriptional signature that is dependent on the lung environment but not bacterial virulence. Computational approaches including pathway analysis and transcription factor motif enrichment analysis identify NRF2 as a master regulator of the response. Using knockout mouse models, we demonstrate that NRF2 drives expression of the cell-protective signature in AMs and impairs the control of early bacterial growth. AMs up-regulate a substantial pro-inflammatory response to M.tb. infection only 10 days after infection, yet comparisons with bystander AMs from the same infected animals demonstrate that M.tb.-infected AMs generate a less robust inflammatory response than the uninfected cells around them. Our findings demonstrate that the initial macrophage response to M.tb. in the lung is far less inflammatory than has previously been described by in vitro systems and may impede the overall host response to infection.

Transcription factor motif enrichment in whole transcriptome analysis identifies STAT4 and BCL6 as the most prominent binding motif in systemic juvenile idiopathic arthritis

BackgroundThe term systemic juvenile idiopathic arthritis (sJIA) describes an autoinflammatory condition characterized by arthritis and severe systemic inflammation, which in later stages can transform into interleukin (IL)-17-driven autoimmune arthritis. IL-1 antagonists have been used with good efficacy in the early stages of sJIA.MethodsA whole transcriptome analysis of peripheral blood RNA samples was performed in six patients with sJIA and active systemic disease, before initiating treatment with the IL-1β receptor antagonist anakinra, and after induction of inactive disease, compared with a single-sample control cohort of 21 patients in several clinical stages of sJIA activity. Whole transcriptomes were compared longitudinally and interindividually including gene ontology and motif enrichment analysis of differentially expressed genes.ResultsThere were 741 transcripts were identified using a threshold with a p value <0.01 and a fold change > 2. HLADRB1 and CD74 were identified as the most strongly upregulated genes in inactive compared to active disease; CD177 expression was significantly enhanced in active disease compared to inactive disease. Motif enrichment analysis revealed STAT4, BCL6, and STAT3 as the most prominent transcription factors that were present during active disease. In addition, strong upregulation of the major histocompatability complex II (MHCII) ligand CD74 was found in both active and inactive sJIA compared to healthy controls.ConclusionUsing transcription factor motif enrichment, this study identifies novel putative pathways in sJIA (STAT4, BCL6) implicating B cell activation at an earlier stage than predicted in refractory disease. The implication of BCL-6 dependent pathways argues for occurrence of autoimmunity early within the process of sJIA chronification. Transcriptional regulation of HLA-DRB1, a recently described independent genetic risk factor, in combination with its cooperating partner CD74 in patients where sJIA is confirmed, supports pathogenic involvement in alterations in antigen presentation during sJIA.

Genome-Wide Temporal Profiling of Transcriptome and Open Chromatin of Early Cardiomyocyte Differentiation Derived From hiPSCs and hESCs.

Recent advances have improved our ability to generate cardiomyocytes from human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). However, our understanding of the transcriptional regulatory networks underlying early stages (ie, from mesoderm to cardiac mesoderm) of cardiomyocyte differentiation remains limited. To characterize transcriptome and chromatin accessibility during early cardiomyocyte differentiation from hiPSCs and hESCs. We profiled the temporal changes in transcriptome and chromatin accessibility at genome-wide levels during cardiomyocyte differentiation derived from 2 hiPSC lines and 2 hESC lines at 4 stages: pluripotent stem cells, mesoderm, cardiac mesoderm, and differentiated cardiomyocytes. Overall, RNA sequencing analysis revealed that transcriptomes during early cardiomyocyte differentiation were highly concordant between hiPSCs and hESCs, and clustering of 4 cell lines within each time point demonstrated that changes in genome-wide chromatin accessibility were similar across hiPSC and hESC cell lines. Weighted gene co-expression network analysis (WGCNA) identified several modules that were strongly correlated with different stages of cardiomyocyte differentiation. Several novel genes were identified with high weighted connectivity within modules and exhibited coexpression patterns with other genes, including noncoding RNA LINC01124 and uncharacterized RNA AK127400 in the module related to the mesoderm stage; E-box-binding homeobox 1 (ZEB1) in the module correlated with postcardiac mesoderm. We further demonstrated that ZEB1 is required for early cardiomyocyte differentiation. In addition, based on integrative analysis of both WGCNA and transcription factor motif enrichment analysis, we determined numerous transcription factors likely to play important roles at different stages during cardiomyocyte differentiation, such as T and eomesodermin (EOMES; mesoderm), lymphoid enhancer-binding factor 1 (LEF1) and mesoderm posterior BHLH transcription factor 1 (MESP1; from mesoderm to cardiac mesoderm), meis homeobox 1 (MEIS1) and GATA-binding protein 4 (GATA4) (postcardiac mesoderm), JUN and FOS families, and MEIS2 (cardiomyocyte). Both hiPSCs and hESCs share similar transcriptional regulatory mechanisms underlying early cardiac differentiation, and our results have revealed transcriptional regulatory networks and new factors (eg, ZEB1) controlling early stages of cardiomyocyte differentiation.

