Low Kit expression identifies Hematopoietic Stem Cell subsets with enhanced lymphoid potential in mice and humans.

Hematopoietic stem cells (HSCs) with multilineage potential are critical for effective T cell reconstitution and restoration of the adaptive immune system after allogeneic Hematopoietic Cell Transplantation (allo-HCT). Our current understanding of lymphoid-poised HSC subsets is limited due to a lack of reliable markers. Through comprehensive molecular profiling of HSCs, we identify Kit lo HSCs-a subset with enhanced lymphoid potential, which declines with age. Using a preclinical allo-HCT model, we demonstrate that Kit lo HSCs exhibit superior lymphoid progenitor output, enhanced thymic recovery, and T-cell reconstitution resulting in improved T cell responses to infection post-HCT. Furthermore, Kit lo HSCs with augmented BM lymphopoiesis mitigate age-associated thymic alterations, thus enhancing T-cell recovery in middle-aged hosts. Chromatin profiling revealed that Kit lo HSCs exhibit higher activity of lymphoid-specifying transcription factors (TFs), including Zbtb1 . Deletion of Zbtb1 in Kit lo HSCs, diminished their T-cell potential, while reinstating Zbtb1 in megakaryocytic-biased Kit hi HSCs rescued T-cell potential, in vitro and in vivo . Finally, we discover an analogous Kit lo HSC subset with enhanced lymphoid potential in human bone marrow. Our results demonstrate that Kit lo HSCs identify HSC subsets with enhanced lymphoid potential mediated by an underlying epigenetic program.

Single-cell multi-ome regression models identify functional and disease-associated enhancers and enable chromatin potential analysis.

We present a gene-level regulatory model, single-cell ATAC + RNA linking (SCARlink), which predicts single-cell gene expression and links enhancers to target genes using multi-ome (scRNA-seq and scATAC-seq co-assay) sequencing data. The approach uses regularized Poisson regression on tile-level accessibility data to jointly model all regulatory effects at a gene locus, avoiding the limitations of pairwise gene-peak correlations and dependence on peak calling. SCARlink outperformed existing gene scoring methods for imputing gene expression from chromatin accessibility across high-coverage multi-ome datasets while giving comparable to improved performance on low-coverage datasets. Shapley value analysis on trained models identified cell-type-specific gene enhancers that are validated by promoter capture Hi-C and are 11× to 15× and 5× to 12× enriched in fine-mapped eQTLs and fine-mapped genome-wide association study (GWAS) variants, respectively. We further show that SCARlink-predicted and observed gene expression vectors provide a robust way to compute a chromatin potential vector field to enable developmental trajectory analysis.

Does immune dysregulation contribute towards development of hypopigmentation in Indian post kala-azar dermal leishmaniasis?

Post kala-azar dermal leishmaniasis (PKDL), a sequel of apparently cured visceral leishmaniasis (VL) presents with papulonodular (polymorphic) or hypopigmented lesions (macular) and is the proposed disease reservoir. As hypopigmentation appears consistently in PKDL, especially the macular form, this study aimed to delineate immune factors that singly or in combination could contribute towards this hypopigmentation. At lesional sites, the presence of melanocytes and CD8+ T-cells was assessed by immunohistochemistry and mRNA expression of melanogenic markers (tyrosinase, tyrosinase-related protein-1 and MITF) by droplet digital PCR, while plasma levels of cytokines and chemokines were measured by a multiplex assay. In comparison with skin from healthy individuals, macular PKDL demonstrated a near total absence of Melan-A+ cells at dermal sites, while the polymorphic cases demonstrated a 3.2-fold decrease, along with a dramatic reduction in the expression of key enzymes related to the melanogenesis signalling pathway in both forms. The levels of circulating IFN-γ, IL-6, IL-2, IL-1ß, TNF-α and IFN-γ-inducible chemokines (CXCL9/10/11) were elevated and was accompanied by an increased lesional infiltration of CD8+ T-cells. The proportion of CD8+ T-cells correlated strongly with plasma levels of IFN-γ (r = 0.8), IL-6 (r = 0.9, p < 0.05), IL-2 (r = 0.7), TNF-α (r = 0.9, p < 0.05) and IL-1ß (r = 0.7), as also with CXCL9 (r = 0.5) and CXCL10 (r = 0.6). Taken together, the absence/reduction in Melan-A suggested hypopigmentation in PKDL was associated with the destruction of melanocytes, following the impairment of the melanogenesis pathway. Furthermore, the presence of CD8+ T-cells and an enhanced IFN-γ-associated immune milieu suggested the generation of a pro-inflammatory landscape that facilitated melanocyte dysfunction/destruction.

