RESUMEN
The Hippo/YAP pathway plays a critical role in tissue homeostasis. Our previous work demonstrated that renal tubular YAP activation induced by double knockout (dKO) of the upstream Hippo kinases Mst1 and Mst2 promotes tubular injury and renal inflammation under basal conditions. However, the importance of tubular YAP activation remains to be established in injured kidneys in which many other injurious pathways are simultaneously activated. Here, we show that tubular YAP was already activated 6 h after unilateral ureteral obstruction (UUO). Tubular YAP deficiency greatly attenuated tubular cell overproliferation, tubular injury, and renal inflammation induced by UUO or cisplatin. YAP promoted the transcription of the transcription factor KLF5. Consistent with this, the elevated expression of KLF5 and its target genes in Mst1/2 dKO or UUO kidneys was blocked by ablation of Yap in tubular cells. Inhibition of KLF5 prevented tubular cell overproliferation, tubular injury, and renal inflammation in Mst1/2 dKO kidneys. Therefore, our results demonstrate that tubular YAP is a key player in kidney injury. YAP and KLF5 form a transcriptional cascade, where tubular YAP activation induced by kidney injury promotes KLF5 transcription. Activation of this cascade induces tubular cell overproliferation, tubular injury, and renal inflammation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Túbulos Renales , Factores de Transcripción de Tipo Kruppel , Ratones Noqueados , Proteínas Señalizadoras YAP , Animales , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Proteínas Señalizadoras YAP/metabolismo , Proteínas Señalizadoras YAP/genética , Ratones , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Túbulos Renales/metabolismo , Túbulos Renales/patología , Túbulos Renales/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Serina-Treonina Quinasa 3 , Transducción de Señal , Proliferación Celular , Regulación de la Expresión Génica , Modelos Animales de Enfermedad , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología , Cisplatino/farmacologíaRESUMEN
Histone H3 lysine 9 (H3K9) methylation, as a hallmark of heterochromatin, has a central role in cell lineage and fate determination. Although evidence of a cooperation between H3K9 methylation writers and their readers has started to emerge, their actual interplay remains elusive. Here, we show that loss of H3K9 methylation readers, the Hp1 family, causes reduced expression of H3K9 methyltransferases, and that this subsequently leads to the exit of embryonic stem cells (ESCs) from pluripotency and a reciprocal gain of lineage-specific characteristics. Importantly, the phenotypes of Hp1-null ESCs can be rescued by ectopic expression of Setdb1, Nanog, and Oct4. Furthermore, Setdb1 ablation results in loss of ESC identity, which is accompanied by a reduction in the expression of Hp1 genes. Together, our data support a model in which the safeguarding of ESC identity involves the cooperation between the H3K9 methylation writers and their readers.
Asunto(s)
Fenómenos Fisiológicos Celulares , Células Madre Embrionarias , Metilación , Linaje de la Célula , Proteínas Cromosómicas no Histona/genéticaRESUMEN
Chronic kidney disease (CKD) is a major health problem. Kidney fibrosis is a hallmark and final common pathway of CKD. The Hippo/yes-associated protein (YAP) pathway regulates organ size, inflammation, and tumorigenesis. Our previous study demonstrated tubular YAP activation by tubule-specific double knockout of mammalian STE20-like protein kinase 1/2 (Mst1/2) induced CKD in mice, but the underlying mechanisms remain to be fully elucidated. Activator protein (AP)-1 activation was found to promote tubular atrophy and tubulointerstitial fibrosis. Therefore, we studied whether YAP regulates AP-1 expression in the kidney. We found that expression of various AP-1 components was induced in kidneys subjected to unilateral ureteric obstruction and in Mst1/2 double knockout kidneys, and these inductions were blocked by deletion of Yap in tubular cells, with Fosl1 being most affected compared with other AP-1 genes. Inhibition of Yap also most highly suppressed Fosl1 expression among AP-1 genes in HK-2 and IMCD3 renal tubular cells. YAP bound to the Fosl1 promoter and promoted Fosl1 promoter-luciferase activity. Our results suggest that YAP controls AP-1 expression and that Fosl1 is the primary target of YAP in renal tubular cells.NEW & NOTEWORTHY Yes-associated protein (YAP) activation leads to tubular injury, renal inflammation, and fibrosis, but the underlying mechanisms are not fully understood. We now provide genetic evidence that YAP promotes activator protein-1 expression and that Fosl1 is the primary target of YAP in renal tubular cells.
