RESUMEN
BACKGROUND: We reasoned that unraveling the dynamic changes in accessibility of genomic regulatory elements and gene expression at single-cell resolution will inform the basic mechanisms of nephrogenesis. METHODS: We performed single-cell ATAC-seq and RNA-seq both individually (singleomes; Six2GFP cells) and jointly in the same cells (multiomes; kidneys) to generate integrated chromatin and transcriptional maps in mouse embryonic and neonatal nephron progenitor cells. RESULTS: We demonstrate that singleomes and multiomes are comparable in assigning most cell states, identification of new cell type markers, and defining the transcription factors driving cell identity. However, multiomes are more precise in defining the progenitor population. Multiomes identified a "pioneer" bHLH/Fox motif signature in nephron progenitor cells. Moreover, we identified a subset of Fox factors exhibiting high chromatin activity in podocytes. One of these Fox factors, Foxp1, is important for nephrogenesis. Key nephrogenic factors are distinguished by strong correlation between linked gene regulatory elements and gene expression. CONCLUSION: Mapping the regulatory landscape at single-cell resolution informs the regulatory hierarchy of nephrogenesis. Paired single-cell epigenomes and transcriptomes of nephron progenitors should provide a foundation to understand prenatal programming, regeneration after injury, and ex vivo nephrogenesis.
Asunto(s)
Cromatina , Podocitos , Animales , Diferenciación Celular/genética , Cromatina/metabolismo , Femenino , Proteínas de Homeodominio/genética , Riñón/metabolismo , Ratones , Nefronas/metabolismo , Organogénesis/genética , Podocitos/metabolismo , Embarazo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Developmental changes in cell fate are tightly regulated by cell-type specific transcription factors. Chromatin reorganization during organismal development ensures dynamic access of developmental regulators to their cognate DNA sequences. Thus, understanding the epigenomic states of promoters and enhancers is of key importance. Recent years have witnessed significant advances in our knowledge of the transcriptional mechanisms of kidney development. Emerging evidence suggests that histone deacetylation by class I HDACs and H3 methylation on lysines 4, 27 and 79 play important roles in regulation of early and late gene expression in the developing kidney. Equally exciting is the realization that nephrogenesis genes in mesenchymal nephron progenitors harbor bivalent chromatin domains which resolve upon differentiation implicating chromatin bivalency in developmental control of gene expression. Here, we review current knowledge of the epigenomic states of nephric cells and current techniques used to study the dynamic chromatin states. These technological advances will provide an unprecedented view of the enhancer landscape during cell fate commitment and help in defining the complex transcriptional networks governing kidney development and disease.
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Epigénesis Genética , Epigenómica/métodos , Riñón/metabolismo , Nefronas/metabolismo , Animales , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Riñón/citología , Riñón/embriología , Enfermedades Renales/embriología , Enfermedades Renales/genética , Enfermedades Renales/patología , Nefronas/citología , Nefronas/embriología , Organogénesis/genéticaRESUMEN
SIX2 (SIX homeobox 2)-positive nephron progenitor cells (NPCs) give rise to all epithelial cell types of the nephron, the filtering unit of the kidney. NPCs have a limited lifespan and are depleted near the time of birth. Epigenetic factors are implicated in the maintenance of organ-restricted progenitors such as NPCs, but the chromatin-based mechanisms are incompletely understood. Here, using a combination of gene targeting, chromatin profiling, and single-cell RNA analysis, we examined the role of the murine histone 3 Lys-27 (H3K27) methyltransferases EZH1 (enhancer of zeste 1) and EZH2 in NPC maintenance. We found that EZH2 expression correlates with NPC growth potential and that EZH2 is the dominant H3K27 methyltransferase in NPCs and epithelial descendants. Surprisingly, NPCs lacking H3K27 trimethylation maintained their progenitor state but cycled slowly, leading to a smaller NPC pool and formation of fewer nephrons. Unlike Ezh2 loss of function, dual inactivation of Ezh1 and Ezh2 triggered overexpression of the transcriptional repressor Hes-related family BHLH transcription factor with YRPW motif 1 (Hey1), down-regulation of Six2, and unscheduled activation of Wnt4-driven differentiation, resulting in early termination of nephrogenesis and severe renal dysgenesis. Double-mutant NPCs also overexpressed the SIX family member Six1 However, in this context, SIX1 failed to maintain NPC stemness. At the chromatin level, EZH1 and EZH2 restricted accessibility to AP-1-binding motifs, and their absence promoted a regulatory landscape akin to differentiated and nonlineage cells. We conclude that EZH2 is required for NPC renewal potential and that tempering of the differentiation program requires cooperation of both EZH1 and EZH2.
