RESUMO
Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12-dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT-AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT-AN minor introns in Ë 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans.
Assuntos
Instabilidade Cromossômica/genética , Cromossomos/genética , Mutação/genética , Spliceossomos/genética , Sequência de Aminoácidos , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Células HeLa , Humanos , Íntrons/genéticaRESUMO
Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1BQ287*, cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 - REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1BQ287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1BQ287*-induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1BQ287* including cell division and interferon signaling. Proteome studies on platelets from GFI1BQ287* patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1BQ287* deregulates this program through LSD1-RCOR-HDAC sequestering.
Assuntos
Transtornos da Coagulação Sanguínea/patologia , Plaquetas/patologia , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/patologia , Megacariócitos/patologia , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Transtornos da Coagulação Sanguínea/genética , Transtornos da Coagulação Sanguínea/metabolismo , Plaquetas/metabolismo , Diferenciação Celular , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Megacariócitos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Mapas de Interação de Proteínas , Proteoma/análise , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismoRESUMO
Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states.
Assuntos
Células-Tronco Pluripotentes/metabolismo , Fator de Transcrição TFIID/metabolismo , Transcrição Gênica , Animais , Reprogramação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIID/deficiência , Fator de Transcrição TFIID/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Vitamin D plays an important role in renal (patho)physiology. Patients with glomerular diseases have an injured renal filtration barrier, leading to proteinuria and reduced renal function. An impaired renal function also leads to 1,25-vitamin D3 deficiency as a result of reduced renal 1α-hydroxylase activity. Vitamin D treatment to reduce proteinuria remains controversial, although there is an inverse correlation between vitamin D levels and proteinuria. Herein, we showed that 1,25-vitamin D3-deficient 25-hydroxy-vitamin-D3-1α-hydroxylase knockout mice and 1,25-vitamin D3-deficient rats develop podocyte injury and renal dysfunction. Glomerular injury was characterized by proteinuria and partial podocyte foot process effacement. Expression of nephrin, podocin, desmin, and transient receptor potential channel C6 in the podocyte was significantly altered in 1,25-vitamin D3-deficient animals. Supplementation with 1,25-vitamin D3 or 1,25-vitamin D2 prevented podocyte effacement or reversed glomerular and tubulointerstitial damage in 1,25-vitamin D3-deficient animals, thereby preserving and restoring renal function, respectively. The effect of 1,25-vitamin D3 deficiency and 1,25-vitamin D3 and 1,25-vitamin D2 repletion on proteinuria could not be explained by hypocalcemia, changes in parathyroid hormone, or fibroblast growth factor 23. This study demonstrates that 1,25-vitamin D3 deficiency directly leads to renal injury in rodents. Translated to human subjects, this would underline the need for early vitamin D supplementation in patients with glomerular disease and chronic renal insufficiency, which might inhibit or potentially reverse renal injury.
Assuntos
Albuminúria/etiologia , Albuminúria/metabolismo , Colecalciferol/deficiência , Nefropatias/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Animais , Glomérulos Renais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hormônio Paratireóideo/metabolismo , Ratos , Ratos WistarRESUMO
Slit diaphragm and podocyte damage is crucial in the pathogenesis of proteinuria in diabetic nephropathy (DNP). Gain-of-function mutations in TRPC6, a slit diaphragm-associated ion channel, cause glomerulosclerosis; TRPC6 expression is increased in acquired glomerular disease. Hyperglycemia and high intrarenal angiotensin II (AngII) levels could contribute to podocyte injury in DNP. We determined whether glucose regulates TRPC6 expression and TRPC6-mediated Ca(2+) influx into the podocyte and whether these effects are AngII dependent. High glucose levels increased TRPC6 mRNA and protein expression in cultured podocytes; however, TRPC1 and TRPC5 mRNA expression was unaltered. AngII and inducing podocyte injury also specifically increased TRPC6 expression. Angiotensin receptor blockade and inhibition of local AngII production through angiotensin-converting enzyme inhibition prevented glucose-mediated increased TRPC6 expression. In addition, high glucose concentration pretreatment enhanced Ca(2+) influx in podocytes, which was prevented by concomitant angiotensin receptor blockade application and TRPC6 knockdown. Studies with a TRPC6 luciferase promoter construct demonstrated a glucose concentration-dependent effect on TRPC6 promoter activity. In vivo, podocyte TRPC6 protein expression was increased in proteinuric streptozotocin-induced diabetic rats. These data suggest that glucose can activate a local renin-angiotensin system in the podocyte, leading to increased TRPC6 expression, which enhances TRPC6-mediated Ca(2+) influx. Regulation of TRPC6 expression could be an important factor in podocyte injury due to chronic hyperglycemia and the antiproteinuric effect of angiotensin receptor blockade or angiotensin-converting enzyme inhibition in DNP.
