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1.
Nat Commun ; 12(1): 4919, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389725

RESUMO

BRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of ß-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed ß-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, ß-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that ß-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Oncogenes/genética , Transcrição Genética/genética , beta Catenina/genética , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Feminino , Perfilação da Expressão Gênica/métodos , Células HeLa , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA-Seq/métodos , beta Catenina/metabolismo
2.
Nat Commun ; 12(1): 4737, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362910

RESUMO

Glucocorticoid hormones (GCs) - acting through hippocampal mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) - are critical to physiological regulation and behavioural adaptation. We conducted genome-wide MR and GR ChIP-seq and Ribo-Zero RNA-seq studies on rat hippocampus to elucidate MR- and GR-regulated genes under circadian variation or acute stress. In a subset of genes, these physiological conditions resulted in enhanced MR and/or GR binding to DNA sequences and associated transcriptional changes. Binding of MR at a substantial number of sites however remained unchanged. MR and GR binding occur at overlapping as well as distinct loci. Moreover, although the GC response element (GRE) was the predominant motif, the transcription factor recognition site composition within MR and GR binding peaks show marked differences. Pathway analysis uncovered that MR and GR regulate a substantial number of genes involved in synaptic/neuro-plasticity, cell morphology and development, behavior, and neuropsychiatric disorders. We find that MR, not GR, is the predominant receptor binding to >50 ciliary genes; and that MR function is linked to neuronal differentiation and ciliogenesis in human fetal neuronal progenitor cells. These results show that hippocampal MRs and GRs constitutively and dynamically regulate genomic activities underpinning neuronal plasticity and behavioral adaptation to changing environments.


Assuntos
Hipocampo/metabolismo , Plasticidade Neuronal/fisiologia , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Receptores de Esteroides/metabolismo , Animais , Regulação da Expressão Gênica , Genoma , Hipocampo/patologia , Humanos , Masculino , Ligação Proteica , RNA/metabolismo , Ratos , Ratos Wistar , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Elementos de Resposta , Fatores de Transcrição
3.
PLoS Comput Biol ; 17(8): e1009254, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34343164

RESUMO

Driven by the necessity to survive environmental pathogens, the human immune system has evolved exceptional diversity and plasticity, to which several factors contribute including inheritable structural polymorphism of the underlying genes. Characterizing this variation is challenging due to the complexity of these loci, which contain extensive regions of paralogy, segmental duplication and high copy-number repeats, but recent progress in long-read sequencing and optical mapping techniques suggests this problem may now be tractable. Here we assess this by using long-read sequencing platforms from PacBio and Oxford Nanopore, supplemented with short-read sequencing and Bionano optical mapping, to sequence DNA extracted from CD14+ monocytes and peripheral blood mononuclear cells from a single European individual identified as HV31. We use this data to build a de novo assembly of eight genomic regions encoding four key components of the immune system, namely the human leukocyte antigen, immunoglobulins, T cell receptors, and killer-cell immunoglobulin-like receptors. Validation of our assembly using k-mer based and alignment approaches suggests that it has high accuracy, with estimated base-level error rates below 1 in 10 kb, although we identify a small number of remaining structural errors. We use the assembly to identify heterozygous and homozygous structural variation in comparison to GRCh38. Despite analyzing only a single individual, we find multiple large structural variants affecting core genes at all three immunoglobulin regions and at two of the three T cell receptor regions. Several of these variants are not accurately callable using current algorithms, implying that further methodological improvements are needed. Our results demonstrate that assessing haplotype variation in these regions is possible given sufficiently accurate long-read and associated data. Continued reductions in the cost of these technologies will enable application of these methods to larger samples and provide a broader catalogue of germline structural variation at these loci, an important step toward making these regions accessible to large-scale genetic association studies.

4.
Sci Rep ; 11(1): 6408, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33742045

RESUMO

Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies PromethION platform is a now a viable solution for genome sequencing. Much of the validation of bioinformatic tools for this long-read data has focussed on calling germline variants (including structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated by the effects of tumour purity/subclonality. Here, we evaluate the extent to which Nanopore sequencing enables detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma and comparing results with 150 bp short-read sequencing of the same samples. Calling germline single nucleotide variants (SNVs) from specific chromosomes of the long-read data achieved good specificity and sensitivity. However, results of somatic SNV calling highlight the need for the development of specialised joint calling algorithms. We find the comparative genome-wide performance of different tools varies significantly between structural variant types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants, confirming that a somatic 1.6 Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans.