Asunto(s)
Cromatina/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Nefronas/citología , Complejo Represivo Polycomb 2/metabolismo , Células Madre/citología , Animales , Supervivencia Celular , Células Cultivadas , Ratones , Nefronas/metabolismo , Células Madre/metabolismoRESUMEN
Human kidney organoid technology holds promise for novel kidney disease treatment strategies and utility in pharmacological and basic science. Given the crucial roles of the intrarenal renin-angiotensin system (RAS) and angiotensin II (ANG II) in the progression of kidney development and injury, we investigated the expression of RAS components and effects of ANG II on cell differentiation in human kidney organoids. Human induced pluripotent stem cell-derived kidney organoids were induced using a modified 18-day Takasato protocol. Gene expression analysis by digital PCR and immunostaining demonstrated the formation of renal compartments and expression of RAS components. The ANG II type 1 receptor (AT1R) was strongly expressed in the early phase of organoid development (around day 0), whereas ANG II type 2 receptor (AT2R) expression levels peaked on day 5. Thus, the organoids were treated with 100 nM ANG II in the early phase on days 0-5 (ANG II-E) or during the middle phase on days 5-10 (ANG II-M). ANG II-E was observed to decrease levels of marker genes for renal tubules and proximal tubules, and the downregulation of renal tubules was inhibited by an AT1R antagonist. In contrast, ANG II-M increased levels of markers for podocytes, the ureteric tip, and the nephrogenic mesenchyme, and an AT2R blocker attenuated the ANG II-M-induced augmentation of podocyte formation. These findings demonstrate RAS expression and ANG II exertion of biphasic effects on cell differentiation through distinct mediatory roles of AT1R and AT2R, providing a novel strategy to establish and further characterize the developmental potential of human induced pluripotent stem cell-derived kidney organoids.NEW & NOTEWORTHY This study demonstrates angiotensin II exertion of biphasic effects on cell differentiation through distinct mediatory roles of angiotensin II type 1 receptor and type 2 receptor in human induced pluripotent stem cell-derived kidney organoids, providing a novel strategy to establish and further characterize the developmental potential of the human kidney organoids.
Asunto(s)
Angiotensina II/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Riñón/efectos de los fármacos , Organoides/efectos de los fármacos , Sistema Renina-Angiotensina/efectos de los fármacos , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Riñón/citología , Riñón/metabolismo , Organoides/citología , Organoides/metabolismo , Receptor de Angiotensina Tipo 1/agonistas , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/agonistas , Receptor de Angiotensina Tipo 2/genética , Receptor de Angiotensina Tipo 2/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Nephron progenitor cells (NPCs) are Six2-positive metanephric mesenchyme cells, which undergo self-renewal and differentiation to give rise to nephrons until the end of nephrogenesis. Histone deacetylases (HDACs) are a group of epigenetic regulators that control cell fate, but their role in balancing NPC renewal and differentiation is unknown. Here, we report that NPC-specific deletion of Hdac1 and Hdac2 genes in mice results in early postnatal lethality owing to renal hypodysplasia and loss of NPCs. HDAC1/2 interact with the NPC renewal regulators Six2, Osr1 and Sall1, and are co-bound along with Six2 on the Six2 enhancer. Although the mutant NPCs differentiate into renal vesicles (RVs), Hdac1/2 mutant kidneys lack nascent nephrons or mature glomeruli, a phenocopy of Lhx1 mutants. Transcriptional profiling and network analysis identified disrupted expression of Lhx1 and its downstream genes, Dll1 and Hnf1a/4a, as key mediators of the renal phenotype. Finally, although HDAC1/2-deficient NPCs and RVs overexpress hyperacetylated p53, Trp53 deletion failed to rescue the renal dysgenesis. We conclude that the epigenetic regulators HDAC1 and HDAC2 control nephrogenesis via interactions with the transcriptional programs of nephron progenitors and renal vesicles.