Assuntos
Angiotensina II/metabolismo , Nefropatias Diabéticas/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Podócitos/metabolismo , Canais de Cátion TRPC/biossíntese , Animais , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Camundongos , Camundongos Knockout , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Podócitos/patologia , Ratos , Ratos Wistar , Sistema Renina-Angiotensina/genética , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6RESUMO
Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.
Assuntos
Linhagem da Célula , Desenvolvimento Embrionário , Proteômica , Animais , Camundongos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Análise de Célula Única , Diferenciação Celular , Gástrula/metabolismo , GastrulaçãoRESUMO
BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a leading cause of steroid-resistant nephrotic syndrome. Hereditary FSGS is frequently caused by mutations in important structural podocyte proteins, including the slit diaphragm-associated transient receptor potential channel C6 (TRPC6). METHODS: In five patients with biopsy-proven autosomal-dominant FSGS from five different Dutch families, all 13 exons of TRPC6 were sequenced. Upon identification of a novel TRPC6 sequence variant, the resultant amino acid change was introduced in the wild-type TRPC6 protein and functionally tested using patch-clamp analyses and cell-surface biotinylation experiments. RESULTS: None of the previously described TRPC6 mutations were found in our cohort. In one family, we identified a novel c.524G>A sequence variant resulting in a p.Arg175Gln (R175Q) substitution in the TRPC6 protein. This sequence variant was absent in 449 control subjects and from public SNP databases. The mutation was located in the third ankyrin repeat domain (ANK3) in the cytoplasmic N-tail of TRPC6, important for protein-protein interaction and regulation of ion channel activity. Patch-clamp analyses of the mutant channel indeed showed an increased TRPC6 channel-mediated current. However, cell-surface expression of the mutant channel was not increased. CONCLUSIONS: We identified a novel TRPC6 p.Arg175Gln gain-of-function mutation that shows increased TRPC6-mediated current, which is not due to altered cell-surface expression. This is the first mutation identified in ANK3 of the TRPC6 N-tail and is most likely responsible for the late-onset autosomal dominant FSGS in this family.
Assuntos
Consanguinidade , Glomerulosclerose Segmentar e Focal/etiologia , Mutação/genética , Canais de Cátion TRPC/genética , Adulto , Idade de Início , Idoso , Sequência de Aminoácidos , Pré-Escolar , Eletrofisiologia , Família , Feminino , Seguimentos , Taxa de Filtração Glomerular , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Países Baixos , Linhagem , Prognóstico , Homologia de Sequência de Aminoácidos , Canal de Cátion TRPC6 , Fatores de TempoRESUMO
The multi-subunit nucleosome remodeling and deacetylase (NuRD) complex consists of seven subunits, each of which comprises two or three paralogs in vertebrates. These paralogs define mutually exclusive and functionally distinct complexes. In addition, several proteins in the complex are multimeric, which complicates structural studies. Attempts to purify sufficient amounts of endogenous complex or recombinantly reconstitute the complex for structural studies have proven quite challenging. Until now, only substructures of individual domains or proteins and low-resolution densities of (partial) complexes have been reported. In this study, we comprehensively investigated the relative orientation of different subunits within the NuRD complex using multiple cross-link IP mass spectrometry (xIP-MS) experiments. Our results confirm that the core of the complex is formed by MTA, RBBP, and HDAC proteins. Assembly of a copy of MBD and GATAD2 onto this core enables binding of the peripheral CHD and CDK2AP proteins. Furthermore, our experiments reveal that not only CDK2AP1 but also CDK2AP2 interacts with the NuRD complex. This interaction requires the C terminus of CHD proteins. Our data provide a more detailed understanding of the topology of the peripheral NuRD subunits relative to the core complex. DATABASE: Proteomics data are available in the PRIDE database under the accession numbers PXD017244 and PXD017378.
Assuntos
Quinases Ciclina-Dependentes/química , Fatores de Transcrição GATA/química , Histona Desacetilases/química , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/química , Nucleossomos/ultraestrutura , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/química , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Células HeLa , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Espectrometria de Massas/métodos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Modelos Moleculares , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
A genomic locus 8 kb downstream of the transcription factor GFI1B (Growth Factor Independence 1B) predisposes to clonal hematopoiesis and myeloproliferative neoplasms. One of the most significantly associated polymorphisms in this region is rs621940-G. GFI1B auto-represses GFI1B, and altered GFI1B expression contributes to myeloid neoplasms. We studied whether rs621940-G affects GFI1B expression and growth of immature cells. GFI1B ChIP-seq showed clear binding to the rs621940 locus. Preferential binding of various hematopoietic transcription factors to either the rs621940-C or -G allele was observed, but GFI1B showed no preference. In gene reporter assays the rs621940 region inhibited GFI1B promoter activity with the G-allele having less suppressive effects compared to the C-allele. However, CRISPR-Cas9 mediated deletion of the locus in K562 cells did not alter GFI1B expression nor auto-repression. In healthy peripheral blood mononuclear cells GFI1B expression did not differ consistently between the rs621940 alleles. Long range and targeted deep sequencing did not detect consistent effects of rs621940-G on allelic GFI1B expression either. Finally, we observed that myeloid colony formation was not significantly affected by either rs621940 allele in 193 healthy donors. Together, these findings show no evidence that rs621940 or its locus affect GFI1B expression, auto-repression or growth of immature myeloid cells.
