RESUMO
Allele-specific expression of imprinted gene clusters is governed by gametic DNA methylation at master regulators called imprinting control regions (ICRs). Non-gametic or secondary differentially methylated regions (DMRs) at promoters and exonic regions reinforce monoallelic expression but do not control an entire cluster. Here, we unveil an unconventional secondary DMR that is indispensable for tissue-specific imprinting of two previously unlinked genes, Grb10 and Ddc. Using polymorphic mice, we mapped an intronic secondary DMR at Grb10 with paternal-specific CTCF binding (CBR2.3) that forms contacts with Ddc. Deletion of paternal CBR2.3 removed a critical insulator, resulting in substantial shifting of chromatin looping and ectopic enhancer-promoter contacts. Destabilized gene architecture precipitated abnormal Grb10-Ddc expression with developmental consequences in the heart and muscle. Thus, we redefine the Grb10-Ddc imprinting domain by uncovering an unconventional intronic secondary DMR that functions as an insulator to instruct the tissue-specific, monoallelic expression of multiple genes-a feature previously ICR exclusive.
Assuntos
Impressão Genômica , RNA Longo não Codificante , Alelos , Animais , Cromatina/genética , Metilação de DNA , Proteína Adaptadora GRB10/genética , Coração , CamundongosRESUMO
Obesity is characterized by an accumulation of macrophages in adipose, some of which form distinct crown-like structures (CLS) around fat cells. While multiple discrete adipose tissue macrophage (ATM) subsets are thought to exist, their respective effects on adipose tissue, and the transcriptional mechanisms that underlie the functional differences between ATM subsets, are not well understood. We report that obese fat tissue of mice and humans contain multiple distinct populations of ATMs with unique tissue distributions, transcriptomes, chromatin landscapes, and functions. Mouse Ly6c ATMs reside outside of CLS and are adipogenic, while CD9 ATMs reside within CLS, are lipid-laden, and are proinflammatory. Adoptive transfer of Ly6c ATMs into lean mice activates gene programs typical of normal adipocyte physiology. By contrast, adoptive transfer of CD9 ATMs drives gene expression that is characteristic of obesity. Importantly, human adipose tissue contains similar ATM populations, including lipid-laden CD9 ATMs that increase with body mass. These results provide a higher resolution of the cellular and functional heterogeneity within ATMs and provide a framework within which to develop new immune-directed therapies for the treatment of obesity and related sequela.
Assuntos
Tecido Adiposo/citologia , Inflamação/fisiopatologia , Macrófagos , Animais , Exossomos/química , Feminino , Humanos , Inflamação/genética , Macrófagos/química , Macrófagos/classificação , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Obesidade/fisiopatologia , Tetraspanina 29/análise , Tetraspanina 29/metabolismo , Transcriptoma/genéticaRESUMO
TET1/2/3 dioxygenases iteratively demethylate 5-methylcytosine, beginning with the formation of 5-hydroxymethylcytosine (5hmC). The post-mitotic brain maintains higher levels of 5hmC than most peripheral tissues, and TET1 ablation studies have underscored the critical role of TET1 in brain physiology. However, deletion of Tet1 precludes the disentangling of the catalytic and non-catalytic functions of TET1. Here, we dissect these functions of TET1 by comparing adult cortex of Tet1 wildtype (Tet1 WT), a novel Tet1 catalytically dead mutant (Tet1 HxD), and Tet1 knockout (Tet1 KO) mice. Using DNA methylation array, we uncover that Tet1 HxD and KO mutations perturb the methylation status of distinct subsets of CpG sites. Gene ontology (GO) analysis on specific differential 5hmC regions indicates that TET1's catalytic activity is linked to neuronal-specific functions. RNA-Seq further shows that Tet1 mutations predominantly impact the genes that are associated with alternative splicing. Lastly, we performed High-performance Liquid Chromatography Mass-Spectrometry lipidomics on WT and mutant cortices and uncover accumulation of lysophospholipids lysophosphatidylethanolamine and lysophosphatidylcholine in Tet1 HxD cortex. In summary, we show that Tet1 HxD does not completely phenocopy Tet1 KO, providing evidence that TET1 modulates distinct cortical functions through its catalytic and non-catalytic roles.