Asunto(s)
Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Nefronas/embriología , Organogénesis/genética , Células Madre/citología , Transcripción Genética/genética , Animales , Proteínas de Unión al Calcio , Línea Celular , Proliferación Celular/genética , Células HEK293 , Factor Nuclear 1-alfa del Hepatocito/biosíntesis , Factor Nuclear 4 del Hepatocito/biosíntesis , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Enfermedades Renales/genética , Proteínas con Homeodominio LIM/genética , Ratones , Ratones Noqueados , Nefronas/citología , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Histone deacetylases (HDACs) regulate a broad range of biological processes through removal of acetyl groups from histones as well as non-histone proteins. Our previous studies showed that Hdac1 and Hdac2 are bound to promoters of key renal developmental regulators and that HDAC activity is required for embryonic kidney gene expression. However, the existence of many HDAC isoforms in embryonic kidneys raises questions concerning the possible specificity or redundancy of their functions. We report here that targeted deletion of both the Hdac1 and Hdac2 genes from the ureteric bud (UB) cell lineage of mice causes bilateral renal hypodysplasia. One copy of either Hdac1 or Hdac2 is sufficient to sustain normal renal development. In addition to defective cell proliferation and survival, genome-wide transcriptional profiling revealed that the canonical Wnt signaling pathway is specifically impaired in UB(Hdac1,2-/-) kidneys. Our results also demonstrate that loss of Hdac1 and Hdac2 in the UB epithelium leads to marked hyperacetylation of the tumor suppressor protein p53 on lysine 370, 379 and 383; these post-translational modifications are known to boost p53 stability and transcriptional activity. Genetic deletion of p53 partially rescues the development of UB(Hdac1,2-/-) kidneys. Together, these data indicate that Hdac1 and Hdac2 are crucial for kidney development. They perform redundant, yet essential, cell lineage-autonomous functions via p53-dependent and -independent pathways.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Uréter/embriología , Proteínas Wnt/metabolismo , Acetilación , Animales , Western Blotting , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Técnicas de Inactivación de Genes , Técnicas Histológicas , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la PolimerasaAsunto(s)
Cromatina , Factores de Transcripción , Riñón , Mesodermo , Factores de Transcripción/genéticaRESUMEN
Appreciation for the role of epigenetic modifications in the diagnosis and treatment of diseases is fast gaining attention. Treatment of chronic kidney disease stemming from diabetes or hypertension as well as Wilms tumor will all profit from knowledge of the changes in the epigenomic landscapes. To do so, it is essential to characterize the epigenomic modifiers and their modifications under normal physiological conditions. The transcription factor Pax2 was identified as a major epigenetic player in the early specification of the kidney. Notably, the progenitors of all nephrons that reside in the cap mesenchyme display a unique bivalent histone signature (expressing repressive epigenetic marks alongside activation marks) on lineage-specific genes. These cells are deemed poised for differentiation and commitment to the nephrogenic lineage. In response to the appropriate inducing signal, these genes lose their repressive histone marks, which allow for their expression in nascent nephron precursors. Such knowledge of the epigenetic landscape and the resultant cell fate or behavior in the developing kidney will greatly improve the overall success in designing regenerative strategies and tissue reprogramming methodologies from pluripotent cells.
