RESUMO
Type I diabetes is an autoimmune disease mediated by T-cell destruction of ß 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.
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Linfócitos T CD4-Positivos , Diabetes Mellitus Tipo 1 , Elementos Facilitadores Genéticos , Predisposição Genética para Doença , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Humanos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Elementos Facilitadores Genéticos/genética , Proteína 1 Supressora da Sinalização de Citocina/genética , Estudo de Associação Genômica Ampla , Lectinas Tipo C/genética , Variação Genética , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
Cells exist in different phenotypes and can transition between them. A phenotype may be characterized by many different aspects. Here, we focus on the example of whether the cell is adhered or suspended and choose particular parameters related to the structure and mechanics of the actin cortex. The cortex is essential to cell mechanics, morphology, and function, such as for adhesion, migration, and division of animal cells. To predict and control cellular functions and prevent malfunctioning, it is necessary to understand the actin cortex. The structure of the cortex governs cell mechanics; however, the relationship between the architecture and mechanics of the cortex is not yet well enough understood to be able to predict one from the other. Therefore, we quantitatively measured structural and mechanical cortex parameters, including cortical thickness, cortex mesh size, actin bundling, and cortex stiffness. These measurements required developing a combination of measurement techniques in scanning electron, expansion, confocal, and atomic force microscopy. We found that the structure and mechanics of the cortex of cells in interphase are different depending on whether the cell is suspended or adhered. We deduced general correlations between structural and mechanical properties and show how these findings can be explained within the framework of semiflexible polymer network theory. We tested the model predictions by perturbing the properties of the actin within the cortex using compounds. Our work provides an important step toward predictions of cell mechanics from cortical structures and suggests how cortex remodeling between different phenotypes impacts the mechanical properties of cells.
Assuntos
Actinas , Adesão Celular , Adesão Celular/fisiologia , Actinas/metabolismo , Animais , Microscopia de Força Atômica/métodos , Fenômenos Biomecânicos , Modelos BiológicosRESUMO
OBJECTIVE: There is a relative lack of consensus regarding the optimal management of hyperglycemia in patients receiving continuous enteral nutrition (EN), with or without a diagnosis of diabetes. METHODS: This retrospective study examined 475 patients (303 with known diabetes) hospitalized in critical care setting units in 2019 in a single center who received continuous EN. Rates of hypoglycemia, hyperglycemia, and glucose levels within the target range (70-180 mg/dL) were compared between patients with and without diabetes, and among patients treated with intermediate-acting (IA) biphasic neutral protamine Hagedorn 70/30, long-acting (LA) insulin, or rapid-acting insulin only. RESULTS: Among those with type 2 diabetes mellitus, IA and LA insulin regimens were associated with a significantly higher proportion of patient-days in the target glucose range and fewer hyperglycemic days. Level 1 (<70 mg/dL) and level 2 (<54 mg/dL) hypoglycemia occurred rarely, and there were no significant differences in level 2 hypoglycemia frequency across the different insulin regimens. CONCLUSION: Administration of IA and LA insulin can be safe and effective for those receiving insulin doses for EN-related hyperglycemia.
