Applying Bioinformatics to Research in Immune, Muscle, and Bone Diseases

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

Investigators

Linked publications, trials & patents

Abstract

The BMDS projects are summarized below: - Epigenetic Regulation of Muscle Stem Cells in Aging-Associated Fibrosis and Sarcopenia This study investigates how aging-associated changes in muscle stem cells (MuSCs) contribute to fibrosis in sarcopenia. Using aged and Ezh2-/- mouse models, co-culture assays, and chromatin profiling, we found that aged MuSCs promote fibro-adipogenic progenitor (FAP) proliferation and fibrogenic differentiation through the secretion of IL-6 and Spp1. Loss of the H3K27me3 repressive mark at the NF-kB locus led to enhanced NF-kB activity and subsequent cytokine activation. Blocking IL-6 and Spp1 signaling reduced FAP expansion and fibrosis, identifying MuSC-driven epigenetic dysregulation as a key contributor to age-related muscle degeneration. Unbiased, genome-wide H3K27me3 CUT&RUN analysis further confirmed that the Nfkb1 locus is demethylated in aged MuSCs compared to adult cells, while increased H3K27me3 was observed at the Hist1h4h cluster. These results highlight gene- and region-specific alterations in H3K27 methylation in aged MuSCs, consistent with the upregulation of multiple Polycomb target genes despite continued expression of Ezh2. This work has been accepted for publication in Nature Aging. - Modulation of Murine Lupus by Aconitate Decarboxylase 1 Mitochondrial synthesis of itaconate in myeloid cells, including monocytes and macrophages, is mediated by the Krebs cycle enzyme Aconitate Decarboxylase 1 (Acod1). To investigate Acod1’s role in systemic lupus erythematosus (SLE), we analyzed bulk RNA-seq data to assess the impact of endogenous itaconate modulation on disease development and severity in the Imiquimod (IMQ)-induced murine lupus model. Our analysis demonstrated Acod1 induction following IMQ treatment, with enrichment in interferon signaling pathways and interferon-responsive genes. This study has been published in The Journal of Immunology. - Reprogramming of Epidermal Keratinocytes by PITX1 We applied the CUT&Tag analysis pipeline to study the transcription factor PITX1, characterizing its genome-wide distribution in both wild-type (WT) and PITX1 transgenic mice. This approach enabled us to investigate how PITX1 reprograms epidermal keratinocytes and contributes to enhanced cutaneous wound healing. This work has been published in JCI Insight. - Gene Regulation in ILC2 and Th2 Lymphocytes To investigate the mechanisms underlying the distinct transcriptional programs of innate (ILC2) and adaptive (Th2) lymphocytes, we employed chromosome conformation capture, bulk RNA-seq, and ATAC-seq to analyze the structural and functional dynamics of the type 2 cytokine locus before and after activation. Our findings reveal that remodeling of the Il4–Il13–Il5 locus drives selective gene expression in these cells. This work has been published in Nature Immunology - Transcriptional Regulation of CCL21 During Inflammation CCL21 plays a critical role in lymphocyte entry from the bloodstream into lymph nodes during homeostasis; however, its expression is downregulated during inflammation. Using our ATAC-seq analysis pipeline, we examined relevant datasets and contributed to motif enrichment and transcription factor footprinting analyses. This work identified SpiB and Smad2/3 as key transcription factors involved in the repressive regulation of CCL21. This study has been published in Cell. - Role of Activating STAT1 Mutations in Innate Lymphocyte Programming This project investigates how gain-of-function (GOF) mutations in STAT1 impair antiviral immunity, using conditional knock-in mouse models and multi-platform next-generation sequencing (NGS) approaches, including bulk RNA-seq, scMultiome, CITE-seq, and CUT&Tag. These analyses revealed defective early IFN-gamma production, skewed ISGF3-driven transcriptional programs, and lethal multi-cytokine responses during viral infection. CUT&Tag profiling showed that, despite normal STAT4 phosphorylation, Stat1-T385M/+ cells exhibited fewer STAT4 peaks overall and reduced STAT4 occupancy at the Ifng locus. In contrast, STAT1 peaks increased genome-wide without enhanced binding at Ifng. This diminished STAT4 recruitment provides a mechanistic explanation for the impaired IFN-gamma production in Stat1-T385M/+ mice. This study has been published in Science Immunology. - Deciphering Transcriptional Regulation in Diffuse Large B Cell Lymphoma (DLBCL) Using Genome-Wide CRISPR/Cas9 Screens To better understand gene regulation in DLBCL, the most common aggressive lymphoma, we performed CRISPR/Cas9 screens targeting 47 super-enhancer–controlled genes critical to B cell biology in a DLBCL cell line. The screens identified approximately 1,500 gene activators and repressors, along with 