Ets Family Research Articles

TMIC-43. BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF NOVEL GLIOBLASTOMA TME SUBTYPES USING TRANSCRIPTOMIC ANALYSIS AND NETWORK MODELLING TO IDENTIFY NOVEL TARGETS OF VULNERABILITY

Abstract New precision medicine therapies are urgently required for glioblastoma (GBM). Recently, we developed a novel GBM classification system, identifying three patient clusters uniquely characterized by tumor microenvironment (TME) composition: TMELow, TMEMedium, and TMEHigh1. Our objective now is to further investigate these subtypes employing transcriptomic analysis and network modelling approaches to identify novel subtype-specific targets of vulnerability. We analyzed transcriptomic data from >600 GBM samples from publicly available and in-house datasets. All samples underwent TME subtyping. Next, we performed differential gene expression (using DESeq2, edgeR) and pathway analysis (using PROGENy). The ‘TRANSFAC’ tool 2 was used to identify potential transcription factors enriched in each TME subtype. TMELow tumours manifested highly upregulated NLGN3 (P=5.777e-5) and HES5 (P=2.233e-3) expression compared to TMEHigh. Moreover, TMEHigh compared to TMELow tumours were enriched for genes associated with tissue remodelling, and showed elevated MMP7 (P=3.051e-6), CLCL13 (P= 3.843e-4), and CXCL5 expression (P= 1.592e-6). PROGENy analysis suggested that the WNT, TRAIL, and JAK/STAT pathways were significantly enriched in TMEHigh, while the PI3K pathway was significantly upregulated in TMEMedium. Additionally, VEGF pathway activity was significantly upregulated in TMELow. TRANSFAC analysis measured by regulatory score (a measure of a transcription factor effect on gene expression) revealed that in TMELow, key transcription factors includes ETS2, NANOG, and MECP2 (regulatory score: 1.97, 1.45, and 1.4, respectively). In TMEMedium, transcription factors HIF1A, PPARA, and CDX2 were identified (regulatory score: 2.52, 2.08 and 1.96, respectively). In TMEHigh, TRANSFAC indicated activiation of transcription factors SMAD3, ETS2, and NFKB1 regulatory score: 2.56, 2.41, 2.24, respectively). Overall these findings highlight distinct molecular signatures across TME subtypes, and provide a deeper understanding of associated molecular mechanisms. Ongoing work is focused on validating key subtype specific genes and associated pathways using spatial proteomics. Prioritised targets will ultimately be interrogated in vivo, as novel subtype-specific treatments. 1. White et al. Ann Oncol. 2023;34(3):300–314. 2. Matys et al. Nucleic Acids Res. 2003;31.

Chromatin structure and 3D architecture define the differential functions of PU.1 regulatory elements in blood cell lineages

The precise spatiotemporal expression of the hematopoietic ETS transcription factor PU.1, a key determinant of hematopoietic cell fates, is tightly regulated at the chromatin level. However, how chromatin signatures are linked to this dynamic expression pattern across different blood cell lineages remains uncharacterized. Here, we performed an in-depth analysis of the relationships between gene expression, chromatin structure, 3D architecture, and trans-acting factors at PU.1 cis-regulatory elements (PCREs). By identifying phylogenetically conserved DNA elements within chromatin-accessible regions in primary human blood lineages, we discovered multiple novel candidate PCREs within the upstream region of the human PU.1 locus. A subset of these elements localizes within an 8-kb-wide cluster exhibiting enhancer features, including open chromatin, demethylated DNA, enriched enhancer histone marks, present enhancer RNAs, and PU.1 occupation, presumably mediating PU.1 autoregulation. Importantly, we revealed the presence of a common 35-kb-wide CTCF-flanked insulated neighborhood that contains the PCRE cluster (PCREC), forming a chromatin territory for lineage-specific and PCRE-mediated chromatin interactions. These include functional PCRE-promoter interactions in myeloid and B cells that are absent in erythroid and T cells. By correlating chromatin structure and 3D architecture with PU.1 expression in various lineages, we were able to attribute enhancer versus silencer functions to individual elements. Our findings provide mechanistic insights into the interplay between dynamic chromatin structure and 3D architecture in the chromatin regulation of PU.1 expression. This study lays crucial groundwork for additional experimental studies that validate and dissect the role of PCREs in epigenetic regulation of normal and malignant hematopoiesis.