Assuntos
Diabetes Mellitus Tipo 2 , Hiperglicemia , Hipoglicemia , Humanos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/efeitos adversos , Estudos Retrospectivos , Nutrição Enteral , Estado Terminal/terapia , Glicemia , Insulina/efeitos adversos , Hipoglicemia/induzido quimicamente , Hipoglicemia/epidemiologia , Hipoglicemia/tratamento farmacológico , Insulina de Ação Prolongada/uso terapêutico , Hiperglicemia/tratamento farmacológico , Hiperglicemia/prevenção & controle , Hiperglicemia/induzido quimicamente , Glucose/uso terapêutico , Insulina Isófana/efeitos adversosRESUMO
INTRODUCTION: In recent years, health psychology has received significant attention within the health sector, due to its application to understanding influences on health and well-being and translation of health psychology into interventions to support behaviour change. The number of health psychologists in public health and healthcare settings is growing but remains limited, and is it unclear why. This study aimed to explore the views of potential and current employers of health psychologists, to elucidate barriers and facilitators of employing health psychologists in healthcare settings. METHODS: Semi-structured interviews were carried out to explore the experiences of working with and/or employing health psychologists. Opportunities and barriers were explored for increasing access to health psychology expertise in the NHS and public health. Interviews were analysed using inductive thematic analysis. RESULTS: Fifteen participants took part in interviews. Participants were mid-senior-level professionals working in varied healthcare settings and/or academic institutions. The majority had experience of health psychology/working with health psychologists, whilst others had limited experience but an interest in employing health psychologists. Three key themes were identified: (1) the organizational fit of health psychologists, (2) perception of competition for roles and (3) ideas for changing hearts, minds and processes. CONCLUSION: Barriers exist to employing health psychologists in healthcare settings. These barriers include misunderstandings of the role of health psychologists and the need to preserve other disciplines due to perceived competition. Recommendations for change included showcasing the benefits and skills of health psychologists and having transparent conversations with employees and multi-disciplinary colleagues about roles.
RESUMO
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.
Assuntos
Dermatite Atópica , Animais , Progressão da Doença , Cães , Epiderme/metabolismo , Humanos , Queratinócitos/metabolismo , Pele/metabolismoRESUMO
Epigenetic mechanisms regulate osteogenic lineage differentiation of mesenchymal stromal cells. Histone methylation is controlled by multiple lysine demethylases and is an important step in controlling local chromatin structure and gene expression. Here, we show that the lysine-specific histone demethylase Kdm1A/Lsd1 is abundantly expressed in osteoblasts and that its suppression impairs osteoblast differentiation and bone nodule formation in vitro. Although Lsd1 knockdown did not affect global H3K4 methylation levels, genome-wide ChIP-Seq analysis revealed high levels of Lsd1 at gene promoters and its binding was associated with di- and tri-methylation of histone 3 at lysine 4 (H3K4me2 and H3K4me3). Lsd1 binding sites in osteoblastic cells were enriched for the Runx2 consensus motif suggesting a functional link between the two proteins. Importantly, inhibition of Lsd1 activity decreased osteoblast activity in vivo. In support, mesenchymal-targeted knockdown of Lsd1 led to decreased osteoblast activity and disrupted primary spongiosa ossification and reorganization in vivo. Together, our studies demonstrate that Lsd1 occupies Runx2-binding cites at H3K4me2 and H3K4me3 and its activity is required for proper bone formation.
Assuntos
Histonas , Osteogênese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Osteoblastos/metabolismo , Osteogênese/genéticaRESUMO
Bifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box >500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation.
Assuntos
Histonas , Complexo Repressor Polycomb 2 , Cromatina , Computadores , Histonas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , TATA BoxRESUMO
The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.
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Histonas/metabolismo , RNA Polimerase II/genética , Sumoilação , Transcrição Gênica , Extratos Celulares , Humanos , RNA Polimerase II/metabolismoRESUMO
While the chicken (Gallus gallus) is the most consumed agricultural animal worldwide, the chicken transcriptome remains understudied. We have characterized the transcriptome of 10 cell and tissue types from the chicken using RNA-seq, spanning intestinal tissues (ileum, jejunum, proximal cecum), immune cells (B cells, bursa, macrophages, monocytes, spleen T cells, thymus), and reproductive tissue (ovary). We detected 17,872 genes and 24,812 transcripts across all cell and tissue types, representing 73% and 63% of the current gene annotation, respectively. Further quantification of RNA transcript biotypes revealed protein-coding and lncRNAs specific to an individual cell/tissue type. Each cell/tissue type also has an average of around 1.2 isoforms per gene, however, they all have at least one gene with at least 11 isoforms. Differential expression analysis revealed a large number of differentially expressed genes between tissues of the same category (immune and intestinal). Many of these differentially expressed genes in immune cells were involved in cellular processes relating to differentiation and cell metabolism as well as basic functions of immune cells such as cell adhesion and signal transduction. The differential expressed genes of the different segments of the chicken intestine (jejunum, ileum, proximal cecum) correlated to the metabolic processes in nutrient digestion and absorption. These data should provide a valuable resource in understanding the chicken genome.
