The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of osteogenesis ...imperfecta (OI) and the Ehlers–Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and a zebrafish model resembling human EDS, which harbors a number of soft connective tissues defects, typical of EDS. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen-related disease models. Our studies reveal a high degree of intergenotype variability in phenotypic expressivity that closely correlates with associated OI severity. Furthermore, we demonstrate the potential for select mutations to give rise to phenotypic variability, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the future potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention.
Eukaryotic gene transcription is accompanied by acetylation and methylation of nucleosomes near promoters, but the locations and roles of histone modifications elsewhere in the genome remain unclear. ...We determined the chromatin modification states in high resolution along 30 Mb of the human genome and found that active promoters are marked by trimethylation of Lys4 of histone H3 (H3K4), whereas enhancers are marked by monomethylation, but not trimethylation, of H3K4. We developed computational algorithms using these distinct chromatin signatures to identify new regulatory elements, predicting over 200 promoters and 400 enhancers within the 30-Mb region. This approach accurately predicted the location and function of independently identified regulatory elements with high sensitivity and specificity and uncovered a novel functional enhancer for the carnitine transporter SLC22A5 (OCTN2). Our results give insight into the connections between chromatin modifications and transcriptional regulatory activity and provide a new tool for the functional annotation of the human genome.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Disorders involving β-globin gene mutations, primarily β-thalassemia and sickle cell disease, represent a major target for hematopoietic stem/progenitor cell (HSPC) gene therapy. This includes ...CRISPR/Cas9-mediated genome editing approaches in adult CD34+ cells aimed toward the reactivation of fetal γ-globin expression in red blood cells. Because models involving erythroid differentiation of CD34+ cells have limitations in assessing γ-globin reactivation, we focused on human β-globin locus-transgenic (β-YAC) mice. We used a helper-dependent human CD46-targeting adenovirus vector expressing CRISPR/Cas9 (HDAd-HBG-CRISPR) to disrupt a repressor binding region within the γ-globin promoter. We transduced HSPCs from β-YAC/human CD46–transgenic mice ex vivo and subsequently transplanted them into irradiated recipients. Furthermore, we used an in vivo HSPC transduction approach that involves HSPC mobilization and the intravenous injection of HDAd-HBG-CRISPR into β-YAC/CD46–transgenic mice. In both models, we demonstrated efficient target site disruption, resulting in a pronounced switch from human β- to γ-globin expression in red blood cells of adult mice that was maintained after secondary transplantation of HSPCs. In long-term follow-up studies, we did not detect hematological abnormalities, indicating that HBG promoter editing does not negatively affect hematopoiesis. This is the first study that shows successful in vivo HSPC genome editing by CRISPR/Cas9.
•CRISPR/Cas9-mediated disruption of a BCL11A binding site in HSCs of β-YAC mice results in the reactivation of γ-globin in erythrocytes.•Our approach for in vivo HSC genome editing that does not require HSC transplantation and myeloablation should simplify HSC gene therapy.
Display omitted
Abstract
Atopic dermatitis (AD) is a common pruritic inflammatory skin disease with unclear molecular and cellular contributions behind the complex etiology. To unravel these differences between ...healthy control and AD skin we employed single-cell transcriptomics, utilizing the canine AD model for its resemblance to human clinical and molecular phenotypes. In this study, we show that there are overall increases in keratinocytes and T cells and decreases in fibroblast populations in AD dogs. Within immune cell types, we identified an enriched γδ T cell population in AD, which may contribute to cutaneous inflammation. A prominent IL26-positive fibroblast subpopulation in AD was detected, which may activate neighboring cells in the dermal–epidermal niche. Lastly, by comparing dogs with different disease severities, we found genes that follow disease progression and may serve as potential biomarkers. In this study, we characterized key AD cell types and cellular processes that can be further leveraged in diagnosis and treatment.
