RESUMEN
Within the pancreas, Keratin 19 (KRT19) labels the ductal lineage and is a determinant of pancreatic ductal adenocarcinoma (PDAC). To investigate KRT19 expression dynamics, we developed a human pluripotent stem cell (PSC)-based KRT19-mCherry reporter system in different genetic backgrounds to monitor KRT19 expression from its endogenous gene locus. A differentiation protocol to generate mature pancreatic duct-like organoids was applied. While KRT19/mCherry expression became evident at the early endoderm stage, mCherry signal was present in nearly all cells at the pancreatic endoderm (PE) and pancreatic progenitor (PP) stages. Interestingly, despite homogenous KRT19 expression, mCherry positivity dropped to 50% after ductal maturation, indicating a permanent switch from biallelic to monoallelic expression. DNA methylation profiling separated the distinct differentiation intermediates, with site-specific DNA methylation patterns occurring at the KRT19 locus during ductal maturation. Accordingly, the monoallelic switch was partially reverted upon treatment with a DNA-methyltransferase inhibitor. In human PDAC cohorts, high KRT19 levels correlate with low locus methylation and decreased survival. At the same time, activation of oncogenic KRASG12D signalling in our reporter system reversed monoallelic back to biallelic KRT19 expression in pancreatic duct-like organoids. Allelic reactivation was also detected in single-cell transcriptomes of human PDACs, which further revealed a positive correlation between KRT19 and KRAS expression. Accordingly, KRAS mutant PDACs had higher KRT19 mRNA but lower KRT19 gene locus DNA methylation than wildtype counterparts. KRT19 protein was additionally detected in plasma of PDAC patients, with higher concentrations correlating with shorter progression-free survival in gemcitabine/nabPaclitaxel-treated and opposing trends in FOLFIRINOX-treated patients. Apart from being an important pancreatic ductal lineage marker, KRT19 appears tightly controlled via a switch from biallelic to monoallelic expression during ductal lineage entry and is aberrantly expressed after oncogenic KRASG12D expression, indicating a role in PDAC development and malignancy. Soluble KRT19 might serve as a relevant biomarker to stratify treatment. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patología , Protocolos de Quimioterapia Combinada Antineoplásica , Queratina-19/genética , Queratina-19/metabolismo , Metilación de ADN , Proteínas Proto-Oncogénicas p21(ras)/genética , Carcinogénesis/genética , Carcinoma Ductal Pancreático/patología , Expresión Génica , Neoplasias PancreáticasRESUMEN
Amyotrophic Lateral Sclerosis (ALS) is a complex and incurable neurodegenerative disorder in which genetic and epigenetic factors contribute to the pathogenesis of all forms of ALS. The interplay of genetic predisposition and environmental footprints generates epigenetic signatures in the cells of affected tissues, which then alter transcriptional programs. Epigenetic modifications that arise from genetic predisposition and systemic environmental footprints should in theory be detectable not only in affected CNS tissue but also in the periphery. Here, we identify an ALS-associated epigenetic signature ('epiChromALS') by chromatin accessibility analysis of blood cells of ALS patients. In contrast to the blood transcriptome signature, epiChromALS includes also genes that are not expressed in blood cells; it is enriched in CNS neuronal pathways and it is present in the ALS motor cortex. By combining simultaneous ATAC-seq and RNA-seq with single-cell sequencing in PBMCs and motor cortex from ALS patients, we demonstrate that epigenetic changes associated with the neurodegenerative disease can be found in the periphery, thus strongly suggesting a mechanistic link between the epigenetic regulation and disease pathogenesis.