RoboCOP: jointly computing chromatin occupancy profiles for numerous factors from chromatin accessibility data.

Chromatin is a tightly packaged structure of DNA and protein within the nucleus of a cell. The arrangement of different protein complexes along the DNA modulates and is modulated by gene expression. Measuring the binding locations and occupancy levels of different transcription factors (TFs) and nucleosomes is therefore crucial to understanding gene regulation. Antibody-based methods for assaying chromatin occupancy are capable of identifying the binding sites of specific DNA binding factors, but only one factor at a time. In contrast, epigenomic accessibility data like MNase-seq, DNase-seq, and ATAC-seq provide insight into the chromatin landscape of all factors bound along the genome, but with little insight into the identities of those factors. Here, we present RoboCOP, a multivariate state space model that integrates chromatin accessibility data with nucleotide sequence to jointly compute genome-wide probabilistic scores of nucleosome and TF occupancy, for hundreds of different factors. We apply RoboCOP to MNase-seq and ATAC-seq data to elucidate the protein-binding landscape of nucleosomes and 150 TFs across the yeast genome, and show that our model makes better predictions than existing methods. We also compute a chromatin occupancy profile of the yeast genome under cadmium stress, revealing chromatin dynamics associated with transcriptional regulation.

Linking the dynamics of chromatin occupancy and transcription with predictive models.

Though the sequence of the genome within each eukaryotic cell is essentially fixed, it exists within a complex and changing chromatin state. This state is determined, in part, by the dynamic binding of proteins to the DNA. These proteins-including histones, transcription factors (TFs), and polymerases-interact with one another, the genome, and other molecules to allow the chromatin to adopt one of exceedingly many possible configurations. Understanding how changing chromatin configurations associate with transcription remains a fundamental research problem. We sought to characterize at high spatiotemporal resolution the dynamic interplay between transcription and chromatin in response to cadmium stress. Whereas gene regulatory responses to environmental stress in yeast have been studied, how the chromatin state changes and how those changes connect to gene regulation remain unexplored. By combining MNase-seq and RNA-seq data, we found chromatin signatures of transcriptional activation and repression involving both nucleosomal and TF-sized DNA-binding factors. Using these signatures, we identified associations between chromatin dynamics and transcriptional regulation, not only for known cadmium response genes, but across the entire genome, including antisense transcripts. Those associations allowed us to develop generalizable models that predict dynamic transcriptional responses on the basis of dynamic chromatin signatures.

RoboCOP: Multivariate State Space Model Integrating Epigenomic Accessibility Data to Elucidate Genome-Wide Chromatin Occupancy.

Chromatin is the tightly packaged structure of DNA and protein within the nucleus of a cell. The arrangement of different protein complexes along the DNA modulates and is modulated by gene expression. Measuring the binding locations and level of occupancy of different transcription factors (TFs) and nucleosomes is therefore crucial to understanding gene regulation. Antibody-based methods for assaying chromatin occupancy are capable of identifying the binding sites of specific DNA binding factors, but only one factor at a time. On the other hand, epigenomic accessibility data like ATAC-seq, DNase-seq, and MNase-seq provide insight into the chromatin landscape of all factors bound along the genome, but with minimal insight into the identities of those factors. Here, we present RoboCOP, a multivariate state space model that integrates chromatin information from epigenomic accessibility data with nucleotide sequence to compute genome-wide probabilistic scores of nucleosome and TF occupancy, for hundreds of different factors at once. RoboCOP can be applied to any epigenomic dataset that provides quantitative insight into chromatin accessibility in any organism, but here we apply it to MNase-seq data to elucidate the protein-binding landscape of nucleosomes and 150 TFs across the yeast genome. Using available protein-binding datasets from the literature, we show that our model more accurately predicts the binding of these factors genome-wide.