884 dual-function regulators, revealing a complex, multilayered regulatory landscape in DLBCL. These findings provide valuable insights into disease mechanisms and highlight potential therapeutic targets. A global analysis revealed enrichment of mTOR signaling–related genes, with Foxo implicated as a contributing factor. The CRISPR library also proved useful for inhibitor screening. A manuscript detailing this work has been accepted by Cell Reports. - Transcriptomic Alterations in Colon Cancer Cells Induced by Doxorubicin The transcriptomic changes induced by doxorubicin (DOX), a widely used chemotherapeutic agent, were investigated in human colon cancer cells. Using single-cell RNA sequencing (scRNA-seq), distinct cell populations and their transcriptional profiles were identified following subtoxic DOX treatment. This revealed cell clusters characterized by differential expression of genes involved in cell cycle regulation and interferon (IFN) signaling. In contrast, DOX-persistent proliferating cells exhibited upregulation of genes activated by the unphosphorylated form of the ISGF3 (U-ISGF3) transcription factor complex. Furthermore, gene co-expression network analysis showed that changes in network architecture, rather than individual gene expression levels, may play a key role in determining cancer cell fate after chemotherapy. This study has been published in Neoplasia. - PD-1 Regulation of T Cells in Chronic Infection and Cancer This study used scRNA-seq to dissect the transcriptional and functional roles of PD-1 in both CD8+ and regulatory T (Treg) cells. In chronic infection, PD-1+ CD8 T cells were found to exist in two major states: progenitor-like and terminally exhausted, with Tox and TCF1 as key regulators. In Treg cells, PD-1 deficiency unexpectedly enhanced suppressive function via upregulation of a compensatory network of coinhibitory receptors, notably CD30. Mechanistically, PD-1 loss increased STAT5 signaling, which in turn induced CD30 expression and amplified Treg-mediated suppression in the tumor microenvironment. These findings uncover PD-1 as a critical checkpoint that modulates both effector and regulatory T cell states, with potential implications for optimizing immunotherapy in chronic infection and cancer. This study has been published in Nature Immunology. - Study on COVID-19 mRNA Vaccine Response in Autoimmune Patients This study investigates immune responses to COVID-19 mRNA vaccination in patients with autoimmune diseases using a multi-omic approach, including bulk RNA-seq, cytokine profiling, immune cell analysis, vascular assessments, and humoral response measurements. RNA-seq data from over 200 patients and healthy controls were analyzed using multiple statistical and computational methods, including ANOVA, paired t-tests, mixed linear modeling, PCA loading, and Random Forest modeling. Both supervised and unsupervised analyses of the RNA-seq data identified two distinct response groups, defined by IFN-gamma expression and a 27-gene interferon-stimulated gene (ISG) composite score. Pathway enrichment analysis of genes distinguishing the two groups revealed activation of immune and antiviral pathways, including interferon and NF-kB signaling, cytokine networks, antigen processing, and SARS-CoV-2–related responses. The 27-ISG signature may serve as a biomarker to stratify vaccine responses and guide personalized vaccination strategies for patients with autoimmune diseases. Ongoing work focuses on integrating molecular findings with clinical outcome measures across platforms. - 3D Modeling of JDM-Associated Peptides Bound to MHC Class II DRB1 Alleles This project aims to model how peptides associated with Juvenile Dermatomyositis (JDM) bind to specific HLA-DRB1 alleles of MHC class II molecules, with the goal of identifying allele-specific binding features. Using genetic data from JDM susceptibility haplotypes, we focused on four DRB1 alleles shown to present relevant peptides. Peptide–MHC binding was modeled using the AlphaFold2-based AlphaPulldown package. Each peptide was scanned using a sliding 9-amino acid window to identify the optimal binding core (anchor) for each DRB1 allele, determined by interface Predicted Aligned Error (PAE) values from the structural models. Peptide sequences were aligned in two ways: Anchor-based alignment, positioning each peptide by its predicted optimal binding core, and Structure-based alignment, using 3D superimposition of peptide-MHC complex models. Ongoing work includes a detailed analysis of the multiple sequence alignments for each DRB1 allele to identify sequence and structural features of peptide residues bound in the four key pockets of the MHC class II binding groove. These findings aim to advance our understanding of autoimmune peptide presentation in JDM. - Characterizing U12-Type Splicing Alterations in Neutrophils from SLE Patients This study investigates