RESUMEN
KDM5C is a histone H3K4-specific demethylase, which has been shown to play a key role in biological disease and development. However, the role of KDM5C in trophoblasts at early pregnancy is currently unknown. Here, we showed that KDM5C was upregulated in placental trophoblasts from recurrent miscarriage (RM) patients compared with healthy controls (HCs). Trophoblast proliferation and invasion was inhibited by KDM5C overexpression and was promoted by KDM5C knockdown. Transcriptome sequencing revealed that elevated KDM5C exerted anti-proliferation and anti-invasion effects by repressing the expression of essential regulatory genes. The combination analysis of RNA-seq, ChIP-seq and CUT&Tag assay showed that KDM5C overexpression leads to the reduction of H3K4me3 on the promoters and the corresponding downregulation of expression of several regulatory genes in trophoblasts. Among these genes, TGFß2 and RAGE are essential for the proliferation and invasion of trophoblasts. Importantly, overexpression of KDM5C by a systemically delivered KDM5C adenovirus vector (Ad-KDM5C) promoted embryo resorption rate in mouse. Our results support that KDM5C is an important regulator of the trophoblast function during early pregnancy, and suggesting that KDM5C activity could be responsible for epigenetic alterations seen RM disease.
RESUMEN
JMJD3, a stress-inducible H3K27 demethylase, plays a critical regulatory role in the initiation and progression of malignant hematopoiesis. However, how this histone modifier affects in a cell type-dependent manner remains unclear. Here, we show that in contrast to its oncogenic effect in preleukemia state and lymphoid malignancies, JMJD3 relieves the differentiation-arrest of certain subtypes (such as M2 and M3) of acute myeloid leukemia (AML) cells. RNA sequencing and ChIP-PCR analyses revealed that JMJD3 exerts anti-AML effect by directly modulating H3K4 and H3K27 methylation levels to activate the expression of a number of key myelopoietic regulatory genes. Mechanistic exploration identified a physical and functional association of JMJD3 with C/EBPß that presides the regulatory network of JMJD3. Thus, the leukemia regulatory role of JMJD3 varies in a disease phase- and lineage-dependent manner, and acts as a potential oncorepressor in certain subsets of AML largely by coupling to C/EBPß-centered myelopoietic program.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Leucemia Mieloide Aguda/metabolismo , Animales , Western Blotting , Proteína beta Potenciadora de Unión a CCAAT/genética , Inmunoprecipitación de Cromatina , Biología Computacional , Citometría de Flujo , Células HL-60 , Humanos , Técnicas In Vitro , Histona Demetilasas con Dominio de Jumonji/genética , Leucemia Mieloide Aguda/genética , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCIDRESUMEN
The histone demethylase Jmjd3 plays a critical role in cell lineage specification and differentiation at various stages of development. However, its function during normal myeloid development remains poorly understood. Here, we carried out a systematic in vivo screen of epigenetic factors for their function in hematopoiesis and identified Jmjd3 as a new epigenetic factor that regulates myelopoiesis in zebrafish. We demonstrated that jmjd3 was essential for zebrafish primitive and definitive myelopoiesis, knockdown of jmjd3 suppressed the myeloid commitment and enhanced the erythroid commitment. Only overexpression of spi1 but not the other myeloid regulators rescued the myeloid development in jmjd3 morphants. Furthermore, preliminary mechanistic studies demonstrated that Jmjd3 could directly bind to the spi1 regulatory region to alleviate the repressive H3K27me3 modification and activate spi1 expression. Thus, our studies highlight that Jmjd3 is indispensable for early zebrafish myeloid development by promoting spi1 expression.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji/genética , Células Mieloides/metabolismo , Mielopoyesis/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Proteínas de Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Metilación , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: The role of cannabinoid receptor type 2 (Cnr2) in regulating immune function had been widely investigated, but the mechanism is not fully understood. RESULTS: Cnr2 activation down-regulates 5-lipoxygenase (Alox5) expression by suppressing the JNK/c-Jun activation. CONCLUSION: The Cnr2-JNK-Alox5 axis modulates leukocyte inflammatory migration. SIGNIFICANCE: Linking two important regulators in leukocyte inflammatory migration and providing a potential therapeutic strategy for treating human inflammation-associated diseases. Inflammatory migration of immune cells is involved in many human diseases. Identification of molecular pathways and modulators controlling inflammatory migration could lead to therapeutic strategies for treating human inflammation-associated diseases. The role of cannabinoid receptor type 2 (Cnr2) in regulating immune function had been widely investigated, but the mechanism is not fully understood. Through a chemical genetic screen using a zebrafish model for leukocyte migration, we found that both an agonist of the Cnr2 and inhibitor of the 