Single-Cell RNA Sequencing Uncovers Pathological Processes and Crucial Targets for Vascular Endothelial Injury in Diabetic Hearts.

Cardiovascular disease remains the leading cause of high mortality in individuals with diabetes mellitus. Endothelial injury is a major contributing factor for vascular dysfunction in diabetes. However, the precise mechanisms underlying endothelial cell injury and their heterogeneity in diabetes remains elusive. In this study, single-cell sequencing is performed in heart tissues from leptin receptor knock-out (db/db) diabetic mice at various pathological stages. Through cell cluster identification, differential gene analysis, intercellular communication analysis, pseudo time analysis, and transcription factor analysis, a novel mechanism of cardiac vascular endothelial damage in diabetes is identified. Specifically, a single-cell transcription map of cardiac vascular endothelial cells is presented in db/db mice. Diverse cellular clusters are found to play vital roles under diabetes-induced damage, highlighting crucial transcription factors involved in their regulation. In addition, the essential transcription factor Ets1 is found to protect against vascular endothelial injury in db/db mice. In summary, the work provides a comprehensive understanding of the development of diabetic cardiac vascular endothelial damage and the heterogeneity of the cells involved. These findings offer valuable insights into potential treatments and assessments of diabetic cardiovascular endothelial damage.

Super-Enhancer-Driven IRF2BP2 is Activated by Master Transcription Factors and Sustains T-ALL Cell Growth and Survival.

Super enhancers (SEs) are large clusters of transcriptional enhancers driving the expression of genes crucial for defining cell identity. In cancer, tumor-specific SEs activate key oncogenes, leading to tumorigenesis. Identifying SE-driven oncogenes in tumors and understanding their functional mechanisms is of significant importance. In this study, a previously unreported SE region is identified in T-cell acute lymphoblastic leukemia (T-ALL) patient samples and cell lines. This SE activates the expression of interferon regulatory factor 2 binding protein 2 (IRF2BP2) and is regulated by T-ALL master transcription factors (TFs) such as ETS transcription factor ERG (ERG), E74 like ETS transcription factor 1 (ELF1), and ETS proto-oncogene 1, transcription factor (ETS1). Hematopoietic system-specific IRF2BP2 conditional knockout mice is generated and showed that IRF2BP2 has minimal impact on normal T cell development. However, in vitro and in vivo experiments demonstrated that IRF2BP2 is crucial for T-ALL cell growth and survival. Loss of IRF2BP2 affects the MYC and E2F pathways in T-ALL cells. Cleavage under targets and tagmentation (CUT&Tag) assays and immunoprecipitation revealed that IRF2BP2 cooperates with the master TFs of T-ALL cells, targeting the enhancer of the T-ALL susceptibility gene recombination activating 1 (RAG1) and modulating its expression. These findings provide new insights into the regulatory network within T-ALL cells, identifying potential new targets for therapeutic intervention.

Dependence of PAX3-FOXO1 chromatin occupancy on ETS1 at important disease-promoting genes exposes new targetable vulnerability in Fusion-Positive Rhabdomyosarcoma