Asunto(s)
Insuficiencia Renal Crónica , Obstrucción Ureteral , Animales , Ratones , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Epiteliales/metabolismo , Fibrosis , Inflamación/metabolismo , Riñón/metabolismo , Mamíferos/metabolismo , Insuficiencia Renal Crónica/metabolismo , Transducción de Señal/fisiología , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Obstrucción Ureteral/genética , Obstrucción Ureteral/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Final urine volume and concentration are defined by water reabsorption through the water channel proteins aquaporin (AQP)-2, -3 and -4 in the collecting duct. However, the transcriptional regulation of these AQPs is not well understood. The Hippo/Yes-associated protein 1 (YAP) pathway plays an important role in organ size control and tissue homeostasis. When the Hippo pathway including the Mst1/Mst2 kinases is inhibited, YAP is activated and functions as a transcription co-activator. Our previous work revealed a pathological role of tubular YAP activation in chronic kidney disease, but the physiological role of YAP in the kidney remains to be established. Here, we found that tubule-specific Yap knockout mice showed increased urine output and decreased urinary osmolality. Decreases in Aqp2, -3 and -4 mRNA and protein abundance in the kidney were evident in Yap knockout mice. Analysis of Mst1/Mst2 double knockout and Mst1/Mst2/Yap triple knockout mice showed that expression of Aqp2 and Aqp4 but not Aqp3 was dependent on YAP. Furthermore, YAP was recruited to the promoters of the Aqp2 and Aqp4 genes and stimulated their transcription. Interestingly, YAP was found to interact with transcription factors GATA2, GATA3 and NFATc1. These three factors promoted Aqp2 transcription in a YAP dependent manner in collecting duct cells. These three factors also promoted Aqp4 transcription whereas only GATA2 and GATA3 enhanced Aqp3 transcription. Thus, our results suggest that YAP promotes Aqp2 and Aqp4 transcription, interacts with GATA2, GATA3 and NFATc1 to control Aqp2 expression, while Aqp-2, -3 and -4 exploit overlapping mechanisms for their baseline transcriptional regulation.
Asunto(s)
Acuaporina 2 , Túbulos Renales Colectores , Ratones , Animales , Acuaporina 2/metabolismo , Proteínas Señalizadoras YAP , Riñón/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Factores de Transcripción/metabolismo , Ratones Noqueados , Agua/metabolismo , Homeostasis , Túbulos Renales Colectores/metabolismoRESUMEN
Follistatin (FS)-like 1 (FSTL1) is a member of the FS-SPARC (secreted protein, acidic and rich in cysteine) family of secreted and extracellular matrix proteins. The functions of FSTL1 have been studied in heart and lung injury as well as in wound healing; however, the role of FSTL1 in the kidney is largely unknown. Here, we show using single-cell RNA-Seq that Fstl1 was enriched in stromal cells in obstructed mouse kidneys. In addition, immunofluorescence demonstrated that FSTL1 expression was induced in fibroblasts during kidney fibrogenesis in mice and human patients. We demonstrate that FSTL1 overexpression increased renal fibrosis and activated the Wnt/ß-catenin signaling pathway, known to promote kidney fibrosis, but not the transforming growth factor ß (TGF-ß), Notch, Hedgehog, or Yes-associated protein (YAP) signaling pathways in obstructed mouse kidneys, whereas inhibition of FSTL1 lowered Wnt/ß-catenin signaling. Importantly, we show that FSTL1 interacted with Wnt ligands and the Frizzled (FZD) receptors but not the coreceptor lipoprotein receptor-related protein 6 (LRP6). Specifically, we found FSTL1 interacted with Wnt3a through its extracellular calcium-binding (EC) domain and von Willebrand factor type C-like (VWC) domain, and with FZD4 through its EC domain. Furthermore, we show that FSTL1 increased the association of Wnt3a with FZD4 and promoted Wnt/ß-catenin signaling and fibrogenesis. The EC domain interacting with both Wnt3a and FZD4 also enhanced Wnt3a signaling. Therefore, we conclude that FSTL1 is a novel extracellular enhancer of the Wnt/ß-catenin pathway.
