Biology subjects

Granja, J. M.

Publications and source records attributed to Granja, J. M..

11 recordsLinked to original sources

bioRxiv2021

Dynamic chromatin regulatory landscape of human CAR T cell exhaustion

Dysfunction in T cells limits the efficacy of cancer immunotherapy1-6. We profiled the epigenome, transcriptome, and enhancer connectome of exhaustion-prone GD2-targeting HA-28z chimeric antigen receptor (CAR) T cells and control CD19-targeting CAR T cells, which present less exhaustion-inducing tonic signaling, at multiple points during their ex vivo expansion7. We found widespread, dynamic changes in chromatin accessibility and 3D chromosome conformation preceding changes in gene expression, notably at loci proximal to exhaustion-associated genes such as PDCD1, CTLA4, and HAVCR2, and increased DNA motif access for AP-1 family transcription factors, which are known to promote exhaustion. Although T cell exhaustion has been studied in detail in mouse, we find that the regulatory networks of T cell exhaustion differ between the species and involve distinct loci of accessible chromatin and cis-regulated target genes in human CAR T cell exhaustion. Deletion of exhaustion-specific candidate enhancers of PDCD1 suppress the expression of PD-1 in an in vitro model of T cell dysfunction and in HA-28z CAR T cells, suggesting enhancer editing as a path forward in improving cancer immunotherapy.

genomics↗

bioRxiv2021

LKB1 inactivation modulates chromatin accessibility to drive metastatic progression

Metastasis is the leading cause of cancer-related deaths, enabling cancer cells to expand to secondary sites and compromise organ function1. Given that primary tumors and metastases often share the same constellation of driver mutations2-4, the mechanisms driving their distinct phenotypes are unclear. Here, we show that inactivation of the frequently mutated tumor suppressor gene, liver kinase B1 (LKB1), has evolving effects throughout lung cancer progression, leading to the differential epigenetic re-programming of early-stage primary tumors compared to late-stage metastases. By integrating genome-scale CRISPR/Cas9 screening with bulk and single-cell multi-omic analyses, we unexpectedly identify LKB1 as a master regulator of chromatin accessibility in lung adenocarcinoma primary tumors. Using an in vivo model of metastatic progression, we further reveal that loss of LKB1 activates the early endoderm transcription factor SOX17 in metastases and a metastatic-like sub-population of cancer cells within primary tumors. SOX17 expression is necessary and sufficient to drive a second wave of epigenetic changes in LKB1-deficient cells that enhances metastatic ability. Overall, our study demonstrates how the downstream effects of an individual driver mutation can appear to change throughout cancer development, with implications for stage-specific therapeutic resistance mechanisms and the gene regulatory underpinnings of metastatic evolution.

cancer biology↗

bioRxiv2020

EcDNA hubs drive cooperative intermolecular oncogene expression

Extrachromosomal DNAs (ecDNAs) are prevalent in human cancers and mediate high oncogene expression through elevated copy number and altered gene regulation1. Gene expression typically involves distal enhancer DNA elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs, comprised of ~10-100 ecDNAs clustered in the nucleus of interphase cells, drive intermolecular enhancer input for amplified oncogene expression. Single-molecule sequencing, single-cell multiome, and 3D enhancer connectome reveal subspecies of MYC-PVT1 ecDNAs lacking enhancers that access intermolecular and ectopic enhancer-promoter interactions in ecDNA hubs. ecDNA hubs persist without transcription and are tethered by BET protein BRD4. BET inhibitor JQ1 disperses ecDNA hubs, preferentially inhibits ecDNA oncogene transcription, and kills ecDNA+ cancer cells. Two amplified oncogenes MYC and FGFR2 intermix in ecDNA hubs, engage in intermolecular enhancer-promoter interactions, and transcription is uniformly sensitive to JQ1. Thus, ecDNA hubs are nuclear bodies of many ecDNAs tethered by proteins and platforms for cooperative transcription, leveraging the power of oncogene diversification and combinatorial DNA interactions. We suggest ecDNA hubs, rather than individual ecDNAs, as units of oncogene function, cooperative evolution, and new targets for cancer therapy.

cancer biology↗

bioRxiv2020

High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer

Spear-ATAC is a modified droplet-based single-cell ATAC-seq (scATAC-seq) protocol that enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at a time. Spear-ATAC profiling of 104,592 cells representing 414 sgRNA knock-down populations revealed the temporal dynamics of epigenetic responses to regulatory perturbations in cancer cells and the associations between transcription factor binding profiles, demonstrating a high-throughput method for perturbing and evaluating dynamic single-cell epigenetic states.

