Summary The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem (ES) cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence, and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres, and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.
Therapeutic antibodies blocking programmed death-1 and its ligand (PD-1/PD-L1) induce durable responses in a substantial fraction of melanoma patients. We sought to determine whether the number and/or type of mutations identified using a next generation sequencing (NGS) panel available in the clinic were correlated with response to anti–PD-1 in melanoma. Using archival melanoma samples from anti–PD-1/PD-L1-treated patients, we performed hybrid capture-based NGS on 236–315 genes and T-cell receptor (TCR) sequencing on initial and validation cohorts from two centers. Patients who responded to anti–PD-1/PD-L1 had higher mutational loads in an initial cohort (median 45.6 vs. 3.9 mutations/MB; P = 0.003), and a validation cohort (37.1 vs. 12.8 mutations/MB; P = 0.002) compared to nonresponders. Response rate, progression-free survival, and overall survival was superior in the high, compared to intermediate and low, mutation load groups. Melanomas with NF1 mutations harbored high mutational loads (median 62.7 mutations/MB) and high response rates (74%) whereas BRAF/NRAS/NF1 wild-type melanomas had a lower mutational load. In these archival samples, TCR clonality did not predict response. Mutation numbers in the 315 genes in the NGS platform strongly correlated with those detected by whole exome sequencing in The Cancer Genome Atlas samples, but was not associated with survival. In conclusion, mutational load, as determined by an NGS platform available in the clinic, effectively stratified patients by likelihood of response. This approach may provide a clinically feasible predictor of response to anti–PD-1/PD-L1.
Gastrointestinal stromal tumour (GIST) is the most common human sarcoma and is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases1,2. KIT is highly expressed in interstitial cells of Cajal (ICCs)—the presumed cell of origin for GIST—as well as in hematopoietic stem cells, melanocytes, mast cells and germ cells2,3. Yet, families harbouring germline activating KIT mutations and mice with knock-in Kit mutations almost exclusively develop ICC hyperplasia and GIST4–7, suggesting that the cellular context is important for KIT to mediated oncogenesis. Here we show that the ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT mediated transformation8, and is required for their development. In addition, ETV1 is universally highly expressed in GISTs and is required for growth of imatinib-sensitive and resistant GIST cell lines. Transcriptome profiling and global analyses of ETV1-binding sites suggest that ETV1 is a master regulator of an ICC-GIST-specific transcription network mainly through enhancer binding. The ETV1 transcriptional program is further regulated by activated KIT, which prolongs ETV1 protein stability and cooperates with ETV1 to promote tumourigenesis. We propose that GIST arises from ICCs with high levels of endogenous ETV1 expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcription program. This differs from other ETS-dependent tumours such as prostate cancer, melanoma, and Ewing sarcoma where genomic translocation or amplification drives aberrant ETS expression9–11 and represents a novel mechanism of oncogenic transcription factor activation.
Summary Using mouse skin, where bountiful reservoirs of synchronized hair follicle stem cells (HF-SCs) fuel cycles of regeneration, we explore how adult SCs remodel chromatin in response to activating cues. By profiling global mRNA and chromatin changes in quiescent and activated HF-SCs and their committed, transit-amplifying (TA) progeny, we show that polycomb-group(PcG)-mediated H3K27-trimethylation features prominently in HF-lineage progression by mechanisms distinct from embryonic-SCs. In HF-SCs, PcG represses non-skin lineages and HF-differentiation. In TA-progeny, non-skin regulators remain PcG-repressed, HF-SC regulators acquire H3K27me3-marks and HF-lineage regulators lose them. Interestingly, genes poised in embryonic-SCs, active in HF-SCs and PcGrepressed in TA-progeny, encode not only key transcription factors, but also signaling regulators. We document their importance in balancing HF-SC quiescence, underscoring the power of chromatin mapping in dissecting SC behavior. Our findings explain how HF-SCs cycle through quiescent and activated states without losing stemness, and define roles for PcG-mediated repression in governing a fate switch irreversibly.