RESUMO
There have been substantial advances in computed tomography (CT) technology since its introduction in the 1970s. More recently, these advances have focused on image reconstruction. Deep learning reconstruction (DLR) is the latest complex reconstruction algorithm to be introduced, which harnesses advances in artificial intelligence (AI) and affordable supercomputer technology to achieve the previously elusive triad of high image quality, low radiation dose, and fast reconstruction speeds. The dose reductions achieved with DLR are redefining ultra-low-dose into the realm of plain radiographs whilst maintaining image quality. This review aims to demonstrate the advantages of DLR over other reconstruction methods in terms of dose reduction and image quality in addition to being able to tailor protocols to specific clinical situations. DLR is the future of CT technology and should be considered when procuring new scanners.
Assuntos
Aprendizado Profundo , Interpretação de Imagem Radiográfica Assistida por Computador/métodos , Tomografia Computadorizada por Raios X/métodos , HumanosRESUMO
Base editors are capable of installing precise genomic alterations without creating double-strand DNA breaks. In this study, we targeted critical motifs regulating γ-globin reactivation with base editors delivered via HDAd5/35++ vectors. Through optimized design, we successfully produced a panel of cytidine and adenine base editor (ABE) vectors targeting the erythroid BCL11A enhancer or recreating naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations in the HBG1/2 promoter. All 5 tested vectors efficiently installed target base conversion and led to γ-globin reactivation in human erythroid progenitor cells. We observed ~23% γ-globin protein production over ß-globin, when using an ABE vector (HDAd-ABE-sgHBG-2) specific to the -113A>G HPFH mutation. In a ß-YAC mouse model, in vivo hematopoietic progenitor/stem cell (HSPC) transduction with HDAd-ABE-sgHBG-2 followed by in vivo selection resulted in >40% γ-globin+ erythrocytes in the peripheral blood. This result corresponded to 21% γ-globin production over human ß-globin. The average -113A>G conversion in total bone marrow cells was 20%. No alterations in hematological parameters, erythropoiesis, and bone marrow cellular composition were observed after treatment. No detectable editing was found at top-scoring, off-target genomic sites. Bone marrow lineage-negative cells from primary mice were capable of reconstituting secondary transplant-recipient mice with stable γ-globin expression. Importantly, the advantage of base editing over CRISPR/Cas9 was reflected by the markedly lower rates of intergenic HBG1/2 deletion and the absence of detectable toxicity in human CD34+ cells. Our observations suggest that HDAd-vectorized base editors represent a promising strategy for precise in vivo genome engineering for the treatment of ß-hemoglobinopathies.
Assuntos
Hemoglobina Fetal , gama-Globinas , Animais , Hemoglobina Fetal/genética , Terapia Genética , Células-Tronco Hematopoéticas , Camundongos , Globinas beta/genética , gama-Globinas/genéticaRESUMO
Oocyte maturation is a coordinated process that is tightly linked to reproductive potential. A better understanding of gene regulation during human oocyte maturation will not only answer an important question in biology, but also facilitate the development of in vitro maturation technology as a fertility treatment. We generated single-cell transcriptome and used our previously published single-cell methylome data from human oocytes at different maturation stages to investigate how genes are regulated during oocyte maturation, focusing on the potential regulatory role of non-CpG methylation. DNMT3B, a gene encoding a key non-CpG methylation enzyme, is one of the 1,077 genes upregulated in mature oocytes, which may be at least partially responsible for the increased non-CpG methylation as oocytes mature. Non-CpG differentially methylated regions (DMRs) between mature and immature oocytes have multiple binding motifs for transcription factors, some of which bind with DNMT3B and may be important regulators of oocyte maturation through non-CpG methylation. Over 98% of non-CpG DMRs locate in transposable elements, and these DMRs are correlated with expression changes of the nearby genes. Taken together, this data indicates that global non-CpG hypermethylation during oocyte maturation may play an active role in gene expression regulation, potentially through the interaction with transcription factors.
