RESUMEN
The dysregulation of striatal gene expression and function is linked to multiple diseases, including Huntington's disease (HD), Parkinson's disease, X-linked dystonia-parkinsonism (XDP), addiction, autism, and schizophrenia. Striatal medium spiny neurons (MSNs) make up 90% of the neurons in the striatum and are critical to motor control. The transcription factor, Bcl11b (also known as Ctip2), is required for striatal development, but the function of Bcl11b in adult MSNs in vivo has not been investigated. We conditionally deleted Bcl11b specifically in postnatal MSNs and performed a transcriptomic and behavioral analysis on these mice. Multiple enrichment analyses showed that the D9-Cre-Bcl11btm1.1Leid transcriptional profile was similar to the HD gene expression in mouse and human data sets. A Gene Ontology enrichment analysis linked D9-Cre-Bcl11btm1.1Leid to calcium, synapse organization, specifically including the dopaminergic synapse, protein dephosphorylation, and HDAC-signaling, commonly dysregulated pathways in HD. D9-Cre-Bcl11btm1.1Leid mice had decreased DARPP-32/Ppp1r1b in MSNs and behavioral deficits, demonstrating the dysregulation of a subtype of the dopamine D2 receptor expressing MSNs. Finally, in human HD isogenic MSNs, the mislocalization of BCL11B into nuclear aggregates points to a mechanism for BCL11B loss of function in HD. Our results suggest that BCL11B is important for the function and maintenance of mature MSNs and Bcl11b loss of function drives, in part, the transcriptomic and functional changes in HD.
RESUMEN
Many diseases are linked to dysregulation of the striatum. Striatal function depends on neuronal compartmentation into striosomes and matrix. Striatal projection neurons are GABAergic medium spiny neurons (MSNs), subtyped by selective expression of receptors, neuropeptides, and other gene families. Neurogenesis of the striosome and matrix occurs in separate waves, but the factors regulating compartmentation and neuronal differentiation are largely unidentified. We performed RNA- and ATAC-seq on sorted striosome and matrix cells at postnatal day 3, using the Nr4a1-EGFP striosome reporter mouse. Focusing on the striosome, we validated the localization and/or role of Irx1, Foxf2, Olig2, and Stat1/2 in the developing striosome and the in vivo enhancer function of a striosome-specific open chromatin region 4.4 Kb downstream of Olig2. These data provide novel tools to dissect and manipulate the networks regulating MSN compartmentation and differentiation, including in human iPSC-derived striatal neurons for disease modeling and drug discovery.
Asunto(s)
Diferenciación Celular/genética , Neostriado/fisiología , Neuronas/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Humanos , Ratones , Neostriado/patologíaRESUMEN
The major surfactant protein, SP-A (a product of the SFTPA gene), serves as a marker of type II pneumocyte differentiation and surfactant synthesis. SFTPA expression in cultured human fetal lung (HFL) epithelial cells is upregulated by hormones that increase cyclic AMP (cAMP) and activate TTF-1/NKX2.1 and NF-κB. To further define mechanisms for type II cell differentiation and induction of SP-A, we investigated roles of microRNAs (miRNAs). Using microarray to identify differentially expressed miRNAs in HFL epithelial cells during type II cell differentiation in culture, we observed that members of the miRNA 199a (miR-199a)/miR-214 cluster were significantly downregulated during differentiation. Validated and predicted targets of miR-199a-3p/miR-199a-5p and miR-214, which serve roles in type II cell differentiation (COX-2, NF-κB p50/p65, and CREB1), and the CREB1 target, C/EBPß, were coordinately upregulated. Accordingly, overexpression of miR-199a-5p, miR-199a-3p, or miR-214 mimics in cultured HFL epithelial cells decreased COX-2, NF-κB p50/p65, CREB1, and C/EBPß proteins, with an associated inhibition of SP-A expression. Interestingly, overexpression of the EMT factor, ZEB1, which declines during cAMP-induced type II cell differentiation, increased pri-miR-199a and reduced the expression of the targets NF-κB/p50 and COX-2. Collectively, these findings suggest that the developmental decline in miR-199a/miR-214 in HFL causes increased expression of critical targets that enhance type II cell differentiation and SP-A expression.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , MicroARNs/genética , Factores de Transcripción/genética , AMP Cíclico/metabolismo , Regulación hacia Abajo , Humanos , FN-kappa B/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Regulación hacia ArribaRESUMEN
