SummaryHeterozygous mutations in the gene encoding CHD7, an ATP-dependent chromatin remodeler result in a complex constellation of congenital anomalies called CHARGE syndrome. Here we show that in humans and in Xenopus, CHD7 is essential for the formation of multipotent migratory neural crest cells, a transient cell population that is ectodermal in origin, but undergoes a major gene expression reprogramming to acquire a remarkably broad differentiation potential and ability to migrate throughout the body to give rise to bones, cartilages, nerves, and cardiac structures. We demonstrate that CHD7 function is essential for activation of core components of neural crest transcriptional circuitry, including Sox9, Twist and Slug. Moreover, the major features of CHARGE are recapitulated in Xenopus embryo by the downregulation of CHD7 levels or overexpression of its catalytically inactive ATP-ase mutant. We further show that in human multipotent neural crest cells, CHD7 associates with a BRG1-containing complex PBAF, and both factors co-occupy a neural crestspecific distal SOX9 enhancer, as well as a novel genomic element located upstream from TWIST1 gene and marked by H3K4me1. Furthermore, in the embryo CHD7 and PBAF act synergistically to promote neural crest gene expression and cell migration. Our work identifies an evolutionary conserved role for CHD7 in orchestrating neural crest gene expression programs, provides insights into the synergistic regulation of distal genomic elements by two distinct chromatin remodelers, and illuminates the patho-embryology of CHARGE syndrome.Recent studies demonstrate that unique chromatin states are associated with retained or restricted differentiation potential. 1 During organismal development, cells gradually restrict their differentiation potential to produce specialized tissues and organs. One exception is germ cell formation, which is accompanied by reacquisition of the pluripotent state. Another major developmental reprogramming event occurs in vertebrate organisms during formation of the §

SummaryThe role of long noncoding RNA (lncRNA) in adult hearts is unknown; also unclear is how lncRNA modulates nucleosome remodeling. An estimated 70% of mouse genes undergo antisense transcription1, including myosin heavy chain 7 (Myh7) that encodes molecular motor proteins for heart contraction2. Here, we identify a cluster of lncRNA transcripts from Myh7 loci and show a new lncRNA–chromatin mechanism for heart failure. In mice, these transcripts, which we named Myosin Heavy Chain Associated RNA Transcripts (MyHEART or Mhrt), are cardiac-specific and abundant in adult hearts. Pathological stress activates the Brg1-Hdac-Parp chromatin repressor complex3 to inhibit Mhrt transcription in the heart. Such stress-induced Mhrt repression is essential for cardiomyopathy to develop: restoring Mhrt to the pre-stress level protects the heart from hypertrophy and failure. Mhrt antagonizes the function of Brg1, a chromatin-remodeling factor that is activated by stress to trigger aberrant gene expression and cardiac myopathy3. Mhrt prevents Brg1 from recognizing its genomic DNA targets, thus inhibiting chromatin targeting and gene regulation by Brg1. Mhrt binds to the helicase domain of Brg1, and this domain is crucial for tethering Brg1 to chromatinized DNA targets. Brg1 helicase has dual nucleic acid-binding specificities: it is capable of binding lncRNA (Mhrt) and chromatinized—but not naked—DNA. This dual-binding feature of helicase enables a competitive inhibition mechanism by which Mhrt sequesters Brg1 from its genomic DNA targets to prevent chromatin remodeling. A Mhrt-Brg1 feedback circuit is thus crucial for heart function. Human MHRT also originates from MYH7 loci and is repressed in various types of myopathic hearts, suggesting a conserved lncRNA mechanism in human cardiomyopathy. Our studies identify the first cardioprotective lncRNA, define a new targeting mechanism for ATP-dependent chromatin-remodeling factors, and establish a new paradigm for lncRNA–chromatin interaction.

Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including “vascular disease,” “disorder of artery,” and “occlusion of artery,” as well as disease-related cellular functions including “cellular movement” and “cellular growth and proliferation.” In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE-/- and Ldlr-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall.