splicing anomalies in U12-type intron-containing genes in the context of systemic lupus erythematosus (SLE). Unlike the more prevalent U2 spliceosome, the U12 spliceosome is a rare but highly conserved RNA splicing system in vertebrates. Disruptions in U12-type splicing may contribute to increased pathogenic risk in SLE. We applied an in-house Snakemake pipeline to process RNA-seq data from 27 patients, including peripheral blood mononuclear cells (PBMCs), low-density granulocytes (LDGs), and normal-density granulocytes (NDGs) derived from both SLE patients and healthy controls. RNA splicing patterns were systematically analyzed, revealing notable alterations in U12-type splicing events, particularly in LDGs. These findings may point to previously unrecognized mechanisms contributing to disease pathogenesis in SLE. - Investigating the Role of SWT1 in RNA Splicing and Pathogenesis of cSLE Systemic lupus erythematosus (SLE) is a systemic autoimmune disorder that tends to be more severe in children than in adults. Due to the earlier onset and increased severity in childhood-onset SLE (cSLE), identifying genetic associations in cSLE may provide critical insights into disease pathogenesis. In this study, we identified a SWT1 variant in a child with SLE. RNA-seq analysis revealed that SWT1 expression levels were negatively correlated with exon–intron junction reads, suggesting a possible role in RNA splicing regulation. We are currently conducting a systematic analysis of SWT1 to understand its role in RNA splicing and to identify specific splicing patterns associated with different experimental conditions. This study may uncover novel mechanisms of RNA dysregulation in cSLE and contribute to the identification of potential molecular targets for diagnosis and therapy. - Cell Type-Dependent Transcriptional Response to Glucocorticoids in SLE Patients This study aimed to investigate the cell type–specific transcriptional responses to glucocorticoids in patients with SLE. A dedicated RNA-seq pipeline was developed to analyze control-spiked transcriptomic data collected from patients at three time points following drug administration. We implemented a customized, automated workflow for quality control, read mapping, gene quantification, and differential expression analysis across more than 2,000 samples from both patients and healthy controls. Understanding the cell-specific effects of glucocorticoids in SLE may help refine treatment strategies and identify biomarkers for therapeutic responsiveness. - Benchmarking Third-Generation Tools for Isoform and Gene Fusion Detection Several vendor- and community-developed tools are available for analyzing long-read transcriptomic data; however, their accuracy and performance remain to be systematically evaluated. The primary goal of this study is to identify the most reliable tools and develop an automated workflow for large-scale processing and analysis. We are benchmarking two dozen tools for isoform detection and gene fusion analysis, with plans to integrate the top-performing tools into Nextflow pipelines for reproducible and scalable use. This work is essential for advancing transcriptome analysis in biomedical research, enabling more accurate detection of alternative splicing and gene fusions -- key features in cancer, rare diseases, and immune disorders. - Gene Expression Studies of Arthritis Patients After Stopping Drug Treatment This project aims to identify gene expression signatures that predict disease relapse or sustained remission in arthritis patients following treatment cessation. Gene expression profiles from peripheral blood mononuclear cell (PBMC) samples of 101 arthritis patients treated with three different drugs were examined using RNA-seq. Samples were collected at multiple time points, including at the time of treatment cessation and three months afterward. These gene expression data were used to develop computational models to predict patient outcomes, specifically, whether they experienced disease flares or remained in remission three months after stopping therapy. Promising models have been developed, with prediction accuracies exceeding 90%. These models are currently under further analysis to identify potential biomarkers. In parallel, functional module–based analyses of the gene expression data are being conducted. This study may enable the development of clinically useful biomarkers to guide personalized treatment strategies and reduce unnecessary long-term immunosuppression in arthritis patients. - Investigating Tubulin Regulation in Muscular Dystrophy This project aims to understand the regulation of tubulin protein expression in the context of Muscular Dystrophy (MD), a debilitating and often fatal muscle disease caused by mutations or absence of dystrophin. The MDX mouse model, which harbors a natural mutation in the Dmd gene, was used to study how tubulin expression differs