AP-1 signaling modulates cardiac fibroblast stress responses.

Matrix remodeling outcomes largely dictate patient survival post myocardial infarction. Moreover, human-restricted noncoding regulatory elements have been shown to worsen fibrosis, but their mechanism of action remains elusive. Here, we demonstrate, using induced pluripotent stem cell-derived cardiac fibroblasts (iCFs), that inflammatory ligands abundant in the remodeling heart after infarction activate AP-1 transcription factor signaling pathways resulting in fibrotic responses. This observed signaling induces deposition of fibronectin matrix and is further capable of supporting immune cell adhesion; pathway inhibition blocks iCF matrix production and cell adhesion. Polymorphisms in the noncoding regulatory elements within the 9p21 locus (also referred to as ANRIL) redirect stress programs, and in iCFs, they transcriptionally silence the AP-1 inducible transcription factor GATA5. The presence of these polymorphisms modulate iCF matrix production and assembly and reduce cell-cell signaling. These data suggest that this signaling axis is a critical modulator of cardiac disease models and might be influenced by noncoding regulatory elements.

DNA architectural protein CTCF facilitates subset-specific chromatin interactions to limit the formation of memory CD8+ T cells.

Although the importance of genome organization for transcriptional regulation of cell-fate decisions and function is clear, the changes in chromatin architecture and how these impact effector and memory CD8+ T cell differentiation remain unknown. Using Hi-C, we studied how genome configuration is integrated with CD8+ T cell differentiation during infection and investigated the role of CTCF, a key chromatin remodeler, in modulating CD8+ T cell fates through CTCF knockdown approaches and perturbation of specific CTCF-binding sites. We observed subset-specific changes in chromatin organization and CTCF binding and revealed that weak-affinity CTCF binding promotes terminal differentiation of CD8+ T cells through the regulation of transcriptional programs. Further, patients with de novo CTCF mutations had reduced expression of the terminal-effector genes in peripheral blood lymphocytes. Therefore, in addition to establishing genome architecture, CTCF regulates effector CD8+ T cell heterogeneity through altering interactions that regulate the transcription factor landscape and transcriptome.

Age-dependent Lamin changes induce cardiac dysfunction via dysregulation of cardiac transcriptional programs.

As we age, structural changes contribute to progressive decline in organ function, which in the heart act through poorly characterized mechanisms. Taking advantage of the short lifespan and conserved cardiac proteome of the fruit fly, we found that cardiomyocytes exhibit progressive loss of Lamin C (mammalian Lamin A/C homologue) with age, coincident with decreasing nuclear size and increasing nuclear stiffness. Premature genetic reduction of Lamin C phenocopies aging's effects on the nucleus, and subsequently decreases heart contractility and sarcomere organization. Surprisingly, Lamin C reduction downregulates myogenic transcription factors and cytoskeletal regulators, possibly via reduced chromatin accessibility. Subsequently, we find a role for cardiac transcription factors in regulating adult heart contractility and show that maintenance of Lamin C, and cardiac transcription factor expression, prevents age-dependent cardiac decline. Our findings are conserved in aged non-human primates and mice, demonstrating that age-dependent nuclear remodeling is a major mechanism contributing to cardiac dysfunction.

Improved epicardial cardiac fibroblast generation from iPSCs.

Since the initial isolation of human embryonic stem cells and subsequent discovery of reprogramming methods for somatic cells, thousands of protocols have been developed to create each of the hundreds of cell types found in-vivo with significant focus on disease-prone systems, e.g., cardiovascular. Robust protocols exist for many of these cell types, except for cardiac fibroblasts (CF). Very recently, several competing methods have been developed to generate these cells through a developmentally conserved epicardial pathway. Such methods generate epicardial cells, but here we report that prolonged exposure to growth factors such as bFGF induces fibroblast spindle-like morphology and similar chromatin architecture to primary CFs. Media conditions for growth and assays are provided, as well as suggestions for seeding densities and timepoints for protein harvest of extracellular matrix. We demonstrate marker expression and matrix competency of resultant cells as shown next to primary human cardiac fibroblasts. These methods provide additional guidance to the original protocol and result in an increasingly stable phenotype.

A single-cell atlas of chromatin accessibility in the human genome.

Current catalogs of regulatory sequences in the human genome are still incomplete and lack cell type resolution. To profile the activity of gene regulatory elements in diverse cell types and tissues in the human body, we applied single-cell chromatin accessibility assays to 30 adult human tissue types from multiple donors. We integrated these datasets with previous single-cell chromatin accessibility data from 15 fetal tissue types to reveal the status of open chromatin for ∼1.2 million candidate cis-regulatory elements (cCREs) in 222 distinct cell types comprised of >1.3 million nuclei. We used these chromatin accessibility maps to delineate cell-type-specificity of fetal and adult human cCREs and to systematically interpret the noncoding variants associated with complex human traits and diseases. This rich resource provides a foundation for the analysis of gene regulatory programs in human cell types across tissues, life stages, and organ systems.

Cardiac cell type-specific gene regulatory programs and disease risk association.

Misregulated gene expression in human hearts can result in cardiovascular diseases that are leading causes of mortality worldwide. However, the limited information on the genomic location of candidate cis-regulatory elements (cCREs) such as enhancers and promoters in distinct cardiac cell types has restricted the understanding of these diseases. Here, we defined >287,000 cCREs in the four chambers of the human heart at single-cell resolution, which revealed cCREs and candidate transcription factors associated with cardiac cell types in a region-dependent manner and during heart failure. We further found cardiovascular disease-associated genetic variants enriched within these cCREs including 38 candidate causal atrial fibrillation variants localized to cardiomyocyte cCREs. Additional functional studies revealed that two of these variants affect a cCRE controlling KCNH2/HERG expression and action potential repolarization. Overall, this atlas of human cardiac cCREs provides the foundation for illuminating cell type-specific gene regulation in human hearts during health and disease.