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Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Epigénesis Genética , Cromatina , Predisposición Genética a la Enfermedad , Enfermedades Neurodegenerativas/genética , Células Sanguíneas/metabolismo , Células Sanguíneas/patologíaRESUMEN
Adult zebrafish are frequently described to be able to "completely" regenerate the heart. Yet, the extent to which cardiomyocytes lost to injury are replaced is unknown, since existing evidence for cardiomyocyte proliferation is indirect or non-quantitative. We established stereological methods to quantify the number of cardiomyocytes at several time-points post cryoinjury. Intriguingly, after cryoinjuries that killed about 1/3 of the ventricular cardiomyocytes, pre-injury cardiomyocyte numbers were restored already within 30 days. Yet, many hearts retained small residual scars, and a subset of cardiomyocytes bordering these fibrotic areas remained smaller, lacked differentiated sarcomeric structures, and displayed defective calcium signaling. Thus, a subset of regenerated cardiomyocytes failed to fully mature. While lineage-tracing experiments have shown that regenerating cardiomyocytes are derived from differentiated cardiomyocytes, technical limitations have previously made it impossible to test whether cardiomyocyte trans-differentiation contributes to regeneration of non-myocyte cell lineages. Using Cre responder lines that are expressed in all major cell types of the heart, we found no evidence for cardiomyocyte transdifferentiation into endothelial, epicardial, fibroblast or immune cell lineages. Overall, our results imply a refined answer to the question whether zebrafish can completely regenerate the heart: in response to cryoinjury, preinjury cardiomyocyte numbers are indeed completely regenerated by proliferation of lineage-restricted cardiomyocytes, while restoration of cardiomyocyte differentiation and function, as well as resorption of scar tissue, is less robustly achieved.
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Corazón/fisiología , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Regeneración , Pez Cebra/metabolismo , Animales , Fibrosis , Miocardio/patología , Miocitos Cardíacos/patologíaRESUMEN
Owing to the local enrichment of factors that influence its dynamics and organization, the actin cytoskeleton displays different shapes and functions within the same cell. In yeast cells, post-Golgi vesicles ride on long actin cables to the bud tip. The proteins Boi1 and Boi2 (Boi1/2) participate in tethering and docking these vesicles to the plasma membrane. Here, we show in Saccharomyces cerevisiae that Boi1/2 also recruit nucleation and elongation factors to form actin filaments at sites of exocytosis. Disrupting the connection between Boi1/2 and the nucleation factor Bud6 impairs filament formation, reduces the directed movement of the vesicles to the tip and shortens the vesicles' tethering time at the cortex. Transplanting Boi1 from the bud tip to the peroxisomal membrane partially redirects the actin cytoskeleton and the vesicular flow towards the peroxisome, and creates an alternative, rudimentary vesicle-docking zone. We conclude that Boi1/2, through interactions with Bud6 and Bni1, induce the formation of a cortical actin structure that receives and aligns incoming vesicles before fusion with the membrane.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Polaridad Celular , Exocitosis , Proteínas de Microfilamentos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Yeast cells select the position of their new bud at the beginning of each cell cycle. The recruitment of septins to this prospective bud site is one of the critical events in a complex assembly pathway that culminates in the outgrowth of a new daughter cell. During recruitment, septin rods follow the high concentration of Cdc42GTP that is generated by the focused localization of the Cdc42 guanine-nucleotide-exchange factor Cdc24. We show that, shortly before budding, Cdc24 not only activates Cdc42 but also transiently interacts with Cdc11, the septin subunit that caps both ends of the septin rods. Mutations in Cdc24 that reduce affinity to Cdc11 impair septin recruitment and decrease the stability of the polarity patch. The interaction between septins and Cdc24 thus reinforces bud assembly at sites where septin structures are formed. Once the septins polymerize to form the septin ring, Cdc24 is found at the cortex of the bud and directs further outgrowth from this position.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Retroalimentación , Factores de Intercambio de Guanina Nucleótido , Estudios Prospectivos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Septinas/genética , Septinas/metabolismoRESUMEN
Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain homeostasis. With aging, the frequency of polar HSCs decreases. Cell polarity in HSCs is controlled by the activity of the small RhoGTPase cell division control protein 42 (Cdc42). Here we demonstrate-using a comprehensive set of paired daughter cell analyses that include single-cell 3D confocal imaging, single-cell transplants, single-cell RNA-seq, and single-cell transposase-accessible chromatin sequencing (ATAC-seq)-that the outcome of HSC divisions is strongly linked to the polarity status before mitosis, which is in turn determined by the level of the activity Cdc42 in stem cells. Aged apolar HSCs undergo preferentially self-renewing symmetric divisions, resulting in daughter stem cells with reduced regenerative capacity and lymphoid potential, while young polar HSCs undergo preferentially asymmetric divisions. Mathematical modeling in combination with experimental data implies a mechanistic role of the asymmetric sorting of Cdc42 in determining the potential of daughter cells via epigenetic mechanisms. Therefore, molecules that control HSC polarity might serve as modulators of the mode of stem cell division regulating the potential of daughter cells.