Hair production is fueled by stem cells (SCs), which transition between cyclical bouts of rest and activity. Here, we explore why hair growth wanes with age. We show that aged hair follicle SCs (HFSCs) in mice exhibit enhanced resting and abbreviated growth phases and are delayed in response to tissue-regenerating cues. Aged HFSCs are poor at initiating proliferation and show diminished self-renewing capacity upon extensive use. Only modestly restored by parabiosis, these features are rooted in elevated cell-intrinsic sensitivity and local elevation in bone morphogenic protein (BMP) signaling. Transcriptional profiling presents differences consistent with defects in aged HFSC activation. Notably, BMP-/calcium-regulated, nuclear factor of activated T-cell c1 (NFATc1) in HFSCs becomes recalcitrant to its normal down-regulating cues, and NFATc1 ChIP-sequencing analyses reveal a marked enrichment of NFATc1 target genes within the age-related signature. Moreover, aged HFSCs display more youthful levels of hair regeneration when BMP and/or NFATc1 are inhibited. These results provide unique insights into how skin SCs age.BMP signaling | hair cycle | quiescence I n adult tissues, stem cells (SCs) must replace cells lost to acute injury and normal biological activity (homeostasis). Aging can be viewed as a failure to maintain proper tissue homeostasis, resulting in a decline in tissue function and delayed response to tissue damage (1). Age-related extrinsic changes in external, systemic, and/or local tissue environment, coupled with intrinsic changes from repetitive use, are all potential underlying causes for SC malfunction. However, the relative contributions of these factors on SC aging vary among SC populations. Studies on hematopoietic and melanocyte SCs show that age-related intrinsic perturbations can impair SC function (2-4). Mesenchymal SCs, cardiac SCs, and liver progenitor cells also show age-related declines in performance (5-7). The impact of extrinsic perturbations is evident from studies on muscle and neural SCs, where exposure to a youthful systemic environment can restore SC functional capabilities (7-10). Most recently, it was shown that cardiomyocytes rely upon systemic growth and differentiation factor 11 (GDF11), a member of the transforming growth factor β (TGF-β) superfamily, which declines with age (11).The skin has some of the most recognizable age-associated changes. In humans and other mammals, skin shows an agerelated decline in homeostasis, with both dermal and epidermal thinning, reductions in epidermal proliferation and injury repair, loss of dermal elasticity, wrinkling, and notably, hair thinning and eventual loss (12). Periods of rest in hair follicles (HFs) also become longer as animals age, and in humans, hair density declines with age. It has been suggested that the progressive dormancy of HFs during aging is a reflection of a declining capacity of SCs to initiate a new hair cycle, but this has not been formally tested and the underlying mechanisms remain largely unexplored.HFs underg...
Recent genetic association studies have identified 55 genetic loci associated with obesity or body mass index (BMI). The vast majority, 51 loci, however, were identified in European-ancestry populations. We conducted a meta-analysis of associations between BMI and ∼2.5 million genotyped or imputed single nucleotide polymorphisms among 86 757 individuals of Asian ancestry, followed by in silico and de novo replication among 7488-47 352 additional Asian-ancestry individuals. We identified four novel BMI-associated loci near the KCNQ1 (rs2237892, P = 9.29 × 10(-13)), ALDH2/MYL2 (rs671, P = 3.40 × 10(-11); rs12229654, P = 4.56 × 10(-9)), ITIH4 (rs2535633, P = 1.77 × 10(-10)) and NT5C2 (rs11191580, P = 3.83 × 10(-8)) genes. The association of BMI with rs2237892, rs671 and rs12229654 was significantly stronger among men than among women. Of the 51 BMI-associated loci initially identified in European-ancestry populations, we confirmed eight loci at the genome-wide significance level (P < 5.0 × 10(-8)) and an additional 14 at P < 1.0 × 10(-3) with the same direction of effect as reported previously. Findings from this analysis expand our knowledge of the genetic basis of obesity.
