Assay For Transposase-accessible Chromatin Sequencing Research Articles

BACH2 regulates diversification of regulatory and proinflammatory chromatin states in TH17 cells.

Interleukin-17 (IL-17)-producing helper T (TH17) cells are heterogenous and consist of nonpathogenic TH17 (npTH17) cells that contribute to tissue homeostasis and pathogenic TH17 (pTH17) cells that mediate tissue inflammation. Here, we characterize regulatory pathways underlying TH17 heterogeneity and discover substantial differences in the chromatin landscape of npTH17 and pTH17 cells both in vitro and in vivo. Compared to other CD4+ T cell subsets, npTH17 cells share accessible chromatin configurations with regulatory T cells, whereas pTH17 cells exhibit features of both npTH17 cells and type 1 helper T (TH1) cells. Integrating single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq), we infer self-reinforcing and mutually exclusive regulatory networks controlling different cell states and predicted transcription factors regulating TH17 cell pathogenicity. We validate that BACH2 promotes immunomodulatory npTH17 programs and restrains proinflammatory TH1-like programs in TH17 cells in vitro and in vivo. Furthermore, human genetics implicate BACH2 in multiple sclerosis. Overall, our work identifies regulators of TH17 heterogeneity as potential targets to mitigate autoimmunity.

Genome-wide analysis of histone modifications can contribute to the identification of candidate cis-regulatory regions in the threespine stickleback fish

BackgroundCis-regulatory mutations often underlie phenotypic evolution. However, because identifying the locations of promoters and enhancers in non-coding regions is challenging, we have fewer examples of identified causative cis-regulatory mutations that underlie naturally occurring phenotypic variations than of causative amino acid-altering mutations. Because cis-regulatory elements have epigenetic marks of specific histone modifications, we can detect cis-regulatory elements by mapping and analyzing them. Here, we investigated histone modifications and chromatin accessibility with cleavage under targets and tagmentation (CUT&Tag) and assay for transposase-accessible chromatin-sequencing (ATAC-seq).ResultsUsing the threespine stickleback (Gasterosteus aculeatus) as a model, we confirmed that the genes for which nearby regions showed active marks, such as H3K4me1, H3K4me3, and high chromatin accessibility, were highly expressed. In contrast, the expression levels of genes for which nearby regions showed repressive marks, such as H3K27me3, were reduced, suggesting that our chromatin analysis protocols overall worked well. Genomic regions with peaks of histone modifications showed higher nucleotide diversity within and between populations. By comparing gene expression in the gills of the marine and stream ecotypes, we identified several insertions and deletions (indels) with transposable element fragments in the candidate cis-regulatory regions.ConclusionsThus, mapping and analyzing histone modifications can help identify cis-regulatory elements and accelerate the identification of causative mutations in the non-coding regions underlying naturally occurring phenotypic variations.

Single cell regulatory architecture of human pancreatic islets suggests sex differences in β cell function and the pathogenesis of type 2 diabetes.

Type 2 and type 1 diabetes (T2D, T1D) exhibit sex differences in insulin secretion, the mechanisms of which are unknown. We examined sex differences in human pancreatic islets from 52 donors with and without T2D combining single cell RNA-seq (scRNA-seq), single nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), hormone secretion, and bioenergetics. In nondiabetic (ND) donors, sex differences in islet cells gene accessibility and expression predominantly involved sex chromosomes. Islets from T2D donors exhibited similar sex differences in sex chromosomes differentially expressed genes (DEGs), but also exhibited sex differences in autosomal genes. Comparing β cells from T2D vs. ND donors, gene enrichment of female β cells showed suppression in mitochondrial respiration, while male β cells exhibited suppressed insulin secretion. Thus, although sex differences in gene accessibility and expression of ND β cells predominantly affect sex chromosomes, the transition to T2D reveals sex differences in autosomes highlighting mitochondrial failure in females.

A single-cell chromatin accessibility dataset of human primed and naïve pluripotent stem cell-derived teratoma

Teratoma, due to its remarkable ability to differentiate into multiple cell lineages, is a valuable model for studying human embryonic development. The similarity of the gene expression and chromatin accessibility patterns in these cells to those observed in vivo further underscores its potential as a research tool. Notably, teratomas derived from human naïve (pre-implantation epiblast-like) pluripotent stem cells (PSCs) have larger embryonic cell diversity and contain extraembryonic lineages, making them more suitable to study developmental processes. However, the cell type-specific epigenetic profiles of naïve PSC teratomas have not been yet characterized. Using single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), we analyzed 66,384 cell profiles from five teratomas derived from human naïve PSCs and their post-implantation epiblast-like (primed) counterparts. We observed 17 distinct cell types from both embryonic and extraembryonic lineages, resembling the corresponding cell types in human fetal tissues. Additionally, we identified key transcription factors specific to different cell types. Our dataset provides a resource for investigating gene regulatory programs in a relevant model of human embryonic development.