Assuntos
Epigenoma/genética , Oócitos/metabolismo , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Metilação de DNA/fisiologia , Impressão Genômica/genética , Humanos , Análise de Célula Única , Transcriptoma/genética , DNA Metiltransferase 3BRESUMO
OBJECTIVES: The United States has the highest number of coronavirus disease 2019 (COVID-19) in the world, with high variability in cases and mortality between communities. We aimed to quantify the associations between socio-economic status and COVID-19-related cases and mortality in the U.S. STUDY DESIGN: The study design includes nationwide COVID-19 data at the county level that were paired with the Distressed Communities Index (DCI) and its component metrics of socio-economic status. METHODS: Severely distressed communities were classified by DCI>75 for univariate analyses. Adjusted rate ratios were calculated for cases and fatalities per 100,000 persons using hierarchical linear mixed models. RESULTS: This cohort included 1,089,999 cases and 62,298 deaths in 3127 counties for a case fatality rate of 5.7%. Severely distressed counties had significantly fewer deaths from COVID-19 but higher number of deaths per 100,000 persons. In risk-adjusted analysis, the two socio-economic determinants of health with the strongest association with both higher cases per 100,000 persons and higher fatalities per 100,000 persons were the percentage of adults without a high school degree (cases: RR 1.10; fatalities: RR 1.08) and proportion of black residents (cases and fatalities: Relative risk(RR) 1.03). The percentage of the population aged older than 65 years was also highly predictive for fatalities per 100,000 persons (RR 1.07). CONCLUSION: Lower education levels and greater percentages of black residents are strongly associated with higher rates of both COVID-19 cases and fatalities. Socio-economic factors should be considered when implementing public health interventions to ameliorate the disparities in the impact of COVID-19 on distressed communities.
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COVID-19/epidemiologia , COVID-19/mortalidade , SARS-CoV-2 , Classe Social , Adulto , Negro ou Afro-Americano/estatística & dados numéricos , Idoso , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mortalidade/tendências , Prevalência , Risco , Estados Unidos/epidemiologiaRESUMO
The study of gene regulation is dominated by a focus on the control of gene activation or increase in the level of expression. Just as critical is the process of gene repression or silencing. Chromatin signatures have identified enhancers, however, genome-wide identification of silencers by computational or experimental approaches are lacking. Here, we first define uncharacterized cis-regulatory elements likely containing silencers and find that 41.5% of ~7500 tested elements show silencer activity using massively parallel reporter assay (MPRA). We trained a support vector machine classifier based on MPRA data to predict candidate silencers in over 100 human and mouse cell or tissue types. The predicted candidate silencers exhibit characteristics expected of silencers. Leveraging promoter-capture HiC data, we find that over 50% of silencers are interacting with gene promoters having very low to no expression. Our results suggest a general strategy for genome-wide identification and characterization of silencer elements.
Assuntos
Regulação da Expressão Gênica/genética , Proteínas Repressoras/genética , Elementos Silenciadores Transcricionais/genética , Transcrição Gênica/genética , Animais , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , Ativação Transcricional/genéticaRESUMO
BACKGROUND AND PURPOSE: Graph theory uses structural similarity to analyze cortical structural connectivity. We used a voxel-based definition of cortical covariance networks to quantify and assess the relationship of network characteristics to cognition in a cohort of patients with relapsing-remitting MS with and without cognitive impairment. MATERIALS AND METHODS: We compared subject-specific structural gray matter network properties of 18 healthy controls, 25 patients with MS with cognitive impairment, and 55 patients with MS without cognitive impairment. Network parameters were compared, and predictive value for cognition was assessed, adjusting for confounders (sex, education, gray matter volume, network size and degree, and T1 and T2 lesion load). Backward stepwise multivariable regression quantified predictive factors for 5 neurocognitive domain test scores. RESULTS: Greater path length (r = -0.28, P < .0057) and lower normalized path length (r = 0.36, P < .0004) demonstrated a correlation with average cognition when comparing healthy controls with patients with MS. Similarly, MS with cognitive impairment demonstrated a correlation between lower normalized path length (r = 0.40, P < .001) and reduced average cognition. Increased normalized path length was associated with better performance for processing (P < .001), learning (P < .001), and executive domain function (P = .0235), while reduced path length was associated with better executive (P = .0031) and visual domains. Normalized path length improved prediction for processing (R 2 = 43.6%, G2 = 20.9; P < .0001) and learning (R 2 = 40.4%, G2 = 26.1; P < .0001) over a null model comprising confounders. Similarly, higher normalized path length improved prediction of average z scores (G2 = 21.3; P < .0001) and, combined with WM volume, explained 52% of average cognition variance. CONCLUSIONS: Patients with MS and cognitive impairment demonstrate more random network features and reduced global efficiency, impacting multiple cognitive domains. A model of normalized path length with normal-appearing white matter volume improved average cognitive z score prediction, explaining 52% of variance.