Lung alveolar type II cells uniquely synthesize surfactant, a developmentally regulated lipoprotein that is essential for breathing. Expression of the gene (SFTPA) encoding the major surfactant protein, SP-A, in midgestation human fetal lung (HFL) is dramatically induced by cyclic AMP (cAMP). cAMP induction of SP-A expression is repressed by transforming growth factor ß (TGF-ß) and by hypoxia. In this study, we found that expression of the microRNA 29 (miR-29) family was significantly upregulated in epithelial cells isolated from mouse fetal lung during late gestation and in epithelial cells isolated from HFL explants during type II cell differentiation in culture. miR-29 expression in cultured HFL epithelial cells was increased by cAMP and inhibited by hypoxia, whereas the miR-29 target, TGF-ß2, was coordinately decreased. Knockdown of the miR-29 family in cultured HFL type II cells blocked cAMP-induced SP-A expression and accumulation of surfactant-containing lamellar bodies, suggesting their physiological relevance. This occurred through derepression of TGF-ß signaling. Notably, cAMP increased binding of endogenous thyroid transcription factor 1 (TTF-1/Nkx2.1) to the miR-29ab1 promoter in HFL type II cells, and TTF-1 increased miR-29ab1 promoter-driven luciferase activity in cotransfection assays. Together, these findings identify miR-29 family members as TTF-1-driven mediators of SP-A expression and type II cell differentiation through repression of TGF-ß signaling.
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Células Epiteliales Alveolares/citología , Pulmón/embriología , MicroARNs/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Células A549 , Animales , Diferenciación Celular , Células Cultivadas , AMP Cíclico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Pulmón/citología , Ratones , Regiones Promotoras Genéticas , Transducción de Señal , Factor Nuclear Tiroideo 1 , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia ArribaRESUMEN
Type II cell differentiation and expression of the major surfactant protein, SP-A, in mid-gestation human fetal lung (HFL) are induced by cAMP and inhibited by TGF-ß. cAMP induction of SP-A promoter activity is mediated by increased phosphorylation and DNA binding of thyroid transcription factor-1 (TTF-1/Nkx2.1), a master regulator of lung development. To further define mechanisms for developmental induction of surfactant synthesis in HFL, herein, we investigated the potential roles of microRNAs (miRNAs, miRs). To identify and characterize differentially regulated miRNAs in mid-gestation HFL explants during type II pneumocyte differentiation in culture, we performed miRNA microarray of RNA from epithelial cells isolated from mid-gestation HFL explants before and after culture with or without Bt2cAMP. Interestingly, the miR-200 family was significantly up-regulated during type II cell differentiation; miR-200 induction was inversely correlated with expression of known targets, transcription factors ZEB1/2 and TGF-ß2. miR-200 antagonists inhibited TTF-1 and surfactant proteins and up-regulated TGF-ß2 and ZEB1 expression in type II cells. Overexpression of ZEB1 in type II cells decreased DNA binding of endogenous TTF-1, blocked cAMP stimulation of surfactant proteins, and inhibited miR-200 expression, whereas cAMP markedly inhibited ZEB1/2 and TGF-ß. Importantly, overexpression of ZEB1 or miR-200 antagonists in HFL type II cells also inhibited LPCAT1 and ABCA3, enzymes involved in surfactant phospholipid synthesis and trafficking, and blocked lamellar body biogenesis. Our findings suggest that the miR-200 family and ZEB1, which exist in a double-negative feedback loop regulated by TGF-ß, serve important roles in the developmental regulation of type II cell differentiation and function in HFL.