BACKGROUND & AIMS: Genome-wide association studies (GWASs) have associated approximately 50 loci with risk of colorectal cancer (CRC)-nearly one third of these loci were initially associated with CRC in studies conducted in East Asian populations. We conducted a GWAS of East Asians to identify CRC risk loci and evaluate the generalizability of findings from GWASs of European populations to Asian populations. METHODS: We analyzed genetic data from 22,775 patients with CRC (cases) and 47,731 individuals without cancer (controls) from 14 studies in the Asia Colorectal Cancer Consortium. First, we performed a meta-analysis of 7 GWASs (10,625 cases and 34,595 controls) and identified 46,554 promising risk variants for replication by adding them to the Multi-Ethnic Global Array (MEGA) for genotype analysis in 6445 cases and 7175 controls. These data were analyzed, along with data from an additional 5705 cases and 5961 controls genotyped using the OncoArray. We also obtained data from 57,976 cases and 67,242 controls of European descent. Variants at identified risk loci were functionally annotated and evaluated in correlation with gene expression levels. RESULTS: A meta-analyses of all Gastroenterology 2019;156:1455-1466 BASIC AND TRANSLATIONAL AT samples from people of Asian descent identified 13 loci and 1 new variant at a known locus (10q24.2) associated with risk of CRC at the genome-wide significance level of P < 5 Â 10-8. We did not perform experiments to replicate these associations in additional individuals of Asian ancestry. However, the lead risk variant in 6 of these loci was also significantly associated with risk of CRC in European descendants. A strong association (44%-75% increase in risk per allele) was found for 2 lowfrequency variants: rs201395236 at 1q44 (minor allele frequency, 1.34%) and rs77969132 at 12p11.21 (minor allele frequency, 1.53%). For 8 of the 13 associated loci, the variants with the highest levels of significant association were located inside or near the protein-coding genes L1TD1, EFCAB2, PPP1R21, SLCO2A1, HLA-G, NOTCH4, DENND5B, and GNAS. For other intergenic loci, we provided evidence for the possible involvement of the genes ALDH7A1, PRICKLE1, KLF5, WWOX, and GLP2R. We replicated findings for 41 of 52 previously reported risk loci. CONCLUSIONS: We showed that most of the risk loci previously associated with CRC risk in individuals of European descent were also associated with CRC risk in East Asians. Furthermore, we identified 13 loci significantly associated with risk for CRC in Asians. Many of these loci contained genes that regulate the immune response, Wnt signaling to bcatenin, prostaglandin E2 catabolism, and cell pluripotency and proliferation. Further analyses of these genes and their variants is warranted, particularly for the 8 loci for which the lead CRC risk variants were not replicated in persons of European descent.
Key Points• Analysis of CSF-1R pTyrregulated messenger RNAs identifies novel signaling nodes and networks that can be targeted to modulate macrophage functions.• miR-21 is a novel CSF-1R pTyr-721-induced molecule that suppresses the macrophage M1 phenotype and enhances the M2 phenotype.Macrophage polarization between the M2 (repair, protumorigenic) and M1 (inflammatory) phenotypes is seen as a continuum of states. The detailed transcriptional events and signals downstream of colony-stimulating factor 1 receptor (CSF-1R) that contributes to amplification of the M2 phenotype and suppression of the M1 phenotype are largely unknown. Macrophage CSF-1R pTyr-721 signaling promotes cell motility and enhancement of tumor cell invasion in vitro. Combining analysis of cellular systems for CSF-1R gain of function and loss of function with bioinformatic analysis of the macrophage CSF-1R pTyr-721-regulated transcriptome, we uncovered microRNA-21 (miR-21) as a downstream molecular switch controlling macrophage activation and identified extracellular signal-regulated kinase 1/2 and nuclear factor-kB as CSF-1R pTyr-721-regulated signaling nodes. We show that CSF-1R pTyr-721 signaling suppresses the inflammatory phenotype, predominantly by induction of miR-21. Profiling of the miR-21-regulated messenger RNAs revealed that 80% of the CSF-1-regulated canonical miR-21 targets are proinflammatory molecules. Additionally, miR-21 positively regulates M2 marker expression. Moreover, miR-21 feeds back to positively regulate its own expression and to limit CSF-1R-mediated activation of extracellular signal-regulated kinase 1/2 and nuclear factor-kB. Consistent with an anti-inflammatory role of miRNA-21, intraperitoneal injection of mice with a miRNA-21 inhibitor increases the recruitment of inflammatory monocytes and enhances the peritoneal monocyte/macrophage response to lipopolysaccharide. These results identify the CSF-1R-regulated miR-21 network that modulates macrophage polarization. (Blood. 2015;125(8):e1-e13) IntroductionMacrophages protect the host against infection and injury and facilitate tissue remodeling.1 However, they frequently accumulate in pathological settings, including cancers, 2 atherosclerosis, 3 metabolic disease, 4 and sepsis, 5 where they respond to microenvironmental cues that can be detrimental to the host. Two distinct extreme states of polarized activation have been described in macrophages:6,7 the classically activated (M1) and the alternatively activated (M2) macrophage phenotypes, each characterized by well-described markers. 5,6,[8][9][10][11] M1 macrophages produce proinflammatory cytokines, elevate the expression of inducible nitric oxide synthase 2 (iNOS) and major histocompatibility complex class II (MHC II), 12 and can play antitumorigenic roles. 5,9 In contrast, the M2 macrophages have increased expression of scavenger receptors, increased activation of the arginase pathway, low expression of interleukin-12 (IL-12), high expression of IL-10 and IL-1RA, and increased anti-inflammatory responses a...
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