5.
Front Immunol ; 12: 640837, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33746983

RESUMO

Inflammatory cardiomyopathy covers a group of diseases characterized by inflammation and dysfunction of the heart muscle. The immunosuppressive agents such as prednisolone, azathioprine and cyclosporine are modestly effective treatments, but a molecular rationale underpinning such therapy or the development of new therapeutic strategies is lacking. We aimed to develop a network-based approach to identify therapeutic targets for inflammatory cardiomyopathy from the evolving myocardial transcriptome in a mouse model of the disease. We performed bulk RNA sequencing of hearts at early, mid and late time points from mice with experimental autoimmune myocarditis. We identified a cascade of pathway-level events involving early activation of cytokine and chemokine-signaling pathways that precede leucocyte infiltration and are followed by innate immune, antigen-presentation, complement and cell-adhesion pathway activation. We integrated these pathway events into a network-like representation from which we further identified a 50-gene subnetwork that is predominantly induced during the course of autoimmune myocardial inflammation. We developed a combinatorial attack strategy where we quantify network tolerance to combinatorial node removal to determine target-specific therapeutic potential. We find that combinatorial attack of Traf2, Nfkb1, Rac1, and Vav1 disconnects 80% of nodes from the largest network component. Two of these nodes, Nfkb1 and Rac1, are directly targeted by prednisolone and azathioprine respectively, supporting the idea that the methodology developed here can identify valid therapeutic targets. Whereas Nfkb1 and Rac1 removal disconnects 56% of nodes, we show that additional removal of Btk and Pik3cd causes 72% node disconnection. In conclusion, transcriptome profiling, pathway integration, and network identification of autoimmune myocardial inflammation provide a molecular signature applicable to the diagnosis of inflammatory cardiomyopathy. Combinatorial attack provides a rationale for immunosuppressive therapy of inflammatory cardiomyopathy and provides an in silico prediction that the approved therapeutics, ibrutinib and idelalisib targeting Btk and Pik3cd respectively, could potentially be re-purposed as adjuncts to immunosuppression.


Assuntos
Doenças Autoimunes , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Miocardite , Animais , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/imunologia , Miocárdio/metabolismo , Miocárdio/patologia , Transcriptoma
6.
Life (Basel) ; 10(5)2020 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-32403239

RESUMO

MicroRNAs (miRNAs) play key roles in cartilage development and homeostasis and are dysregulated in osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs, critical if miR-145 is to be considered a target for cartilage repair. We performed RIP-seq (RNA-immunoprecipitation and high-throughput sequencing) of miRISC (miRNA-induced silencing complex) in HACs overexpressing miR-145 to identify miR-145 direct targets and used cWords to assess enrichment of miR-145 seed matches in the identified targets. Further validations were performed by RT-qPCR, Western immunoblot, and luciferase assays. MiR-145 affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3'UTR or, more commonly, the coding region. MiR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion leads to MMP13 upregulation, suggesting a contribution towards the effect of miR-145 on MMP13 expression. In conclusion, miR-145 directly targets several genes involved in the expression of the extracellular matrix and inflammation in primary chondrocytes. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

7.
Malar J ; 18(1): 418, 2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31835999

RESUMO

BACKGROUND: Transcriptional profiling of the human immune response to malaria has been used to identify diagnostic markers, understand the pathogenicity of severe disease and dissect the mechanisms of naturally acquired immunity (NAI). However, interpreting this body of work is difficult given considerable variation in study design, definition of disease, patient selection and methodology employed. This work details a comprehensive review of gene expression profiling (GEP) of the human immune response to malaria to determine how this technology has been applied to date, instances where this has advanced understanding of NAI and the extent of variability in methodology between studies to allow informed comparison of data and interpretation of results. METHODS: Datasets from the gene expression omnibus (GEO) including the search terms; 'plasmodium' or 'malaria' or 'sporozoite' or 'merozoite' or 'gametocyte' and 'Homo sapiens' were identified and publications analysed. Datasets of gene expression changes in relation to malaria vaccines were excluded. RESULTS: Twenty-three GEO datasets and 25 related publications were included in the final review. All datasets related to Plasmodium falciparum infection, except two that related to Plasmodium vivax infection. The majority of datasets included samples from individuals infected with malaria 'naturally' in the field (n = 13, 57%), however some related to controlled human malaria infection (CHMI) studies (n = 6, 26%), or cells stimulated with Plasmodium in vitro (n = 6, 26%). The majority of studies examined gene expression changes relating to the blood stage of the parasite. Significant heterogeneity between datasets was identified in terms of study design, sample type, platform used and method of analysis. Seven datasets specifically investigated transcriptional changes associated with NAI to malaria, with evidence supporting suppression of the innate pro-inflammatory response as an important mechanism for this in the majority of these studies. However, further interpretation of this body of work was limited by heterogeneity between studies and small sample sizes. CONCLUSIONS: GEP in malaria is a potentially powerful tool, but to date studies have been hypothesis generating with small sample sizes and widely varying methodology. As CHMI studies are increasingly performed in endemic settings, there will be growing opportunity to use GEP to understand detailed time-course changes in host response and understand in greater detail the mechanisms of NAI.