Assuntos
Disfunção Cognitiva/etiologia , Disfunção Cognitiva/fisiopatologia , Esclerose Múltipla Recidivante-Remitente/complicações , Esclerose Múltipla Recidivante-Remitente/fisiopatologia , Adulto , Disfunção Cognitiva/patologia , Feminino , Substância Cinzenta/patologia , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/patologia , Substância Branca/patologiaRESUMO
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.
Assuntos
Adenoviridae/genética , Dependovirus/genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Transdução Genética , Transgenes/fisiologia , Integração Viral , Animais , Sistemas CRISPR-Cas , Feminino , Genes Reporter , Terapia Genética , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , gama-Globinas/antagonistas & inibidores , gama-Globinas/genéticaRESUMO
During mammalian embryogenesis, changes in morphology and gene expression are concurrent with epigenomic reprogramming. Using human embryonic stem cells representing the preimplantation blastocyst (naive) and postimplantation epiblast (primed), our data in 2iL/I/F naive cells demonstrate that a substantial portion of known human enhancers are premarked by H3K4me1, providing an enhanced open chromatin state in naive pluripotency. The 2iL/I/F enhancer repertoire occupies 9% of the genome, three times that of primed cells, and can exist in broad chromatin domains over 50 kb. Enhancer chromatin states are largely poised. Seventy-seven percent of 2iL/I/F enhancers are decommissioned in a stepwise manner as cells become primed. While primed topologically associating domains are largely unaltered upon differentiation, naive 2iL/I/F domains expand across primed boundaries, affecting three-dimensional genome architecture. Differential topologically associating domain edges coincide with 2iL/I/F H3K4me1 enrichment. Our results suggest that naive-derived 2iL/I/F cells have a unique chromatin landscape, which may reflect early embryogenesis.
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Blastocisto/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Camadas Germinativas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Animais , Blastocisto/citologia , Diferenciação Celular/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Células-Tronco Embrionárias Humanas/citologia , HumanosRESUMO
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.
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Metilação de DNA , Superovulação/genética , Sequenciamento Completo do Genoma/veterinária , Animais , Ilhas de CpG , Desenvolvimento Embrionário , Epigênese Genética , Feminino , Camundongos , Técnicas de Reprodução AssistidaRESUMO
Recent developments in the nucleic acid editing technologies have provided a powerful tool to precisely engineer the genome and epigenome for studying many aspects of immune cell differentiation and development as well as several immune mediated diseases (IMDs) including autoimmunity and cancer. Here, we discuss the recent technological achievements of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based RNA-guided genome and epigenome editing toolkit and provide an insight into how CRISPR/Cas9 (CRISPR Associated Protein 9) toolbox could be used to examine genetic and epigenetic mechanisms underlying IMDs. In addition, we will review the progress in CRISPR/Cas9-based genome-wide genome and epigenome screens in various cell types including immune cells. Finally, we will discuss the potential of CRISPR/Cas9 in defining the molecular function of disease associated SNPs overlapping gene regulatory elements.