Asunto(s)
Proteínas Relacionadas con la Folistatina , Receptores Frizzled , Riñón , Vía de Señalización Wnt , Animales , Proteínas Relacionadas con la Folistatina/genética , Proteínas Relacionadas con la Folistatina/metabolismo , Receptores Frizzled/metabolismo , Humanos , Riñón/metabolismo , Riñón/fisiopatología , Ligandos , Ratones , Proteína Wnt3ARESUMEN
Polycomb group proteins assemble into multi-protein complexes, known as Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), that guide cell fate decisions during embryonic development. PRC1 forms an array of biochemically distinct canonical PRC1 (cPRC1) or non-canonical PRC1 (ncPRC1) complexes characterized by the mutually exclusive presence of PCGF (PCGF1-PCGF6) paralog subunit; however, whether each one of these subcomplexes fulfills a distinct role remains largely controversial. Here, by performing a CRISPR-based loss-of-function screen in embryonic stem cells (ESCs), we uncovered a previously unappreciated functional redundancy among PRC1 subcomplexes. Disruption of ncPRC1, but not cPRC1, displayed severe defects in ESC pluripotency. Remarkably, coablation of non-canonical and canonical PRC1 in ESCs resulted in exacerbation of the phenotype observed in the non-canonical PRC1-null ESCs, highlighting the importance of functional redundancy among PRC1 subcomplexes. Together, our studies demonstrate that PRC1 subcomplexes act redundantly to silence lineage-specific genes and ensure robust maintenance of ESC identity.
Asunto(s)
Proteínas de Drosophila , Células Madre Embrionarias , Diferenciación Celular/genética , Proteínas de Drosophila/metabolismo , Células Madre Embrionarias/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas del Grupo Polycomb/genéticaRESUMEN
Increasing evidences indicate that unlimited capacity for self-renewal and pluripotency, two unique properties of embryonic stem cells (ESCs), are intrinsically linked to cell cycle control. However, the precise mechanisms coordinating cell fate decisions and cell cycle regulation remain to be fully explored. Here, using CRISPR/Cas9-mediated genome editing, we show that in ESCs, deficiency of components of the cell cycle regulatory MuvB complex Lin54 or Lin52, but not Lin9 or Lin37, triggers G2/M arrest, loss of pluripotency, and spontaneous differentiation. Further dissection of these phenotypes demonstrated that this cell cycle arrest is accompanied by the gradual activation of mesoendodermal lineage-specifying genes. Strikingly, the abnormalities observed in Lin54-null ESCs were partially but significantly rescued by ectopic coexpression of genes encoding G2/M proteins Cyclin B1 and Cdk1. Thus, our study provides new insights into the mechanisms by which the MuvB complex determines cell fate through regulation of the cell cycle machinery.
