Yasuhiro Murakawa

More than twenty years ago, the FANTOM consortium uncovered that mammalian genomes are pervasively transcribed, revealing multitudes of RNAs with unknown functions. A subset of these transcripts has since then been linked to transcriptional control and to chromatin organization via their ability to interact with DNA, suggesting that chromatin-associated RNAs could be key players in genome regulation. Although recent technological advances now enable the mapping of genome-wide RNA-DNA contacts, a lack of analyses integrating these methods with other genomic features and across multiple cellular contexts hinders our comprehensive understanding of the principles underlying RNA-DNA interactions and of their biological importance. As part of the FANTOM6 project, we thus generated RNA-DNA interaction maps in 16 different human cell types, then combined these contacts with multiple layers of other genomic data to investigate how patterns of interaction between RNA and DNA relate to chromatin organization and function. We show that the RNA-DNA interactome is highly dynamic yet reproducibly organized in cell-type specific networks, constituted of a great diversity of interactions that vary in function of their distance, the nature of their sources and the chromatin state of their targets. In particular, we detected numerous regulatory elements that exhibit marked changes in activity when differentially bound by transcripts, implying that thousands of RNA-DNA interactions can play a mechanistic role in gene expression. This regulatory function correlates with RNA-protein interactions and significantly associates with cell type-relevant and disorder-related traits. In addition to providing essential resources for future research in RNA-mediated chromatin regulation, cellular biology and human diseases, our study thus establishes the RNA-DNA interactome as a new genome regulatory layer that defines and maintains cellular identity and behavior.

Studies using mouse models of viral infection have shown that viral antigen-reactive regulatory T cells (Tregs) play multiple context-dependent roles, including attenuating the immune response against the pathogen and limiting inflammatory damage. By contrast, evidence for the presence and function of viral antigen-specific Tregs in humans remains very limited. Here, we investigated whether BNT162b2 mRNA COVID-19 vaccination activates spike (S)-reactive Tregs in healthy adults. We performed an integrated analysis combining single-cell RNA sequencing (scRNA-seq), T-cell receptor repertoire profiling, and flow cytometry of activation-induced marker (AIM)-expressing S-reactive CD4+ T cells isolated from peripheral blood mononuclear cells cultured with trimeric S protein. We found that CD39+ cells coexpressing CTLA4 and TIGIT within the S-reactive AIM+ CD4+ T-cell population were preferentially enriched for Tregs relative to their CD39− counterparts. In subjects vaccinated with the BNT162b2 mRNA vaccine, the frequencies of CD39+CTLA4+ and CD39+TIGIT+ S-reactive Treg-like cells were significantly increased following vaccination. Using scRNA-seq, we identified two FoxP3+ Treg clusters among S-reactive CD4+ T cells: one characterized by IFN-activated gene signatures and another by high HLA class II, CD39, and CTLA4 expression. Both subsets were transcriptionally activated following vaccination, with upregulation of genes associated with T-cell activation and inflammatory cytokines, including TNF and IFNG. Consistent with these findings, flow cytometry detected increased proportions of CD39+ and FoxP3+ S-reactive Tregs expressing IFN-γ and/or TNF-α following vaccination. Collectively, these findings support a model in which BNT162b2 mRNA vaccination activates both an S-reactive effector T-cell response and a Treg response, suggesting Treg coinduction may be critical for balancing antiviral immunity with immunoregulation.

Cells of the retina, photoreceptors especially, are highly metabolically active with substantial energy requirements. Accordingly, the effective delivery of oxygen and nutrients to the retina via the retinal vasculature is key to tissue function. Indeed, a disturbed retinal blood supply, especially if prolonged, can result in a loss of vision. Vascular endothelial cells, which form the inner lining of blood vessels, are pivotal in ensuring a healthy blood circulation in many tissues, including the light-sensitive retina at the back of the eye. However, the mechanism by which vascular endothelial cells maintain a functional retina (and aid the healing of a damaged one) is not understood. Here, through single-cell RNA sequencing, colony-forming assays and lineage tracing analysis, we provide data that point to a new regulatory system by which vascular endothelial cells are supplied to the retina by a reservoir of stem/progenitor cells in the optic nerve.

Naïve pluripotency represents the ground state of mammalian development. A comprehensive understanding of the molecular mechanisms governing its establishment is crucial for elucidating the unique properties of embryonic cells and the regulatory mechanisms controlling cell fate determination. However, the key molecule to robustly achieve naïve pluripotency with minimal manipulation remains unclear. We found that acetylation status of lysine 49 (K49) of Catenin beta-1 (Ctnnb1) plays a critical role in naïve pluripotency of murine stem cells. Deacetylated Ctnnb1 at K49 binds to transcription factor Nanog, impeding its repressor function and thereby promoting differentiation. Remarkably, treatment with IQ1, an inhibitor of interaction between acetyltransferase Ep300 and Ctnnb1, enhances acetylation at K49 of Ctnnb1, enabling establishment and long-term maintenance of embryonic stem cells (ESCs) independently of leukemia inhibitory factor, and also driving complete conversion of epiblast stem cells (EpiSCs) to naïve state. This study reveals the critical role of Ctnnb1 in naïve pluripotency and introduces a novel strategy for its induction and maintenance.

