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1.
Am J Hum Genet ; 108(2): 337-345, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33434492

RESUMO

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.


Assuntos
Transtornos 46, XX do Desenvolvimento Sexual/genética , Anormalidades Congênitas/genética , Ductos Paramesonéfricos/anormalidades , Ductos Paramesonéfricos/crescimento & desenvolvimento , Mutação , Ductos Mesonéfricos/crescimento & desenvolvimento , Adulto , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 7/genética , Códon sem Sentido , Feminino , Estudos de Associação Genética , Pleiotropia Genética , Proteínas Homeobox A10/genética , Proteínas de Homeodomínio/genética , Humanos , Fator de Transcrição PAX8/genética , Herança Paterna , Penetrância , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Proteínas Wnt/genética , Ductos Mesonéfricos/anormalidades
3.
Nat Commun ; 10(1): 4495, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31582743

RESUMO

Aneuploidy is a major source of gene dosage imbalance due to copy number alterations (CNA), and viable human trisomies are model disorders of altered gene expression. We study gene and allele-specific expression (ASE) of 9668 single-cell fibroblasts from trisomy 21 (T21) discordant twins and from mosaic T21, T18, T13 and T8. We examine 928 single cells with deep scRNAseq. Expected and observed overexpression of trisomic genes in trisomic vs. diploid bulk RNAseq is not detectable in trisomic vs. diploid single cells. Instead, for trisomic genes with low-to-average expression, their altered gene dosage is mainly due to the higher fraction of trisomic cells simultaneously expressing these genes, in agreement with a stochastic 2-state burst-like model of transcription. These results, confirmed in a further analysis of 8740 single fibroblasts with shallow scRNAseq, suggest that the specific transcriptional profile of each gene contributes to the phenotypic variability of trisomies. We propose an improved model to understand the effects of CNA and, generally, of gene regulation on gene dosage imbalance.


Assuntos
Variações do Número de Cópias de DNA , Dosagem de Genes , Modelos Genéticos , Transcriptoma/genética , Trissomia/genética , Alelos , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 8/genética , Síndrome de Down/genética , Feminino , Fibroblastos , Perfilação da Expressão Gênica , Humanos , Masculino , Mosaicismo , Fenótipo , RNA-Seq , Análise de Célula Única
4.
Nat Biotechnol ; 37(3): 314-322, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30778230

RESUMO

Reproducibility in research can be compromised by both biological and technical variation, but most of the focus is on removing the latter. Here we investigate the effects of biological variation in HeLa cell lines using a systems-wide approach. We determine the degree of molecular and phenotypic variability across 14 stock HeLa samples from 13 international laboratories. We cultured cells in uniform conditions and profiled genome-wide copy numbers, mRNAs, proteins and protein turnover rates in each cell line. We discovered substantial heterogeneity between HeLa variants, especially between lines of the CCL2 and Kyoto varieties, and observed progressive divergence within a specific cell line over 50 successive passages. Genomic variability has a complex, nonlinear effect on transcriptome, proteome and protein turnover profiles, and proteotype patterns explain the varying phenotypic response of different cell lines to Salmonella infection. These findings have implications for the interpretation and reproducibility of research results obtained from human cultured cells.


Assuntos
Variações do Número de Cópias de DNA/genética , Genoma Humano/genética , Células HeLa , Transcriptoma/genética , Genômica/normas , Humanos , Proteoma/genética , Reprodutibilidade dos Testes
5.
Proc Natl Acad Sci U S A ; 115(51): 13015-13020, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30510006

RESUMO

X-chromosome inactivation (XCI) provides a dosage compensation mechanism where, in each female cell, one of the two X chromosomes is randomly silenced. However, some genes on the inactive X chromosome and outside the pseudoautosomal regions escape from XCI and are expressed from both alleles (escapees). We investigated XCI at single-cell resolution combining deep single-cell RNA sequencing with whole-genome sequencing to examine allelic-specific expression in 935 primary fibroblast and 48 lymphoblastoid single cells from five female individuals. In this framework we integrated an original method to identify and exclude doublets of cells. In fibroblast cells, we have identified 55 genes as escapees including five undescribed escapee genes. Moreover, we observed that all genes exhibit a variable propensity to escape XCI in each cell and cell type and that each cell displays a distinct expression profile of the escapee genes. A metric, the Inactivation Score-defined as the mean of the allelic expression profiles of the escapees per cell-enables us to discover a heterogeneous and continuous degree of cellular XCI with extremes represented by "inactive" cells, i.e., cells exclusively expressing the escaping genes from the active X chromosome and "escaping" cells expressing the escapees from both alleles. We found that this effect is associated with cell-cycle phases and, independently, with the XIST expression level, which is higher in the quiescent phase (G0). Single-cell allele-specific expression is a powerful tool to identify novel escapees in different tissues and provide evidence of an unexpected cellular heterogeneity of XCI.