Assuntos
Expressão Gênica/imunologia , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Malária/imunologia , Plasmodium/imunologia , Perfilação da Expressão Gênica , Humanos
8.
Nat Commun ; 10(1): 3143, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31316060

RESUMO

Heterozygous germline mutations in BRCA2 predispose to breast and ovarian cancer. Contrary to non-cancerous cells, where BRCA2 deletion causes cell cycle arrest or cell death, tumors carrying BRCA2 inactivation continue to proliferate. Here we set out to investigate adaptation to loss of BRCA2 focusing on genome-wide transcriptome alterations. Human cells in which BRCA2 expression is inhibited for 4 or 28 days are subjected to RNA-seq analyses revealing a biphasic response to BRCA2 abrogation. The early, acute response consists of downregulation of genes involved in cell cycle progression, DNA replication and repair and is associated with cell cycle arrest in G1. Surprisingly, the late, chronic response consists predominantly of upregulation of interferon-stimulated genes (ISGs). Activation of the cGAS-STING-STAT pathway detected in these cells further substantiates the concept that BRCA2 abrogation triggers cell-intrinsic immune signaling. Importantly, we find that treatment with PARP inhibitors stimulates the interferon response in cells and tumors lacking BRCA2.


Assuntos
Proteína BRCA2/genética , Neoplasias da Mama/genética , Animais , Neoplasias da Mama/imunologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Dano ao DNA , Reparo do DNA , Feminino , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Inata , Camundongos SCID , Ftalazinas/farmacologia , Piperazinas/farmacologia
9.
Nat Commun ; 10(1): 1869, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015479

RESUMO

Whole-genome sequencing (WGS) is becoming widely used in clinical medicine in diagnostic contexts and to inform treatment choice. Here we evaluate the potential of the Oxford Nanopore Technologies (ONT) MinION long-read sequencer for routine WGS by sequencing the reference sample NA12878 and the genome of an individual with ataxia-pancytopenia syndrome and severe immune dysregulation. We develop and apply a novel reference panel-free analytical method to infer and then exploit phase information which improves single-nucleotide variant (SNV) calling performance from otherwise modest levels. In the clinical sample, we identify and directly phase two non-synonymous de novo variants in SAMD9L, (OMIM #159550) inferring that they lie on the same paternal haplotype. Whilst consensus SNV-calling error rates from ONT data remain substantially higher than those from short-read methods, we demonstrate the substantial benefits of analytical innovation. Ongoing improvements to base-calling and SNV-calling methodology must continue for nanopore sequencing to establish itself as a primary method for clinical WGS.


Assuntos
Testes Genéticos/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nanoporos , Sequenciamento Completo do Genoma/métodos , Adulto , Ataxia Cerebelar/diagnóstico , Ataxia Cerebelar/genética , Feminino , Genoma Humano/genética , Genômica/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Lactente , Masculino , Nanotecnologia , Pancitopenia/diagnóstico , Pancitopenia/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Supressoras de Tumor/genética , Sequenciamento Completo do Genoma/instrumentação
10.
Nat Commun ; 9(1): 5409, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30573728

RESUMO

Inducible nitric oxide synthase (iNOS) plays a crucial role in controlling growth of Mycobacterium tuberculosis (M.tb), presumably via nitric oxide (NO) mediated killing. Here we show that leukocyte-specific deficiency of NO production, through targeted loss of the iNOS cofactor tetrahydrobiopterin (BH4), results in enhanced control of M.tb infection; by contrast, loss of iNOS renders mice susceptible to M.tb. By comparing two complementary NO-deficient models, Nos2-/- mice and BH4 deficient Gch1fl/flTie2cre mice, we uncover NO-independent mechanisms of anti-mycobacterial immunity. In both murine and human leukocytes, decreased Gch1 expression correlates with enhanced cell-intrinsic control of mycobacterial infection in vitro. Gene expression analysis reveals that Gch1 deficient macrophages have altered inflammatory response, lysosomal function, cell survival and cellular metabolism, thereby enhancing the control of bacterial infection. Our data thus highlight the importance of the NO-independent functions of Nos2 and Gch1 in mycobacterial control.


