Unwinding chromatin at the right places: how BAF is targeted to specific genomic locations during development.

The BAF (SWI/SNF) chromatin remodeling complex plays a crucial role in modulating spatiotemporal gene expression during mammalian development. Although its remodeling activity was characterized in vitro decades ago, the complex actions of BAF in vivo have only recently begun to be unraveled. In living cells, BAF only binds to and remodels a subset of genomic locations. This selectivity of BAF genomic targeting is crucial for cell-type specification and for mediating precise responses to environmental signals. Here, we provide an overview of the distinct molecular mechanisms modulating BAF chromatin binding, including its combinatory assemblies, DNA/histone modification-binding modules and post-translational modifications, as well as its interactions with proteins, RNA and lipids. This Review aims to serve as a primer for future studies to decode the actions of BAF in developmental processes.

Exploring Extracellular Matrix Crosslinking as a Therapeutic Approach to Fibrosis.

The extracellular matrix (ECM) provides structural support for tissues and regulatory signals for resident cells. ECM requires a careful balance between protein accumulation and degradation for homeostasis. Disruption of this balance can lead to pathological processes such as fibrosis in organs across the body. Post-translational crosslinking modifications to ECM proteins such as collagens alter ECM structure and function. Dysregulation of crosslinking enzymes as well as changes in crosslinking composition are prevalent in fibrosis. Because of the crucial roles these ECM crosslinking pathways play in disease, the enzymes that govern crosslinking events are being explored as therapeutic targets for fibrosis. Here, we review in depth the molecular mechanisms underlying ECM crosslinking, how ECM crosslinking contributes to fibrosis, and the therapeutic strategies being explored to target ECM crosslinking in fibrosis to restore normal tissue structure and function.

Modulating in vitro lung fibroblast activation via senolysis of senescent human alveolar epithelial cells.

Idiopathic pulmonary fibrosis (IPF) is an age-related disease with poor prognosis and limited therapeutic options. Activation of lung fibroblasts and differentiation to myofibroblasts are the principal effectors of disease pathology, but damage and senescence of alveolar epithelial cells, specifically type II (ATII) cells, has recently been identified as a potential trigger event for the progressive disease cycle. Targeting ATII senescence and the senescence-associated secretory phenotype (SASP) is an attractive therapeutic strategy; however, translatable primary human cell models that enable mechanistic studies and drug development are lacking. Here, we describe a novel system of conditioned medium (CM) transfer from bleomycin-induced senescent primary alveolar epithelial cells (AEC) onto normal human lung fibroblasts (NHLF) that demonstrates an enhanced fibrotic transcriptional and secretory phenotype compared to non-senescent AEC CM treatment or direct bleomycin damage of the NHLFs. In this system, the bleomycin-treated AECs exhibit classical hallmarks of cellular senescence, including SASP and a gene expression profile that resembles aberrant epithelial cells of the IPF lung. Fibroblast activation by CM transfer is attenuated by pre-treatment of senescent AECs with the senolytic Navitoclax and AD80, but not with the standard of care agent Nintedanib or senomorphic JAK-targeting drugs (e.g., ABT-317, ruxolitinib). This model provides a relevant human system for profiling novel senescence-targeting therapeutics for IPF drug development.

Multi-omics integration identifies cell-state-specific repression by PBRM1-PIAS1 cooperation.

PBRM1 is frequently mutated in cancers of epithelial origin. How PBRM1 regulates normal epithelial homeostasis, prior to cancer initiation, remains unclear. Here, we show that PBRM1's gene regulatory roles differ drastically between cell states, leveraging human skin epithelium (epidermis) as a research platform. In progenitors, PBRM1 predominantly functions to repress terminal differentiation to sustain progenitors' regenerative potential; in the differentiation state, however, PBRM1 switches toward an activator. Between these two cell states, PBRM1 retains its genomic binding but associates with differential interacting proteins. Our targeted screen identified the E3 SUMO ligase PIAS1 as a key interactor. PIAS1 co-localizes with PBRM1 on chromatin to directly repress differentiation genes in progenitors, and PIAS1's chromatin binding drastically diminishes in differentiation. Furthermore, SUMOylation contributes to PBRM1's repressive function in progenitor maintenance. Thus, our findings highlight PBRM1's cell-state-specific regulatory roles influenced by its protein interactome despite its stable chromatin binding.

NUP98 and RAE1 sustain progenitor function through HDAC-dependent chromatin targeting to escape from nucleolar localization.

Self-renewing somatic tissues rely on progenitors to support the continuous tissue regeneration. The gene regulatory network maintaining progenitor function remains incompletely understood. Here we show that NUP98 and RAE1 are highly expressed in epidermal progenitors, forming a separate complex in the nucleoplasm. Reduction of NUP98 or RAE1 abolishes progenitors' regenerative capacity, inhibiting proliferation and inducing premature terminal differentiation. Mechanistically, NUP98 binds on chromatin near the transcription start sites of key epigenetic regulators (such as DNMT1, UHRF1 and EZH2) and sustains their expression in progenitors. NUP98's chromatin binding sites are co-occupied by HDAC1. HDAC inhibition diminishes NUP98's chromatin binding and dysregulates NUP98 and RAE1's target gene expression. Interestingly, HDAC inhibition further induces NUP98 and RAE1 to localize interdependently to the nucleolus. These findings identified a pathway in progenitor maintenance, where HDAC activity directs the high levels of NUP98 and RAE1 to directly control key epigenetic regulators, escaping from nucleolar aggregation.