Single cell regulatory architecture of human pancreatic islets suggests sex differences in β cell function and the pathogenesis of type 2 diabetes.

Biological sex affects the pathogenesis of type 2 and type 1 diabetes (T2D, T1D) including the development of β cell failure observed more often in males. The mechanisms that drive sex differences in β cell failure is unknown. Studying sex differences in islet regulation and function represent a unique avenue to understand the sex-specific heterogeneity in β cell failure in diabetes. Here, we examined sex and race differences in human pancreatic islets from up to 52 donors with and without T2D (including 37 donors from the Human Pancreas Analysis Program [HPAP] dataset) using an orthogonal series of experiments including single cell RNA-seq (scRNA-seq), single nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), dynamic hormone secretion, and bioenergetics. In cultured islets from nondiabetic (ND) donors, in the absence of the in vivo hormonal environment, sex differences in islet cell type gene accessibility and expression predominantly involved sex chromosomes. Of particular interest were sex differences in the X-linked KDM6A and Y-linked KDM5D chromatin remodelers in female and male islet cells respectively. Islets from T2D donors exhibited similar sex differences in differentially expressed genes (DEGs) from sex chromosomes. However, in contrast to islets from ND donors, islets from T2D donors exhibited major sex differences in DEGs from autosomes. Comparing β cells from T2D and ND donors revealed that females had more DEGs from autosomes compared to male β cells. Gene set enrichment analysis of female β cell DEGs showed a suppression of oxidative phosphorylation and electron transport chain pathways, while male β cell had suppressed insulin secretion pathways. Thus, although sex-specific differences in gene accessibility and expression of cultured ND human islets predominantly affect sex chromosome genes, major differences in autosomal gene expression between sexes appear during the transition to T2D and which highlight mitochondrial failure in female β cells.

Mapping cell diversity in human sporadic cerebral cavernous malformations

BackgroundCerebral cavernous malformation (CCM) is a low-flow, bleeding-prone vascular disease that can cause cerebral hemorrhage, seizure and neurological deficits. Its inheritance mode includes sporadic or autosomal dominant inheritance with incomplete penetrance, namely sporadic CCM (SCCM) and familial CCM. SCCM is featured by single lesion and single affection in a family. Among CCM patients especially SCCM, the pathogenesis of the corresponding phenotypes and pathological features or candidate genes have not been fully elucidated yet. MethodsHere, we performed in-depth single-cell RNA sequencing (scRNA-Seq) and bulk assay for transposase-accessible chromatin sequencing (ATAC-Seq) in SCCM and control patients. Further validation was conducted for the gene of interest using qPCR and RNA in situ hybridization (RNA FISH) techniques to provide further atlas and evidence for SCCM generative process. ResultsWe identified six cell types in the SCCM and control vessels and found that the expression of NEK1, RNPC3, FBRSL1, IQGAP2, MCUB, AP3B1, ESCO1, MYO9B and PVT1 were up-regulated in SCCM tissues. Among the six cell types, we found that compared with control conditions, PVT1 showed a rising peak which followed the pseudo-time axis in endothelial cell clusters of SCCM samples, while showed an increasing trend in smooth muscle cell clusters of SCCM samples. Further experiments indicated that, compared with the control vessels, PVT1 exhibited significantly elevated expression in SCCM samples. ConclusionIn SCCM conditions, We found that in the process of development from control to lesion conditions, PVT1 showed a rising peak in endothelial cells and showed an increasing trend in smooth muscle cells at the same time. Overall, there was a significantly elevated expression of NEK1, RNPC3, FBRSL1, IQGAP2, MCUB, AP3B1, ESCO1, MYO9B and PVT1 in SCCM specimens compared to control samples.