Assuntos
Biopterina/análogos & derivados , GTP Cicloidrolase/fisiologia , Óxido Nítrico Sintase Tipo II/fisiologia , Óxido Nítrico/biossíntese , Tuberculose/imunologia , Animais , Biopterina/genética , Biopterina/metabolismo , Biopterina/fisiologia , Sobrevivência Celular , GTP Cicloidrolase/genética , GTP Cicloidrolase/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo
11.
Sci Rep ; 8(1): 10439, 2018 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-29992973

RESUMO

ZIC2 mutation is known to cause holoprosencephaly (HPE). A subset of ZIC2 HPE probands harbour cardiovascular and visceral anomalies suggestive of laterality defects. 3D-imaging of novel mouse Zic2 mutants uncovers, in addition to HPE, laterality defects in lungs, heart, vasculature and viscera. A strong bias towards right isomerism indicates a failure to establish left identity in the lateral plate mesoderm (LPM), a phenotype that cannot be explained simply by the defective ciliogenesis previously noted in Zic2 mutants. Gene expression analysis showed that the left-determining NODAL-dependent signalling cascade fails to be activated in the LPM, and that the expression of Nodal at the node, which normally triggers this event, is itself defective in these embryos. Analysis of ChiP-seq data, in vitro transcriptional assays and mutagenesis reveals a requirement for a low-affinity ZIC2 binding site for the activation of the Nodal enhancer HBE, which is normally active in node precursor cells. These data show that ZIC2 is required for correct Nodal expression at the node and suggest a model in which ZIC2 acts at different levels to establish LR asymmetry, promoting both the production of the signal that induces left side identity and the morphogenesis of the cilia that bias its distribution.


Assuntos
Mesoderma/embriologia , Morfogênese , Proteína Nodal/metabolismo , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Padronização Corporal , Cílios , Holoprosencefalia/genética , Camundongos , Mutação , Proteínas Nucleares/genética , Fenótipo , Transdução de Sinais , Fatores de Transcrição/genética
12.
Blood ; 132(12): 1225-1240, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-29930011

RESUMO

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.


Assuntos
Mutação , Síndromes Mielodisplásicas/genética , Fatores de Processamento de RNA/genética , Splicing de RNA , Spliceossomos/genética , Estudos de Coortes , Reparo do DNA , Regulação da Expressão Gênica , Humanos , Síndromes Mielodisplásicas/epidemiologia , Fosfoproteínas/genética , Fatores de Processamento de Serina-Arginina/genética , Fator de Processamento U2AF/genética , Análise de Sobrevida
13.
Sci Rep ; 8(1): 2151, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29391536

RESUMO

The design and implementation of single-cell experiments is often limited by their requirement for fresh starting material. We have adapted a method for histological tissue fixation using dithio-bis(succinimidyl propionate) (DSP), or Lomant's Reagent, to stabilise cell samples for single-cell transcriptomic applications. DSP is a reversible cross-linker of free amine groups that has previously been shown to preserve tissue integrity for histology while maintaining RNA integrity and yield in bulk RNA extractions. Although RNA-seq data from DSP-fixed single cells appears to be prone to characteristic artefacts, such as slightly reduced yield of cDNA and a detectable 3' bias in comparison with fresh cells, cell preservation using DSP does not appear to substantially reduce RNA complexity at the gene level. In addition, there is evidence that instantaneous fixation of cells can reduce inter-cell technical variability. The ability of DSP-fixed cells to retain commonly used dyes, such as propidium iodide, enables the tracking of experimental sub-populations and the recording of cell viability at the point of fixation. Preserving cells using DSP will remove several barriers in the staging of single-cell experiments, including the transport of samples and the scheduling of shared equipment for downstream single-cell isolation and processing.