Assuntos
Cromossomos Humanos X/genética , Fibroblastos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Célula Única/métodos , Inativação do Cromossomo X , Alelos , Células Cultivadas , Feminino , Fibroblastos/citologia , Humanos , Transcriptoma
6.
Cell Rep ; 22(6): 1589-1599, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29425512

RESUMO

The gonad is a unique biological system for studying cell-fate decisions. However, major questions remain regarding the identity of somatic progenitor cells and the transcriptional events driving cell differentiation. Using time-series single-cell RNA sequencing on XY mouse gonads during sex determination, we identified a single population of somatic progenitor cells prior to sex determination. A subset of these progenitors differentiates into Sertoli cells, a process characterized by a highly dynamic genetic program consisting of sequential waves of gene expression. Another subset of multipotent cells maintains their progenitor state but undergoes significant transcriptional changes restricting their competence toward a steroidogenic fate required for the differentiation of fetal Leydig cells. Our findings confirm the presence of a unique multipotent progenitor population in the gonadal primordium that gives rise to both supporting and interstitial lineages. These also provide the most granular analysis of the transcriptional events occurring during testicular cell-fate commitment.


Assuntos
Diferenciação Celular/fisiologia , Células Intersticiais do Testículo/citologia , Células de Sertoli/citologia , Processos de Determinação Sexual/fisiologia , Testículo/citologia , Animais , Linhagem da Célula , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Multipotentes/citologia , Análise de Sequência de RNA
7.
Nat Commun ; 8(1): 1212, 2017 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-29089484

RESUMO

Down syndrome (DS) is mostly caused by a trisomy of the entire Chromosome 21 (Trisomy 21, T21). Here, we use SWATH mass spectrometry to quantify protein abundance and protein turnover in fibroblasts from a monozygotic twin pair discordant for T21, and to profile protein expression in 11 unrelated DS individuals and matched controls. The integration of the steady-state and turnover proteomic data indicates that protein-specific degradation of members of stoichiometric complexes is a major determinant of T21 gene dosage outcome, both within and between individuals. This effect is not apparent from genomic and transcriptomic data. The data also reveal that T21 results in extensive proteome remodeling, affecting proteins encoded by all chromosomes. Finally, we find broad, organelle-specific post-transcriptional effects such as significant downregulation of the mitochondrial proteome contributing to T21 hallmarks. Overall, we provide a valuable proteomic resource to understand the origin of DS phenotypic manifestations.


Assuntos
Fibroblastos/metabolismo , Fibroblastos/patologia , Proteoma/metabolismo , Proteostase , Trissomia/patologia , Bases de Dados de Proteínas , Compensação de Dosagem (Genética) , Regulação da Expressão Gênica , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Organelas/metabolismo , Proteólise , Proteostase/genética , Transdução de Sinais , Trissomia/genética
8.
Elife ; 62017 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-28869038

RESUMO

The importance of natural gene expression variation for human behavior is undisputed, but its impact on circadian physiology remains mostly unexplored. Using umbilical cord fibroblasts, we have determined by genome-wide association how common genetic variation impacts upon cellular circadian function. Gene set enrichment points to differences in protein catabolism as one major source of clock variation in humans. The two most significant alleles regulated expression of COPS7B, a subunit of the COP9 signalosome. We further show that the signalosome complex is imported into the nucleus in timed fashion to stabilize the essential circadian protein BMAL1, a novel mechanism to oppose its proteasome-mediated degradation. Thus, circadian clock properties depend in part upon a genetically-encoded competition between stabilizing and destabilizing forces, and genetic alterations in these mechanisms provide one explanation for human chronotype.