Type I diabetes is an autoimmune disease mediated by T-cell destruction of β cells in pancreatic islets. Currently, there is no known cure, and treatment consists of daily insulin injections. ...Genome-wide association studies and twin studies have indicated a strong genetic heritability for type I diabetes and implicated several genes. As most strongly associated variants are noncoding, there is still a lack of identification of functional and, therefore, likely causal variants. Given that many of these genetic variants reside in enhancer elements, we have tested 121 CD4+ T-cell enhancer variants associated with T1D. We found four to be functional through massively parallel reporter assays. Three of the enhancer variants weaken activity, while the fourth strengthens activity. We link these to their cognate genes using 3D genome architecture or eQTL data and validate them using CRISPR editing. Validated target genes include CLEC16A and SOCS1. While these genes have been previously implicated in type 1 diabetes and other autoimmune diseases, we show that enhancers controlling their expression harbor functional variants. These variants, therefore, may act as causal type 1 diabetic variants.
DNA methylation is one of several epigenetic mechanisms that contribute to the regulation of gene expression; however, the extent to which methylation of CpG dinucleotides correlates with gene ...expression at the genome-wide level is still largely unknown. Using purified primary monocytes from subjects in a large community-based cohort (n = 1264), we characterized methylation (>485 000 CpG sites) and mRNA expression (>48K transcripts) and carried out genome-wide association analyses of 8370 expression phenotypes. We identified 11 203 potential cis-acting CpG loci whose degree of methylation was associated with gene expression (eMS) at a false discovery rate threshold of 0.001. Most of the associations were consistent in effect size and direction of effect across sex and three ethnicities. Contrary to expectation, these eMS were not predominately enriched in promoter regions, or CpG islands, but rather in the 3' UTR, gene bodies, CpG shores or 'offshore' sites, and both positive and negative correlations between methylation and expression were observed across all locations. eMS were enriched for regions predicted to be regulatory by ENCODE (Encyclopedia of DNA Elements) data in multiple cell types, particularly enhancers. One of the strongest association signals detected (P < 2.2 × 10(-308)) was a methylation probe (cg17005068) in the promoter/enhancer region of the glutathione S-transferase theta 1 gene (GSTT1, encoding the detoxification enzyme) with GSTT1 mRNA expression. Our study provides a detailed description of the epigenetic architecture in human monocytes and its relationship to gene expression. These data may help prioritize interrogation of biologically relevant methylation loci and provide new insights into the epigenetic basis of human health and diseases.
Activating germline mutations in STAT3 were recently identified as a cause of neonatal diabetes mellitus associated with beta-cell autoimmunity. We have investigated the effect of an activating ...mutation, STAT3K392R, on pancreatic development using induced pluripotent stem cells (iPSCs) derived from a patient with neonatal diabetes and pancreatic hypoplasia. Early pancreatic endoderm differentiated similarly from STAT3K392R and healthy-control cells, but in later stages, NEUROG3 expression was upregulated prematurely in STAT3K392R cells together with insulin (INS) and glucagon (GCG). RNA sequencing (RNA-seq) showed robust NEUROG3 downstream targets upregulation. STAT3 mutation correction with CRISPR/Cas9 reversed completely the disease phenotype. STAT3K392R-activating properties were not explained fully by altered DNA-binding affinity or increased phosphorylation. Instead, reporter assays demonstrated NEUROG3 promoter activation by STAT3 in pancreatic cells. Furthermore, proteomic and immunocytochemical analyses revealed increased nuclear translocation of STAT3K392R. Collectively, our results demonstrate that the STAT3K392R mutation causes premature endocrine differentiation through direct induction of NEUROG3 expression.
Display omitted
•iPSCs derived from a neonatal diabetes patient with an activating STAT3 mutation•Mutant cells show premature endocrine differentiation through NEUROG3 upregulation•Disease phenotype normalized by CRISPR/Cas9 mutation correction•Mechanism involves increased nuclear shuttling of mutant STAT3
Saarimäki-Vire et al. use iPSCs derived from a patient with permanent neonatal diabetes to demonstrate that an activating STAT3 mutation leads to premature NEUROG3 expression and concomitant differentiation of pancreatic progenitors through increased nuclear shuttling of the mutant protein.