Author Correction: Impaired activation of lesional CD8+ T-cells is associated with enhanced expression of Programmed Death-1 in Indian Post Kala-azar Dermal Leishmaniasis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In-situ immune profile of polymorphic vs. macular Indian Post Kala-azar dermal leishmaniasis.

Post Kala-azar Dermal Leishmaniasis (PKDL), a sequel of apparently cured Visceral Leishmaniasis presents in South Asia with papulonodular (polymorphic) or hypomelanotic lesions (macular). Till date, the polymorphic variant was considered predominant, constituting 85-90%. However, following active-case surveillance, the proportion of macular PKDL has increased substantially to nearly 50%, necessitating an in-depth analysis of this variant. Accordingly, this study aimed to delineate the cellular infiltrate in macular vis-à-vis polymorphic PKDL. To study the overall histopathology, hematoxylin and eosin staining was performed on lesional sections and phenotyping by immunohistochemistry done in terms of dendritic cells (CD1a), macrophages (CD68), HLA-DR, T-cells (CD8, CD4), B-cells (CD20) and Ki67 along with assessment of the status of circulating homing markers CCL2, CCL7 and CXCL13. In polymorphic cases (n = 20), the cellular infiltration was substantial, whereas in macular lesions (n = 20) it was mild and patchy with relative sparing of the reticular dermis. Although parasite DNA was identified in both variants by ITS-1 PCR, the parasite load was significantly higher in the polymorphic variant and Leishman-Donovan bodies were notably minimally present in macular cases. Both variants demonstrated a decrease in CD1a+ dendritic cells, HLA-DR expression and CD4+ T-cells. In macular cases, the proportion of CD68+ macrophages, CD8+ T-cells and CD20+ B-cells was 4.6 fold, 17.0 fold and 1.6 fold lower than polymorphic cases. The absence of Ki67 positivity and increased levels of chemoattractants suggested dermal homing of these cellular subsets. Taken together, as compared to the polymorphic variant, patients with macular PKDL demonstrated a lower parasite load along with a lesser degree of cellular infiltration, suggesting differences in host-pathogen interactions, which in turn can impact on their disease transmitting potential and responses to chemotherapy.

Role of redox imbalance and cytokines in mediating oxidative damage and disease progression of patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disorder wherein the contributory role of oxidative stress has been established in the synovial fluid. As availability of synovial fluid is limited, this study aimed to evaluate in the peripheral blood of patients with RA, the relationship if any, between the extent of oxidative stress in terms of generation of reactive oxygen species (ROS) in neutrophils, plasma NADPH oxidase and myeloperoxidase activity with markers of oxidative damage, circulating cytokines and disease activity score (DAS28). In patients with RA, neutrophils in peripheral blood demonstrated an enhanced generation of ROS, coupled with depletion of free radical scavenging activity. Furthermore, the NADPH oxidase and myeloperoxidase activity was enhanced as were markers of damage. There was a positive correlation between the DAS 28 and generation of ROS, NADPH oxidase and myeloperoxidase activity as also with oxidative stress mediated protein carbonylation. Patients with RA demonstrated an increase in proinflammatory (IL-17, IL-23, and IFN-γ) and some anti-inflammatory (IL-4, IL-5, and TGF-ß) cytokines. Although the levels of IL-17 correlated positively with generation of ROS, myeloperoxidase, markers of protein damage and DAS28, IL-23 correlated positively only with protein damage, and negatively with free radical scavenging activity. Importantly, incubation of neutrophils from healthy donors with plasma or SF from patients with RA translated into an enhanced generation of ROS, along with an elevation of intracellular proinflammatory cytokines. Taken together, in patients with RA, circulating neutrophils mediated a shift in the oxidant/antioxidant balance favouring the former, which translated into protein damage and contributed towards disease progression.