Assuntos
Variação Biológica da População , Ritmo Circadiano , Regulação da Expressão Gênica , Variação Genética , Fatores de Transcrição ARNTL/metabolismo , Complexo do Signalossomo COP9/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Estabilidade Proteica , Proteínas/metabolismo
9.
EMBO Mol Med ; 9(8): 1132-1149, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28554943

RESUMO

Azoospermia, characterized by the absence of spermatozoa in the ejaculate, is a common cause of male infertility with a poorly characterized etiology. Exome sequencing analysis of two azoospermic brothers allowed the identification of a homozygous splice mutation in SPINK2, encoding a serine protease inhibitor believed to target acrosin, the main sperm acrosomal protease. In accord with these findings, we observed that homozygous Spink2 KO male mice had azoospermia. Moreover, despite normal fertility, heterozygous male mice had a high rate of morphologically abnormal spermatozoa and a reduced sperm motility. Further analysis demonstrated that in the absence of Spink2, protease-induced stress initiates Golgi fragmentation and prevents acrosome biogenesis leading to spermatid differentiation arrest. We also observed a deleterious effect of acrosin overexpression in HEK cells, effect that was alleviated by SPINK2 coexpression confirming its role as acrosin inhibitor. These results demonstrate that SPINK2 is necessary to neutralize proteases during their cellular transit toward the acrosome and that its deficiency induces a pathological continuum ranging from oligoasthenoteratozoospermia in heterozygotes to azoospermia in homozygotes.


Assuntos
Astenozoospermia/genética , Astenozoospermia/fisiopatologia , Azoospermia/genética , Azoospermia/fisiopatologia , Glicoproteínas/deficiência , Inibidores de Serinopeptidase do Tipo Kazal/deficiência , Animais , Modelos Animais de Doenças , Heterozigoto , Homozigoto , Masculino , Camundongos , Camundongos Knockout
10.
Am J Hum Genet ; 100(3): 444-453, 2017 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-28190458

RESUMO

Genomic imprinting results in parental-specific gene expression. Imprinted genes are involved in the etiology of rare syndromes and have been associated with common diseases such as diabetes and cancer. Standard RNA bulk cell sequencing applied to whole-tissue samples has been used to detect imprinted genes in human and mouse models. However, lowly expressed genes cannot be detected by using RNA bulk approaches. Here, we report an original and robust method that combines single-cell RNA-seq and whole-genome sequencing into an optimized statistical framework to analyze genomic imprinting in specific cell types and in different individuals. Using samples from the probands of 2 family trios and 3 unrelated individuals, 1,084 individual primary fibroblasts were RNA sequenced and more than 700,000 informative heterozygous single-nucleotide variations (SNVs) were genotyped. The allele-specific coverage per gene of each SNV in each single cell was used to fit a beta-binomial distribution to model the likelihood of a gene being expressed from one and the same allele. Genes presenting a significant aggregate allelic ratio (between 0.9 and 1) were retained to identify of the allelic parent of origin. Our approach allowed us to validate the imprinting status of all of the known imprinted genes expressed in fibroblasts and the discovery of nine putative imprinted genes, thereby demonstrating the advantages of single-cell over bulk RNA-seq to identify imprinted genes. The proposed single-cell methodology is a powerful tool for establishing a cell type-specific map of genomic imprinting.


Assuntos
Alelos , Expressão Gênica , Impressão Genômica , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Estudos de Associação Genética , Técnicas de Genotipagem , Heterozigoto , Humanos , Modelos Teóricos , Polimorfismo de Nucleotídeo Único , Transcriptoma
12.
Sex Dev ; 9(5): 289-95, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26544196

RESUMO

We report the case of a female patient suffering from a 46,XY disorder of sexual development (DSD) with complete gonadal dysgenesis and Wiedemann-Steiner Syndrome (WDSTS). The coexistence of these 2 conditions has not yet been reported. Using whole exome sequencing and comparative genome hybridization array, we identified a de novo MLL/KMT2A gene nonsense mutation which explains the WDSTS phenotype. In addition, we discovered novel genetic variants, which could explain the testicular dysgenesis observed in the patient, a maternally inherited 167-kb duplication of DAAM2 and MOCS1 genes and a de novo LRRC33/NRROS gene mutation. These genes, some of which are expressed during mouse gonadal development, could be considered as potentially new candidate genes for DSD.


Assuntos
Transtorno 46,XY do Desenvolvimento Sexual/genética , Disgenesia Gonadal 46 XY/genética , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Códon sem Sentido , Anormalidades Craniofaciais/genética , Deficiências do Desenvolvimento/genética , Feminino , Seguimentos , Genitália/patologia , Humanos , Recém-Nascido , Síndromes de Malabsorção/genética , Masculino , Linhagem , Síndrome
13.
PLoS One ; 10(8): e0135555, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26317209

RESUMO

DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes.