Remodeling ceramide homeostasis promotes functional maturation of human pluripotent stem cell-derived β cells

Human pluripotent stem cell-derived β cells (hPSC-β cells) show the potential to restore euglycemia. However, the immature functionality of hPSC-β cells has limited their efficacy in application. Here, by deciphering the continuous maturation process of hPSC-β cells post transplantation via single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), we show that functional maturation of hPSC-β cells is an orderly multistep process during which cells sequentially undergo metabolic adaption, removal of negative regulators of cell function, and establishment of a more specialized transcriptome and epigenome. Importantly, remodeling lipid metabolism, especially downregulating the metabolic activity of ceramides, the central hub of sphingolipid metabolism, is critical for β cell maturation. Limiting intracellular accumulation of ceramides in hPSC-β cells remarkably enhanced their function, as indicated by improvements in insulin processing and glucose-stimulated insulin secretion. In summary, our findings provide insights into the maturation of human pancreatic β cells and highlight the importance of ceramide homeostasis in function acquisition.

Abstract 1247: PPARsing epigenetic memory in intestinal stem cells: High-fat diet and oncogenic susceptibility

Abstract Diet has a profound impact on health and susceptibility to cancer. Adaptation to a Western diet is strongly associated with increased colorectal cancer (CRC) and obesity prevalence. This study demonstrates the epigenetic changes in intestinal stem cells (ISCs) induced by a pro-obesity high-fat Western diet (HFD) and the heightened risk of oncogenic transformation. A HFD induces a phenotype characterized by increased ISC frequency, proliferation, regenerative capacity, and ability to initiate adenomas. We hypothesized that a HFD generates lasting epigenetic alterations in ISCs thereby enhancing oncogenic susceptibility. We evaluated the chromatin landscape of intestinal stem cells (ISCs) and the reversibility of the epigenetic changes. Mice carrying a fluorescent reporter of the ISC marker Lgr5, were subjected to a control diet, HFD, or HFD with a return to control. We isolated ISCs by flow cytometry and used an assay for transposase-accessible chromatin sequencing (ATAC-seq) to identify differentially accessible genomic regions correlated with the ISC phenotype. We found that a HFD induces distinct changes in the chromatin landscape that persist after removal from the HFD. These accessible regions are associated with genes involved in lipid metabolism and are enriched with PPAR (Peroxisome Proliferator-Activated Receptor) binding motifs. We demonstrate that these lipid metabolism regulators, PPAR-delta and PPAR-alpha, are necessary for the majority of significant accessible regions. We further show that HFD-induced accessibility significantly correlates with early chromatin alterations seen by loss of tumor suppressor APC. Our data supports a mechanistic pathway for diet-induced epigenetic reprogramming due to PPAR activity and suggests that the HFD imposes a preneoplastic stem cell chromatin state that stokes the risk of oncogenic change. Citation Format: Dominic R. Saiz, Thomas Hartley-McDerrmott, Yesenia Barrera-Millan, Karla Fabiola Castro-Ochoa, Abhigyan Shukla, Matthew Torel, Miyeko D. Mana. PPARsing epigenetic memory in intestinal stem cells: High-fat diet and oncogenic susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1247.

Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells.

Induced pluripotent stem cell (iPSC)-based models are excellent platforms to understand blood development, and iPSC-derived blood cells have translational utility as clinical testing reagents and transfusable cell therapeutics. The advent and expansion of multiomics analysis, including but not limited to single nucleus RNA sequencing (snRNAseq) and Assay for Transposase-Accessible Chromatin sequencing (snATACseq), offers the potential to revolutionize our understanding of cell development. This includes developmental biology using in vitro hematopoietic models. However, it can be technically challenging to isolate intact nuclei from cultured or primary cells. Different cell types often require tailored nuclear preparations depending on cellular rigidity and content. These technical difficulties can limit data quality and act as a barrier to investigators interested in pursuing multiomics studies. Specimen cryopreservation is often necessary due to limitations with cell collection and/or processing, and frozen samples can present additional technical challenges for intact nuclear isolation. In this manuscript, we provide a detailed method to isolate high-quality nuclei from iPSC-derived cells at different stages of in vitro hematopoietic development for use in single-nucleus multiomics workflows. We have focused the method development on the isolation of nuclei from iPSC-derived adherent stromal/endothelial cells and non-adherent hematopoietic progenitor cells, as these represent very different cell types with regard to structural and cellular identity. The described troubleshooting steps limited nuclear clumping and debris, allowing the recovery of nuclei in sufficient quantity and quality for downstream analyses. Similar methods may be adapted to isolate nuclei from other cryopreserved cell types.

Variation of gene expression of fatty acid acyl CoA reductase associated with wax secretion of a scale insect, Ericerus pela, and identification of its regulation factors through the accessible chromatin analyses and yeast one-hybrid.