Semi-synthetic sapogenin exerts neuroprotective effects by skewing the brain ischemia reperfusion transcriptome towards inflammatory resolution

Stroke represents one of the first causes of mortality and morbidity worldwide. We evaluated the therapeutic potential of a novel semi-synthetic spirosteroid sapogenin derivative “S15” in a transient middle cerebral artery occlusion (tMCAO) focal ischemia model in rat. S15-treated rats had significantly reduced infarct volumes and improved neurological functions at 24h post-reperfusion, compared with ischemia. Corresponding gene expression changes in brain were characterized by mRNA sequencing and qPCR approaches. Next, we applied geneset, pathway and transcription factor motif enrichment analysis to identify relevant signaling networks responsible for neuronal damage upon ischemia-reperfusion or neuroprotection upon pretreatment with S15. As expected, ischemia-reperfusion brain damage strongly modulates transcriptional programs associated with immune responses, increased differentiation of immune cells as well as reduced (cat)ion transport and synaptic activity. Interestingly, S15-dependent neuroprotection regulates inflammation-associated genes involved in phagosome specific resolution of tissue damage, chemotaxis and anti-inflammatory alternative activation of microglia. Altogether our transcriptome wide RNA sequencing and integrated pathway analysis provides new clues in the neuroprotective properties of a novel spirosteroid S15 or neuronal damage in rat brains subjected to ischemia, which opens new perspectives for successful treatment of stroke.

Epigenetic Signatures of Salivary Gland Inflammation in Sjögren's Syndrome.

ObjectiveSjögren's syndrome (SS) is a complex multisystem autoimmune disease that results in progressive destruction of the exocrine glands. The purpose of this study was to characterize epigenetic changes in affected gland tissue and describe the relationship of these changes to known inflammatory processes.MethodsA genome‐wide DNA methylation study was performed on human labial salivary gland (LSG) biopsy samples obtained from 28 female members of the Sjögren's International Collaborative Clinical Alliance (SICCA) Registry. Gland tissue was methylotyped using the Illumina HumanMethylation450 BeadChip platform, followed by rigorous probe‐filtering and data‐normalization procedures.ResultsA genome‐wide case–control study of 26 of the 28 subjects revealed 7,820 differentially methylated positions (DMPs) associated with disease status, including 5,699 hypomethylated and 2,121 hypermethylated DMPs. Further analysis identified 57 genes that were enriched for DMPs in their respective promoters; many are involved in immune response, including 2 previously established SS genetic risk loci. Bioinformatics analysis highlighted an extended region of hypomethylation surrounding PSMB8 and TAP1, consistent with an increased frequency of antigen‐presenting cells in LSG tissue from the SS cases. Transcription factor motif enrichment analysis revealed the specific nature of the genome‐wide methylation differences, demonstrating colocalization of SS‐associated DMPs with stress‐ and immune response–related motifs.ConclusionOur findings underscore the utility of CpG methylotyping as an independent probe of active disease processes in SS, offering unique insights into the composition of disease‐relevant tissue. Methylation profiling implicated several genes and pathways previously thought to be involved in disease‐related processes, as well as a number of new candidates.

Characterizing the Global Transcriptional Effects of TEL-AML1-Fusion in Childhood Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer affecting thousands of children worldwide each year. Most patients are cured but only after long chemotherapy treatment and still 15 % of them relapse. Therefore, better understanding of the molecular biology behind the disease is fundamental. TEL-AML1 fusion - comprised of two transcription factors important in hematopoiesis regulation - is the most frequent genetic variation found in 25 % of pediatric ALL-patients. The translocation occurs during pregnancy and is suggested to serve as a “first hit” enabling secondary changes that lead to cancer formation. Despite its frequency, genetic targets of this aberrant transcription factor are unclear, and its function in leukemia initiation and progression remains elusive.Here we aim at solving the genome-wide effects of TEL-AML1 on transcription using pre-B-leukemic cell models. After a time-series induction of wild-type or DNA-binding compromised mutated TEL-AML1, or a constitutive expression of shRNA silencing the fusion, the cells were studied using global nuclear run-on sequencing (GRO-seq). GRO-seq technology can map RNA polymerase II location and identify actively transcribed coding and non-coding genes and their directionality. Thus, GRO-seq provides a snapshot of global active transcription and was used here to identify bona fide targets of the TEL-AML1-fusion protein.In addition to transcriptional changes in known genes, we identified a notable number of novel transcripts including antisense-, lincRNA-, and microRNAs. We also characterized genomic locations where the fusion functions as a direct regulator by taking advantage of non-coding RNA transcripts known as enhancer RNAs. Furthermore, we performed transcription factor motif enrichment analysis on these active regulatory eRNA regions. The results uncover early transcriptional changes induced by TEL-AML1-fusion and may reveal critical steps in leukemia initiation and development. The findings also highlight the importance of non-coding RNAs in the pathogenesis of pediatric leukemia. DisclosuresNo relevant conflicts of interest to declare.