Peripheral helper T (T _PH ) cells can play pathogenic roles in human autoimmune diseases. T _PH cells are proposed to be the major B cell helpers in inflamed joints in rheumatoid arthritis (RA), but whether and how T _PH cells are engaged in tissue inflammation remains unclear. We demonstrate that T _PH cells comprise two subsets in RA: stem-like T _PH (S-T _PH ) and effector T _PH (E-T _PH ) cells. These two subsets differed in transcriptome, epigenome, B cell helper capacity, spatial localization, and cell interactions. S-T _PH cells displayed self-renewal capacity and were mainly found within tertiary lymphoid structures (TLSs) in synovial tissue together with B cells. S-T _PH cells potently induced B cells to produce immunoglobulins. By contrast, E-T _PH cells expressed effector molecules and colocalized with proinflammatory macrophages and CD8 ⁺ T cells outside TLSs. S-T _PH cells could differentiate into E-T _PH cells upon TCR stimulation and coculture with B cells. Collectively, our study shows that S-T _PH cells play a central role in promoting T _PH responses by undergoing self-renewal and seeding E-T _PH cells.

High-grade sarcomas often lack typical morphological features and exhibit no clear differentiation, often leading to a diagnosis of undifferentiated sarcoma (US). Pleomorphic leiomyosarcoma (PLMS) is a high-grade sarcoma consisting of a typical leiomyosarcoma (LMS) component alongside dedifferentiated high-grade areas. A few decades ago, PLMS was regarded as a subtype of high-grade sarcoma previously referred to as malignant fibrous histiocytoma; it is now classified as a variant of LMS. The mechanisms underlying myogenic differentiation and their relevance to the pathological diagnosis of high-grade sarcomas remain poorly understood. To investigate the gene expression networks associated with myogenic differentiation, we employed Cap Analysis of Gene Expression (CAGE) to distinguish PLMS from other high-grade sarcoma subtypes. We analyzed 27 frozen high-grade sarcoma samples, comprising 10 PLMSs, 11 high-grade myxofibrosarcomas, 3 dedifferentiated liposarcomas, 2 USs, and 1 high-grade sarcoma not otherwise specified, using CAGE profiling. Hierarchical clustering based on differentially expressed genes identified by CAGE separated 7 of the 10 PLMSs from other high-grade sarcomas, while the remaining 3 PLMSs clustered with a single US case. CAGE analysis also revealed that the myostatin (MSTN) promoter (false discovery rate [FDR] < 0.05) was more strongly activated in the high-grade sarcoma group lacking morphological and immunohistochemical smooth muscle differentiation than in the PLMS group, whereas the alpha smooth muscle actin (ACTA2) promoter (FDR < 0.05) was more prominently activated in the PLMS group. Immunohistochemical analysis showed reduced or absent myostatin expression in PLMSs, in contrast to diffuse myostatin expression in other high-grade sarcomas. Smooth muscle actin, encoded by ACTA2, was expressed in all 10 PLMS cases but only in 11 of 17 other high-grade sarcomas. Furthermore, both conventional immunohistochemistry and double immunostaining revealed that myostatin and myogenic markers exhibited largely mutually exclusive expression patterns within these tumors. A validation study was performed using 59 soft tissue sarcoma cases, including 27 PLMSs and 16 LMSs. Loss or reduction in myostatin expression was confirmed in both LMS and PLMS, and the ratio of myostatin loss was comparable (62.5% in LMS vs. 63% in PLMS). Collectively, these findings suggest that myostatin contributes to smooth muscle differentiation in high-grade sarcomas and has potential utility as a diagnostic marker.

Human CD4 ⁺ T cells play a central role in the pathogenesis of autoimmune diseases, but their immunoregulatory mechanisms driving pathogenesis remain to be elucidated. We show that human T peripheral helper cells (T _PH cells) regulate peripheral immune responses via insulin-like growth factor–like family member 2 (IGFL2), an inflammatory factor found exclusively in primates. Single-cell RNA sequencing of seropositive rheumatoid arthritis (RA) synovium showed that IGFL2 is specifically expressed by CD4 ⁺ T cells, predominantly T _PH cells. IGFL2 promotes transforming growth factor–β–induced CXCL13 production in CD4 ⁺ T cells, activates nuclear factor κB signaling, and induces monocyte gene signatures like those of pathogenic macrophages. CRISPR-Cas9 knockout of IGFL2 in synovial T _PH cells suppressed this gene signature in cocultured monocytes. Blood IGFL2 protein levels correlated with RA disease severity and could be used as a potential biomarker. These findings highlight the involvement of IGFL2 in RA pathogenesis, emphasizing how human T _PH cells regulate local immune responses via IGFL2.