between healthy (WT) and dystrophic muscle. Tubulins, which are highly conserved across eukaryotes, displayed distinct isotype-specific expression patterns across different muscle types at the protein level, though these patterns were not well correlated with mRNA abundance. To investigate this discrepancy, we analyzed bulk RNA-seq and mass spectrometry–based proteomics data from 56 muscle samples. We assessed gene–protein correlations and performed detailed transcriptomic analysis of genes showing discordant expression, aiming to uncover post-transcriptional or translational regulatory mechanisms governing tubulin expression in muscle health and disease. - Gene Expression Profiling of IL-10–Stimulated NK Cells in STAT4 Knockout and Wild-Type Mice This project aims to characterize the global transcriptional response of natural killer (NK) cells to IL-10 stimulation and to identify STAT4-dependent genes involved in this response. Using an in-house data processing pipeline, we analyzed gene expression profiles from 18 samples derived from wild-type and STAT4 knockout mice. This work established a robust baseline for identifying STAT4-regulated targets under IL-10 stimulation, supporting future functional validation through flow cytometry (FC). To extend these findings, we plan to replicate the study using human NK cell lines, enabling translational relevance. This study provides valuable insights into the immunoregulatory role of STAT4 under anti-inflammatory cytokine stimulation and lays the groundwork for future investigations into NK cell–mediated immune modulation. - Characterizing HIV RRE Region Mutations and Their Impact on Viral RNA Export This project investigates mutations in the Rev Response Element (RRE) region of the HIV genome, a critical regulatory element required for nuclear export of full-length viral RNA during HIV replication. The Rev protein binds to the RRE and recruits host export factors to mediate this essential step. Disrupting this process presents a potential therapeutic strategy for HIV treatment. We analyzed longitudinal HIV genomic data (N=117) from six patients, both untreated and treated, to identify evolving mutations in the RRE region and assess their potential structural and functional consequences. This analysis was supported by extensive literature review and expert consultation to establish a foundational understanding of RRE sequence variability. Ongoing work involves expanding the dataset by incorporating additional HIV sequences from patients with diverse antiretroviral therapy (ART) exposure histories. This study aims to inform novel therapeutic approaches targeting HIV RNA export mechanisms. - Uncovering the Pathogenic Role of IgA Autoantibodies in Hidradenitis Suppurativa (HS) Lesions The pathogenic role of IgA autoantibodies in hidradenitis suppurativa (HS) has been investigated through multi-modal profiling, including qPCR, immunofluorescence, western blotting, RNA-seq, and autoantigen analysis. Elevated expression of IGHA1, IGHA2, and J chain was observed in HS lesions, with plasma cells and B cells identified as the primary sources of IgA. IgA autoantibodies were found to bind macrophages, promote neutrophil extracellular trap (NET) formation, activate dendritic cells, and drive fibroblast activation, collectively amplifying type I interferon, Th17, and profibrotic pathways. To assess the functional impact, primary human M2-like macrophages were treated with IgA purified from HS lesions, control IgA, or vehicle, followed by RNA-seq analysis. Treatment with HS-derived IgA led to substantial upregulation of genes involved in inflammation, leukocyte chemotaxis, fibroblast activation, and the NLRP3 inflammasome pathway. These findings suggest that IgA autoantibodies act as key mediators of inflammation and fibrosis in hidradenitis suppurativa. - Multiomic and Spatial Transcriptomic Profiling of Lupus Nephritis Progression in a Murine Model To elucidate the molecular pathways and tissue niches that govern lupus nephritis progression, temporal bulk RNA-seq was performed on NZB/W F1 mouse kidneys collected at 10, 20, and 30 weeks, capturing stage-specific transcriptional changes. Spatial transcriptomics was also applied to 30-week kidneys, enabling these signatures to be mapped to precise intrarenal locations. Integration of the two datasets revealed late-stage activation of inflammatory (TNFR1, CCR5) and oxidative stress (NRF2/ARE) pathways, which were localized to immune cell–rich clusters within the glomerular and interstitial compartments. This approach delineated both the timing and spatial distribution of key drivers of renal damage in lupus nephritis. - Impact of STAT3 Loss-of-Function in Vascular Endothelial Cells This project explores how loss of STAT3 function in vascular endothelial cells (VECs) disrupts gene regulatory networks and triggers inflammatory responses, using multiome