5-lipoxygenase (Alox5, encoded by alox5) inhibit leukocyte migration in response to acute injury. These agents have a similar effect on migration of human myeloid cells. Consistent with these results, we found that inactivation of Cnr2 by zinc finger nuclease-mediated mutagenesis enhances leukocyte migration, while inactivation of Alox5 blocks leukocyte migration. Further investigation indicates that there is a signaling link between Cnr2 and Alox5 and that alox5 is a target of c-Jun. Cnr2 activation down-regulates alox5 expression by suppressing the JNK/c-Jun activation. These studies demonstrate that Cnr2, JNK, and Alox5 constitute a pathway regulating leukocyte migration. The cooperative effect between the Cnr2 agonist and Alox5 inhibitor also provides a potential therapeutic strategy for treating human inflammation-associated diseases.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Movimiento Celular/efectos de los fármacos , Leucocitos/fisiología , Sistema de Señalización de MAP Quinasas , Receptor Cannabinoide CB2/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Araquidonato 5-Lipooxigenasa/genética , Secuencia de Bases , Agonistas de Receptores de Cannabinoides/farmacología , Regulación Enzimológica de la Expresión Génica , Técnicas de Inactivación de Genes , Indoles/farmacología , Leucocitos/efectos de los fármacos , Datos de Secuencia Molecular , Unión Proteica , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/genética , Cola (estructura animal) , Imagen de Lapso de Tiempo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
The primitive hematopoietic stem/progenitor cells (HSPCs) during embryonic hematopoiesis are thought to be short-lived (SL) with limited self-renewal potential. The fate and consequence of these short-lived HSPCs, once reprogrammed into "long-lived" in a living animal body, remain unknown. Here we show that targeted expression of a dominant-negative C/ebpα (C/ebpαDN) in the primitive SL-HSPCs during zebrafish embryogenesis extends their life span, allowing them to survive to later developmental stage to colonize the definitive hematopoietic sites, where they undergo a proliferative expansion followed by erythropoietic dysplasia and embryonic lethality because of circulation congestion. Mechanistically, C/ebpαDN binds to a conserved C/EBP-binding motif in the promoter region of bmi1 gene, associated with a specific induction of bmi1 transcription in the transgenic embryos expressing C/ebpαDN. Targeted expression of Bmi1 in the SL-HSPCs recapitulates nearly all aberrant phenotypes induced by C/ebpαDN, whereas knockdown of bmi1 largely rescues these abnormalities. The results indicate that Bmi1 acts immediately downstream of C/ebpαDN to regulate the survival and self-renewal of HSPCs and contribute to the erythropoietic dysplasia.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/genética , Eritropoyesis , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/citología , Sistema Hematopoyético/embriología , Proteínas Nucleares/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Embrión no Mamífero/anomalías , Embrión no Mamífero/irrigación sanguínea , Células Madre Hematopoyéticas/metabolismo , Sistema Hematopoyético/metabolismo , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 1 , Proteínas de Pez Cebra/metabolismoRESUMEN
Loss of function of tumor suppressor genes, such as PTEN, CEBPAlpha, and CTNNA1 (encoding the alpha-catenin protein), has been found to play an essential role in leukemogenesis. However, whether these genes genetically interact remains largely unknown. Here, we show that PTEN-mammalian target of rapamycin signaling acts upstream to dictate the ratio of wild-type p42 C/EBPalpha to its dominant-negative p30 isoform, which critically determines whether p30 C/EBPalpha (lower p42/p30 ratio) or p42 C/EBPalpha (higher p42/p30 ratio) binds to the proximal promoter of the retained CTNNA1 allele. Binding of p30 C/EBPalpha recruits the polycomb repressive complex 2 to suppress CTNNA1 transcription through repressive H3K27me3 modification, whereas binding of p42 C/EBPalpha relieves this repression and promotes CTNNA1 expression through activating H3K4me3 modification. Loss of Pten function in mice and zebrafish induces myelodysplasia with abnormal invasiveness of myeloid progenitors accompanied by significant reductions in both wild-type C/EBPalpha and alpha-catenin protein. Importantly, frame-shift mutations in either PTEN or CEBPA were detected exclusively in the primary LICs with low CTNNA1 expression. This study uncovers a novel molecular pathway, PTEN-C/EBPalpha-CTNNA1, which is evolutionarily conserved and might be therapeutically targeted to eradicate LICs with low CTNNA1 expression.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Transformación Celular Neoplásica/metabolismo , Leucemia/metabolismo , Mielopoyesis , Células Madre Neoplásicas/metabolismo , Fosfohidrolasa PTEN/metabolismo , alfa Catenina/metabolismo , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Transformación Celular Neoplásica/genética , Mutación del Sistema de Lectura , Regulación Leucémica de la Expresión Génica/genética , Células HL-60 , Humanos , Leucemia/genética , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/genética , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal/genética , Transcripción Genética/genética , Pez Cebra , alfa Catenina/genéticaRESUMEN