The Chinese white wax scale insect (CWWSI), Ericerus pela, can secret an amount of wax equivalent to their body weight. Previous studies demonstrated the fatty acyl-CoA reductase (far3) plays a pivotal role in wax secretion of CWWSI. The high expression of far3 is crucial for the massive wax secretion. However, the transcription regulation of far3 was not clear. To identify regulatory factors that control the expression of far3, the assay for transposase-accessible chromatin (ATAC) and yeast one-hybrid (Y1H) were carried out in this study. The ATAC sequencing of the CWWSI at the early wax-secretion stage ATAC-seq resulted in 22.75 GB raw data, generated 75,827,225 clean reads and revealed 142,771 peaks. There was one significant peak in the 3 kb upstream regulation regions. The peak sequence is located between -1000 and -670 bp upstream of the far3 transcription start site, spanning a length of 331 bp. This peak sequence served as bait for creating the pAbAi-peak recombinant vector, used in Y1H screenings to identify proteins interacting with far3 gene. The results indicate a successful CWWSI cDNA library construction with a capacity of 1.2 × 107 colony forming unit, a 95.8% recombination rate, and insert sizes between 1,000 and 2,000 bp. Self-activation tests established that 100 ng/mL of AbA effectively inhibited bait vector self-activation. Finally, a total of 88 positive clones were selected. After sequencing and removal of duplication, 63 unique clones were obtained from these screened colonies. By aligning the clone sequences with full-length transcriptome and genome of CWWSI, the full-length coding sequences of these clones were obtained. BlastX analysis identified a transcription factor, nuclear transcription factor Y beta, and two co-activators, cAMP-response-element-binding-protein-binding protein and WW domain binding protein 2. Reverse transcription quantitative polymerase chain reaction analysis confirmed that their expression patterns were consistent with the developmental stages preceding wax secretion and matched the wax secretion characteristics during ovulation periods. These results are beneficial for further research into the regulatory mechanisms of wax secretion of CWWSI.

Gene co-expression network analysis in areca floral organ and the potential role of the AcMADS17 and AcMADS23 in transgenic Arabidopsis

Areca catechu L., a monocot belonging to the palm family, is monoecious, with female and male flowers separately distributed on the same inflorescence. To discover the molecular mechanism of flower development in Areca, we sequenced different floral samples to generate tissue-specific transcriptomic profiles. We conducted a comparative analysis of the transcriptomic profiles of apical sections of the inflorescence with male flowers and the basal section of the inflorescence with female flowers. Based on the RNA sequencing dataset, we applied weighted gene co-expression network analysis (WGCNA) to identify sepal, petal, stamen, stigma and other specific modules as well as hub genes involved in specific floral organ development. The syntenic and expression patterns of AcMADS-box genes were analyzed in detail. Furthermore, we analyzed the open chromatin regions and transcription factor PI binding sites in male and female flowers by assay for transposase-accessible chromatin sequencing (ATAC-seq) assay. Heterologous expression revealed the important role of AcMADS17 and AcMADS23 in floral organ development. Our results provide a valuable genomic resource for the functional analysis of floral organ development in Areca.

Single-cell ATAC sequencing identifies sleepy macrophages during reciprocity of cytokines in L. major infection.

The hallmark characteristic of macrophages lies in their inherent plasticity, allowing them to adapt to dynamic microenvironments. Leishmania strategically modulates the phenotypic plasticity of macrophages, creating a favorable environment for intracellular survival and persistent infection through regulatory cytokine such as interleukin (IL)-10. Nevertheless, these effector cells can counteract infection by modulating crucial cytokines like IL-12 and key components involved in its production. Using sophisticated tool of single-cell assay for transposase accessible chromatin (ATAC) sequencing, we systematically examined the regulatory axis of IL-10 and IL-12 in a time-dependent manner during Leishmania major infection in macrophages Our analysis revealed the cellular heterogeneity post-infection with the regulators of IL-10 and IL-12, unveiling a reciprocal relationship between these cytokines. Notably, our significant findings highlighted the presence of sleepy macrophages and their pivotal role in mediating reciprocity between IL-10 and IL-12. To summarize, the roles of cytokine expression, transcription factors, cell cycle, and epigenetics of host cell machinery were vital in identification of sleepy macrophages, which is a transient state where transcription factors controlled the epigenetic remodeling and expression of genes involved in pro-inflammatory cytokine expression and recruitment of immune cells.IMPORTANCELeishmaniasis is an endemic affecting 99 countries and territories globally, as outlined in the 2022 World Health Organization report. The disease's severity is compounded by compromised host immune systems, emphasizing the pivotal role of the interplay between parasite and host immune factors in disease regulation. In instances of cutaneous leishmaniasis induced by L. major, macrophages function as sentinel cells. Our findings indicate that the plasticity and phenotype of macrophages can be modulated to express a cytokine profile involving IL-10 and IL-12, mediated by the regulation of transcription factors and their target genes post-L. major infection in macrophages. Employing sophisticated methodologies such as single-cell ATAC sequencing and computational genomics, we have identified a distinctive subset of macrophages termed "sleepy macrophages." These macrophages exhibit downregulated housekeeping genes while expressing a unique set of variable features. This data set constitutes a valuable resource for comprehending the intricate host-parasite interplay during L. major infection.