sequencing to simultaneously profile gene expression and chromatin accessibility. The Cell Ranger pipeline was used to generate metadata, followed by data cleaning, quality control, and cell cluster annotation. Differential analyses of gene expression, chromatin accessibility, and gene activity were conducted by comparing wild-type and STAT3 knockout VECs to identify dysregulated pathways. Pathway enrichment analysis was also performed to further interpret the results. - Investigation of African Ancestry–Specific Genetic Regulation of TGFB3 in Systemic Sclerosis–Associated Fibrosis This project aims to uncover why systemic sclerosis (SSc) manifests more aggressively in African American (AA) patients by identifying African ancestry–specific genetic variants that contribute to heightened fibrosis. An AA-specific genome-wide association study (GWAS) identified a unique intronic enhancer variant in the IFT43/TGFB3 locus. ATAC-seq in iPSC-derived fibroblasts homozygous for this allele revealed increased chromatin accessibility at the locus, and RNA-seq confirmed elevated TGFB3 expression. Emerging motif and EMSA data suggest allele-specific binding by the transcription factor GATA2. Given that recombinant TGFB3 upregulates profibrotic genes such as SERPINE1, COL1A1, and COMP, this variant likely contributes to the severe fibrotic phenotype seen in AA patients. These findings support the potential of TGFB3 isoform–specific inhibition as a promising therapeutic strategy in ancestry-tailored treatment approaches for systemic sclerosis. - Neutrophil Extracellular Trap–Induced Mesangial Remodeling in Lupus Nephritis In kidney specimens from NZB/W F1 lupus-prone mice and from patients with proliferative lupus nephritis, neutrophils and their extracellular traps (NETs) were observed to accumulate predominantly within glomerular mesangial regions as proteinuria progressed. Purified lupus NETs were rapidly internalized by primary human mesangial cells, triggering an extracellular matrix–remodeling transcriptional program characterized by marked upregulation of MMP-2 expression and enzymatic activity. This led to degradation of collagen IV and erosion of the mesangial matrix. Bulk RNA-seq was performed to compare the transcriptomes of mesangial cells exposed to NETs versus unstimulated controls at multiple time points, further delineating NET-driven changes in gene expression. These findings identify NET formation and MMP-2 activity as actionable therapeutic targets in lupus nephritis. - Investigating the Role of Paxbp1 in ILC2 Homeostasis and RNA Splicing Regulation This project aims to identify key regulators of tissue-resident ILC2 maintenance and activation. Single-cell RNA-seq of helminth-infected mice highlighted Paxbp1 as a top candidate. Conditional deletion of Paxbp1 in ILC2s resulted in normal bone marrow precursors but a marked reduction in mature lung ILC2s and impaired IL-33-driven expansion, suggesting a defect in proliferation and/or survival. ATAC-seq comparing Paxbp1-deficient and wild-type ILC2s revealed decreased chromatin accessibility at promoters involved in RNA transcription, splicing, translation, and cell-cycle regulation. Proteomic analysis further mapped Paxbp1 to spliceosome-associated components, suggesting a role for Paxbp1 as an RNA metabolic scaffold. Ongoing work is evaluating how the Paxbp1-spliceosome axis influences mRNA maturation. - Defining the Role of Dyrk1a in Modulating T Cell Receptor Signaling Strength To investigate the role of Dual-specificity tyrosine-regulated kinase 1A (Dyrk1a) in T cell receptor (TCR) signaling, bulk RNA-seq was conducted on naive CD4⁺ T cells stimulated in vitro with CD3 and/or CD28 to assess differential transcriptomic responses. Conditional Dyrk1a knockout (KO) mice, generated at various stages of thymocyte development, are being utilized to further define the kinase’s role in T cell biology. Ongoing multi-omics studies aim to identify Dyrk1a substrates and clarify the signaling pathways it modulates. - Role of Sex Hormones in Neutrophil Development and Autoimmunity To investigate the impact of sex hormones on neutrophil biology in the context of autoimmunity, we analyzed single-cell RNA sequencing (scRNA-seq) data and identified sex-specific differences in neutrophil bioenergetics across tissues, including the spleen, bone marrow, and blood in both mice and humans. In addition, bulk RNA-seq, bulk ATAC-seq, and estrogen receptor alpha (ER-alpha) CUT&RUN were performed to define the estrogen-dependent regulome governing neutrophil development and function. - Evaluating the Impact of DHX36 Variants in cSLE Patients This project investigates the functional consequences of DHX36 variants identified in patients with childhood-onset systemic lupus erythematosus (cSLE). Gene expression profiling was performed using bulk RNA sequencing in doxycycline-inducible HEK293 cells expressing either