Development of the cerebral vessels, pharyngeal arch arteries (PAAs). and cardiac outflow tract (OFT) requires multipotent neural crest cells (NCCs) that migrate from the neural tube to target tissue destinations. Little is known about how mammalian NCC development is orchestrated by gene programming at the chromatin level, however. Here we show that Brahma-related gene 1 (Brg1), an ATPase subunit of the Brg1/Brahma-associated factor (BAF) chromatin-remodeling complex, is required in NCCs to direct cardiovascular development. Mouse embryos lacking Brg1 in NCCs display immature cerebral vessels, aberrant PAA patterning, and shortened OFT. Brg1 suppresses an apoptosis factor, Apoptosis signal-regulating kinase 1 (Ask1), and a cell cycle inhibitor, p21 cip1 , to inhibit apoptosis and promote proliferation of NCCs, thereby maintaining a multipotent cell reservoir at the neural crest. Brg1 also supports Myosin heavy chain 11 (Myh11) expression to allow NCCs to develop into mature vascular smooth muscle cells of cerebral vessels. Within NCCs, Brg1 partners with chromatin remodeler Chromodomain-helicase-DNAbinding protein 7 (Chd7) on the PlexinA2 promoter to activate PlexinA2, which encodes a receptor for semaphorin to guide NCCs into the OFT. Our findings reveal an important role for Brg1 and its downstream pathways in the survival, differentiation, and migration of the multipotent NCCs critical for mammalian cardiovascular development.N eural crest cells (NCCs) originate from the neural crest of the dorsal neural tube and migrate to many regions of the embryo, where they differentiate into a variety of local cells, including cardiovascular tissues (1). NCCs that emigrate from the neural crest of rhombomere 6-8 to pharyngeal arches and the heart are essential for the patterning of pharyngeal arch arteries (PAAs) and the cardiac outflow tract (OFT) (2, 3). These NCCs also differentiate into vascular smooth muscle cells (SMCs) of PAAs and the muscular septum of the aorta and pulmonary trunk (4, 5). In contrast, NCCs from the cephalic neural tube migrate to the face and forebrain to form craniofacial bones, as well as SMCs of facial and forebrain vessels (6). Thus, NCCs are critical for the formation of cardiac OFT and vascular supplies of large areas of the body.Disruption of NCC development, either directly or indirectly, results in many forms of human birth defects with cardiovascular malformations, including Alagille, Carpenter, Ivemark, Leopard, Williams, DiGeorge, and CHARGE syndromes (7). These syndromes involve defects in PAAs or cardiac OFT, such as coarctation of the aorta, interrupted aortic arch, pulmonary artery stenosis, double-outlet right ventricle, tetralogy of Fallot, or persistent truncus arteriosus. During PAA and OFT development, NCCs are regulated by numerous transcription factors, including Pax3, Pbx1/2/3, Tbx1/2/3/20, Msx1/2, Hand2, AP2a, Cited2, Pitx2, Sox4, Foxc1/c2/d3/h1, Fog2, Gata3/4/6, and Notch/NICD (8). Such extensive involvement of transcription factors indicates the importance of gene pr...

Hair follicle stem cells (bulge cells) are essential for hair regeneration and early epidermal repair after wounding. Here we show that Brg1, a key enzyme in the chromatin-remodeling machinery, is dynamically expressed in bulge cells to control tissue regeneration and repair. In mice, sonic hedgehog (Shh) signals Gli to activate Brg1 in bulge cells to begin hair regeneration, whereas Brg1 recruits NF-κB to activate Shh in matrix cells to sustain hair growth. Such reciprocal Brg1-Shh interaction is essential for hair regeneration. Moreover, Brg1 is indispensable for maintaining the bulge cell reservoir. Without Brg1, bulge cells are depleted over time, partly through the ectopic expression of the cell-cycle inhibitor p27(Kip1). Also, bulge Brg1 is activated by skin injury to facilitate early epidermal repair. Our studies demonstrate a molecular circuit that integrates chromatin remodeling (Brg1), transcriptional regulation (NF-κB, Gli), and intercellular signaling (Shh) to control bulge stem cells during tissue regeneration.