CTCF regulates hepatitis B virus cccDNA chromatin topology.

Hepatitis B Virus (HBV) is a small DNA virus that replicates via an episomal covalently closed circular DNA (cccDNA) that serves as the transcriptional template for viral mRNAs. The host protein, CCCTC-binding factor (CTCF), is a key regulator of cellular transcription by maintaining epigenetic boundaries, nucleosome phasing, stabilisation of long-range chromatin loops and directing alternative exon splicing. We previously reported that CTCF binds two conserved motifs within Enhancer I of the HBV genome and represses viral transcription, however, the underlying mechanisms were not identified. We show that CTCF depletion in cells harbouring cccDNA-like HBV molecules and in de novo infected cells resulted in an increase in spliced transcripts, which was most notable in the abundant SP1 spliced transcript. In contrast, depletion of CTCF in cell lines with integrated HBV DNA had no effect on the abundance of viral transcripts and in line with this observation there was limited evidence for CTCF binding to viral integrants, suggesting that CTCF-regulation of HBV transcription is specific to episomal cccDNA. Analysis of HBV chromatin topology by Assay for Transposase Accessible Chromatin Sequencing (ATAC-Seq) revealed an accessible region spanning Enhancers I and II and the basal core promoter (BCP). Mutating the CTCF binding sites within Enhancer I resulted in a dramatic rearrangement of chromatin accessibility where the open chromatin region was no longer detected, indicating loss of the phased nucleosome up- and down-stream of the HBV enhancer/BCP. These data demonstrate that CTCF functions to regulate HBV chromatin conformation and nucleosomal positioning in episomal maintained cccDNA, which has important consequences for HBV transcription regulation.

Leveraging multimodal chromatin profiling to identify a new potential driver of diabetic kidney disease

The 3-dimensional nature of chromatin architecture plays crucial roles in regulating gene expression in development, homeostasis, and disease. Until recently, however, comprehensive chromatin profiling in human kidneys has been lacking. In this issue, Eun and Kim etal. employed a multimodal approach by integrating a single-nucleus assay for transposase-accessible chromatin sequencing, chromatin immunoprecipitation sequencing, and Hi-C (a method to comprehensively detect chromatin interactions) to investigate how the epigenetic landscape is altered in diabetic kidney disease.

Genomic and epigenomic integrative subtypes of renal cell carcinoma in a Japanese cohort

Renal cell carcinoma (RCC) comprises several histological types characterised by different genomic and epigenomic aberrations; however, the molecular pathogenesis of each type still requires further exploration. We perform whole-genome sequencing of 128 Japanese RCC cases of different histology to elucidate the significant somatic alterations and mutagenesis processes. We also perform transcriptomic and epigenomic sequencing to identify distinguishing features, including assay for transposase-accessible chromatin sequencing (ATAC-seq) and methyl sequencing. Genomic analysis reveals that the mutational signature differs among the histological types, suggesting that different carcinogenic factors drive each histology. From the ATAC-seq results, master transcription factors are identified for each histology. Furthermore, clear cell RCC is classified into three epi-subtypes, one of which expresses highly immune checkpoint molecules with frequent loss of chromosome 14q. These genomic and epigenomic features may lead to the development of effective therapeutic strategies for RCC.

Single Nuclei Multiomics Analysis of Transcriptional and Chromatin Accessibility of Tumor Cells Uncovers Molecular Signatures and Regulatory Elements of Malignant Clonal Evolution in Multiple Myeloma