wild-type DHX36 or its disease-associated variants. Impaired DHX36 function is hypothesized to reduce G-quadruplex (G4) unwinding in stress granules, leading to chronic type I interferon (IFN) release and inflammation. The RNA-seq analysis includes data demultiplexing, quality control, read alignment, and gene expression quantification, followed by differential expression and pathway enrichment analyses. The focus is primarily on interferon-stimulated genes (ISGs) and G4-enriched gene sets. - Impact of Stretching on Tumor Growth and Metastasis This study explores the mechanisms underlying tumor size reduction in the MET-1 orthotopic mammary tumor model in FVB mice. Using recently developed Xenium spatial transcriptomics platform for single cell analysis, we mapped the tumor microenvironment to investigate whether tumor shrinkage results from increased immune cell infiltration or from biomechanical stretching-induced changes in the extracellular matrix (ECM) that facilitate immune cell access. The analysis workflow begins with Xenium data preprocessing and quality control, followed by cell type annotation and spatial mapping. Immune and stromal cell populations are quantified, key gene expression programs are identified, and spatial interaction metrics are computed to characterize in situ cellular organization. Differential expression and pathway enrichment analyses will then be conducted to integrate molecular insights with phenotypic data and explain the observed tumor regression. - Unraveling Cochlear Bone Tonotopy Through Transcriptomic Profiling This project investigates how cochlear bone quality varies along the tonotopic (frequency) axis and how these region-specific properties are biologically regulated. Bulk RNA-seq is used to compare apical (low-frequency) and basal (high-frequency) cochlear bone to identify differential gene expression and signaling pathways. The analysis workflow includes quality control, principal component analysis (PCA), differentially expressed gene (DEG) analysis, and pathway enrichment analysis. These transcriptomic data will directly complement spatial proteomics from the same regions, together providing a comprehensive, multi-layered view of the cellular and molecular programs underlying cochlear bone specialization. - Effects of Short-Term Exercise on Gene Expression and Bone Quality in Aging Bone fragility increases with age, yet many fractures occur in individuals with normal bone mineral density (BMD), highlighting the importance of bone quality (BQ). BQ depends on matrix composition, structure, and material properties - features partially maintained by osteocytes through perilacunar/canalicular remodeling (PLR), a process that declines with aging. This project investigates whether short-term exercise can restore PLR-related gene expression toward a more youthful profile. Bulk RNA-seq was performed to identify differentially expressed genes (DEGs) and pathway alterations across key comparisons: young vs. aged bone, and young/aged bone vs. aged bone following short-term exercise at multiple time points. The analysis workflow includes quality control, principal component analysis (PCA), DEG analysis, and pathway enrichment analysis. - Epigenetic Analysis in Systemic Lupus Erythematosus (SLE) Using Oxford Nanopore (ONT) Long-Read Sequencing Technology Epigenetic events, such as X-chromosome inactivation, have long been suspected to play a role in SLE. We evaluated analysis methods for variant calling and methylation detection using ONT long-read sequencing data. We developed and implemented a bioinformatics workflow to analyze a cohort of more than 50 SLE samples sequenced with ONT. Additionally, we provided guidance on the ongoing cohort analysis to help uncover the role of X-chromosome inactivation in SLE. - Investigation of Complement Component 4 (C4) Genes Using ONT Long Reads The human C4 gene loci are critical regulators of immunity. Due to the high complexity of the C4 genomic regions, standard mutation analysis methods often fail to accurately genotype C4 genes. We investigated various tools and methods with the goal of developing a custom pipeline for C4 genotyping. A promising workflow that combines ONT long reads with hifiasm-based targeted diploid assembly is currently under active development. - Structural Variant Analysis in Whole Genome Sequencing (WGS) Standard WGS data analysis primarily targets small variants up to 50 base pairs in size. However, larger variants, such as structural variants (SVs) and copy number variants (CNVs), are increasingly recognized as important contributors to disease. Several publicly available tools now support SV/CNV detection. We have evaluated and tested most of the commonly used software tools and have implemented a robust SV/CNV analysis pipeline. This pipeline has been successfully applied to detect both somatic and germline variants. Most notably, recurrent