Asunto(s)
Epigénesis Genética , Riñón/embriología , Organogénesis/genética , Animales , HumanosRESUMEN
We elucidated the role of collecting duct kinin B2 receptor (B2R) in the development of salt-sensitivity and angiotensin II (ANG II)-induced hypertension. To this end, we used a Cre-Lox recombination strategy to generate mice lacking Bdkrb2 gene for B2R in the collecting duct (Hoxb7-Cre(tg/+):Bdkrb2(flox/flox)). In 3 groups of control (Bdkrb2(flox/flox)) and 3 groups of UB(Bdkrb2-/-) mice, systolic blood pressure (SBP) responses to high salt intake (4 or 8% NaCl; HS) were monitored by radiotelemetry in comparison with standard salt diet (0.4% NaCl) prior to and during subcutaneous ANG II infusion (1000 ng/min/kg) via osmotic minipumps. High salt intakes alone for 2 weeks did not alter SBP in either strain. ANG II significantly increased SBP equally in control (121 ± 2 to 156 ± 3 mmHg) and UB(Bdkrb2-/-) mice (120 ± 2 to 153 ± 2 mmHg). The development of ANG II-induced hypertension was exacerbated by 4%HS in both control (125 ± 3 to 164 ± 5 mmHg) and UB(Bdkrb2-/-) mice (124 ± 2 to 162 ± 3 mmHg) during 2 weeks. Interestingly, 8%HS caused a more profound and earlier ANG II-induced hypertension in UB(Bdkrb2-/-) (129 ± 2 to 166 ± 3 mmHg) as compared to control (128 ± 2 to 158 ± 2 mmHg) and it was accompanied by body weight loss and increased mortality. In conclusion, targeted inactivation of B2R in the renal collecting duct does not cause salt-sensitivity; however, collecting duct B2R attenuates the hypertensive actions of ANG II under conditions of very high salt intake.
Asunto(s)
Angiotensina II/metabolismo , Presión Sanguínea , Hipertensión , Túbulos Renales Colectores , Cloruro de Sodio Dietético/efectos adversos , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Hipertensión/metabolismo , Hipertensión/fisiopatología , Túbulos Renales Colectores/metabolismo , Túbulos Renales Colectores/fisiopatología , Masculino , Ratones , Receptor de Bradiquinina B2/genéticaRESUMEN
An understanding of epigenetics is indispensable to our understanding of gene regulation under normal and pathological states. This knowledge will help with designing better therapeutic approaches in regenerative tissue medicine. Epigenetics allows us to parse out the mechanisms by which transcriptional regulators gain access to specific gene loci thereby imprinting epigenetic information affecting chromatin function. This epigenetic memory forms the basis of cell lineage specification in multicellular organisms. Post-translational modifications to DNA and histones in the nucleosome core form characteristic epigenetic codes which are distinct for self-renewing and primed progenitor cell populations. Studies of chromatin modifiers and modifications in renal development and disease have been gaining momentum. Both congenital and adult renal diseases have a gene-environment component, which involves alterations to the epigenetic information imprinted during development. This epigenetic memory must be characterized to establish optimal treatment of both acute and chronic renal diseases.
Asunto(s)
Epigénesis Genética/genética , Animales , Cromatina/metabolismo , Histonas/metabolismo , Humanos , Riñón/metabolismo , Procesamiento Proteico-Postraduccional/genéticaRESUMEN
The balance between nephron progenitor cell (NPC) renewal, survival and differentiation ultimately determines nephron endowment and thus susceptibile to chronic kidney disease and hypertension. Embryos lacking the p53-E3 ubiquitin ligase, Murine double minute 2 (Mdm2), die secondary to p53-mediated apoptosis and growth arrest, demonstrating the absolute requirement of Mdm2 in embryogenesis. Although Mdm2 is required in the maintenance of hematopoietic stem cells, its role in renewal and differentiation of stem/progenitor cells during kidney organogenesis is not well defined. Here we examine the role of the Mdm2-p53 pathway in NPC renewal and fate in mice. The Six2-GFP::Cre(tg/+) mediated inactivation of Mdm2 in the NPC (NPC(Mdm)2(-/-)) results in perinatal lethality. NPC(Mdm)2(-/-) neonates have hypo-dysplastic kidneys, patchy depletion of the nephrogenic zone and pockets of superficially placed, ectopic, well-differentiated proximal tubules. NPC(Mdm2-/-) metanephroi exhibit thinning of the progenitor GFP(+)/Six2(+) population and a marked reduction or loss of progenitor markers Amphiphysin, Cited1, Sall1 and Pax2. This is accompanied by aberrant accumulation of phospho-γH2AX and p53, and elevated apoptosis together with reduced cell proliferation. E13.5-E15.5 NPC(Mdm2-/-) kidneys show reduced expression of Eya1, Pax2 and Bmp7 while the few surviving nephron precursors maintain expression of Wnt4, Lhx1, Pax2, and Pax8. Lineage fate analysis and section immunofluorescence revealed that NPC(Mdm2-/-) kidneys have severely reduced renal parenchyma embedded in an expanded stroma. Six2-GFP::Cre(tg/+); Mdm2(f/f) mice bred into a p53 null background ensures survival of the GFP-positive, self-renewing progenitor mesenchyme and therefore restores normal renal development and postnatal survival of mice. In conclusion, the Mdm2-p53 pathway is essential to the maintenance of the nephron progenitor niche.