Chromatin structure is determined by nucleosome positioning, histone modifications, and DNA methylation. How chromatin modifications are coordinately altered under pathological conditions remains elusive. Here we describe a stress-activated mechanism of concerted chromatin modification in the heart. In mice, pathological stress activates cardiomyocytes to express Brg1 (nucleosome-remodeling factor), G9a/Glp (histone methyltransferase), and Dnmt3 (DNA methyltransferase). Once activated, Brg1 recruits G9a and then Dnmt3 to sequentially assemble repressive chromatin-marked by H3K9 and CpG methylation-on a key molecular motor gene (Myh6), thereby silencing Myh6 and impairing cardiac contraction. Disruption of Brg1, G9a or Dnmt3 erases repressive chromatin marks and de-represses Myh6, reducing stress-induced cardiac dysfunction. In human hypertrophic hearts, BRG1-G9a/GLP-DNMT3 complex is also activated; its level correlates with H3K9/CpG methylation, Myh6 repression, and cardiomyopathy. Our studies demonstrate a new mechanism of chromatin assembly in stressed hearts and novel therapeutic targets for restoring Myh6 and ventricular function. The stress-induced Brg1-G9a-Dnmt3 interactions and sequence of repressive chromatin assembly on Myh6 illustrates a molecular mechanism by which the heart epigenetically responds to environmental signals. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

BackgroundExtrapulmonary small cell carcinomas (ESCC) are rare but aggressive tumors. Relapses are common despite treatment with chemotherapy and/or radiotherapy. Prospective data for treatment of ESCC are lacking; treatment of these cancers usually incorporates lung small cell carcinoma treatment recommendations. Cancer staging remains the most important prognostic factor. Cancer immunotherapy targeting the PD-1/PD-L1 pathway has shown efficacy in multiple tumor types, and could be an appealing treatment strategy for these rare tumors.MethodsWe investigated PD-L1 expression by immunochemistry (IHC) in ESCCs diagnosed at University of Massachusetts Medical Center, from 1999 to 2016. 34 cases with sufficient material were selected for PD-L1 IHC analysis using clone E1L3N. PD-L1 expression was evaluated using the combined positive score (CPS). Retrospective chart review was performed. We evaluated the incidence and prognostic value of PD-L1 expression in ESCC at our institution.ResultsTwelve out 34 cases (35%) had PD-L1 CPS scores ≥1. Ten cases had CPS scores ranging 1–5, whereas 2 cases had CPS scores > 80. The overall response rate to the standard chemotherapy with/without radiotherapy in the PD-L1 positive group was 80% versus 67% for the PDL-1 negative group (p-value 0.67). The median overall survival for the PD-L1 positive group, regardless of stage, was 11.5 months versus 7 months for PD-L1 negative group (p-value 0.34). Patients with limited stage disease with positive PD-L1 had a median survival of 53 months compared to 15 months for patients with PD-L1 negative limited stage (p-value 0.80).ConclusionsThis study showed that at least one third of our ESCC tissue samples expressed PD-L1. There was a trend for higher response rates to the standard chemotherapy with/without radiotherapy and improved survival in PD-L1 positive patients. Further studies are required to understand the implications of immune dysregulation in these aggressive tumors. PD-L1/PD-1 inhibitors should be investigated in this group of patients.

Objectives. Transducer-like enhancer of split 1 (TLE1) immunohistochemistry is widely used as a biomarker of synovial sarcoma. Spindle cell or desmoplastic melanoma can morphologically mimic synovial sarcoma. The aim of this study was to investigate the expression of TLE1 in melanomas with a spindle cell morphology. Methods. A search of the surgical pathology files resulted in 57 cases of melanomas diagnosed with a spindle cell or desmoplastic component. After review, 8 cases had no definitive dermal spindle cell component and 7 cases had insufficient tissue remaining and were excluded from the study. A total of 42 melanomas were examined for TLE1 immunohistochemistry using a mouse monoclonal antibody (Cell Marque, clone 1F5). Strength and percentage of nuclear TLE1 positivity was graded on a scale from 0 to 3+. Staining for TLE1 was considered positive for 2 to 3+ and negative for 0 to 1+. Results. Nuclear TLE1 expression was identified in 24 (57%) of the 42 melanoma cases with spindle cell morphology (2+, n = 14; 3+, n = 10). TLE1 was considered negative in 18 cases (43%), of which most contained weak staining (1+, n = 14 [33%]) and only a small subset did not show any staining (0, n = 4 [10%]). Conclusion. TLE1 frequently highlights melanomas with spindle cell morphology and is a potential diagnostic pitfall. Therefore, when evaluating spindle cell tumors in which the differential may include both a melanoma and synovial sarcoma, TLE1 expression should be interpreted with caution and in conjunction with an immunohistochemical panel.