Assuntos
Predisposição Genética para Doença , Transtornos Mieloproliferativos/genética , Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Adulto , Idoso , Alelos , Sistemas CRISPR-Cas/genética , Feminino , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células K562 , Masculino , Pessoa de Meia-Idade , Células Mieloides/metabolismo , Células Mieloides/patologia , Transtornos Mieloproliferativos/patologia , Neoplasias/patologia , Fagocitose/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto JovemRESUMO
Experience-dependent plasticity (EDP) powerfully shapes neural circuits by inducing long-lasting molecular changes in the brain. Molecular mechanisms of EDP have been traditionally studied by identifying single or small subsets of targets along the biochemical pathways that link synaptic receptors to nuclear processes. Recent technological advances in large-scale analysis of gene transcription and translation now allow systematic observation of thousands of molecules simultaneously. Here we employed label-free quantitative mass spectrometry to address experience-dependent changes in the proteome after sensory deprivation of the primary somatosensory cortex. Cortical column- and layer-specific tissue samples were collected from control animals, with all whiskers intact, and animals whose C-row whiskers were bilaterally plucked for 11-14 days. Thirty-three samples from cortical layers (L) 2/3 and L4 spanning across control, deprived, and first- and second-order spared columns yielded at least 10 000 peptides mapping to â¼5000 protein groups. Of these, 4676 were identified with high confidence, and >3000 were found in all samples. This comprehensive database provides a snapshot of the proteome after whisker deprivation, a protocol that has been widely used to unravel the synaptic, cellular, and network mechanisms of EDP. Complementing the recently made available transcriptome for identical experimental conditions (see the accompanying article by Kole et al.), the database can be used to (i) mine novel targets whose translation is modulated by sensory organ use, (ii) cross-validate experimental protocols from the same developmental time point, and (iii) statistically map the molecular pathways of cortical plasticity at a columnar and laminar resolution.
Assuntos
Proteômica , Privação Sensorial/fisiologia , Córtex Somatossensorial/metabolismo , Animais , Feminino , Camundongos Endogâmicos C57BLRESUMO
RNA modifications are integral to the regulation of RNA metabolism. One abundant mRNA modification is N6-methyladenosine (m6A), which affects various aspects of RNA metabolism, including splicing, translation and degradation. Current knowledge about the proteins recruited to m6A to carry out these molecular processes is still limited. Here we describe comprehensive and systematic mass-spectrometry-based screening of m6A interactors in various cell types and sequence contexts. Among the main findings, we identified G3BP1 as a protein that is repelled by m6A and positively regulates mRNA stability in an m6A-regulated manner. Furthermore, we identified FMR1 as a sequence-context-dependent m6A reader, thus revealing a connection between an mRNA modification and an autism spectrum disorder. Collectively, our data represent a rich resource and shed further light on the complex interplay among m6A, m6A interactors and mRNA homeostasis.
Assuntos
Adenosina/análogos & derivados , Homeostase , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Animais , Linhagem Celular , Humanos , Espectrometria de Massas , Ligação ProteicaRESUMO
Trimethylation of histone H3 at lysine 4 (H3K4me3) is regarded as a hallmark of active human promoters, but it remains unclear how this posttranslational modification links to transcriptional activation. Using a stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic screening we show that the basal transcription factor TFIID directly binds to the H3K4me3 mark via the plant homeodomain (PHD) finger of TAF3. Selective loss of H3K4me3 reduces transcription from and TFIID binding to a subset of promoters in vivo. Equilibrium binding assays and competition experiments show that the TAF3 PHD finger is highly selective for H3K4me3. In transient assays, TAF3 can act as a transcriptional coactivator in a PHD finger-dependent manner. Interestingly, asymmetric dimethylation of H3R2 selectively inhibits TFIID binding to H3K4me3, whereas acetylation of H3K9 and H3K14 potentiates TFIID interaction. Our experiments reveal crosstalk between histone modifications and the transcription factor TFIID. This has important implications for regulation of RNA polymerase II-mediated transcription in higher eukaryotes.