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División Celular/genética , Senescencia Celular/genética , Epigénesis Genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Envejecimiento/metabolismo , Animales , División Celular Asimétrica/genética , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Agregación Celular , Linaje de la Célula/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Cromatina , Ratones Endogámicos C57BL , Transcriptoma/genética , Proteína Wnt-5a/farmacología , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
There is high interest in understanding the mechanisms that drive self-renewal of stem cells. HOXB4 is one of the few transcription factors that can amplify long-term repopulating hematopoietic stem cells in a controlled way. Here we show in mice that this characteristic of HOXB4 depends on a proline-rich sequence near the N terminus, which is unique among HOX genes and highly conserved in higher mammals. Deletion of this domain substantially enhanced the oncogenicity of HOXB4, inducing acute leukemia in mice. Conversely, insertion of the domain into Hoxa9 impaired leukemogenicity of this homeobox gene. These results indicate that proline-rich stretches attenuate the potential of stem cell active homeobox genes to acquire oncogenic properties.
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Autorrenovación de las Células , Células Madre Hematopoyéticas/fisiología , Proteínas de Homeodominio/fisiología , Leucemia/etiología , Factores de Transcripción/fisiología , Enfermedad Aguda , Animales , Carcinógenos , Proteínas de Homeodominio/genética , Ratones , Prolina , Análisis de Secuencia de Proteína , Factores de Transcripción/genéticaRESUMEN
Recent data indicate that a variety of regulatory molecules active in embryonic development may also play a role in the regulation of early hematopoiesis. Here we report that the human Vent-like homeobox gene VENTX, a putative homolog of the Xenopus xvent2 gene, is a unique regulatory hematopoietic gene that is aberrantly expressed in CD34(+) leukemic stem-cell candidates in human acute myeloid leukemia (AML). Quantitative RT-PCR documented expression of the gene in lineage positive hematopoietic subpopulations, with the highest expression in CD33(+) myeloid cells. Notably, expression levels of VENTX were negligible in normal CD34(+)/CD38(-) or CD34(+) human progenitor cells. In contrast to this, leukemic CD34(+)/CD38(-) cells from AML patients with translocation t(8,21) and normal karyotype displayed aberrantly high expression of VENTX. Gene expression and pathway analysis demonstrated that in normal CD34(+) cells enforced expression of VENTX initiates genes associated with myeloid development and down-regulates genes involved in early lymphoid development. Functional analyses confirmed that aberrant expression of VENTX in normal CD34(+) human progenitor cells perturbs normal hematopoietic development, promoting generation of myeloid cells and impairing generation of lymphoid cells in vitro and in vivo. Stable knockdown of VENTX expression inhibited the proliferation of human AML cell lines. Taken together, these data extend our insights into the function of embryonic mesodermal factors in human postnatal hematopoiesis and indicate a role for VENTX in normal and malignant myelopoiesis.