somatic SVs in the SYK gene have been discovered in patients with Hypereosinophilic Syndrome (HES) with vasculitis. Ongoing work is applying this pipeline to a cohort of patients with autoinflammatory diseases. - Mutation Analysis in NIAMS Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS) Cohorts The goal of this project is to discover the genetic causes of disease by analyzing patient WES and WGS data. We processed over 400 samples and identified quality control (QC) issues. Joint variant calling was performed, followed by either family-based or somatic mutation analysis. Additional analyses included rare variant enrichment, targeted gene analysis, and HLA typing. - Role of the IDR Region of NIPBL in Genome Architecture and Transcription NIPBL, a protein comprising over 2,800 amino acid residues, displays a distinctive structure: its N-terminal half contains an intrinsically disordered region (IDR), while the C-terminal half forms a hook-like structure. Deletion of the IDR does not affect loop formation or domain interactions within genome architecture; however, it does result in compartmental changes in specific genomic regions, typically gene deserts. The IDR appears to play a key role in promoting the formation of NIPBL hubs at transcriptionally active sites, thereby facilitating the preferential loading of cohesin at active promoters and enhancers. Our global mapping of the NIPBL interactome has revealed a functional link between the IDR and the transcription machinery. We are also leveraging AlphaFold2 to predict potential protein interactors of the IDR. - Bursting Kinetics and Regulatory Effects of STAT4 Activating Variants To investigate the transcriptional bursting behavior of STAT4 activating variants, we employed computational modeling of single-cell transcriptomes, aiming to uncover regulatory mechanisms without the need for fluorescent labeling of individual genes. We conducted a comprehensive set of genomic and epigenomic assays, including scRNA-seq, bulk mRNA-seq, ChIP-seq, ATAC-seq, and CUT&Tag, to explore the functional relationship between transcriptional bursting and gene regulation. To further elucidate variant-specific mechanisms, we plan to incorporate Hi-C (High-throughput chromosome conformation capture), single-molecule tracking, and proteomics to identify interacting partners unique to these STAT4 variants. - Transcriptional Regulation of Dyrk1A in Mouse CD4 and CD8 T Cells Using a conditional knockout (KO) mouse model targeting the Dyrk1A gene, we aim to elucidate its role as a transcription factor and/or kinase in T cell regulation. By analyzing time-course RNA expression patterns, alternative splicing events, and changes in chromatin accessibility following stimulation, we seek to identify affected pathways and uncover the key mechanisms regulated by Dyrk1A. We performed scRNA-seq, scTCR-seq, and scATAC-seq on thymic T cells, as well as bulk RNA-seq and ATAC-seq on splenic CD4 and CD8 T cells under IL-12 activation conditions. Ongoing work includes generating additional replicates for RNA-seq and ATAC-seq, along with planned ChIP-seq and western blot experiments to validate our findings. - Roles of 53BP1 in DNA Replication and DNA Double-Strand Break Repair Our previous work showed that cohesin prevents interactions between DNA double-strand break (DSB) domains. To investigate what causes damaged genomic regions to interact, we generated deletion mutants of DNA repair factors involved in non-homologous end joining. In 53BP1-deficient cells, we observed that cohesin depletion did not result in the diffusive and interactive foci seen in wild-type cells. To elucidate the functional mechanisms and key domains of 53BP1, we plan to perform additional microscopic analyses, high-throughput genome translocation sequencing (HTGTS), Hi-C, and ChIP-seq. We are currently troubleshooting technical challenges with the Hi-C experiments. - Characterizing Endothelial Cell Populations in PBMCs from Takayasu Arteritis (TAK) Patients Using flow cytometry, we found that CD31+ terminally differentiated endothelial cells (CECs) from Takayasu arteritis (TAK) patients with active disease exhibited a reduced capacity to form tubular structures in vitro. Additionally, TNF-alpha treatment demonstrated a pronounced anti-angiogenic effect on these cells. To further investigate rare endothelial cell populations in peripheral blood mononuclear cells (PBMCs) from patients with Takayasu arteritis (TAK), we performed single-cell RNA sequencing (scRNA-seq) with and without CD45-negative selection to enrich for non-immune cells. While a distinct endothelial cell cluster has not yet been identified, our findings are guiding the refinement of our approach. We now plan to culture the CD45-depleted PBMC fraction over several passages, followed by bulk RNA-seq and/or scRNA-seq, to advance the characterization of endothelial populations.