AbstractRhabdomyosarcoma (RMS), a malignancy of impaired myogenic differentiation, is the most common soft tissue pediatric cancer. PAX3-FOXO1 oncofusions drive the majority of the clinically more aggressive fusion-positive rhabdomyosarcoma (FP-RMS). Recent studies have established an epigenetic basis for PAX3-FOXO1-driven oncogenic processes. However, details of PAX3-FOXO1 epigenetic mechanisms, including interactions with, and dependence on, other chromatin and transcription factors, are incompletely understood. We previously identified a novel disease-promoting epigenetic axis in RMS, involving the histone demethylase KDM3A and the ETS1 transcription factor, and demonstrated that this epigenetic axis interfaces with PAX3-FOXO1 both phenotypically and transcriptomically, including co-regulation of biological processes and genes important to FP-RMS progression. In this study, we demonstrate that KDM3A and ETS1 colocalize with PAX3-FOXO1 to enhancers of important disease-promoting genes in FP-RMS, including FGF8, IL4R, and MEST, as well as PODXL, which we define herein as a new FP-RMS-promoting gene. We show that ETS1, which is induced by both PAX3-FOXO1 and KDM3A, exists in complex with PAX3-FOXO1, and augments PAX3-FOXO1 chromatin occupancy. We further show that the PAX3-FOXO1/ETS1 complex can be disrupted by the clinically relevant small molecule inhibitor YK-4-279. YK-4-279 displaces PAX3-FOXO1 from chromatin and interferes with PAX3-FOXO1-dependent gene regulation, resulting in potent inhibition of growth and invasive properties in FP-RMS, along with downregulation of FGF8, IL4R, MEST and PODXL expression. We additionally show that, in some FP-RMS, KDM3A also increases PAX3-FOXO1 levels. Together, our studies illuminate mechanisms of action of the KDM3A/ETS1 regulatory module, and reveal novel targetable mechanisms of PAX3-FOXO1 chromatin complex regulation, in FP-RMS.

Suppressing DUSP16 overexpression induced by ELK1 promotes neural progenitor cell differentiation in mouse models of Alzheimer's disease.

Emerged evidence indicated that stimulating hippocampal neurogenesis is a potential strategy for restoring cognition in AD. Mitogen-activated protein kinases (MAPKs) play an essential role in neurogenesis. Meanwhile, the enzymatic power of the phosphatases is much greater than that of kinases. Dual-specificity phosphatase 16 (DUSP16), known to as a phosphatase negatively regulate MAPKs, may be implicated in neural differentiation. Nevertheless, the effect of DUSP16 on cognitive disorders by stimulating neural progenitor cell (NPC) differentiation in AD mice remains unclear. Our study demonstrates an association between DUSP16 SNPs and clinical progression in individuals with mild cognitive impairment (MCI). Besides, increased DUSP16 expression was detected in both 3xTg and SAMP8 mouse models of AD, accompanied by NPC neural differentiation impairments. By silencing DUSP16, the induction of neural differentiation, synaptic transmission, and cognitive benefits were observed in both AD mice. Furthermore, DUSP16 was involved in the process of NPC differentiation through regulating c-Jun N-terminal kinase (JNK) phosphorylation and SOX2 expression. Moreover, ETS transcription factor (ELK1) was involved in the DUSP16 transcription, which resulted in the upregulation of DUSP16 at the state of AD. Our data uncovers a potential regulatory role for DUSP16 in adult hippocampal neurogenesis (AHN) and provides a possibility to find a novel strategy for AD intervention.

Disruption of blood-brain barrier and endothelial-to-mesenchymal transition are attenuated by Astragalus polysaccharides mediated through upregulation of ETS1 expression in experimental autoimmune encephalomyelitis

Blood-brain barrier (BBB) breakdown, an early hallmark of multiple sclerosis (MS), remains crucial for MS progression. Our previous works have confirmed that Astragalus polysaccharides (APS) can significantly ameliorate demyelination and disease progression in experimental autoimmune encephalomyelitis (EAE) mice. However, it remains unclear whether APS protects BBB and the potential mechanism. In this study, we found that APS effectively reduced BBB leakage in EAE mice, which was accompanied by a decreased level of endothelial-to-mesenchymal transition (EndoMT) in the central nervous system (CNS). We further induced EndoMT in the mouse brain endothelial cells (bEnd.3) by interleukin-1β (IL-1β) in vitro. The results showed that APS treatment could inhibit IL-1β-induced EndoMT and endothelial cell dysfunction. In addition, the transcription factor ETS1 is a central regulator of EndoMT related to the compromise of BBB. We tested the regulation of APS on ETS1 and identified the expression of ETS1 was upregulated in both EAE mice and bEnd.3 cells by APS. ETS1 knockdown facilitated EndoMT and endothelial cell dysfunction, which completely abolished the regulatory effect of APS. Collectively, APS treatment could protect BBB integrity by inhibiting EndoMT, which might be associated with upregulating ETS1 expression. Our findings indicated that APS has potential value in the prevention of MS.