Asunto(s)
Proteínas de Ciclo Celular , Células Madre Embrionarias , Animales , Apoptosis/fisiología , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/fisiología , División Celular/fisiología , Línea Celular Tumoral , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular , Humanos , Ratones , Factores de Transcripción/metabolismoRESUMEN
Polycomb group (PcG) proteins form multiprotein complexes that affect stem cell identity and fate decisions by still largely unexplored mechanisms. Here, by performing a CRISPR-based loss-of-function screen in embryonic stem cells (ESCs), we identify PcG gene Mga involved in the repression of endodermal transcription factor Gata6 We report that deletion of Mga results in peri-implantation embryonic lethality in mice. We further demonstrate that Mga-null ESCs exhibit impaired self-renewal and spontaneous differentiation to primitive endoderm (PE). Our data support a model in which Mga might serve as a scaffold for PRC1.6 assembly and guide this multimeric complex to specific genomic targets including genes that encode endodermal factors Gata4, Gata6, and Sox17. Our findings uncover an unexpected function of Mga in ESCs, where it functions as a gatekeeper to prevent ESCs from entering into the PE lineage by directly repressing expression of a set of endoderm differentiation master genes.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células Madre Embrionarias , Endodermo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/metabolismo , Regulación del Desarrollo de la Expresión Génica , RatonesRESUMEN
Polycomb group (PcG) proteins exist in distinct multi-protein complexes and play a central role in silencing developmental genes, yet the underlying mechanisms remain elusive. Here, we show that deficiency of retinoblastoma binding protein 4 (RBBP4), a component of the Polycomb repressive complex 2 (PRC2), in embryonic stem cells (ESCs) leads to spontaneous differentiation into mesendodermal lineages. We further show that Rbbp4 and core PRC2 share an important number of common genomic targets, encoding regulators involved in early germ layer specification. Moreover, we find that Rbbp4 is absolutely essential for genomic targeting of PRC2 to a subset of developmental genes. Interestingly, we demonstrate that Rbbp4 is necessary for sustaining the expression of Oct4 and Sox2 and that the forced co-expression of Oct4 and Sox2 fully rescues the pluripotency of Rbbp4-null ESCs. Therefore, our study indicates that Rbbp4 links maintenance of the pluripotency regulatory network with repression of mesendoderm lineages.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/fisiología , Regulación del Desarrollo de la Expresión Génica , Complejo Represivo Polycomb 2/fisiología , Proteína 4 de Unión a Retinoblastoma/fisiología , Animales , Línea Celular , Autorrenovación de las Células , Secuenciación de Inmunoprecipitación de Cromatina , Técnicas de Inactivación de Genes , Células HEK293 , Histonas/metabolismo , Humanos , Metilación , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/metabolismoRESUMEN
Polycomb group (PcG) proteins are essential for maintenance of lineage fidelity by coordinating developmental gene expression programs. Polycomb group ring finger 6 (PCGF6) has been previously reported to repress expression of lineage-specific genes, especially germ cell-related genes in mouse embryonic stem cells (ESCs) via the noncanonical polycomb repressive complex PRC1.6. However, the molecular mechanism of this repression remains largely unknown. Here, using RNA-Seq, real-time RT-PCR, immunohistochemistry, immunoprecipitation, and ChIP analyses, we demonstrate that PCGF6 plays an essential role in embryonic development, indicated by the partially penetrant embryonic lethality in homozygous PCGF6 (Pcgf6-/-)-deficient mice. We also found that surviving Pcgf6-deficient mice exhibit reduced fertility. Using the Pcgf6-deficient mice, we observed that ablation of Pcgf6 in somatic tissues robustly derepresses germ cell-related genes. We further provide evidence that these genes are direct targets of PCGF6 in ESCs and that endogenous PCGF6 co-localizes with the histone-modifying proteins G9A histone methyltransferase (G9A)/G9a-like protein (GLP) and histone deacetylase 1/2 (HDAC1/2) on the promoters of the germ cell-related genes. Moreover, the binding of these proteins to their target genes correlated with methylation of Lys-9 of histone 3 and with the status of histone acetylation at these genes. Moreover, the recruitment of G9A/GLP and HDAC1/2 to target promoters depended on the binding of PCGF6. Our findings indicate that PCGF6 has a critical role in safeguarding lineage decisions and in preventing aberrant expression of germ cell-related genes.