Impaired activation of lesional CD8+ T-cells is associated with enhanced expression of Programmed Death-1 in Indian Post Kala-azar Dermal Leishmaniasis.

Post Kala-azar dermal leishmaniasis (PKDL), caused by Leishmania donovani is the dermal sequel of Visceral Leishmaniasis and importantly, is the proposed disease reservoir. The survival of Leishmania parasites within monocytes/macrophages hinges on its ability to effectively nullify immune activation mechanisms. Thus, delineating the disease-promoting immune mechanisms can facilitate development of immunotherapeutic strategies. Accordingly, in the absence of an animal model, this study aimed to delineate the status of CD8+ T-cells in patients with PKDL. At disease presentation, the absence of CD4+ T-cells at lesional sites was concomitant with an overwhelming infiltration of CD8+ T-cells that demonstrated an absence of Perforin, Granzyme and Zap-70, along with an enhanced expression of Programmed Death-1 (PD-1) and the skin-homing CCL17. Additionally, the lesional CCR4+CD8+ population was associated with an enhanced expression of IL-10 and IL-5. In circulation, the enhanced CD8+CCR4+ T-cell population and raised levels of CCL17/22 was associated with an increased frequency of PD-1, while CD127 was decreased. Taken together, in PKDL, the enhanced plasma and lesional CCL17 accounted for the dermal homing of CD8+CCR4+ T-cells, that along with a concomitant upregulation of PD-1 and IL-10 mediated immune inactivation, emphasizing the need for designing immunotherapies capable of reinvigorating T-cell potency.

DIVERSITY in binding, regulation, and evolution revealed from high-throughput ChIP.

Genome-wide in vivo protein-DNA interactions are routinely mapped using high-throughput chromatin immunoprecipitation (ChIP). ChIP-reported regions are typically investigated for enriched sequence-motifs, which are likely to model the DNA-binding specificity of the profiled protein and/or of co-occurring proteins. However, simple enrichment analyses can miss insights into the binding-activity of the protein. Note that ChIP reports regions making direct contact with the protein as well as those binding through intermediaries. For example, consider a ChIP experiment targeting protein X, which binds DNA at its cognate sites, but simultaneously interacts with four other proteins. Each of these proteins also binds to its own specific cognate sites along distant parts of the genome, a scenario consistent with the current view of transcriptional hubs and chromatin loops. Since ChIP will pull down all X-associated regions, the final reported data will be a union of five distinct sets of regions, each containing binding sites of one of the five proteins, respectively. Characterizing all five different motifs and the corresponding sets is important to interpret the ChIP experiment and ultimately, the role of X in regulation. We present diversity which attempts exactly this: it partitions the data so that each partition can be characterized with its own de novo motif. Diversity uses a Bayesian approach to identify the optimal number of motifs and the associated partitions, which together explain the entire dataset. This is in contrast to standard motif finders, which report motifs individually enriched in the data, but do not necessarily explain all reported regions. We show that the different motifs and associated regions identified by diversity give insights into the various complexes that may be forming along the chromatin, something that has so far not been attempted from ChIP data. Webserver at http://diversity.ncl.res.in/; standalone (Mac OS X/Linux) from https://github.com/NarlikarLab/DIVERSITY/releases/tag/v1.0.0.

Levels of oxidative damage and proinflammatory cytokines are enhanced in patients with active vitiligo.