EphA-Mediated Regulation of Stomatin Expression in Prostate Cancer Cells.

Tumor growth and progression are affected by interactions between tumor cells and stromal cells within the tumor microenvironment. We previously showed that the expression of an integral membrane protein, called stomatin, was increased in cancer cells following their association with stromal cells. Additionally, stomatin impaired the Akt signaling pathway to suppress tumor growth. However, it remains unclear how stomatin expression is regulated. To explore this, we examined the cell surface molecules that can transduce the intercellular communication signals between cancer cells and stromal cells. Among these molecules, EphA3 and EphA7 receptors and their ligand ephrin-A5 were found to be expressed in prostate cancer cells, but not in prostate stromal cells. Cell-to-cell contact of prostate cancer cells through the EphA-ephrin-A interaction suppressed stomatin expression, while knockdown of EphA3/7 or ephrin-A5 increased stomatin expression. This increase contributed to an inhibition of prostate cancer cell proliferation. Intracellularly, the binding of ephrin-A to EphA attenuated extracellular signaling-regulated kinase (ERK) activation that promoted stomatin expression. Furthermore, ELK1 and ELK4, which are Ets family transcription factors phosphorylated by ERK, were involved in the induction of stomatin expression. We also found that higher Gleason score prostate cancer tissue samples had increased activation of EphA, while the stomatin expression and activated ERK and ELK levels were all low. In the mouse xenograft tumor samples generated by implantation of prostate cancer cells, EphA3 phosphorylation was attenuated and the ERK-ELK signaling and stomatin expression were enhanced in the area where stromal cells infiltrated the tumor. The EphA-mediated signaling suppresses the ERK-ELK pathway, leading to the reduction of stomatin expression that affects prostate cancer malignancy.

Transcription factor ETS1‑mediated ECT2 expression promotes the malignant behavior of prostate cancer cells.

Prostate cancer remains the most prevalent malignancy diagnosed in men worldwide. Epithelial cell transforming sequence 2 (ECT2) is an oncogene involved in the progression of human tumors. The present study aimed to explore the involvement of ECT2 in prostate cancer and its participation in the malignant progression of prostate cancer. ECT2 expression in prostate cancer cell lines was examined via reverse transcription-quantitative PCR and western blotting. The effects of knockdown of ECT2 expression in PC-3 cells on cellular biological behaviors, including proliferation, migration and invasion, were examined using Cell Counting Kit-8, colony formation, wound healing and Transwell assays. The glycolysis level was determined based on the lactate release, glucose uptake, oxygen consumption rate and extracellular acidification rate. The binding relationship between ECT2 and ETS1 was verified using luciferase reporter and chromatin immunoprecipitation assays. The results indicated that ECT2 was highly expressed in prostate cancer cell lines. Knockdown of ECT2 expression could inhibit cell proliferation, migration, invasion and glycolysis. In addition, the transcription factor ETS1 could directly bind to the ECT2 promoter and positively regulate ECT2 expression. These data were combined with the results of rescue experiments and demonstrated that the inhibitory effects of the knockdown of ECT2 expression on the malignant behavior and glycolysis of prostate cancer cells were partially reversed by ETS1 overexpression. In conclusion, ETS1 induced transcriptional upregulation of ECT2 and enhanced the malignant biological behaviors of prostate cancer cells, thereby promoting the progression of prostate cancer. This evidence provides a theoretical basis for the treatment of prostate cancer.

Loss of the ETS Transcription Factor ERG Disrupts Fate-defining Programs in the Aortic Endothelium and Promotes Expansion of Endothelial Lineage Cells in Atherosclerotic Plaque

Loss of the ETS Transcription Factor ERG Disrupts Fate-defining Programs in the Aortic Endothelium and Promotes Expansion of Endothelial Lineage Cells in Atherosclerotic Plaque

Prostate Cancer: A Review of Genetics, Current Biomarkers and Personalised Treatments.