Category Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational Introduction Multiple myeloma (MM) is a hematological cancer resulting from abnormal proliferation of plasma cells (PC) in bone marrow. The progression of the disease from Monoclonal Gammopathy of Undetermined Significance (MGUS), Smoldering Multiple Myeloma (SMM), to MM involves multiple drivers and dynamic interactions of premalignant/malignant cells. In the advancement of single-cell technologies, the same-cell readouts of nucleus RNA sequencing (snRNA-seq) and Assay for Transposase-Accessible Chromatin sequencing (snATAC-seq) provide a less biased measurement of gene expressions and chromatin accessible regions. Profiling and analyzing both omics data at different stages of MM may reveal essential molecular signatures and regulatory elements for microenvironmental modification and evolution. These insights could benefit MM patients as novel targets for early therapeutic intervention at different stages of progression. Methods Bone marrow aspirates were collected from 33 individuals: Healthy donors (n=3), patients with MGUS (n=2), SMM (n=5), MM (n=21), and Plasma Cell Leukemia (PCL; n=2). Viable mononuclear cells underwent CD138+ sorting and nuclei isolation prior to library preparation. Libraries were prepared with the Chromium Multiome kit (10X Genomics) and sequenced on NovaSeq S4 flow cells at UAMS. The clonal V(D)J arrangements of each sample and expression level from HD samples were used to distinguish malignant (MM) from normal plasma cells (PC). The MM data from snATAC-seq and snRNA-seq were integrated using Signac with the weighted nearest neighbor methods from Seurat. All inter- and intra-tumor clusters were evaluated for markers of high-risk disease, differential gene expression, differentially accessible regions, and transcription factor activities. Results We performed snRNA-seq and snATAC-seq sequencing to generate multi-stage, molecular classified-specific chromatin accessibility and transcriptional profiles of plasma cell neoplasms comprising 439,134 cells across 33 samples. The malignant plasma cells for each sample were extracted by the unique clonotypic signature of the immunoglobulin gene rearrangement. Of these, we detected the highest clonality in MGUS samples, suggesting clonal competition at the premalignant stage. Based on our primary microarray data, we detected the overexpression of molecular signatures for most cells in each sample, confirming the intratumor cell heterogeneity. Furthermore, we used the ratio of 70-gene signature of aggressive disease and proliferation signatures to investigate the clonal composition of MM cells corresponding to the risk of relapse. Surprisingly, the proportion of high-risk cells increased respectively from low to high-risk samples. The expansion of high-risk clonality might exhibit competition among malignant cell subpopulations regarding microenvironmental adaptation. The multiomics integration generated less distinct clusters between MGUS and SMM, indicating the gradual complex alterations of subclonal gene expression and chromatin accessibility in malignant transformation. Interestingly, most accessible regions were found in intronic/distal intergenic regions implying the long-range gene regulation from cis-regulatory elements. Based on the positive/negative correlation of binding motifs within differentially accessible regions, we identified unique enriched transcription factors for each disease stage, suggesting the mechanism patterns in the evolutionary process of malignant cells. Conclusion In the largest multiomics study to date of CD138+ cells, we demonstrate the malignant clonal evolution using gene expression and chromatin accessibility profiling from patients with early-stage disease to highly aggressive form. Throughout the time of disease progression, we found an increase in the proportion of malignant cells with high-risk signatures. Also, we uncover unique signatures of chromatin accessibility for each molecular classification that can further refine our understanding of functional heterogeneity in multiple myeloma. Collectively, our joint single nuclei profiling reveals additional insights into the malignant clonal evolution and investigates novel regulatory elements involved in disease progression.

NFκB Dysregulation in the Bone Marrow Niche Drives Epigenomic Reprogramming of Hematopoietic Stem Cells and Myeloid Bias

Introduction Aging and chronic inflammation are known to skew hematopoietic output by favoring myelopoiesis and disfavoring lymphopoiesis. In addition, the bone marrow niche plays critical roles in supporting steady-state hematopoiesis, and alterations in the microenvironment may similarly influence disordered hematopoiesis. The NFkB system is a central, pleiotropic regulator of inflammatory signaling in many cell types. Thus, we reasoned that experimental perturbations of NFκB signaling may be used to study how inflammatory dysregulation affects hematopoiesis. Methods We generated a mouse model of inflammatory immune dysregulation, the IκB - mouse ( Nfkbia +/- Nfkbib -/-Nfkbie -/-), and then compared the peripheral blood and bone marrow inflammatory proteins and cell compositions betweenyoung WT, young IκB -,and aged WT mice. Using bone marrow transplantation, we next assayed the effect of an inflamed IκB - recipient niche on the hematopoietic output of WT donor bone marrow. Finally, we performed single cell RNA sequencing (scRNA-seq) and assay for transposase-accessible chromatin sequencing (ATAC-seq) on sorted hematopoietic stem and progenitor cells (HSPC) to characterize the epigenomic and transcriptional changes occurring in WT HSPC from an immune dysregulated environment. Results We observed an increase in inflammatory cytokines in both young IκB - and aged WT mice compared to young WT controls, as well as myeloid bias in the peripheral blood leukocytes and bone marrow HPSC. Transplantation studies showed that inflammatory dysregulation in the radio-resistant microenvironment resulted in myeloid-biased hematopoiesis. However, this myeloid bias was lost upon secondary transplantation of bone marrow into a WT recipient. Performed on HSPC from an inflamed microenvironment, our scRNA-seq studies showed myeloid bias at the transcriptional level, and ATAC-seq studies further revealed epigenomic changes, with an increased transcription factor (TF) binding motif accessibility for myeloid-priming TFs in long term HSC. Conclusions Our studies establish the IκB - mouse as a new model of chronic inflammatory dysregulation that can be used to study the effects of an inflamed bone marrow microenvironment on hematopoiesis. Furthermore, our findings indicate that dysregulated NFκB signaling in the bone marrow microenvironment leads to myeloid bias in HSPC, and is associated with epigenomic reprogramming at the stem cell level. However, despite this epigenomic reprogramming, we observed that myeloid bias could be reversed upon bone marrow re-transplant into a non-dysregulated niche. Together, these data suggest that further understanding niche-derived inflammatory dysregulation may be a useful in targeting disordered hematopoiesis in chronic inflammatory states such as aging.