Promoter-proximal CTCF binding promotes distal enhancer-dependent gene activation.

The CCCTC-binding factor (CTCF) works together with the cohesin complex to drive the formation of chromatin loops and topologically associating domains, but its role in gene regulation has not been fully defined. Here, we investigated the effects of acute CTCF loss on chromatin architecture and transcriptional programs in mouse embryonic stem cells undergoing differentiation to neural precursor cells. We identified CTCF-dependent enhancer-promoter contacts genome-wide and found that they disproportionately affect genes that are bound by CTCF at the promoter and are dependent on long-distance enhancers. Disruption of promoter-proximal CTCF binding reduced both long-range enhancer-promoter contacts and transcription, which were restored by artificial tethering of CTCF to the promoter. Promoter-proximal CTCF binding is correlated with the transcription of over 2,000 genes across a diverse set of adult tissues. Taken together, the results of our study show that CTCF binding to promoters may promote long-distance enhancer-dependent transcription at specific genes in diverse cell types.

miR-155 harnesses Phf19 to potentiate cancer immunotherapy through epigenetic reprogramming of CD8+ T cell fate.

T cell senescence and exhaustion are major barriers to successful cancer immunotherapy. Here we show that miR-155 increases CD8+ T cell antitumor function by restraining T cell senescence and functional exhaustion through epigenetic silencing of drivers of terminal differentiation. miR-155 enhances Polycomb repressor complex 2 (PRC2) activity indirectly by promoting the expression of the PRC2-associated factor Phf19 through downregulation of the Akt inhibitor, Ship1. Phf19 orchestrates a transcriptional program extensively shared with miR-155 to restrain T cell senescence and sustain CD8+ T cell antitumor responses. These effects rely on Phf19 histone-binding capacity, which is critical for the recruitment of PRC2 to the target chromatin. These findings establish the miR-155-Phf19-PRC2 as a pivotal axis regulating CD8+ T cell differentiation, thereby paving new ways for potentiating cancer immunotherapy through epigenetic reprogramming of CD8+ T cell fate.

The transcription factor c-Myb regulates CD8+ T cell stemness and antitumor immunity.

Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here, we found that the transcription factor c-Myb was essential for generating and maintaining stem cells in the CD8+ T cell memory compartment. Following viral infection, CD8+ T cells lacking Myb underwent terminal differentiation and generated fewer stem cell-like central memory cells than did Myb-sufficient T cells. c-Myb acted both as a transcriptional activator of Tcf7 (which encodes the transcription factor Tcf1) to enhance memory development and as a repressor of Zeb2 (which encodes the transcription factor Zeb2) to hinder effector differentiation. Domain-mutagenesis experiments revealed that the transactivation domain of c-Myb was necessary for restraining differentiation, whereas its negative regulatory domain was critical for cell survival. Myb overexpression enhanced CD8+ T cell memory formation, polyfunctionality and recall responses that promoted curative antitumor immunity after adoptive transfer. These findings identify c-Myb as a pivotal regulator of CD8+ T cell stemness and highlight its therapeutic potential.

Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies.

Long-lived, self-renewing, multipotent T memory stem cells (TSCM) can trigger profound and sustained tumor regression but their rareness poses a major hurdle to their clinical application. Presently, clinically compliant procedures to generate relevant numbers of this T-cell population are undefined. Here, we provide a strategy for deriving large numbers of clinical-grade tumor-redirected TSCM starting from naive precursors. CD8(+)CD62L(+)CD45RA(+) naive T cells enriched by streptamer-based serial-positive selection were activated by CD3/CD28 engagement in the presence of interleukin-7 (IL-7), IL-21, and the glycogen synthase-3ß inhibitor TWS119, and genetically engineered to express a CD19-specific chimeric antigen receptor (CD19-CAR). These conditions enabled the generation of CD19-CAR-modified CD8(+) TSCM that were phenotypically, functionally, and transcriptomically equivalent to their naturally occurring counterpart. Compared with CD8(+) T cells generated with clinical protocols currently under investigation, CD19-CAR-modified CD8(+) TSCM exhibited enhanced metabolic fitness and mediated robust, long-lasting antitumor responses against systemic acute lymphoblastic leukemia xenografts. This clinical-grade platform provides the basis for a phase 1 trial evaluating the activity of CD19-CAR-modified CD8(+) TSCM in patients with B-cell malignancies refractory to prior allogeneic hematopoietic stem cell transplantation.

Enhancing adoptive T cell immunotherapy with microRNA therapeutics.

Adoptive T cell-based immunotherapies can mediate complete and durable regressions in patients with advanced cancer, but current response rates remain inadequate. Maneuvers to improve the fitness and antitumor efficacy of transferred T cells have been under extensive exploration in the field. Small non-coding microRNAs have emerged as critical modulators of immune system homeostasis and T cell immunity. Here, we summarize recent advances in our understanding of the role of microRNAs in regulating T cell activation, differentiation, and function. We also discuss how microRNA therapeutics could be employed to fine-tune T cell receptor signaling and enhance T cell persistence and effector functions, paving the way for the next generation of adoptive immunotherapies.