Prostate cancer is the second leading cause of cancer deaths in men, second only to lung cancer. Despite this, diagnosis and prognosis methods remain limited, with effective treatments being few and far between. Traditionally, prostate cancer is initially tested for through a prostate serum antigen (PSA) test and a digital rectum examination (DRE), followed by confirmation through an invasive prostate biopsy. The DRE and biopsy are uncomfortable for the patient, so less invasive, accurate diagnostic tools are needed. Current diagnostic tools, along with genes that hold possible biomarker uses in diagnosis, prognosis and indications for personalised treatment plans, were reviewed in this article. Several genes from multiple families have been identified as possible biomarkers for disease, including those from the MYC and ETS families, as well as several tumour suppressor genes, Androgen Receptor signalling genes and DNA repair genes. There have also been advances in diagnostic tools, including MRI-targeted and liquid biopsies. Several personalised treatments have been developed over the years, including those that target metabolism-driven prostate cancer or those that target inflammation-driven cancer. Several advances have been made in prostate cancer diagnosis and treatment, but the disease still grows year by year, leading to more and more deaths annually. This calls for even more research into this disease, allowing for better diagnosis and treatment methods and a better chance of patient survival.

Single-cell transcriptomes and chromatin accessibility of endothelial cells unravel transcription factors associated with dysregulated angiogenesis in systemic sclerosis

ObjectivesVasculopathy emerges early in systemic sclerosis (SSc) and links to endothelial cell (EC) injury and angiogenesis. Understanding EC transcriptomes and epigenomes is crucial for unravelling the mechanisms involved.MethodsTranscriptomes and chromatin...

Decreased PU.1 expression in mature B cells induces lymphomagenesis.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma, accounting for 30% of non-Hodgkin lymphomas. Although comprehensive analysis of genetic abnormalities has led to the classification of lymphomas, the exact mechanism of lymphomagenesis remains elusive. The Ets family transcription factor, PU.1, encoded by Spi1, is essential for the development of myeloid and lymphoid cells. Our previous research illustrated the tumor suppressor function of PU.1 in classical Hodgkin lymphoma and myeloma cells. In the current study, we found that patients with DLBCL exhibited notably reduced PU.1 expression in their lymphoma cells, particularly in the non-germinal center B-cell-like (GCB) subtype. This observation suggests that downregulation of PU.1 may be implicated in DLBCL tumor growth. To further assess PU.1's role in mature B cells invivo, we generated conditional Spi1 knockout mice using Cγ1-Cre mice. Remarkably, 13 of the 23 knockout mice (56%) showed splenomegaly, lymphadenopathy, or masses, with some having histologically confirmed B-cell lymphomas. In contrast, no wild-type mice developed B-cell lymphoma. In addition, RNA-seq analysis of lymphoma cells from Cγ1-Cre Spi1F/F mice showed high frequency of each monoclonal CDR3 sequence, indicating that these lymphoma cells were monoclonal tumor cells. When these B lymphoma cells were transplanted into immunodeficient recipient mice, all mice died within 3 weeks. Lentiviral-transduced Spi1 rescued 60% of the recipient mice, suggesting that PU.1 has a tumor suppressor function invivo. Collectively, PU.1 is a tumor suppressor in mature B cells, and decreased PU.1 results in mature B-cell lymphoma development.

Assembly of mTORC3 Involves Binding of ETV7 to Two Separate Sequences in the mTOR Kinase Domain.

mTOR plays a crucial role in cell growth by controlling ribosome biogenesis, metabolism, autophagy, mRNA translation, and cytoskeleton organization. It is a serine/threonine kinase that is part of two distinct extensively described protein complexes, mTORC1 and mTORC2. We have identified a rapamycin-resistant mTOR complex, called mTORC3, which is different from the canonical mTORC1 and mTORC2 complexes in that it does not contain the Raptor, Rictor, or mLST8 mTORC1/2 components. mTORC3 phosphorylates mTORC1 and mTORC2 targets and contains the ETS transcription factor ETV7, which binds to mTOR and is essential for mTORC3 assembly in the cytoplasm. Tumor cells that assemble mTORC3 have a proliferative advantage and become resistant to rapamycin, indicating that inhibiting mTORC3 may have a therapeutic impact on cancer. Here, we investigate which domains or amino acid residues of ETV7 and mTOR are involved in their mutual binding. We found that the mTOR FRB and LBE sequences in the kinase domain interact with the pointed (PNT) and ETS domains of ETV7, respectively. We also found that forced expression of the mTOR FRB domain in the mTORC3-expressing, rapamycin-resistant cell line Karpas-299 out-competes mTOR for ETV7 binding and renders these cells rapamycin-sensitive in vivo. Our data provide useful information for the development of molecules that prevent the assembly of mTORC3, which may have therapeutic value in the treatment of mTORC3-positive cancer.