Asunto(s)
Silenciador del Gen , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Animales , Línea Celular , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , N-Metiltransferasa de Histona-Lisina/genética , Histonas , Ratones , Ratones Noqueados , Complejo Represivo Polycomb 1/genética , RNA-SeqRESUMEN
BACKGROUND: The serine/threonine kinases MST1 and MST2 are core components of the Hippo pathway, which has been found to be critically involved in embryonic kidney development. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the pathway's main effectors. However, the biologic functions of the Hippo/YAP pathway in adult kidneys are not well understood, and the functional role of MST1 and MST2 in the kidney has not been studied. METHODS: We used immunohistochemistry to examine expression in mouse kidneys of MST1 and MST2, homologs of Hippo in Drosophila. We generated mice with tubule-specific double knockout of Mst1 and Mst2 or triple knockout of Mst1, Mst2, and Yap. PCR array and mouse inner medullary collecting duct cells were used to identify the primary target of Mst1/Mst2 deficiency. RESULTS: MST1 and MST2 were predominantly expressed in the tubular epithelial cells of adult kidneys. Deletion of Mst1/Mst2 in renal tubules increased activity of YAP but not TAZ. The kidneys of mutant mice showed progressive inflammation, tubular and glomerular damage, fibrosis, and functional impairment; these phenotypes were largely rescued by deletion of Yap in renal tubules. TNF-α expression was induced via both YAP-dependent and YAP-independent mechanisms, and TNF-α and YAP amplified the signaling activities of each other in the tubules of kidneys with double knockout of Mst1/Mst2. CONCLUSIONS: Our findings show that tubular Mst1/Mst2 deficiency leads to CKD through both the YAP and non-YAP pathways and that tubular YAP activation induces renal fibrosis. The pathogenesis seems to involve the reciprocal stimulation of TNF-α and YAP signaling activities.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas de Ciclo Celular/fisiología , Túbulos Renales/enzimología , Proteínas Serina-Treonina Quinasas/deficiencia , Insuficiencia Renal Crónica/enzimología , Animales , Células Cultivadas , Fibrosis , Regulación de la Expresión Génica , Vía de Señalización Hippo , Etiquetado Corte-Fin in Situ , Riñón/embriología , Riñón/enzimología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Serina-Treonina Quinasa 3 , Transducción de Señal , Transactivadores/fisiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/fisiología , Proteínas Señalizadoras YAPRESUMEN
Although the roles of the Hippo pathway in organogenesis and tumorigenesis have been well studied in multiple organs, its role in sperm maturation and male fertility has not been investigated. The initial segment (IS) of the epididymis plays a critical role in sperm maturation. IS differentiation is governed by ERK1/2, but the mechanisms of ERK1/2 activation in IS are not fully understood. Here we show that double knockout (dKO) of mammalian sterile 20-like kinases 1 and 2 (Mst1 and Mst2), homologs of Hippo in Drosophila, in the epididymal epithelium led to male infertility in mice. Sperm in the cauda epididymides of mutant mice were immotile with flagellar angulation and severely disorganized structures. Loss of Mst1/2 activated YAP and increased proliferation and cell death in all the segments of epididymis. The mutant mice showed substantially suppressed MEK/ERK signaling in the IS and failed IS differentiation. Deletion of Yap restored the reduced MEK/ERK signaling, and partially rescued the defective IS differentiation and fertility in Mst1/2 dKO mice. Our results demonstrate that YAP inhibits the MEK/ERK pathway in IS epithelial cells, and MST1/2 control IS differentiation and fertility at least partially by repressing YAP. Taken together, the Hippo pathway is essential for sperm maturation and male fertility.
Asunto(s)
Epidídimo , Células Epiteliales , Infertilidad Masculina/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Diferenciación Celular , Epidídimo/citología , Epidídimo/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Noqueados , Serina-Treonina Quinasa 3RESUMEN
BACKGROUND: Proinflammatory cytokines, which can upregulate the expression of matrix-degrading enzymes in chondrocytes, play important roles in the development of osteoarthritis. BET family proteins, acting as the "readers" of acetylated modifications on histones, have been linked to transcriptional regulation. And a BET protein inhibitor, I-BET151, has been shown to inhibit the induction of matrix-degrading enzymes by proinflammatory cytokines in chondrocytes. Our objective is to clarify the role and mechanism of BET proteins on matrix-degrading enzyme gene expression by using a human chondrosarcoma cell line (SW1353). METHODS: We pretreated SW1353 cells with I-BET151 prior to treatment with IL-1ß or TNF-α and then checked the expression of four matrix-degrading enzyme genes (MMP1, MMP3, MMP13, and ADAMTS4). We performed knockdown of BET protein family members (BRD2, BRD3, and BRD4) with corresponding siRNAs in SW1353 cells prior to treatment with IL-1ß or TNF-α and checked the expression of the matrix-degrading enzyme genes. We evaluated Brd-mediated transcriptional regulation on the matrix-degrading enzyme genes by ChIP assay. RESULTS: We confirmed that I-BET151 could suppress the IL-1ß- or TNF-α-induced expression of MMP1, MMP3, MMP13, and ADAMTS4 in SW1353 cells. Brd3 and Brd4 were required for the IL-1ß- or TNF-α-induced expression of matrix-degrading enzyme genes in SW1353 cells. We revealed that inducible acetylation of H4k5/8/12 and the recruitment of Brd3, Brd4, and p-TEFb to chromatin were involved in IL-1ß- or TNF-α-induced transcription. CONCLUSIONS: Our findings suggested that Brd3 and Brd4 were essential for the IL-1ß- or TNF-α-induced transcription of matrix-degrading enzyme genes, and recruitment of Brd3 and Brd4 to chromatin of these genes played the main role in this process.