Impact of Gamma-Secretase Inhibition on the Multiple Myeloma Immune Microenvironment

Background: Therapy with monoclonal antibodies, chimeric antigen receptor (CAR) T-cells, and bispecific T-cell engagers (BiTE) has significantly changed the treatment landscape for multiple myeloma (MM). B-cell maturation antigen (BCMA), a cell surface protein highly expressed in malignant plasma cells, has been established as an effective MM cell target, however, achieving long-term responses in relapsed or refractory patients remains a challenge. Resistance to BCMA targeting may result from the enzymatic cleavage of BCMA by surface membrane gamma-secretase resulting in reduced target antigen density on MM cells, and from the soluble BCMA ectodomain that may hinder tumor cell recognition through binding to circulating therapeutic antibodies or to CAR T-cells. Methods: We have previously demonstrated that gamma-secretase inhibitors (GSIs) abrogate enzymatic function and increase BCMA surface density on plasma cells with a concomitant decrease in soluble BCMA concentrations [Pont, MJ. et al. Blood, 134(2019)]. In a phase I, first-in-human clinical trial (n=18; relapsed/refractory MM) combining the GSI, crenigacestat, with anti-BCMA CAR T-cell therapy (FCARH143), we recently demonstrated that plasma cell BCMA antibody-binding capacity increased a median of 12-fold among 17/18 (94%) of participants after they received a 5-day GSI “run-in” (25 mg orally administered QOD for 3 doses) [Cowan AJ, et al. Lancet Oncology. 24(7) 2023]. Overall, 78% of participants experienced a very good partial response or better to GSI with FCAR143, but the duration of response was significantly longer in patients without prior anti-BCMA exposure. Here, we report results from an analysis of the bone marrow aspirates collected at enrollment and again within 12 hours of the third “run-in” dose. We leveraged single-cell RNA sequencing (scRNAseq) coupled with the single-cell assay for transposase-accessible chromatin sequencing (scATACseq) to profile the transcriptome and chromatin accessibility of individual cells within the bone marrow microenvironment. Results: Single-cell multi-omic analysis was performed on 32 bone marrow mononuclear cell samples from 16 patients collected before and within 12 hours after the third oral dose of GSI. Six patients had previously been treated with BCMA-directed therapy, including antibody-drug conjugates, CAR T-cells, or BiTEs. After quality filtering, including compensation for ambient RNA, 142,498 cells were analyzed (median 6,373 cells/sample, range 613-17,150 cells/sample). Cells were automatically classified into 92 immune cell types using Celltypist and a reference dataset trained on immune sub-populations from 20 tissues in 18 studies. Comparing samples pre- and post-GSI exposure revealed a significant decrease in the abundance of plasmablasts, non-classical monocytes, germinal center B cells, mid-erythroid cells, and central memory/naïve cytotoxic T cells after GSI treatment. Additionally, gene expression analysis detected increased expression of Notch signaling genes, NEURL1 and HES1, in non-classical monocytes following GSI treatment. Although RNA expression of TNFRSF17 ( BCMA) did not significantly change, chromatin accessibility was considerably higher in patients without a history of prior BCMA-directed therapy following GSI treatment. In non-plasma cells, we observed increased chromatin accessibility after GSI treatment involving genes encoding proteins known to be cleaved by gamma-secretase, such as ERBB4, NOTCH1, NOTCH2, and TNFRSF17. Accessibility of CD38, the target of daratumumab, was significantly increased in B cells, and SLAMF7, the target of elotuzumab, was significantly increased in plasma cells. Conclusions: BCMA cleavage from myeloma cells' surface is a putative resistance mechanism to BCMA-targeting immunotherapy. This study assessed the single-cell transcriptome and chromatin accessibility in the bone marrow environment of 16 patients given GSI monotherapy to ultimately enhance the efficacy of subsequent anti-BCMA CAR T-cell therapy. We found that prior BCMA-targeted therapy resulted in reduced chromatin accessibility within the BCMA epigenome. Surprisingly, GSI administration also modified the epigenome of other gamma-secretase sensitive genes. These data provide insight into the molecular consequences of prior BCMA-targeted therapy and GSI exposure that may inform future studies.