Prostate cancer subtyping and differential methylation analysis based on the ETS family of transcription factors fusion genes

Prostate cancer (PCa) is a highly heterogeneous disease, encompassing various molecular and clinical pathological subtypes. Fusion genes play a facilitating role in the occurrence and progression of PCa. We categorized PCa samples into the ETS family of transcription factors fusion positive and fusion negative subtypes based on fusion genes. This subtyping method is closely related to the epigenomic DNA methylation profiles of PCa, with each sample cluster including more than 85% of the patients. We conducted an analysis of the distribution of the ETS family fusion genes on chromosomes, fusion modes within reading frames, and predictions of structural domains. Among these, the highest frequency of the ETS family related fusion genes occurred on chromosome 21. Compared to the parental genes, fusion genes exhibited new structural domains, such as IG_like, and the most common fusion mode was out-of-frame fusion. The correlation between the methylation levels of hypermethylated CpG sites and the expression levels of their corresponding mRNAs indicates that CD8A and B3GNT5 (with correlations of − 0.388 and − 0.253, respectively) could serve as potential prognostic markers for PCa.

BRAF-induced EHF expression affects TERT in aggressive papillary thyroid cancer.

BRAFV600E and TERT promoter mutations in papillary thyroid carcinoma (PTC) have a synergistic effect on prognosis. This effect is believed to arise from MAPK activation triggered by BRAFV600E, leading to the upregulation of ETS transcription factors that bind to the mutant TERT promoter. To explore the role of ETS factors in relation to clinical features, BRAFV600E and TERT promoter mutations in PTC. Transcriptomic data for 28 ETS factors were analyzed in the PTC cohort of The Cancer Genome Atlas (TCGA, n=399) and subsequently validated in a local cohort (n=93). In vitro experiments were performed to investigate the regulatory role in relation to BRAFV600E and TERT expression. TCGA identified ETS1, ERG, FLI1, GABPA, EHF, ETV6 and SPDEF as differentially expressed genes between stages I+II and III+IV. In both cohorts, EHF was consistently associated with adverse clinical features, BRAFV600E and TERT promoter mutation/expression. Notably, in BRAFV600E mutated PTC, high EHF expression was associated with shorter disease-free survival. Cases harboring concurrent BRAFV600E, TERT promoter mutations and high EHF expression exhibited the shortest disease-free survival. In cells harboring concurrent BRAFV600E and TERT promoter mutation, over-expression of EHF significantly increased TERT expression while knockdown or pharmacological inhibition of BRAF significantly decreased both EHF and TERT expression. In addition, ChIP-qPCR analysis suggested a potential binding of EHF in TERT promoter mutant cells but not in TERT promoter wild-type cells. The ETS transcription factor EHF is associated with poor prognosis in PTC. This is potentially mediated by BRAF-induced upregulation of EHF which in turn increases TERT expression in TERT promoter mutated cells.

Clinical practice guidelines for the treatment of Ewing sarcoma (Spanish Sarcoma Research Group-GEIS).

Ewing sarcoma is a small round-cell sarcoma characterized by gene fusion involving EWSR1 (or another TET family protein like FUS) and an ETS family transcription factor. The estimated incidence of this rare bone tumor, which occurs most frequently in adolescents and young adults, is 0.3 per 100,000/year. Although only 25% of patients with Ewing sarcoma are diagnosed with metastatic disease, historical series show that this is a systemic disease. Patient management requires multimodal therapies-including intensive chemotherapy-in addition to local treatments (surgery and/or radiotherapy). In the recurrent/refractory disease setting, different approaches involving systemic treatments and local therapies are also recommended as well as patient inclusion in clinical trials whenever possible. Because of the complexity of Ewing sarcoma diagnosis and treatment, it should be carried out in specialized centers and treatment plans should be designed upfront by a multidisciplinary tumor board. These guidelines provide recommendations for diagnosis, staging, and multimodal treatment of Ewing sarcoma.