Asunto(s)
Cromatina/metabolismo , Citocinas/metabolismo , Regulación Enzimológica de la Expresión Génica , Mediadores de Inflamación/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Proteínas de Ciclo Celular , Línea Celular Tumoral , Citocinas/genética , Humanos , Proteínas Nucleares/genética , Proteínas de Unión al ARN/genética , Factores de Transcripción/genéticaRESUMEN
Lethal (3) malignant brain tumor like 2 (L3MBTL2) is a member of the MBT-domain proteins, which are involved in transcriptional repression and implicated in chromatin compaction. Our previous study has shown that L3MBTL2 is highly expressed in the testis, but its role in spermatogenesis remains unclear. In the present study, we found that L3MBTL2 was most highly expressed in pachytene spermatocytes within the testis. Germ cell-specific ablation of L3mbtl2 in the testis led to increased abnormal spermatozoa, progressive decrease of sperm counts and premature testicular failure in mice. RNA-sequencing analysis on L3mbtl2 deficient testes confirmed that L3MBTL2 was a transcriptional repressor but failed to reveal any significant changes in spermatogenesis-associated genes. Interestingly, L3mbtl2 deficiency resulted in increased γH2AX deposition in the leptotene spermatocytes, subsequent inappropriate retention of γH2AX on autosomes, and defective crossing-over and synapsis during the pachytene stage of meiosis I, and more germ cell apoptosis and degeneration in aging mice. L3MBTL2 interacted with the histone ubiquitin ligase RNF8. Inhibition of L3MBTL2 reduced nuclear RNF8 and ubH2A levels in GC2 cells. L3mbtl2 deficiency led to decreases in the levels of the RNF8 and ubH2A pathway and in histone acetylation in elongating spermatids, and in protamine 1 deposition and chromatin condensation in sperm. These results suggest that L3MBTL2 plays important roles in chromatin remodeling during meiosis and spermiogenesis.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Cromatina/metabolismo , Proteínas Nucleares/genética , Espermatocitos/metabolismo , Espermatogénesis/genética , Factores de Transcripción/genética , Acetilación , Animales , Apoptosis/genética , Ensamble y Desensamble de Cromatina/fisiología , Histonas/metabolismo , Masculino , Profase Meiótica I/fisiología , Ratones , Ratones Noqueados , Proteínas Nucleares/metabolismo , Fase Paquiteno/fisiología , Proteínas del Grupo Polycomb/metabolismo , Recuento de Espermatozoides , Testículo/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The polycomb group (PcG) proteins are key epigenetic regulators in stem cell maintenance. PcG proteins have been thought to act through one of two polycomb repressive complexes (PRCs), but more recent biochemical analyses have challenged this model in the identification of noncanonical PRC1 (nc-PRC1) complexes characterized by the presence of Rybp or Yaf2 in place of the canonical Chromobox proteins. However, the biological significance of these nc-PRC1s and the potential mechanisms by which they mediate gene repression are largely unknown. Here, we explore the functional consequences of Yaf2 disruption on stem cell regulation. We show that deletion of Yaf2 results in compromised proliferation and abnormal differentiation of mouse embryonic stem cells (mESCs). Genome-wide profiling indicates Yaf2 functions primarily as a transcriptional repressor, particularly impacting genes associated with ectoderm cell fate in a manner distinct from Rybp. We confirm that Yaf2 assembles into a noncanonical PRC complex, with deletion analysis identifying the region encompassing amino acid residues 102-150 as required for this assembly. Furthermore, we identified serine 166 as a Yaf2 phosphorylation site, and we demonstrate that mutation of this site to alanine (S166A) compromises Ring1B-mediated H2A monoubiquitination and in turn its ability to repress target gene expression. We therefore propose that Yaf2 and its phosphorylation status serve as dual regulators to maintain the pluripotent state in mESCs.