Autoimmune diseases, among the most common disorders of young adults, are mediated by genetic and environmental factors. Although CD4 ⁺ FOXP3 ⁺ regulatory T cells (T _regs ) play a central role in preventing autoimmunity, the molecular mechanism underlying their dysfunction is unknown. Here, we performed comprehensive transcriptomic and epigenomic profiling of T _regs in the autoimmune disease multiple sclerosis (MS) to identify critical transcriptional programs regulating human autoimmunity. We found that up-regulation of a primate-specific short isoform of PR domain zinc finger protein 1 (PRDM1-S) induces expression of serum and glucocorticoid-regulated kinase 1 (SGK1) independent from the evolutionarily conserved long PRDM1 , which led to destabilization of forkhead box P3 (FOXP3) and T _reg dysfunction. This aberrant PRDM1-S/SGK1 axis is shared among other autoimmune diseases. Furthermore, the chromatin landscape profiling in T _regs from individuals with MS revealed enriched activating protein–1 (AP-1)/interferon regulatory factor (IRF) transcription factor binding as candidate upstream regulators of PRDM1-S expression and T _reg dysfunction. Our study uncovers a mechanistic model where the evolutionary emergence of PRDM1-S and epigenetic priming of AP-1/IRF may be key drivers of dysfunctional T _regs in autoimmune diseases.

Transcribed enhancer maps can reveal nuclear interactions underpinning each cell type and connect specific cell types to diseases. Using a 5′ single-cell RNA sequencing approach, we defined transcription start sites of enhancer RNAs and other classes of coding and noncoding RNAs in human CD4 ⁺ T cells, revealing cellular heterogeneity and differentiation trajectories. Integration of these datasets with single-cell chromatin profiles showed that active enhancers with bidirectional RNA transcription are highly cell type–specific and that disease heritability is strongly enriched in these enhancers. The resulting cell type–resolved multimodal atlas of bidirectionally transcribed enhancers, which we linked with promoters using fine-scale chromatin contact maps, enabled us to systematically interpret genetic variants associated with a range of immune-mediated diseases.

Significance Statement: Ectopic lymphoid structures called tertiary lymphoid tissues (TLTs) develop in several kidney diseases and are associated with poor renal prognosis. However, the mechanisms underlying TLT expansion and their effect on renal regeneration remain unclear. The authors report that single-nucleus RNA sequencing and validation experiments demonstrate that TLTs potentially amplify inflammation in aged injured kidneys. Lymphocytes within TLTs promote proinflammatory phenotypes of the surrounding proximal tubules and fibroblasts within the TLTs via proinflammatory cytokine production. These proinflammatory parenchymal cells then interact with immune cells by chemokine or cytokine production. Such cell-cell interactions potentially increase inflammation, expand TLTs, and exacerbate kidney injury. These findings help illuminate renal TLT pathology and suggest potential therapeutic targets. Background: Ectopic lymphoid structures called tertiary lymphoid tissues (TLTs) develop in several kidney diseases and are associated with poor renal prognosis. However, the mechanisms that expand TLTs and underlie exacerbation of kidney injury remain unclear. Methods: We performed single-nucleus RNA sequencing (snRNA-seq) on aged mouse kidneys with TLTs after ischemia-reperfusion injury. The results were validated using immunostaining, in situ hybridization of murine and human kidneys, and in vitro experiments. Results: Using snRNA-seq, we identified proinflammatory and profibrotic Vcam1 ⁺ injured proximal tubules (PTs) with NFκB and IFN-inducible transcription factor activation. VCAM1⁺ PTs were preferentially localized around TLTs and drove inflammation and fibrosis via the production of multiple chemokines or cytokines. Lymphocytes within TLTs expressed Tnf and Ifng at high levels, which synergistically upregulated VCAM1 and chemokine expression in cultured PT cells. In addition, snRNA-seq also identified proinflammatory and profibrotic fibroblasts, which resided within and outside TLTs, respectively. Proinflammatory fibroblasts exhibited STAT1 activation and various chemokine or cytokine production, including CXCL9/CXCL10 and B cell–activating factor, contributing to lymphocyte recruitment and survival. IFNγ upregulated the expression of these molecules in cultured fibroblasts in a STAT1-dependent manner, indicating potential bidirectional interactions between IFNγ-producing CXCR3⁺ T cells and proinflammatory fibroblasts within TLTs. The cellular and molecular components described in this study were confirmed in human kidneys with TLTs. Conclusions: These findings suggest that TLTs potentially amplify inflammation by providing a microenvironment that allows intense interactions between renal parenchymal and immune cells. These interactions may serve as novel therapeutic targets in kidney diseases involving TLT formation.