Single-nucleus multiome analysis of human cerebellum in Alzheimer's disease-related dementia.

Although human cerebellum is known to be neuropathologically impaired in Alzheimer's disease (AD) and AD-related dementias (ADRD), the cell type-specific transcriptional and epigenomic changes that contribute to this pathology are not well understood. Here, we report single-nucleus multiome (snRNA-seq and snATAC-seq) analysis of 103,861 nuclei isolated from cerebellum from 9 human cases of AD/ADRD and 8 controls, and with frontal cortex of 6 AD donors for additional comparison. Using peak-to-gene linkage analysis, we identified 431,834 significant linkages between gene expression and cell subtype-specific chromatin accessibility regions enriched for candidate cis-regulatory elements (cCREs). These cCREs were associated with AD/ADRD-specific transcriptomic changes and disease-related gene regulatory networks, especially for RAR Related Orphan Receptor A (RORA) and E74 Like ETS Transcription Factor 1 (ELF1) in cerebellar Purkinje cells and granule cells, respectively. Trajectory analysis of granule cell populations further identified disease-relevant transcription factors, such as RORA, and their regulatory targets. Finally, we prioritized two likely causal genes, including Seizure Related 6 Homolog Like 2 (SEZ6L2) in Purkinje cells and KAT8 Regulatory NSL Complex Subunit 1 (KANSL1) in granule cells, through integrative analysis of cCREs derived from snATAC-seq, genome-wide AD/ADRD loci, and Hi-C looping data. This first cell subtype-specific regulatory landscape in the human cerebellum identified here offer novel genomic and epigenomic insights into the neuropathology and pathobiology of AD/ADRD and other neurological disorders if broadly applied.

60 Dissecting the Cellular and Epigenetic Landscape of Clear Cell Renal Cell Carcinoma Through Paired snRNA/ATAC-Seq Analysis