Intestinal damage is required for the pro-inflammatory differentiation of commensal CBir1-specific T cells

Commensal-specific CD4+ T cells are expanded in inflammatory bowel disease (IBD) patients compared to healthy individuals. How and where commensal-specific CD4+ T cells get activated is yet to be fully understood. We used CBir1 TCR-transgenic CD4+ T cells, specific to a commensal bacterial antigen, and different mouse models of IBD to characterize the dynamics of commensal-specific CD4+ T cells activation. We found that CBir1 T cells proliferate following intestinal damage and cognate antigen presentation is mediated by CD11c+ cells in the colon-draining mesenteric lymph nodes (cMLNs). Using assay for transposase-accessible chromatin sequencing (ATAC-seq) and flow cytometry, we showed that activated CBir1 T cells preferentially acquire an effector rather than regulatory phenotype, which is plastic over time. Moreover, CBir1 T cells, while insufficient to initiate intestinal inflammation, contributed to worse disease outcome in the presence of other CD4+ T cells. Our results suggest that the commensal-specific T cell responses observed in IBD exacerbate rather than initiate disease.

A spatially-resolved multiomic cell atlas reveals gene regulatory networks underlying cell specification in the developing human heart

Abstract Introduction Cardiogenesis requires the differentiation and coordinated function of a large variety of cell types. Congenital heart disease (CHD), affecting 1% of live births[1], and several adult-onset heart diseases result when these processes go awry[2–4]. To understand the cellular and molecular mechanisms at play we must define the repertoire of cell types in the developing heart as well as the cellular niches within which they interact. Single-cell RNA sequencing is a powerful means of defining cell populations, however existing studies of the human foetal heart[5,6] have been hampered by low cell numbers which limits detection of rare but important cell types. To create an improved atlas we increased the number of cells, increased the age range of foetal hearts sampled, and generated spatial transcriptomic data to map the defined cells to multicellular niches of co-located and interacting cells. To better infer the transcription factors (TFs) governing cell identity using gene regulatory network (GRN) inference we also generated paired single-cell resolution RNA and chromatin accessibility profiles using Assay for Transposase-Accessible Chromatin (ATAC) sequencing. Methods We performed paired single-nuclei RNA and ATAC sequencing (10X Multiome) and single-cell RNAseq (10X 3’ RNAseq) on 23 human foetal hearts ranging from 4 to 20 post-conception weeks. In addition we generated spatial transcriptomic data (10X Visium) from these hearts. We validated cell types defined in single-cell data using protein-level immunofluorescence imaging. Results Our healthy reference atlas includes 300k cells and nuclei. Using their gene expression profile we resolved 46 fine-grained cell types (FIGURE 1). We used cell2location[7], a machine learning classifier trained on the atlas to map the defined cell types to the spatial data (FIGURE 2) and detected niches of co-locating cells, such as a macrophage population in the walls of the great vessels predicted to interact with mural cells. We were able to define location-specific transcriptional signatures such as cardiomyocytes of each chamber (as well as the compact and trabeculated myocardium) and smooth muscle cells specific to great vessels vs coronary arteries. Using a combination of RNA and ATAC data we inferred cell-specific GRNs, which implicated novel TFs such as MSX2 in sinoatrial node pacemaker cells and the enrichment of CHD genes amongst cardiomyocyte-specific GRNs. In the spatial data we showed that gene modules associated with different forms of CHD enrich in specific anatomical regions, such as the outflow tract. Discussion: Together these data constitute the most comprehensive cardiac developmental cell atlas to date. It sheds new light on the diversity of cell states, their niches, and their vulnerability to the genetic causes of CHD.Figure 1:UMAP showing atlasFigure 2:Spatial transcriptomic mapping