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Nefronas/embriología , Proteínas Proto-Oncogénicas c-mdm2/genética , Células Madre/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis , Proteína Morfogenética Ósea 7/biosíntesis , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Genotipo , Proteínas Fluorescentes Verdes/genética , Histonas/biosíntesis , Histonas/metabolismo , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Proteínas con Homeodominio LIM/biosíntesis , Ratones , Ratones Noqueados , Nefronas/citología , Nefronas/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Organogénesis/genética , Factor de Transcripción PAX2/biosíntesis , Factor de Transcripción PAX2/deficiencia , Factor de Transcripción PAX2/genética , Factor de Transcripción PAX8 , Factores de Transcripción Paired Box/biosíntesis , Proteínas Tirosina Fosfatasas/biosíntesis , Células Madre/citología , Transactivadores/deficiencia , Transactivadores/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/metabolismo , Proteína Wnt4/biosíntesisRESUMEN
In mammals, formation of new nephrons ends perinatally due to consumption of mesenchymal progenitor cells. Premature depletion of progenitors due to prematurity or postnatal loss of nephrons due to injury causes chronic kidney disease and hypertension. Intensive efforts are currently invested in designing regenerative strategies to form new nephron progenitors from pluripotent cells, which upon further differentiation provide a potential source of new nephrons. To know if reprogramed renal cells can maintain their identity and fate requires knowledge of the epigenetic states of native nephron progenitors and their progeny. In this article, we summarize current knowledge and gaps in the epigenomic landscape of the developing kidney. We now know that Pax2/PTIP/H3K4 methyltransferase activity provides the initial epigenetic specification signal to the metanephric mesenchyme. During nephrogenesis, the cap mesenchyme housing nephron progenitors is enriched in bivalent chromatin marks; as tubulogenesis proceeds, the tubular epithelium acquires H3K79me2. The latter mark is uniquely induced during epithelial differentiation. Analysis of histone landscapes in clonal metanephric mesenchyme cell lines and in Wilms tumor and normal fetal kidney has revealed that promoters of poised nephrogenesis genes carry bivalent histone signatures in progenitors. Differentiation or stimulation of Wnt signaling promotes resolution of bivalency; this does not occur in Wilms tumor cells consistent with their developmental arrest. The use of small cell number ChIP-Seq should facilitate the characterization of the chromatin landscape of the metanephric mesenchyme and various nephron compartments during nephrogenesis. Only then we will know if stem and somatic cell reprogramming into kidney progenitors recapitulates normal development.