Asunto(s)
Diferenciación Celular , Histonas/metabolismo , Células Madre Embrionarias de Ratones/citología , Proteínas Musculares/fisiología , Complejo Represivo Polycomb 1/metabolismo , Proteínas Represoras/fisiología , Animales , Células Cultivadas , Cromatina/genética , Perfilación de la Expresión Génica , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/metabolismo , Fosforilación , Complejo Represivo Polycomb 1/genéticaRESUMEN
BACKGROUND: Proinflammatory cytokines, which can upregulate the expression of matrix-degrading enzymes in chondrocytes, play important roles in the development of osteoarthritis. And a BET protein inhibitor, I-BET151, has been shown to exert an anti-inflammatory effect by repressing the BET protein-mediated expression of inflammatory genes. Our objective is to investigate the effect of I-BET151 on a surgical mouse model of osteoarthritis (OA) and human chondrocytes. METHODS: We first treated a surgical mouse model of OA with I-BET151 once per day and evaluated the knee joints at 6 and 8 weeks after treatment. We then pretreated the human chondrocytes with I-BET151 prior to treatment with IL-1ß or TNF-α and checked the expression and activity of the matrix-degrading enzyme genes. We also checked the expression of ACAN, COL2A1, and SOX9. RESULTS: We demonstrated that I-BET151 could prevent articular cartilage damage in the surgical mouse model of OA at an earlier time after treatment, but not at a later time after treatment. I-BET151 could robustly suppress the IL-1ß- and TNF-α-induced expression and activity of several matrix-degrading enzymes in human chondrocytes. I-BET151 could also suppress the expression of ACAN, COL2A1, and SOX9. CONCLUSIONS: Our findings suggested that inhibiting BET proteins could exert a repression effect on both of chondrocyte anabolism and catabolism, and the effect of BET protein inhibitor on surgical mouse model of OA needs further evaluation.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Articulación de la Rodilla/metabolismo , Anciano , Animales , Cartílago Articular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Articulación de la Rodilla/efectos de los fármacos , Articulación de la Rodilla/patología , Masculino , Ratones , Ratones de la Cepa 129 , Persona de Mediana Edad , Osteoartritis de la Rodilla/tratamiento farmacológico , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/patologíaRESUMEN
Though genetic data suggest that Polycomb group proteins (PcGs) are central chromatin modifiers and repressors that have been implicated in control of embryonic stem cell (ESC) pluripotency, the precise mechanism of PcG complex recruitment remains elusive, especially in mammals. We now report that the first and second MBT repeats of L3mbtl2 are important structural and functional features that are necessary and sufficient for L3mbtl2-mediated recruitment of PRC1.6 complex to target promoters. Interestingly, this region of L3mbtl2 harbors the evolutionarily conserved Pho-binding pocket also present in Drosophila Sfmbt, and mutation of the critical residues within this pocket completely abolishes its interaction with target promoters. Additionally, decreased PRC1.6 chromatin occupancy was observed following loss of individual components (L3mbtl2, Pcgf6, and Max) of the complex. Our findings suggest that the recruitment of noncanonical PRC1.6 complex in ESCs might be the result of L3mbtl2's interaction with multiple components of the complex.