Assuntos
Metilação de DNA , Síndrome de Down/genética , Gêmeos Monozigóticos , Ilhas de CpG , Síndrome de Down/metabolismo , Epigênese Genética , Fibroblastos , Regulação da Expressão Gênica , Biblioteca Gênica , Histonas/metabolismo , Humanos , Fenótipo , Regiões Promotoras Genéticas
14.
PLoS One ; 10(5): e0126475, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25955728

RESUMO

The HSA21 encoded Single-minded 2 (SIM2) transcription factor has key neurological functions and is a good candidate to be involved in the cognitive impairment of Down syndrome. We aimed to explore the functional capacity of SIM2 by mapping its DNA binding sites in mouse embryonic stem cells. ChIP-sequencing revealed 1229 high-confidence SIM2-binding sites. Analysis of the SIM2 target genes confirmed the importance of SIM2 in developmental and neuronal processes and indicated that SIM2 may be a master transcription regulator. Indeed, SIM2 DNA binding sites share sequence specificity and overlapping domains of occupancy with master transcription factors such as SOX2, OCT4 (Pou5f1), NANOG or KLF4. The association between SIM2 and these pioneer factors is supported by co-immunoprecipitation of SIM2 with SOX2, OCT4, NANOG or KLF4. Furthermore, the binding of SIM2 marks a particular sub-category of enhancers known as super-enhancers. These regions are characterized by typical DNA modifications and Mediator co-occupancy (MED1 and MED12). Altogether, we provide evidence that SIM2 binds a specific set of enhancer elements thus explaining how SIM2 can regulate its gene network in neuronal features.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , DNA/metabolismo , Elementos Facilitadores Genéticos , Células-Tronco Embrionárias Murinas/metabolismo , Fatores de Transcrição/genética , Animais , Sítios de Ligação , Células Cultivadas , Imunoprecipitação da Cromatina/métodos , DNA/genética , Camundongos , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo
15.
PLoS Genet ; 11(3): e1005062, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25803843

RESUMO

The trisomy of human chromosome 21 (Hsa21), which causes Down syndrome (DS), is the most common viable human aneuploidy. In contrast to trisomy, the complete monosomy (M21) of Hsa21 is lethal, and only partial monosomy or mosaic monosomy of Hsa21 is seen. Both conditions lead to variable physiological abnormalities with constant intellectual disability, locomotor deficits, and altered muscle tone. To search for dosage-sensitive genes involved in DS and M21 phenotypes, we created two new mouse models: the Ts3Yah carrying a tandem duplication and the Ms3Yah carrying a deletion of the Hspa13-App interval syntenic with 21q11.2-q21.3. Here we report that the trisomy and the monosomy of this region alter locomotion, muscle strength, mass, and energetic balance. The expression profiling of skeletal muscles revealed global changes in the regulation of genes implicated in energetic metabolism, mitochondrial activity, and biogenesis. These genes are downregulated in Ts3Yah mice and upregulated in Ms3Yah mice. The shift in skeletal muscle metabolism correlates with a change in mitochondrial proliferation without an alteration in the respiratory function. However, the reactive oxygen species (ROS) production from mitochondrial complex I decreased in Ms3Yah mice, while the membrane permeability of Ts3Yah mitochondria slightly increased. Thus, we demonstrated how the Hspa13-App interval controls metabolic and mitochondrial phenotypes in muscles certainly as a consequence of change in dose of Gabpa, Nrip1, and Atp5j. Our results indicate that the copy number variation in the Hspa13-App region has a peripheral impact on locomotor activity by altering muscle function.


Assuntos
Síndrome de Down/genética , Monossomia/genética , Atividade Motora/genética , Força Muscular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cromossomos Humanos Par 21/genética , Modelos Animais de Doenças , Síndrome de Down/fisiopatologia , Metabolismo Energético/genética , Fator de Transcrição de Proteínas de Ligação GA/genética , Humanos , Camundongos , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/patologia , ATPases Mitocondriais Próton-Translocadoras/genética , Monossomia/fisiopatologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Proteínas Nucleares/genética , Proteína 1 de Interação com Receptor Nuclear
16.
Mol Endocrinol ; 29(4): 627-42, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25710594

RESUMO

Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1ß), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.


Assuntos
Túbulos Seminíferos/metabolismo , Células de Sertoli/metabolismo , Espermatogênese/genética , Transcriptoma , Animais , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos
17.
Am J Hum Genet ; 96(1): 70-80, 2015 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-25557783

RESUMO

The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability.