Asunto(s)
Riñón/citología , Nefronas/citología , Animales , Diferenciación Celular/fisiología , Epigenómica , Femenino , Humanos , Riñón/embriología , Riñón/metabolismo , Mesodermo/citología , Nefronas/metabolismo , Embarazo , Células Madre/citología , Células Madre/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Epigenetic regulation of gene expression is critical to phenotypic maintenance and transition of human breast cancer cells. HOX antisense intergenic RNA (HOTAIR) is a long intergenic non-coding RNA that epigenetically represses gene expression via recruitment of enhancer of zeste homolog 2 (EZH2), a histone methyltransferase. Elevated expression of HOTAIR promotes progression of breast cancer. In the current study we examined the expression and function of HOTAIR in MCF-7-TNR cells, a derivative of the luminal-like breast cancer cell line MCF-7 that acquired resistance to TNF-α-induced cell death. The expression of HOTAIR, markers of the luminal-like and basal-like subtypes, and growth were compared between MCF-7 and MCF-7-TNR cells. These variables were further assessed upon inhibition of HOTAIR, EZH2, p38 MAPK, and SRC kinase in MCF-7-TNR cells. When compared with MCF-7 cells, MCF-7-TNR cells exhibited an increase in the expression of HOTAIR, which correlated with characteristics of a luminal-like to basal-like transition as evidenced by dysregulated gene expression and accelerated growth. MCF-7-TNR cells exhibited reduced suppressive histone H3 lysine27 trimethylation on the HOTAIR promoter. Inhibition of HOTAIR and EZH2 attenuated the luminal-like to basal-like transition in terms of gene expression and growth in MCF-7-TNR cells. Inhibition of p38 and SRC diminished HOTAIR expression and the basal-like phenotype in MCF-7-TNR cells. HOTAIR was robustly expressed in the native basal-like breast cancer cells and inhibition of HOTAIR reduced the basal-like gene expression and growth. Our findings suggest HOTAIR-mediated regulation of gene expression and growth associated with the basal-like phenotype of breast cancer cells.
Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica/genética , ARN Largo no Codificante/genética , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética/genética , Femenino , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Humanos , Células MCF-7 , Complejo Represivo Polycomb 2/genética , Factor de Necrosis Tumoral alfa/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Familia-src Quinasas/genéticaRESUMEN
The G protein-coupled bradykinin B2 receptor (Bdkrb2) plays an important role in regulation of blood pressure under conditions of excess salt intake. Our previous work has shown that Bdkrb2 also plays a developmental role since Bdkrb2(-/-) embryos, but not their wild-type or heterozygous littermates, are prone to renal dysgenesis in response to gestational high salt intake. Although impaired terminal differentiation and apoptosis are consistent findings in the Bdkrb2(-/-) mutant kidneys, the developmental pathways downstream of gene-environment interactions leading to the renal phenotype remain unknown. Here, we performed genome-wide transcriptional profiling on embryonic kidneys from salt-stressed Bdkrb2(+/+) and Bdkrb2(-/-) embryos. The results reveal significant alterations in key pathways regulating Wnt signaling, apoptosis, embryonic development, and cell-matrix interactions. In silico analysis reveal that nearly 12% of differentially regulated genes harbor one or more Pax2 DNA-binding sites in their promoter region. Further analysis shows that metanephric kidneys of salt-stressed Bdkrb2(-/-) have a significant downregulation of Pax2 gene expression. This was corroborated in Bdkrb2(-/-);Pax2(GFP+/tg) mice, demonstrating that Pax2 transcriptional activity is significantly repressed by gestational salt-Bdkrb2 interactions. We conclude that gestational gene (Bdkrb2) and environment (salt) interactions cooperate to impact gene expression programs in the developing kidney. Suppression of Pax2 likely contributes to the defects in epithelial survival, growth, and differentiation in salt-stressed BdkrB2(-/-) mice.
Asunto(s)
Interacción Gen-Ambiente , Genoma , Riñón/embriología , Riñón/metabolismo , Animales , Sitios de Unión , Regulación hacia Abajo/genética , Células Epiteliales/metabolismo , Femenino , Ontología de Genes , Genes Reporteros , Ratones Endogámicos C57BL , Ratones Mutantes , Factor de Transcripción PAX2/genética , Factor de Transcripción PAX2/metabolismo , Embarazo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Bradiquinina B2/deficiencia , Receptor de Bradiquinina B2/genética , Reproducibilidad de los Resultados , Cloruro de Sodio , Estrés Fisiológico/genética , Transcriptoma/genética , Regulación hacia Arriba/genéticaRESUMEN
Type 2 and type 1 diabetes (T2D, T1D) exhibit sex differences in insulin secretion, the mechanisms of which are unknown. We examined sex differences in human pancreatic islets from 52 donors with and without T2D combining single cell RNA-seq (scRNA-seq), single nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), hormone secretion, and bioenergetics. In nondiabetic (ND) donors, sex differences in islet cells gene accessibility and expression predominantly involved sex chromosomes. Islets from T2D donors exhibited similar sex differences in sex chromosomes differentially expressed genes (DEGs), but also exhibited sex differences in autosomal genes. Comparing ß cells from T2D vs. ND donors, gene enrichment of female ß cells showed suppression in mitochondrial respiration, while male ß cells exhibited suppressed insulin secretion. Thus, although sex differences in gene accessibility and expression of ND ß cells predominantly affect sex chromosomes, the transition to T2D reveals sex differences in autosomes highlighting mitochondrial failure in females.