Asunto(s)
Células Epiteliales Alveolares/metabolismo , Diferenciación Celular/fisiología , Feto/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Edad Gestacional , MicroARNs/biosíntesis , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Células Epiteliales Alveolares/citología , Proteínas de Unión al ADN/metabolismo , Feto/citología , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
SP-A gene expression is developmentally regulated in fetal lung. Cyclic AMP (cAMP) induction of SP-A expression in human fetal lung type II cells is O(2) dependent and is mediated by increased binding of TTF-1/Nkx2.1 and NF-κB to a critical response element (TBE). This is associated with increased acetylation and decreased methylation of H3K9 at the TBE. Using chromatin immunoprecipitation analysis of fetal lung between 15.5 and 19.0 days of gestation, we observed that the developmental induction of SP-A was associated with increased recruitment of TTF-1, NF-κB, PCAF, and CBP, as well as enhanced acetylation and decreased methylation of histone H3K9 at the TBE. Importantly, expression and TBE binding of the H3K9 methyltransferases, Suv39h1 and Suv39h2, was inversely correlated with the developmental upregulation of SP-A. In human fetal lung epithelial cells, Suv39H1 and Suv39H2 mRNA levels declined with cAMP induction of SP-A. Moreover, hypoxia, which inhibits cAMP stimulation of SP-A, markedly increased Suv39h1 and Suv39h2 binding to the TBE. Finally, short hairpin RNA knockdown of Suv39H1 or Suv39H2 in fetal lung epithelial cells repressed H3K9 methylation and greatly enhanced SP-A expression. Collectively, our findings suggest that Suv39H1 and Suv39H2 are key hypoxia-induced methyltransferases; their decline in fetal lung during late gestation is critical for epigenetic changes resulting in the developmental induction of SP-A.
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Hipoxia de la Célula , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Metiltransferasas/metabolismo , Proteína A Asociada a Surfactante Pulmonar/genética , Proteínas Represoras/metabolismo , Acetilación , Diferenciación Celular/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , AMP Cíclico/metabolismo , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Pulmón/citología , Metilación , Metiltransferasas/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Oxígeno , ARN Mensajero/análisis , ARN Interferente Pequeño/genética , Proteínas Represoras/genética , Elementos de Respuesta , Factor Nuclear Tiroideo 1 , Factores de Transcripción/metabolismo , Factor Trefoil-1 , Proteínas Supresoras de Tumor/metabolismo , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Estrogen-related receptor (ERRalpha) plays a critical role in basal and cAMP-induced expression of the human surfactant protein-A (SP-A) gene in lung type II cells through direct binding to an ERR response element (ERRE, 5'-TGACCTTA-3') within its 5'-flanking region. Furthermore, protein kinase A (PKA) up-regulates ERRalpha activation of the hSP-A promoter. In the present study, using cultured human fetal lung type II cells, we observed that cAMP enhanced ERRalpha phosphorylation and nuclear expression levels. cAMP/PKA stimulation of ERRalpha activation of the SP-A promoter was blocked by the PKA inhibitor, H89, whereas the MAPK P38 inhibitor, SB203580, and the MAPK kinase inhibitor, PD98059, had negligible to modest effects. This suggests that cAMP acts selectively through PKA to increase ERRalpha transcriptional activity. Of several coactivators tested, steroid receptor coactivator 2 (SRC-2) had the most pronounced effect to increase ERRalpha transcriptional activity at the SP-A promoter; this was enhanced by cotransfection with PKA catalytic subunit (PKAcat). Interestingly, SRC-2, ERRalpha, and PKAcat in type II cell nuclear extracts interacted at the ERRE; this was enhanced by cAMP and inhibited by H89. cAMP increased in vivo binding of PKAcat and SRC-2 to the ERRE genomic region in lung type II cells. In mutagenesis studies, three serines (S87, S114, and S277) were found to be critical for PKA and SRC-2 induction of ERRalpha transcriptional activity. Collectively, these findings indicate that cAMP/PKA signaling enhances ERRalpha phosphorylation and nuclear localization, recruitment to the SP-A promoter, and interaction with PKAcat and SRC-2, resulting in the up-regulation of SP-A gene transcription.