Asunto(s)
Células Madre Embrionarias/metabolismo , Variación Genética/genética , Diferenciación Celular , HumanosRESUMEN
The ovariectomized (OVX) mouse model has been widely accepted to be suitable for the study of postmenopausal osteoporosis. However, whether C57BL/6J mice, a commonly used genetic background mouse strain, is an appropriate model for postmenopausal osteoporosis remains controversial. The present study investigated the effect of the OVX model on alterations in bone density and microarchitecture in C57BL/6J female mice of different ages. C57BL/6J mice were divided into 8-, 12- and 16-week-old groups (OVX8, OVX12 and OVX16) from the beginning of OVX. At 8 weeks post-surgery, the mice were anesthetized and micro-computed tomography was used to analyze the bone density and microarchitecture. The results revealed that OVX-induced loss of cancellous bone was greatest in OVX8, moderate in OVX12, and only a weak bone loss was observed in the OVX16 group when compared with the SHAM16 control group. In addition, the effect of genetic backgrounds in response to the OVX model were examined. Several other strains of mice, including inbred (BALB/c) and outbred (ICR and Kunming), were used in the present study, all of which were subjected to OVX at 8 weeks of age. The present findings revealed that the highest rate of bone loss was detected in C57BL/6J female mice. In addition, treatment with estrogen (17ß-estradiol, 30 µg/kg five times per week) led to a significant increase in bone density in C57BL/6J mice compared with the other strains of mice. Therefore, these results may provide novel insights into the age- and strain-associated effect of OVX on regulating turnover of bone in female mice. The present findings also suggest 8-week-old C57BL/6J mice as an animal model for postmenopausal osteoporosis and preclinical testing of potential therapies for this disease.
RESUMEN
Tubular cell necrosis is a key histological feature of acute kidney injury (AKI). Necroptosis is a type of programed necrosis, which is executed by mixed lineage kinase domain-like protein (MLKL) upon its binding to the plasma membrane. Emerging evidence indicates that necroptosis plays a critical role in the development of AKI. However, it is unclear whether renal tubular cells undergo necroptosis in vivo and how the necroptotic pathway is regulated during AKI. Repulsive guidance molecule (RGM)-b is a member of the RGM family. Our previous study demonstrated that RGMb is highly expressed in kidney tubular epithelial cells, but its biological role in the kidney has not been well characterized. In the present study, we found that RGMb reduced membrane-associated MLKL levels and inhibited necroptosis in cultured cells. During ischemia/reperfusion injury (IRI) or oxalate nephropathy, MLKL was induced to express on the apical membrane of proximal tubular (PT) cells. Specific knockout of Rgmb in tubular cells (Rgmb cKO) increased MLKL expression at the apical membrane of PT cells and induced more tubular cell death and more severe renal dysfunction compared with wild-type mice. Treatment with the necroptosis inhibitor Necrostatin-1 or GSK'963 reduced MLKL expression on the apical membrane of PT cells and ameliorated renal function impairment after IRI in both wild-type and Rgmb cKO mice. Taken together, our results suggest that proximal tubular cell necroptosis plays an important role in AKI, and that RGMb protects against AKI by inhibiting MLKL membrane association and necroptosis in proximal tubular cells.
Asunto(s)
Lesión Renal Aguda/prevención & control , Apoptosis , Túbulos Renales/patología , Necrosis , Proteínas del Tejido Nervioso/fisiología , Proteínas Quinasas/metabolismo , Daño por Reperfusión/complicaciones , Lesión Renal Aguda/etiología , Lesión Renal Aguda/patología , Animales , Moléculas de Adhesión Celular Neuronal , Proteínas Ligadas a GPI , Túbulos Renales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Sustancias Protectoras/farmacología , Proteínas Quinasas/genéticaRESUMEN
DNA damage contributes to renal tubular cell death during kidney injury, but how DNA damage in tubular cells is regulated is not fully understood. Lethal (3) malignant brain tumor-like 2 (L3MBTL2), a novel polycomb group protein, has been implicated in regulating chromatin architecture. However, the biological functions of L3MBTL2 are largely undefined. Here we found that L3MBTL2 was expressed in the nuclei of renal tubular epithelial cells in mice. Ablation of L3mbtl2 in renal tubular cells resulted in increases in nuclear DNA damage, p53 activation, apoptosis, tubular injury and kidney dysfunction after cisplatin treatment or unilateral ureteral obstruction. In vitro, inhibition of L3MBTL2 sequentially promoted histone γH2AX expression, p53 activation and apoptosis in cisplatin-treated mouse proximal tubular TKPTS cells. Inhibition of p53 activity attenuated the apoptosis induced by L3mbtl2 deficiency after cisplatin treatment both in vivo and in vitro. Intriguingly, unlike other polycomb proteins, L3MBTL2 was not recruited to DNA damage sites, but instead increased nuclear chromatin density and reduced initial DNA damage load. Thus, L3MBTL2 plays a protective role in kidney injury, in part by inhibiting the DNA damage-p53-apoptosis pathway.