Assuntos
Reagentes para Ligações Cruzadas/química , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Succinimidas/química , Fixação de Tecidos , Transcriptoma , Separação Celular , Humanos , Células K562 , Análise de Célula Única/instrumentação
14.
Epigenetics ; 12(10): 897-908, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29099281

RESUMO

Genome-wide association studies in the fields of reproductive medicine and endocrinology are yielding robust genetic variants associated with disease. Integrated genomic, transcriptomic, and epigenomic molecular profiling studies are common methodologies used to understand the biologic pathways perturbed by these variants. However, molecular profiling resources do not include the tissue most relevant to many female reproductive traits, the endometrium, while the parameters influencing variability of results from its molecular profiling are unclear. We investigated the sources of DNA methylation and RNA expression profile variability in endometrium (n = 135), endometriotic disease tissue (endometriosis), and subcutaneous abdominal fat samples from 24 women, quantifying between-individual, within-tissue (cellular heterogeneity), and technical variation. DNA samples (n = 96) were analyzed using Illumina HumanMethlylation450 BeadChip arrays; RNA samples (n = 39) were analyzed using H12-expression arrays. Variance-component analyses showed that, for the top 10-50% variable DNA methylation/RNA expression sites, between-individual variation far exceeded within-tissue and technical variation. Menstrual-phase accounted for most variability in methylation/expression patterns in endometrium (Pm = 7.8 × 10-3, Pe = 8.4 × 10-5) but not in fat and endometriotic tissue; age was significantly associated with DNA methylation profile of endometrium (Pm = 9 × 10-5) and endometriotic disease tissue (Pm = 2.4 × 10-5); and smoking was significantly associated with DNA methylation in adipose tissue (Pm = 1.8 × 10-3). Hierarchical cluster analysis showed significantly different methylation signatures between endometrium and endometriotic tissue enriched for WNT signaling, angiogenesis, cadherin signaling, and gonadotropin-releasing-hormone-receptor pathways. Differential DNA methylation/expression analyses suggested detection of a limited number of sites with large fold changes (FC > 4), but power calculations accounting for different sources of variability showed that for robust detection >500 tissue samples are required. These results enable appropriate study design for large-scale expression and methylation tissue-based profiling relevant to many reproductive and endocrine traits.


Assuntos
Metilação de DNA/genética , Doenças do Sistema Endócrino/genética , Endometriose/genética , Reprodução/genética , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Adulto , Fatores Etários , Ilhas de CpG/genética , Doenças do Sistema Endócrino/patologia , Endometriose/patologia , Endométrio/metabolismo , Endométrio/patologia , Epigênese Genética , Feminino , Regulação da Expressão Gênica/genética , Estudo de Associação Genômica Ampla , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/genética
15.
Nat Commun ; 8(1): 1258, 2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-29097735

RESUMO

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αßγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f) and ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Bradicardia/genética , Cálcio/metabolismo , Frequência Cardíaca/genética , Sarcolema/metabolismo , Nó Sinoatrial/metabolismo , Adulto , Animais , Bradicardia/metabolismo , Eletrocardiografia Ambulatorial , Exercício Físico , Coração/diagnóstico por imagem , Humanos , Imagem Cinética por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Camundongos , Microscopia Eletrônica de Transmissão , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/ultraestrutura , Condicionamento Físico Animal , Resistência Física , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Nó Sinoatrial/patologia
16.
BMC Genomics ; 18(1): 53, 2017 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-28061811

RESUMO

BACKGROUND: Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. RESULTS: We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. CONCLUSION: As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.


Assuntos
Interação Gene-Ambiente , Macrófagos/citologia , Análise de Sequência de RNA , Análise de Célula Única , Técnicas de Inativação de Genes , Humanos , Macrófagos/metabolismo , Fenótipo , Proteína 1 com Domínio SAM e Domínio HD/deficiência , Proteína 1 com Domínio SAM e Domínio HD/genética
17.
Cancer Cell ; 30(4): 578-594, 2016 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-27693047

RESUMO

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.


Assuntos
Neoplasias Encefálicas/enzimologia , Glioma/enzimologia , Isocitrato Desidrogenase/biossíntese , Ventrículos Laterais/enzimologia , Células-Tronco Neoplásicas/enzimologia , Nicho de Células-Tronco , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Metilação de DNA , Glioma/genética , Glioma/patologia , Isocitrato Desidrogenase/genética , Ventrículos Laterais/patologia , Camundongos , Camundongos Transgênicos , Mutação , Células-Tronco Neoplásicas/patologia , Transcriptoma
18.
Mol Cell ; 64(1): 51-64, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27546791