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Regulación Leucémica de la Expresión Génica , Proteínas de Homeodominio/biosíntesis , Leucemia Mieloide Aguda/metabolismo , Células Mieloides/citología , Mielopoyesis/genética , Técnicas de Cocultivo , Células Eritroides/citología , Células Eritroides/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Humanos , Leucemia Mieloide Aguda/genética , Células Mieloides/metabolismoRESUMEN
Rationale: Pancreatic lineage specification follows the formation of tripotent pancreatic progenitors (PPs). Current protocols rebuilding PPs in vitro have an endocrine lineage bias and are mostly based on PDX1/NKX6-1 coexpression neglecting other markers decisive for PP heterogeneity and lineage potential. However, true tripotent PPs are of utmost interest to study also exocrine disorders such as pancreatic cancer and to simultaneously generate all three pancreatic lineages from the same ancestor. Methods: Here, we performed a comprehensive compound testing to advance the generation of multipotent progenitors, which were further characterized for their trilineage potential in vitro and in vivo. The heterogeneity and cell-cell communication across the PP subpopulations were analyzed via single-cell transcriptomics. Results: We introduce a novel PP differentiation platform based on a comprehensive compound screening with an advanced design of experiments computing tool to reduce impurities and to increase Glycoprotein-2 expression and subsequent trilineage potential. Superior PP tripotency was proven in vitro by the generation of acinar, endocrine, and ductal cells as well as in vivo upon orthotopic transplantation revealing all three lineages at fetal maturation level. GP2 expression levels at PP stage ascribed varying pancreatic lineage potential. Intermediate and high GP2 levels were superior in generating endocrine and duct-like organoids (PDLO). FACS-based purification of the GP2high PPs allowed the generation of pancreatic acinar-like organoids (PALO) with proper morphology and expression of digestive enzymes. scRNA-seq confirmed multipotent identity, positioned the GP2/PDX1/NKX6-1high population next to human fetal tip and trunk progenitors and identified novel ligand-receptor (LR) interactions in distinct PP subpopulations. LR validation experiments licensed midkine and VEGF signaling to increase markers labelling the single cell clusters with high GP2 expression. Conclusion: In this study, we guide human pluripotent stem cells into multipotent pancreatic progenitors. This common precursor population, which has the ability to mature into acinar, ductal and functional ß-cells, serves as a basis for studying developmental processes and deciphering early cancer formation in a cell type-specific context. Using single-cell RNA sequencing and subsequent validation studies, we were able to dissect PP heterogeneity and specific cell-cell communication signals.
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Células Secretoras de Insulina , Células Madre Pluripotentes , Humanos , Páncreas/metabolismo , Diferenciación Celular/fisiología , Células Secretoras de Insulina/metabolismo , OrganoidesRESUMEN
BACKGROUND AND AIMS: Development of pancreatic ductal adenocarcinoma (PDAC) is a multistep process intensively studied; however, precocious diagnosis and effective therapy still remain unsatisfactory. The role for Notch signaling in PDAC has been discussed controversially, as both cancer-promoting and cancer-antagonizing functions have been described. Thus, an improved understanding of the underlying molecular mechanisms is necessary. Here, we focused on RBPJ, the receiving transcription factor in the Notch pathway, examined its expression pattern in PDAC, and characterized its function in mouse models of pancreatic cancer development and in the regeneration process after acute pancreatitis. METHODS: Conditional transgenic mouse models were used for functional analysis of RBPJ in the adult pancreas, initiation of PDAC precursor lesions, and pancreatic regeneration. Pancreata and primary acinar cells were tested for acinar-to-ductal metaplasia together with immunohistology and comprehensive transcriptional profiling by RNA sequencing. RESULTS: We identified reduced RBPJ expression in a subset of human PDAC specimens. Ptf1α-CreERT-driven depletion of RBPJ in transgenic mice revealed that its function is dispensable for the homeostasis and maintenance of adult acinar cells. However, primary RBPJ-deficient acinar cells underwent acinar-to-ductal differentiation in ex vivo. Importantly, oncogenic KRAS expression in the context of RBPJ deficiency facilitated the development of pancreatic intraepithelial neoplasia lesions with massive fibrotic stroma formation. Interestingly, RNA-sequencing data revealed a transcriptional profile associated with the cytokine/chemokine and extracellular matrix changes. In addition, lack of RBPJ delays the course of acute pancreatitis and critically impairs it in the context of KRASG12D expression. CONCLUSIONS: Our findings imply that downregulation of RBPJ in PDAC patients derepresses Notch targets and promotes KRAS-mediated pancreatic acinar cells transformation and desmoplasia development.