RESUMEN
Despite mounting evidence that p53 senses and responds to physiological cues in vivo, existing knowledge regarding p53 function and target genes is largely derived from studies in cancer or stressed cells. Herein we utilize p53 transcriptome and ChIP-Seq (chromatin immunoprecipitation-high throughput sequencing) analyses to identify p53 regulated pathways in the embryonic kidney, an organ that develops via mesenchymal-epithelial interactions. This integrated approach allowed identification of novel genes that are possible direct p53 targets during kidney development. We find the p53-regulated transcriptome in the embryonic kidney is largely composed of genes regulating developmental, morphogenesis, and metabolic pathways. Surprisingly, genes in cell cycle and apoptosis pathways account for <5% of differentially expressed transcripts. Of 7,893 p53-occupied genomic regions (peaks), the vast majority contain consensus p53 binding sites. Interestingly, 78% of p53 peaks in the developing kidney lie within proximal promoters of annotated genes compared with 7% in a representative cancer cell line; 25% of the differentially expressed p53-bound genes are present in nephron progenitors and nascent nephrons, including key transcriptional regulators, components of Fgf, Wnt, Bmp, and Notch pathways, and ciliogenesis genes. The results indicate widespread p53 binding to the genome in vivo and context-dependent differences in the p53 regulon between cancer, stress, and development. To our knowledge, this is the first comprehensive analysis of the p53 transcriptome and cistrome in a developing mammalian organ, substantiating the role of p53 as a bona fide developmental regulator. We conclude p53 targets transcriptional networks regulating nephrogenesis and cellular metabolism during kidney development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Genoma/genética , Riñón/embriología , Riñón/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Secuencia de Bases , Sitios de Unión , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Genes Reguladores , Genes Reporteros , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Morfogénesis/genética , Nefronas/metabolismo , Motivos de Nucleótidos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica/genética , Reproducibilidad de los Resultados , Transducción de Señal/genética , Transcriptoma/genéticaRESUMEN
Gene-environment interactions are implicated in congenital disorders. Accordingly, there is a pressing need to develop animal models of human disease, which are the product of defined gene-environment interactions. Work from our laboratory demonstrates the presence of genetic interactions between the bradykinin B2 receptor (BdkrB2) and the tumor suppressor protein p53 in the developing kidney. Our studies have shown that the Bdkrb2(-/-) embryos exposed to gestational salt stress develop renal dysgenesis. The underlying mechanism is p53 stabilization and mediated apoptosis and repression of the terminal epithelial differentiation program. We also uncovered a novel functional cross-talk between p53 and BdkrB2. Thus, while BdkrB2 is a target for p53-mediated transcriptional activation, BdkrB2 inactivation results in the upregulation of checkpoint kinase 1 (Chk1) levels, thus potentiating phosphorylation of p53 on Ser23 by Chk1, an essential step in the pathway leading to renal dysgenesis in salt-stressed BdkrB2(-/-) mutant mice. Future studies will now focus on defining how this G-protein-coupled receptor is coupled to the activation of p53, a tumor suppressor gene that is mutated in more than 50% of all human cancers.