RESUMO

The tumor suppressor protein 53BP1, a pivotal regulator of DNA double-strand break (DSB) repair, was first identified as a p53-interacting protein over two decades ago. However, its direct contributions to p53-dependent cellular activities remain undefined. Here, we reveal that 53BP1 stimulates genome-wide p53-dependent gene transactivation and repression events in response to ionizing radiation (IR) and synthetic p53 activation. 53BP1-dependent p53 modulation requires both auto-oligomerization and tandem-BRCT domain-mediated bivalent interactions with p53 and the ubiquitin-specific protease USP28. Loss of these activities results in inefficient p53-dependent cell-cycle checkpoint and exit responses. Furthermore, we demonstrate 53BP1-USP28 cooperation to be essential for normal p53-promoter element interactions and gene transactivation-associated events, yet dispensable for 53BP1-dependent DSB repair regulation. Collectively, our data provide a mechanistic explanation for 53BP1-p53 cooperation in controlling anti-tumorigenic cell-fate decisions and reveal these activities to be distinct and separable from 53BP1's regulation of DNA double-strand break repair pathway choice.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/genética , Endonucleases/metabolismo , Raios gama , Edição de Genes , Regulação da Expressão Gênica , Humanos , Células MCF-7 , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , RNA Guia/genética , RNA Guia/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo
19.
Sci Rep ; 6: 28210, 2016 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-27324164

RESUMO

BMP signalling is negatively autoregulated by several genes including SMAD6, Noggin and Gremlin, and autoregulators are possible targets for enhancing BMP signalling in disorders such as fibrosis and pulmonary hypertension. To identify novel negative regulators of BMP signalling, we used siRNA screening in mouse C2C12 cells with a BMP-responsive luciferase reporter. Knockdown of several splicing factors increased BMP4-dependent transcription and target gene expression. Knockdown of RBM39 produced the greatest enhancement in BMP activity. Transcriptome-wide RNA sequencing identified a change in Sin3b exon usage after RBM39 knockdown. SIN3B targets histone deacetylases to chromatin to repress transcription. In mouse, Sin3b produces long and short isoforms, with the short isoform lacking the ability to recruit HDACs. BMP4 induced a shift in SIN3B expression to the long isoform, and this change in isoform ratio was prevented by RBM39 knockdown. Knockdown of long isoform SIN3B enhanced BMP4-dependent transcription, whereas knockdown of the short isoform did not. We propose that BMP4-dependent transcription is negatively autoregulated in part by SIN3B alternative splicing, and that RBM39 plays a role in this process.


Assuntos
Proteína Morfogenética Óssea 4/genética , Hipertensão Pulmonar/genética , Pulmão/patologia , Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Processamento Alternativo , Animais , Linhagem Celular Tumoral , Cromatina/genética , Retroalimentação Fisiológica , Fibrose , Histona Desacetilases/metabolismo , Homeostase , Humanos , Hipertensão Pulmonar/metabolismo , Camundongos , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Smad6/genética
20.
Wellcome Open Res ; 1: 17, 2016 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-28058287

RESUMO

Background: Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines. It is a potent cytokine, with potential antiviral impact, and has been shown to play a role in modulating functions of diverse cell types, including Th1, Th2, and NK and B cells, demonstrating both pro- and anti-inflammatory roles.  In hepatocytes, it is capable of inducing signal transducer and activator of transcription (STAT)1, STAT3 and interferon-stimulated genes. Methods: To address its role in viral hepatitis, the antiviral activity of IL-27 against hepatitis C virus (HCV) and hepatitis B virus (HBV) was tested in vitro using cell-culture-derived infectious HCV (HCVcc) cell culture system and the HepaRG HBV cell culture model. To further investigate the impact of IL-27 on hepatocytes, Huh7.5 cells were treated with IL-27 to analyse the differentially expressed genes by microarray analysis. Furthermore, by quantitative PCR, we analyzed the up-regulation of chemokine (CXCL)-10 in response to IL-27. Results: In both HCV and HBV infection models, we observed only a modest direct antiviral effect. Microarray analysis showed that the up-regulated genes mostly belonged to antigen presentation and DNA replication pathways, and involved strong up-regulation of CXCL-10, a gene associated with liver inflammation. Overall, gene set enrichment analysis showed a striking correlation of these genes with those up-regulated in response to related cytokines in diverse cell populations. Conclusion: Our data indicate that IL-27 can have a significant pro-inflammatory impact in vitro, although the direct antiviral effect is modest. It may have a potential impact on hepatocyte function, especially chemokine expression and antigen presentation.

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