Abstract Background Clear cell renal cell carcinoma (ccRCC) stands as one of the most prevalent and aggressive forms of renal malignancies, accounting for a substantial portion of kidney cancer-related mortality worldwide. The heterogeneity within ccRCC poses a significant challenge to understanding its molecular mechanisms and devising effective therapeutic strategies. [1] Previous studies utilizing single nucleus RNA sequencing (snRNA-seq) have revealed transcriptional contributions to cell-type specificity in both mature human and mouse kidneys. Moreover, recent advancements have expanded this approach to single-cell profiling of chromatin accessibility. Single-nuclei assay for transposase-accessible chromatin using sequencing (snATAC-seq) has enabled the measurement of chromatin accessibility in thousands of individual cells, providing insights into the dynamic process that drives nephron development and differentiation. Integration and analysis of multimodal single-cell datasets, such as snRNA-seq and snATAC-seq, allows for the prediction of cell-type-specific cis-regulatory DNA interactions and transcription factor activity, complementing the transcriptional information obtained by snRNA-seq.[2] [3] [1] Yang, Wang, and Yang, “Treatment Strategies for Clear Cell Renal Cell Carcinoma.” [2] Muto et al., “Single Cell Transcriptional and Chromatin Accessibility Profiling Redefine Cellular Heterogeneity in the Adult Human Kidney.” [3] Monteagudo-Sánchez, Noordermeer, and Greenberg, “The Impact of DNA Methylation on CTCF-Mediated 3D Genome Organization.” Methods This study leveraged paired snRNA/ATAC-seq in tumor and normal-adjacent samples from the Renal Tumor Biobank at Dartmouth (n= 63) to explore the ccRCC landscape at single-cell resolution using 10x multiome technology. The snRNA-seq dataset was processed with Seurat v5.0 to remove low-quality nuclei. Doublets were removed with DoubletFinder v2.02, and ambient DNA was removed using decontX from celda. Normalization was conducted via SCTransform, and missing data was imputed with ALRA. Clustering was performed by constructing a KNN graph and running the Louvain algorithm. Cell type assignments were predicted using Azimuth with the human kidney reference dataset. The snATAC-seq data was processed with ArchR v1.0.1, which performed dimensionality reduction and clustering. Cluster identities defined in the Seurat workflow were cross-annotated to the ATAT-seq data, and open peaks were identified within these cell-type clusters using MACS3 v3.0.1. Identification of marker peaks, motif enrichment, and motif enrichment deviations were inferred utilizing ArchR’s getmarkerFeatures, peakAnnoEnrichment, and addMotifAnnotations functions, respectively. Results Motif enrichment analysis revealed canonical cancer-associated transcription factor (TF) dysregulation in tumor cells (nephron loop-like), including enrichment of HNF1B, SMARCC1, FOSL1, and JUND. Interestingly, HNF and SMARCC were also enriched in the lymphoid compartment, indicating possible crosstalk between these two cell types. Previous work has investigated the involvement of the HNF1 in ccRCC and in chromophobe RCC for prognosis,and we plan on investigating the relationship of these enrichments to tumor grade, stage and outcome.[1] Additional TF enrichments included BCL families and ELF within the myeloid compartment, and vascularization related TF families in endothelial cell types including enrichment of various ETS TFs. Investigation of cell-cell interactions between immune and endothelial cell populations as well as between tumor and normal adjacent tissues will be performed to further disentangle the role differentially accessable chromatin plays in ccRCC. [1] Buchner et al., “Downregulation of HNF-1B in Renal Cell Carcinoma Is Associated With Tumor Progression and Poor Prognosis.” Conclusions Our multi-omic approach has several advantages. First, it enables the characterization of cellular heterogeneity within the tumor microenvironment, identifying cell populations and their contributions to disease progression. Secondly, by integrating transcriptomic and epigenomic data, we gain insights into the dynamic interplay between gene expression and chromatin accessibility, uncovering potential therapeutic vulnerabilities and biomarkers for personalized treatment strategies. DOD CDMRP Funding: yes

Sequences within and upstream of the mouse Ets1 gene drive high level expression in B cells, but are not sufficient for consistent expression in T cells.

The levels of transcription factor Ets1 are high in resting B and T cells, but are downregulated by signaling through antigen receptors and Toll-like receptors (TLRs). Loss of Ets1 in mice leads to excessive immune cell activation and development of an autoimmune syndrome and reduced Ets1 expression has been observed in human PBMCs in the context of autoimmune diseases. In B cells, Ets1 serves to prevent premature activation and differentiation to antibody-secreting cells. Given these important roles for Ets1 in the immune response, stringent control of Ets1 gene expression levels is required for homeostasis. However, the genetic regulatory elements that control expression of the Ets1 gene remain relatively unknown. Here we identify a topologically-associating domain (TAD) in the chromatin of B cells that includes the mouse Ets1 gene locus and describe an interaction hub that extends over 100 kb upstream and into the gene body. Additionally, we compile epigenetic datasets to find several putative regulatory elements within the interaction hub by identifying regions of high DNA accessibility and enrichment of active enhancer histone marks. Using reporter constructs, we determine that DNA sequences within this interaction hub are sufficient to direct reporter gene expression in lymphoid tissues of transgenic mice. Further analysis indicates that the reporter construct drives faithful expression of the reporter gene in mouse B cells, but variegated expression in T cells, suggesting the existence of T cell regulatory elements outside this region. To investigate how the downregulation of Ets1 transcription is associated with alterations in the epigenetic landscape of stimulated B cells, we performed ATAC-seq in resting and BCR-stimulated primary B cells and identified four regions within and upstream of the Ets1 locus that undergo changes in chromatin accessibility that correlate to Ets1 gene expression. Interestingly, functional analysis of several putative Ets1 regulatory elements using luciferase constructs suggested a high level of functional redundancy. Taken together our studies reveal a complex network of regulatory elements and transcription factors that coordinate the B cell-specific expression of Ets1.