genomics↗

bioRxiv2020

Polycomb-mediated Genome Architecture Enables Long-range Spreading of H3K27 methylation

Polycomb-group proteins play critical roles in gene silencing through the deposition of histone H3 lysine 27 trimethylation (H3K27me3) and chromatin compaction1-5. This process is essential for embryonic stem cell (ESCs) pluripotency, differentiation, and development. Polycomb repressive complex 2 (PRC2) can both read and write H3K27me3, enabling progressive spread of H3K27me3 on the linear genome6. Long-range Polycomb-associated DNA contacts have also been described, but their regulation and role in gene silencing remains unclear7-10. Here, we apply H3K27me3 HiChIP11-13, a protein-directed chromosome conformation method, and optical reconstruction of chromatin architecture14 to profile long-range Polycomb-associated DNA loops that span tens to hundreds of megabases across multiple topological associated domains in mouse ESCs and human induced pluripotent stem cells7-10. We find that H3K27me3 loop anchors are enriched for Polycomb nucleation points and coincide with key developmental genes, such as Hmx1, Wnt6 and Hoxa. Genetic deletion of H3K27me3 loop anchors revealed a coupling of Polycomb-associated genome architecture and H3K27me3 deposition evidenced by disruption of spatial contact between distant loci and altered H3K27me3 in cis, both locally and megabases away on the same chromosome. Further, we find that global alterations in PRC2 occupancy resulting from an EZH2 mutant15 selectively deficient in RNA binding is accompanied by loss of Polycomb-associated DNA looping. Together, these results suggest PRC2 acts as a "genomic wormhole", using RNA binding to enhance long range chromosome folding and H3K27me3 spreading. Additionally, developmental gene loci have novel roles in Polycomb spreading, emerging as important architectural elements of the epigenome.

genetics↗

bioRxiv2020

ArchR: An integrative and scalable software package for single-cell chromatin accessibility analysis

The advent of large-scale single-cell chromatin accessibility profiling has accelerated our ability to map gene regulatory landscapes, but has outpaced the development of robust, scalable software to rapidly extract biological meaning from these data. Here we present a software suite for single-cell analysis of regulatory chromatin in R (ArchR; www.ArchRProject.com) that enables fast and comprehensive analysis of single-cell chromatin accessibility data. ArchR provides an intuitive, user-focused interface for complex single-cell analyses including doublet removal, single-cell clustering and cell type identification, robust peak set generation, cellular trajectory identification, DNA element to gene linkage, transcription factor footprinting, mRNA expression level prediction from chromatin accessibility, and multi-omic integration with scRNA-seq. Enabling the analysis of over 1.2 million single cells within 8 hours on a standard Unix laptop, ArchR is a comprehensive analytical suite for end-to-end analysis of single-cell chromatin accessibility data that will accelerate the understanding of gene regulation at the resolution of individual cells.

genomics↗

bioRxiv2020

Single-cell epigenomic identification of inherited risk loci in Alzheimer's and Parkinson's disease

Genome-wide association studies (GWAS) have identified thousands of variants associated with disease phenotypes. However, the majority of these variants do not alter coding sequences, making it difficult to assign their function. To this end, we present a multi-omic epigenetic atlas of the adult human brain through profiling of the chromatin accessibility landscapes and three-dimensional chromatin interactions of seven brain regions across a cohort of 39 cognitively healthy individuals. Single-cell chromatin accessibility profiling of 70,631 cells from six of these brain regions identifies 24 distinct cell clusters and 359,022 cell type-specific regulatory elements, capturing the regulatory diversity of the adult brain. We develop a machine learning classifier to integrate this multi-omic framework and predict dozens of functional single nucleotide polymorphisms (SNPs), nominating gene and cellular targets for previously orphaned GWAS loci. These predictions both inform well-studied disease-relevant genes, such as BIN1 in microglia for Alzheimers disease (AD) and reveal novel gene-disease associations, such as STAB1 in microglia and MAL in oligodendrocytes for Parkinsons disease (PD). Moreover, we dissect the complex inverted haplotype of the MAPT (encoding tau) PD risk locus, identifying ectopic enhancer-gene contacts in neurons that increase MAPT expression and may mediate this disease association. This work greatly expands our understanding of inherited variation in AD and PD and provides a roadmap for the epigenomic dissection of noncoding regulatory variation in disease.