Assuntos
5-Metilcitosina , Córtex Cerebral , Metilação de DNA , Proteínas Proto-Oncogênicas , Animais , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Córtex Cerebral/metabolismo , Camundongos Knockout , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ilhas de CpG , MutaçãoRESUMO
Loss of imprinting (LOI) at the Dlk1-Dio3 locus is linked to Kagami-Ogata and Temple syndromes, and to cancer, but molecular mechanisms that prevent LOI are under-studied. In this issue of Developmental Cell, Aronson et al. demarcate the bipartite regulation of the Dlk1-Dio3 imprinting control region (ICR) IG-DMR, which maintains locus imprinting.
Assuntos
Impressão Genômica , Neoplasias , Proteínas de Ligação ao Cálcio/genética , Metilação de DNA , Humanos , Proteínas de Membrana/genéticaRESUMO
For mice, a maternal vitamin A (VA)-deficient diet initiated from midgestation (GVAD) produces serum retinol deficiency in mature offspring. We hypothesize that the effects of GVAD arise from preweaning developmental changes. We compare the effect of this GVAD protocol in combination with a postweaning high-fat diet (HFD) or high-carbohydrate diet (LF12). Each is compared to an equivalent VA-sufficient combination. GVAD extensively decreased serum retinol and liver retinol, retinyl esters, and retinoid homeostasis genes (Lrat, Cyp26b1 and Cyp26a1). These suppressions were each more effective with LF12 than with HFD. Postweaning initiation of VA deficiency with LF12 depleted liver retinoids, but serum retinol was unaffected. Liver retinoid depletion, therefore, precedes serum attenuation. Maternal LF12 decreased the obesity response to the HFD, which was further decreased by GVAD. LF12 fed to the mother and offspring extensively stimulated genes marking stellate activation (Col1a1, Timp2 and Cyp1b1) and novel inflammation markers (Ly6d, Trem2 and Nupr1). The GVAD with LF12 diet combination suppressed these responses. GVAD in combination with the HFD increased these same clusters. A further set of expression differences on the HFD when compared to a high-carbohydrate diet was prevented when GVAD was combined with HFD. Most of these GVAD gene changes match published effects from deletion of Nr0b2/Shp, a retinoid-responsive, nuclear co-repressor that modulates metabolic homeostasis. The stellate and inflammatory increases seen with the high-carbohydrate LF12 diet may represent postprandial responses. They depend on retinol and Shp, but the regulation reverses with an HFD.
Assuntos
Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Mediadores da Inflamação/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Receptores Citoplasmáticos e Nucleares/metabolismo , Retinoides/metabolismo , Deficiência de Vitamina A/metabolismo , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Dieta da Carga de Carboidratos/efeitos adversos , Dieta Hiperlipídica/efeitos adversos , Feminino , Células Estreladas do Fígado/imunologia , Células Estreladas do Fígado/patologia , Mediadores da Inflamação/sangue , Lactação , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Gravidez , Receptores Citoplasmáticos e Nucleares/genética , Retinoides/sangue , Índice de Gravidade de Doença , Deficiência de Vitamina A/imunologia , Deficiência de Vitamina A/patologia , Deficiência de Vitamina A/fisiopatologia , DesmameRESUMO
Cyp1b1 deletion and gestational vitamin A deficiency (GVAD) redirect adult liver gene expression. A matched sufficient pre- and post-natal diet, which has high carbohydrate and normal iron content (LF12), increased inflammatory gene expression markers in adult livers that were suppressed by GVAD and Cyp1b1 deletion. At birth on the LF12 diet, Cyp1b1 deletion and GVAD each suppress liver expression of the iron suppressor, hepcidin (Hepc), while increasing stellate cell activation markers and suppressing post-natal increases in lipogenesis. Hepc was less suppressed in Cyp1b1-/- pups with a standard breeder diet, but was restored by iron supplementation of the LF12 diet. CONCLUSIONS: The LF12 diet delivered low post-natal iron and attenuated Hepc. Hepc decreases in Cyp1b1-/- and GVAD mice resulted in stellate activation and lipogenesis suppression. Endothelial BMP6, a Hepc stimulant, is a potential coordinator and Cyp1b1 target. These neonatal changes in Cyp1b1-/- mice link to diminished adult obesity and liver inflammation.