CDK9 activity switch associated with AFF1 and HEXIM1 controls differentiation initiation from epidermal progenitors.

Progenitors in epithelial tissues, such as human skin epidermis, continuously make fate decisions between self-renewal and differentiation. Here we show that the Super Elongation Complex (SEC) controls progenitor fate decisions by directly suppressing a group of "rapid response" genes, which feature high enrichment of paused Pol II in the progenitor state and robust Pol II elongation in differentiation. SEC's repressive role is dependent on the AFF1 scaffold, but not AFF4. In the progenitor state, AFF1-SEC associates with the HEXIM1-containing inactive CDK9 to suppress these rapid-response genes. A key rapid-response SEC target is ATF3, which promotes the upregulation of differentiation-activating transcription factors (GRHL3, OVOL1, PRDM1, ZNF750) to advance terminal differentiation. SEC peptidomimetic inhibitors or PKC signaling activates CDK9 and rapidly induces these transcription factors within hours in keratinocytes. Thus, our data suggest that the activity switch of SEC-associated CDK9 underlies the initial processes bifurcating progenitor fates between self-renewal and differentiation.

Translational implications of Th17-skewed inflammation due to genetic deficiency of a cadherin stress sensor.

Desmoglein 1 (Dsg1) is a cadherin restricted to stratified tissues of terrestrial vertebrates, which serve as essential physical and immune barriers. Dsg1 loss-of-function mutations in humans result in skin lesions and multiple allergies, and isolated patient keratinocytes exhibit increased proallergic cytokine expression. However, the mechanism by which genetic deficiency of Dsg1 causes chronic inflammation is unknown. To determine the systemic response to Dsg1 loss, we deleted the 3 tandem Dsg1 genes in mice. Whole transcriptome analysis of embryonic Dsg1-/- skin showed a delay in expression of adhesion/differentiation/keratinization genes at E17.5, a subset of which recovered or increased by E18.5. Comparing epidermal transcriptomes from Dsg1-deficient mice and humans revealed a shared IL-17-skewed inflammatory signature. Although the impaired intercellular adhesion observed in Dsg1-/- mice resembles that resulting from anti-Dsg1 pemphigus foliaceus antibodies, pemphigus skin lesions exhibit a weaker IL-17 signature. Consistent with the clinical importance of these findings, treatment of 2 Dsg1-deficient patients with an IL-12/IL-23 antagonist originally developed for psoriasis resulted in improvement of skin lesions. Thus, beyond impairing the physical barrier, loss of Dsg1 function through gene mutation results in a psoriatic-like inflammatory signature before birth, and treatment with a targeted therapy significantly improved skin lesions in patients.

Epidermal progenitors suppress GRHL3-mediated differentiation through intronic polyadenylation promoted by CPSF-HNRNPA3 collaboration.

In self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors.

PRMT1 promotes neuroblastoma cell survival through ATF5.

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma.

Brief report: Hope, perceived maternal empathy, medical regimen adherence, and glycemic control in adolescents with type 1 diabetes.

OBJECTIVE: The relationships among hope, perceived maternal empathy, medical regimen adherence, and glycemic control in adolescents with type 1 diabetes were examined. METHOD: Twenty-nine girls and 21 boys with type 1 diabetes completed measures of hope, perceived maternal empathy, and medical regimen adherence. Each participant's most recent hemoglobin A1c, a measure of glycemic control, was obtained from the diabetes clinic database. RESULTS: Significant correlations were found among hope, perceived maternal empathy, and medical regimen adherence. Significant correlations were also found among hope, perceived maternal empathy, and glycemic control. Adolescents' perceptions of maternal empathy were positively correlated with level of hope. Hope appeared to mediate the relationship between perceived maternal empathy and adherence, as well as between perceived maternal empathy and glycemic control. CONCLUSIONS: The results of this study affirm the need for longitudinal research that examines the associations among hope, perceived maternal empathy, medical regimen adherence, and glycemic control.

Pinpointing the Genomic Localizations of Chromatin-Associated Proteins: The Yesterday, Today, and Tomorrow of ChIP-seq.

Chromatin-associated proteins are instrumental for controlling spatiotemporal gene expression. Determining where these proteins bind across the genome is critical for understanding gene regulation. A widely used technique at present is ChIP-seq, which leverages chromatin fragmentation, antibody-mediated enrichment, next-generation sequencing, and data analysis to uncover the genomic sequences and patterns of protein-DNA interactions. In this article, we will provide an overview of how ChIP-seq was developed, the key elements of the experimentation and data analysis pipeline, and the recent variations that push the boundaries of precision and cell number requirements. We will also briefly discuss how future development of ChIP-seq may further advance our understanding of chromatin biology. © 2019 by John Wiley & Sons, Inc.