genomics↗

bioRxiv2019

A single cell framework for multi-omic analysis of disease identifies malignant regulatory signatures in mixed phenotype acute leukemia

In order to identify the molecular determinants of human diseases, such as cancer, that arise from a diverse range of tissue, it is necessary to accurately distinguish normal and pathogenic cellular programs.1-3 Here we present a novel approach for single-cell multi-omic deconvolution of healthy and pathological molecular signatures within phenotypically heterogeneous malignant cells. By first creating immunophenotypic, transcriptomic and epigenetic single-cell maps of hematopoietic development from healthy peripheral blood and bone marrow mononuclear cells, we identify cancer-specific transcriptional and chromatin signatures from single cells in a cohort of mixed phenotype acute leukemia (MPAL) clinical samples. MPALs are a high-risk subtype of acute leukemia characterized by a heterogeneous malignant cell population expressing both myeloid and lymphoid lineage-specific markers.4, 5 Our results reveal widespread heterogeneity in the pathogenetic gene regulatory and expression programs across patients, yet relatively consistent changes within patients even across malignant cells occupying diverse portions of the hematopoietic lineage. An integrative analysis of transcriptomic and epigenetic maps identifies 91,601 putative gene-regulatory interactions and classifies a number of transcription factors that regulate leukemia specific genes, including RUNX1-linked regulatory elements proximal to CD69. This work provides a template for integrative, multi-omic analysis for the interpretation of pathogenic molecular signatures in the context of developmental origin.

genomics↗

bioRxiv2019

Clonal replacement of tumor-specific T cells following PD-1 blockade

Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of cancer patients. However, which tumor-specific T cells are mobilized following checkpoint blockade remains unclear. Here, we performed paired single-cell RNA- and T cell receptor (TCR)-sequencing on 79,046 cells from site-matched tumors from patients with basal cell carcinoma (BCC) or squamous cell carcinoma (SCC) pre- and post-anti-PD-1 therapy. Tracking TCR clones and transcriptional phenotypes revealed a coupling of tumor-recognition, clonal expansion, and T cell dysfunction: the T cell response to treatment was accompanied by clonal expansions of CD8+CD39+ T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, this expansion did not derive from pre-existing tumor infiltrating T cell clones; rather, it comprised novel clonotypes, which were not previously observed in the same tumor. Clonal replacement of T cells was preferentially observed in exhausted CD8+ T cells, compared to other distinct T cell phenotypes, and was evident in BCC and SCC patients. These results, enabled by single-cell multi-omic profiling of clinical samples, demonstrate that pre-existing tumor-specific T cells may be limited in their capacity for re-invigoration, and that the T cell response to checkpoint blockade relies on the expansion of a distinct repertoire of T cell clones that may have just recently entered the tumor.

immunology↗

bioRxiv2019

Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion

Understanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here we present a massively parallel droplet-based platform for mapping transposase-accessible chromatin in tens of thousands of single cells per sample (scATAC-seq). We obtain and analyze chromatin profiles of over 200,000 single cells in two primary human systems. In blood, scATAC-seq allows marker-free identification of cell type-specific cis- and trans-regulatory elements, mapping of disease-associated enhancer activity, and reconstruction of trajectories of differentiation from progenitors to diverse and rare immune cell types. In basal cell carcinoma, scATAC-seq reveals regulatory landscapes of malignant, stromal, and immune cell types in the tumor microenvironment. Moreover, scATAC-seq of serial tumor biopsies before and after PD-1 blockade allows identification of chromatin regulators and differentiation trajectories of therapy-responsive intratumoral T cell subsets, revealing a shared regulatory program driving CD8+ T cell exhaustion and CD4+ T follicular helper cell development. We anticipate that droplet-based single-cell chromatin accessibility will provide a broadly applicable means of identifying regulatory factors and elements that underlie cell type and function.

genomics↗

bioRxiv2017

Enhancer connectome in primary human cells reveals target genes of disease-associated DNA elements

The challenge of linking intergenic mutations to target genes has limited molecular understanding of diverse human diseases. Here, we show H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells to either T helper 17 cells or regulatory T cells create subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a four-fold increase of potential target genes for autoimmune and cardiovascular diseases.

genomics↗