Vitiligo is an autoimmune depigmenting skin disease characterised by loss of melanocytes wherein oxidative stress is proposed to be the initial triggering factor with subsequent immune dysregulation. This study aimed to evaluate the relationship, if any, between the generation of reactive oxygen species (ROS), markers of oxidative damage and circulating cytokines in patients with active vitiligo. The generation of ROS in erythrocytes and neutrophils was significantly higher in patients with active vitiligo than healthy controls. Alongside, markers of oxidative stress-mediated damage namely lipid peroxidation, DNA damage and protein carbonylation were evaluated. Patients with active vitiligo demonstrated increased lipid and DNA damage but minimal protein damage. There was a significant decline in the free radical scavenging capacity of active vitiligo cases. A positive correlation existed between baseline levels of ROS and lipid peroxidation as also DNA damage. Patients with active vitiligo demonstrated an increase in several proinflammatory (IL-6, TNF-α, IL-1ß, IFN-γ and IL-8) and some anti-inflammatory/immunoregulatory (IL-5 and IL-10) cytokines. Importantly, the levels of IFN-γ and IL-10 consistently correlated with the generation of ROS, markers of damage and their free radical scavenging capacity. Taken together, patients with active vitiligo demonstrated an enhanced generation of ROS in erythrocytes and neutrophils which mediated lipid peroxidation, DNA damage and coupled with a decline in their antioxidant capacity created a pro-oxidant milieu that favoured tissue damage and potential generation of neoantigens, accounting for disease progression.

A study of the free radical scavenging effects of Piper betle leaf extract in patients with vitiligo.

BACKGROUND: Vitiligo is an idiopathic skin disease manifested by depigmented macules. It is characterised by melanocyte destruction, and redox imbalance is proposed to play a contributory role. AIM: The aim of this study was to analyze the effects of an ethanolic extract of Piper betle leaves on the generation of reactive oxygen species in erythrocytes sourced from vitiligo patients. METHODS: The effect of Piper betle on the generation of reactive oxygen species in erythrocytes was measured by flow cytometry in patients with active and stable vitiligo versus healthy controls, using 5-(and-6)-chloromethyl-2'-7'-dichlorodihydrofluorescein diacetate. RESULTS: The generation of reactive oxygen species in erythrocytes was higher in patients with vitiligo (n = 23) compared to healthy controls (n = 18). The geometrical mean fluorescence channel was 23.05 ± 2.11 in patients versus 17.77 ± 1.79 in controls, P = 0.039. The levels of reactive oxygen species were higher in patients with active vitiligo. Treatment of erythrocytes with Piper betle in concentrations of 0.5 and 1.0 µg/ml significantly decreased the baseline levels of reactive oxygen species by 31.7% in healthy controls, and 47.6% and 44.3% in patients with active vitiligo, respectively. Piper betle effectively scavenged hydrogen peroxide, which was evident by a decrease in the geometrical mean fluorescence channel by 52.4% and 62.9% in healthy controls, and 45.0% and 57.0% in patients with active vitiligo. LIMITATIONS: The study had a small sample size. Future studies should focus on evaluation of the antioxidant role of Piper betle at the lesional site. CONCLUSION: This pilot study indicates that patients with active vitiligo demonstrate enhanced generation of reactive oxygen species in erythrocytes, which was significantly reduced following ex vivo treatment with Piper betle.

No Promoter Left Behind (NPLB): learn de novo promoter architectures from genome-wide transcription start sites.

UNLABELLED: Promoters have diverse regulatory architectures and thus activate genes differently. For example, some have a TATA-box, many others do not. Even the ones with it can differ in its position relative to the transcription start site (TSS). No Promoter Left Behind (NPLB) is an efficient, organism-independent method for characterizing such diverse architectures directly from experimentally identified genome-wide TSSs, without relying on known promoter elements. As a test case, we show its application in identifying novel architectures in the fly genome. AVAILABILITY AND IMPLEMENTATION: Web-server at http://nplb.ncl.res.in Standalone also at https://github.com/computationalBiology/NPLB/ (Mac OSX/Linux). CONTACT: l.narlikar@ncl.res.in SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.