Assuntos
Alelos , Fibroblastos/citologia , Genoma Humano , Análise de Sequência de RNA , DNA Complementar/genética , DNA Complementar/metabolismo , Haplótipos , Heterozigoto , Humanos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula Única
18.
PLoS Genet ; 11(1): e1004958, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25634236

RESUMO

Understanding how genetic variation affects distinct cellular phenotypes, such as gene expression levels, alternative splicing and DNA methylation levels, is essential for better understanding of complex diseases and traits. Furthermore, how inter-individual variation of DNA methylation is associated to gene expression is just starting to be studied. In this study, we use the GenCord cohort of 204 newborn Europeans' lymphoblastoid cell lines, T-cells and fibroblasts derived from umbilical cords. The samples were previously genotyped for 2.5 million SNPs, mRNA-sequenced, and assayed for methylation levels in 482,421 CpG sites. We observe that methylation sites associated to expression levels are enriched in enhancers, gene bodies and CpG island shores. We show that while the correlation between DNA methylation and gene expression can be positive or negative, it is very consistent across cell-types. However, this epigenetic association to gene expression appears more tissue-specific than the genetic effects on gene expression or DNA methylation (observed in both sharing estimations based on P-values and effect size correlations between cell-types). This predominance of genetic effects can also be reflected by the observation that allele specific expression differences between individuals dominate over tissue-specific effects. Additionally, we discover genetic effects on alternative splicing and interestingly, a large amount of DNA methylation correlating to alternative splicing, both in a tissue-specific manner. The locations of the SNPs and methylation sites involved in these associations highlight the participation of promoter proximal and distant regulatory regions on alternative splicing. Overall, our results provide high-resolution analyses showing how genome sequence variation has a broad effect on cellular phenotypes across cell-types, whereas epigenetic factors provide a secondary layer of variation that is more tissue-specific. Furthermore, the details of how this tissue-specificity may vary across inter-relations of molecular traits, and where these are occurring, can yield further insights into gene regulation and cellular biology as a whole.


Assuntos
Processamento Alternativo/genética , Metilação de DNA/genética , Epigênese Genética , Regulação da Expressão Gênica/genética , Variação Genética , Alelos , Ilhas de CpG , Humanos , Recém-Nascido , Especificidade de Órgãos , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico/genética
19.
PLoS One ; 9(9): e107023, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25244517

RESUMO

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process.


Assuntos
Fertilidade/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Espermatogênese/genética , Espermatozoides/metabolismo , Animais , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Knockout , RNA Interferente Pequeno , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo , Testículo/metabolismo
20.
PLoS Genet ; 10(6): e1004418, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24945355

RESUMO

Tandem repeats are common in eukaryotic genomes, but due to difficulties in assaying them remain poorly studied. Here, we demonstrate the utility of Nanostring technology as a targeted approach to perform accurate measurement of tandem repeats even at extremely high copy number, and apply this technology to genotype 165 HapMap samples from three different populations and five species of non-human primates. We observed extreme variability in copy number of tandemly repeated genes, with many loci showing 5-10 fold variation in copy number among humans. Many of these loci show hallmarks of genome assembly errors, and the true copy number of many large tandem repeats is significantly under-represented even in the high quality 'finished' human reference assembly. Importantly, we demonstrate that most large tandem repeat variations are not tagged by nearby SNPs, and are therefore essentially invisible to SNP-based GWAS approaches. Using association analysis we identify many cis correlations of large tandem repeat variants with nearby gene expression and DNA methylation levels, indicating that variations of tandem repeat length are associated with functional effects on the local genomic environment. This includes an example where expansion of a macrosatellite repeat is associated with increased DNA methylation and suppression of nearby gene expression, suggesting a mechanism termed "repeat induced gene silencing", which has previously been observed only in transgenic organisms. We also observed multiple signatures consistent with altered selective pressures at tandemly repeated loci, suggesting important biological functions. Our studies show that tandemly repeated loci represent a highly variable fraction of the genome that have been systematically ignored by most previous studies, copy number variation of which can exert functionally significant effects. We suggest that future studies of tandem repeat loci will lead to many novel insights into their role in modulating both genomic and phenotypic diversity.


Assuntos
Metilação de DNA/genética , DNA Satélite/genética , Dosagem de Genes/genética , Primatas/genética , Sequências de Repetição em Tandem/genética , Animais , Variações do Número de Cópias de DNA , Inativação Gênica , Genoma Humano , Humanos , Hylobates/genética , Desequilíbrio de Ligação , Macaca/genética , Pan paniscus/genética , Pan troglodytes/genética , Polimorfismo de Nucleotídeo Único
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