Asunto(s)
Interacción Gen-Ambiente , Riñón/embriología , Receptor de Bradiquinina B2/genética , Receptor de Bradiquinina B2/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Humanos , RatonesRESUMEN
Histone deacetylases (HDACs) are an evolutionarily conserved group of enzymes that regulate a broad range of biological processes through removal of acetyl groups from histones as well as non-histone proteins. Recent studies using a variety of pharmacological inhibitors and genetic models of HDACs have revealed a central role of HDACs in control of kidney development. These findings provide new insights into the epigenetic mechanisms underlying congenital anomalies of the kidney and urinary tract (CAKUT) and implicate the potential of HDACs as therapeutic targets in kidney diseases, such as cystic kidney diseases and renal cell cancers. Determining the specific functions of individual HDAC members would be an important task of future research.
Asunto(s)
Histona Desacetilasas/metabolismo , Enfermedades Renales/enzimología , Riñón/enzimología , Animales , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/genética , Humanos , Isoenzimas , Riñón/efectos de los fármacos , Riñón/embriología , Riñón/crecimiento & desarrollo , Enfermedades Renales/diagnóstico , Enfermedades Renales/fisiopatología , Enfermedades Renales/terapia , OrganogénesisRESUMEN
Nephron endowment at birth impacts long-term renal and cardiovascular health, and it is contingent on the nephron progenitor cell (NPC) pool. Glycolysis modulation is essential for determining NPC fate, but the underlying mechanism is unclear. Combining RNA sequencing and quantitative proteomics we identify 267 genes commonly targeted by Wnt activation or glycolysis inhibition in NPCs. Several of the impacted pathways converge at Acetyl-CoA, a co-product of glucose metabolism. Notably, glycolysis inhibition downregulates key genes of the Mevalonate/cholesterol pathway and stimulates NPC differentiation. Sodium acetate supplementation rescues glycolysis inhibition effects and favors NPC maintenance without hindering nephrogenesis. Six2Cre-mediated removal of ATP-citrate lyase (Acly), an enzyme that converts citrate to acetyl-CoA, leads to NPC pool depletion, glomeruli count reduction, and increases Wnt4 expression at birth. Sodium acetate supplementation counters the effects of Acly deletion on cap-mesenchyme. Our findings show a pivotal role of acetyl-CoA metabolism in kidney development and uncover new avenues for manipulating nephrogenesis and preventing adult kidney disease.
Asunto(s)
Riñón , Nefronas , Acetilcoenzima A/metabolismo , Acetato de Sodio/metabolismo , Riñón/metabolismo , Células Madre/metabolismoRESUMEN
Low nephron number correlates with the development of hypertension and chronic kidney disease later in life. While intrauterine growth restriction caused by maternal low protein diet (LPD) is thought to be a significant cause of reduced nephron endowment in impoverished communities, its influence on the cellular and molecular processes which drive nephron formation are poorly understood. We conducted a comprehensive characterization of the impact of LPD on kidney development using tomographic and confocal imaging to quantify changes in branching morphogenesis and the cellular and morphological features of nephrogenic niches across development. These analyses were paired with single-cell RNA sequencing to dissect the transcriptional changes that LPD imposes during renal development. Differences in the expression of genes involved in metabolism were identified in most cell types we analyzed, yielding imbalances and shifts in cellular energy production. We further demonstrate that LPD impedes branching morphogenesis and significantly reduces the number of pretubular aggregates - the initial precursors to nephron formation. The most striking observation was that LPD changes the developmental trajectory of nephron progenitor cells, driving the formation of a partially committed cell population which likely reflects a failure of cells to commit to nephron formation and which ultimately reduces endowment. This unique profile of a fetal programming defect demonstrates that low nephron endowment arises from the pleiotropic impact of changes in branching morphogenesis and nephron progenitor cell commitment, the latter of which highlights a critical role for nutrition in regulating the cell fate decisions underpinning nephron endowment. Significance Statement: While a mother's diet and behavior can negatively impact the number of nephrons in the kidneys of her offspring, the root cellular and molecular drivers of these deficits have not been rigorously explored. In this study we use advanced imaging and gene expression analysis in mouse models to define how a maternal low protein diet, analogous to that of impoverished communities, results in reduced nephron endowment. We find that low protein diet has pleiotropic effects on metabolism and the normal programs of gene expression. These profoundly impact the process of branching morphogenesis necessary to establish niches for nephron generation and change cell behaviors which regulate how and when nephron progenitor cells commit to differentiation.