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Coactivador 2 del Receptor Nuclear/metabolismo , Regiones Promotoras Genéticas/genética , Proteína A Asociada a Surfactante Pulmonar/genética , Receptores de Estrógenos/genética , Transcripción Genética/efectos de los fármacos , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Feto/citología , Genoma Humano/genética , Humanos , Isoquinolinas/farmacología , Pulmón/citología , Ratones , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Receptores de Estrógenos/metabolismo , Elementos de Respuesta/genética , Serina/metabolismo , Sulfonamidas/farmacología , Treonina/metabolismo , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
Op18 (Oncoprotein 18, Stathmin) is a mitotic regulator that is highly expressed in many cancers. We have characterized four functional CCAAT boxes in the Op18 gene located at positions: -980, -745, -599 and -65, relative to the transcriptional start site. NF-Y is a ubiquitously expressed CCAAT-binding transcription factor that regulates a number of cell cycle controlled genes. We have used promoter-reporter assays and mobility shift assays to functionally examine these CCAAT boxes. All sites contribute to the basal expression of Op18, with the sites at -980 and -599 being repressive and the sites at -745 and -65 being stimulatory. Mobility shift assays indicate that all CCAAT box sites bind factors in nuclear extracts from Hek293. However, only the repressive site at -599 and the stimulatory site at -65 are competent to bind NF-Y, suggesting that NF-Y may play a role in promoting both activation and repression of Op18 expression. The NF-Y site at -65 accounts for greater than 60% of the Op18 gene expression. EMSA competition studies indicate that NF-Y binds with a much higher affinity to the -65 site than to the -599 site, suggesting that in asynchronously growing cells NF-Y functions only to stimulate expression through the -65 binding site. These data suggest that NF-Y is a major transcription factor promoting expression of Op18.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Estatmina/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , ADN/genética , ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Expresión Génica , Genes Reporteros , Humanos , Luciferasas/genética , Ratones , Mutagénesis Sitio-Dirigida , Células 3T3 NIH , TransfecciónRESUMEN
Op18 (Oncoprotein 18, Stathmin) is an important mitotic regulator that is highly expressed in many cancers including the metastatic prostate carcinoma cell line PC-3-M. Recent studies indicate that antisense-mediated down-regulation of Op18 can inhibit cellular proliferation. However, the transcriptional mechanisms responsible for its normal regulation and for its high level of expression in proliferating cells remain poorly understood. In the study presented here, we have characterized transcription factor binding sites that together account for nearly 80% of the Op18 expression in PC-3-M cells. The 5' flanking region of the Op18 gene contains four putative E2F sites located at -700 (site 1), -28 (site 2), -19 (site 3), and +720 (site 4) relative to the transcriptional start site. E2F has been implicated in both the c-Jun-mediated up-regulation and the doxorubicin-induced repression of Op18 expression. We have used promoter-reporter assays and mobility shift assays to functionally examine each of these E2F sites. Mutagenesis studies indicate that all sites contribute to the basal expression of Op18. Mutagenesis of either site 1 or 4 reduced the reporter activity by 40%, mutagenesis of site 2 reduced reporter activity by 20%, and mutations in site 3 had no effect on reporter activity. Combinatorial mutagenesis indicates that site 1 and 4 function independently, whereas site 2 functions synergistically with either site 3 or 4. Mobility shift assays indicate that all elements bind factors in the nuclear extracts of PC-3-M cells. Characterization of the sites show that site 1, though a positive element, is not E2F specific; sites 2 and 3 may contain an overlapping binding site for E2F and NF1; and site 4, which resides in intron 1, is the only site shown to interact exclusively with E2F. These studies suggest that the overexpression of Op18 in PC-3-M cells is mediated predominantly through the E2F family of transcription factors.