Our goal is the development of in vivo hematopoietic stem cell (HSC) transduction technology with targeted integration. To achieve this, we modified helper-dependent HDAd5/35++ vectors to express a ...CRISPR/Cas9 specific to the “safe harbor” adeno-associated virus integration site 1 (AAVS1) locus and to provide a donor template for targeted integration through homology-dependent repair. We tested the HDAd-CRISPR + HDAd-donor vector system in AAVS1 transgenic mice using a standard ex vivo HSC gene therapy approach as well as a new in vivo HSC transduction approach that involves HSC mobilization and intravenous HDAd5/35++ injections. In both settings, the majority of treated mice had transgenes (GFP or human γ-globin) integrated into the AAVS1 locus. On average, >60% of peripheral blood cells expressed the transgene after in vivo selection with low-dose O6BG/bis-chloroethylnitrosourea (BCNU). Ex vivo and in vivo HSC transduction and selection studies with HDAd-CRISPR + HDAd-globin-donor resulted in stable γ-globin expression at levels that were significantly higher (>20% γ-globin of adult mouse globin) than those achieved in previous studies with a SB100x-transposase-based HDAd5/35++ system that mediates random integration. The ability to achieve therapeutically relevant transgene expression levels after in vivo HSC transduction and selection and targeted integration make our HDAd5/35++-based vector system a new tool in HSC gene therapy.
Display omitted
We developed a simple in vivo hematopoietic stem and progenitor cell (HSPC) gene therapy approach that involves GSCF/AMD3100-mediated mobilization of HSPCs from the bone marrow. Although HSPCs circulate at high numbers in the periphery, we transduce them with intravenously injected helper-dependent HDAd5/35++ adenovirus vectors. Transduced cells return to the bone marrow, where they persist long term. Using a HDAd5/35++-CRISPR/Cas9 plus HDAd5/35++-γ-globin donor vector system, we demonstrate efficient targeted integration in AAVS1 transgenic mice. γ-Globin expression levels were significantly higher than those achieved with a Sleeping Beauty (SB100x) transposase-based HDAd5/35++ system that mediated random integration.
Assisted reproductive technologies are known to alter the developmental environment of gametes and early embryos during the most dynamic period of establishing the epigenome. This may result in the ...introduction of errors during active DNA methylation reprogramming. Controlled ovarian hyperstimulation, or superovulation, is a ubiquitously used intervention which has been demonstrated to alter the methylation of certain imprinted genes. The objective of this study was to investigate whether ovarian hyperstimulation results in genome-wide DNA methylation changes in mouse early embryos. Ovarian hyperstimulation was induced by treating mice with either low doses (5 IU) or high doses (10 IU) of PMSG and hCG. Natural mating (NM) control mice received no treatment. Zygotes and 8-cell embryos were collected from each group and DNA methylomes were generated by whole-genome bisulfite sequencing. In the NM group, mean CpG methylation levels slightly decreased from zygote to 8-cell stage, whereas a large decrease in mean CpG methylation level was observed in both superovulated groups. A separate analysis of the mean CpG methylation levels within each developmental stage confirmed that significant genome-wide erasure of CpG methylation from the zygote to 8-cell stage only occurred in the superovulation groups. Our results suggest that superovulation alters the genome-wide DNA methylation erasure process in mouse early pre-implantation embryos. It is not clear whether these changes are transient or persistent. Further studies are ongoing to investigate the impact of ovarian hyperstimulation on DNA methylation re-establishment in later stages of embryo development.
The establishment of DNA methylation patterns in oocytes is a highly dynamic process marking gene-regulatory events during fertilization, embryonic development, and adulthood. However, after ...epigenetic reprogramming in primordial germ cells, how and when DNA methylation is re-established in developing human oocytes remains to be characterized. Here, using single-cell whole-genome bisulfite sequencing, we describe DNA methylation patterns in three different maturation stages of human oocytes. We found that while broad-scale patterns of CpG methylation have been largely established by the immature germinal vesicle stage, localized changes continue into later development. Non-CpG methylation, on the other hand, undergoes a large-scale, generalized remodeling through the final stage of maturation, with the net overall result being the accumulation of methylation as oocytes mature. The role of the genome-wide, non-CpG methylation remodeling in the final stage of oocyte maturation deserves further investigation.
•Broad-scale CpG methylation is largely stable during oocyte maturation•Non-CpG methylation accumulates as oocytes mature•Non-CpG methylation changes are genome wide
CpG methylation is established globally in adult immature oocytes and remains stable during maturation. Non-CpG methylation undergoes a genome-wide, generalized remodeling through the final stage of maturation, with the net overall result being accumulation of methylation as oocytes mature.
PDF