Assuntos
Citocromo P-450 CYP1B1/deficiência , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Hepcidinas/genética , Lipogênese/genética , Fígado/metabolismo , Deficiência de Vitamina A/genética , Vitamina A/metabolismo , Adiposidade/efeitos dos fármacos , Envelhecimento , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Dieta , Eritropoese/efeitos dos fármacos , Eritropoese/genética , Ésteres/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepcidinas/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Ferro/farmacologia , Lipogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Obesidade/genética , Obesidade/patologia , Tamanho do Órgão/efeitos dos fármacos , Gravidez , Desmame , alfa-Fetoproteínas/metabolismoRESUMO
Obesity causes insulin resistance, and PPARγ ligands such as rosiglitazone are insulin sensitizing, yet the mechanisms remain unclear. In C57BL/6 (B6) mice, obesity induced by a high-fat diet (HFD) has major effects on visceral epididymal adipose tissue (eWAT). Here, we report that HFD-induced obesity in B6 mice also altered the activity of gene regulatory elements and genome-wide occupancy of PPARγ. Rosiglitazone treatment restored insulin sensitivity in obese B6 mice, yet, surprisingly, had little effect on gene expression in eWAT. However, in subcutaneous inguinal fat (iWAT), rosiglitazone markedly induced molecular signatures of brown fat, including the key thermogenic gene Ucp1. Obesity-resistant 129S1/SvImJ mice (129 mice) displayed iWAT browning, even in the absence of rosiglitazone. The 129 Ucp1 locus had increased PPARγ binding and gene expression that were preserved in the iWAT of B6x129 F1-intercrossed mice, with an imbalance favoring the 129-derived alleles, demonstrating a cis-acting genetic difference. Thus, B6 mice have genetically defective Ucp1 expression in iWAT. However, when Ucp1 was activated by rosiglitazone, or by iWAT browning in cold-exposed or young mice, expression of the B6 version of Ucp1 was no longer defective relative to the 129 version, indicating epigenomic rescue. These results provide a framework for understanding how environmental influences like drugs can affect the epigenome and potentially rescue genetically determined disease phenotypes.
Assuntos
Epigênese Genética , Obesidade/metabolismo , PPAR gama/fisiologia , Animais , Dieta Hiperlipídica/efeitos adversos , Hipoglicemiantes/farmacologia , Gordura Intra-Abdominal/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Ligação Proteica , Elementos Reguladores de Transcrição , Rosiglitazona , Gordura Subcutânea Abdominal/metabolismo , Tiazolidinedionas/farmacologia , Ativação Transcricional , Transcriptoma , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
The steroidogenic acute regulatory protein (StAR) has been proposed to serve as the switch that can turn on/off steroidogenesis. We investigated the events that facilitate dynamic StAR transcription in response to cAMP stimulation in MA-10 Leydig cells, focusing on splicing anomalies at StAR gene loci. We used 3' reverse primers in a single reaction to respectively quantify StAR primary (p-RNA), spliced (sp-RNA/mRNA), and extended 3' untranslated region (UTR) transcripts, which were quantitatively imaged by high-resolution fluorescence in situ hybridization (FISH). This approach delivers spatio-temporal resolution of initiation and splicing at single StAR loci, and transfers individual mRNA molecules to cytoplasmic sites. Gene expression was biphasic, initially showing slow splicing, transitioning to concerted splicing. The alternative 3.5-kb mRNAs were distinguished through the use of extended 3'UTR probes, which exhibited distinctive mitochondrial distribution. Combining quantitative PCR and FISH enables imaging of localization of RNA expression and analysis of RNA processing rates.
Assuntos
Imageamento Tridimensional , Hibridização in Situ Fluorescente/métodos , Microscopia/métodos , Fosfoproteínas/genética , RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Análise de Célula Única/métodos , Transcrição Gênica , Regiões 3' não Traduzidas/genética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Pareamento de Bases/genética , Células Cultivadas , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Poliadenilação/efeitos dos fármacos , Poliadenilação/genética , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb StAR mRNA molecules via dual hybridization at the 3'- and 5'-ends and reveals an unexpectedly high frequency of 1:1 pairing with mitochondria marked by the matrix StAR protein. This pairing may be central to translation-coupled cholesterol transfer. Altogether, our results show that adrenal cells exhibit high-efficiency StAR activity that needs to integrate rapid cholesterol transfer with homeostasis and pulsatile hormonal stimulation. StAR NBD, the extended 3.5-kb mRNA, SIK1, and Tis11b play important roles.