Dorsal organizer formation is one of the most critical steps in early embryonic development. Several genes and signaling pathways that positively regulate the dorsal organizer development have been identified; however, little is known about the factor(s) that negatively regulates the organizer formation. Here, we show that Setdb2, a SET domain-containing protein possessing potential histone H3K9 methyltransferase activity, restricts dorsal organizer development and regulates left-right asymmetry by suppressing fibroblast growth factor 8 (fgf8) expression. Knockdown of Setdb2 results in a massive expansion of dorsal organizer markers floating head (flh), goosecoid (gsc), and chordin (chd), as well as a significant increase of fgf8, but not fgf4 mRNAs. Consequently, disrupted midline patterning and resultant randomization of left-right asymmetry are observed in Setdb2-deficient embryos. These characteristic changes induced by Setdb2 deficiency can be nearly corrected by either overexpression of a dominant-negative fgf receptor or knockdown of fgf8, suggesting an essential role for Setdb2-Fgf8 signaling in restricting dorsal organizer territory and regulating left-right asymmetry. These results provide unique evidence that a SET domain-containing protein potentially involved in the epigenetic control negatively regulates dorsal organizer formation during early embryonic development.
Asunto(s)
Tipificación del Cuerpo/genética , Embrión no Mamífero/metabolismo , Factor 8 de Crecimiento de Fibroblastos/genética , N-Metiltransferasa de Histona-Lisina/genética , Proteínas de Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Embrión no Mamífero/embriología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glicoproteínas/genética , Proteína Goosecoide/genética , Proteínas de Homeodominio/genética , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
Precise transcriptional control of developmental stage-specific expression and switching of alpha- and beta-globin genes is significantly important to understand the general principles controlling gene expression and the pathogenesis of thalassemia. Although transcription factors regulating beta-globin genes have been identified, little is known about the microRNAs and trans-acting mechanism controlling alpha-globin genes transcription. Here, we show that an erythroid lineage-specific microRNA gene, miR-144, expressed at specific developmental stages during zebrafish embryogenesis, negatively regulates the embryonic alpha-globin, but not embryonic beta-globin, gene expression, through physiologically targeting klfd, an erythroid-specific Krüppel-like transcription factor. Klfd selectively binds to the CACCC boxes in the promoters of both alpha-globin and miR-144 genes to activate their transcriptions, thus forming a negative feedback circuitry to fine-tune the expression of embryonic alpha-globin gene. The selective effect of the miR-144-Klfd pathway on globin gene regulation may thereby constitute a novel therapeutic target for improving the clinical outcome of patients with thalassemia.
Asunto(s)
Embrión no Mamífero/metabolismo , Eritropoyesis/fisiología , Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Globinas alfa/genética , Animales , Animales Modificados Genéticamente , Apoptosis , Northern Blotting , Western Blotting , Biología Computacional , Embrión no Mamífero/citología , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Técnicas para Inmunoenzimas , Hibridación in Situ , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/fisiología , MicroARNs/metabolismo , Oligonucleótidos/farmacología , Regiones Promotoras Genéticas/genética , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/fisiología , Globinas alfa/metabolismo , Globinas beta/genética , Globinas beta/metabolismoRESUMEN
Interstitial cell migration through extracellular matrix is a hallmark of the inflammation response, tumor invasion, and metastasis. We have established a stable zebrafish transgenic line expressing enhanced GFP under the lysozyme C promoter for visualizing and measuring primitive macrophage migration in vivo. We show that tissue-resident primitive macrophages migrate rapidly through extracellular matrix to the site of acute injury induced by tail transection. Mechanistically, the specific inhibition of JNK, but not p38 and ERK, dramatically abolished the chemotactic migration in a dose-dependent manner, suppressing the trauma-induced recruitment of phosphorylated C-Jun transcription factor to proximal AP-1 sites in the promoter of matrix metalloproteinase 13 (mmp13), a gene specifically expressed in primitive macrophages during embryogenesis and required for the interstitial migration. Furthermore, dexamethasone suppressed the trauma-induced JNK phosphorylation and macrophage migration accompanied by simultaneous up-regulation of mkp-1, a well-known phosphatase capable of inactivating phosphorylated JNK. The results indicate that the JNK-Mmp13 signaling pathway plays an essential role in regulating the innate immune cell migration in response to severe injury in vivo.