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Carcinoma in Situ , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Pancreatitis , Animales , Humanos , Ratones , Células Acinares/metabolismo , Enfermedad Aguda , Carcinoma in Situ/metabolismo , Carcinoma Ductal Pancreático/patología , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Ratones Transgénicos , Neoplasias Pancreáticas/patología , Pancreatitis/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias PancreáticasRESUMEN
Quantitative 3D imaging of organ-wide cellular and subcellular components is central for revealing and understanding complex interactions between stem cells and their microenvironment. Here, we present a gentle but fast whole-mount immunofluorescence staining protocol for 3D confocal microscopy (iFAST3D) that preserves the 3D structure of the entire tissue and that of subcellular structures with high fidelity. The iFAST3D protocol enables reproducible and high-resolution 3D imaging of stem cells and various niche components for many mouse organs and tissues. For complete details on the use and execution of this protocol, please refer to Saçma et al. (2019).
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Imagenología Tridimensional , Células Madre , Animales , Imagenología Tridimensional/métodos , Ratones , Microscopía Confocal/métodos , Coloración y EtiquetadoRESUMEN
Upon aging, the function of the intestinal epithelium declines with a concomitant increase in aging-related diseases. ISCs play an important role in this process. It is known that ISC clonal dynamics follow a neutral drift model. However, it is not clear whether the drift model is still valid in aged ISCs. Tracking of clonal dynamics by clonal tracing revealed that aged crypts drift into monoclonality substantially faster than young ones. However, ISC tracing experiments, in vivo and ex vivo, implied a similar clonal expansion ability of both young and aged ISCs. Single-cell RNA sequencing for 1,920 high Lgr5 ISCs from young and aged mice revealed increased heterogeneity among subgroups of aged ISCs. Genes associated with cell adhesion were down-regulated in aged ISCs. ISCs of aged mice indeed show weaker adhesion to the matrix. Simulations applying a single cell-based model of the small intestinal crypt demonstrated an accelerated clonal drift at reduced adhesion strength, implying a central role for reduced adhesion for affecting clonal dynamics upon aging.
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Intestinos , Células Madre , Animales , Células Cultivadas , Íleon , Mucosa Intestinal/metabolismo , Ratones , Células Madre/metabolismoRESUMEN
The complexity of signaling events and cellular responses unfolding in neuronal, glial, and immune cells upon traumatic brain injury (TBI) constitutes an obstacle in elucidating pathophysiological links and targets for intervention. We use array phosphoproteomics in a murine mild blunt TBI to reconstruct the temporal dynamics of tyrosine-kinase signaling in TBI and then scrutinize the large-scale effects of perturbation of Met/HGFR, VEGFR1, and Btk signaling by small molecules. We show Met/HGFR as a selective modifier of early microglial response and that Met/HGFR blockade prevents the induction of microglial inflammatory mediators, of reactive microglia morphology, and TBI-associated responses in neurons and vasculature. Both acute and prolonged Met/HGFR inhibition ameliorate neuronal survival and motor recovery. Early elevation of HGF itself in the cerebrospinal fluid of TBI patients suggests that this mechanism has translational value in human subjects. Our findings identify Met/HGFR as a modulator of early neuroinflammation in TBI with promising translational potential.
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Lesiones Traumáticas del Encéfalo , Microglía , Humanos , Ratones , Animales , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
The hematopoietic stem cell (HSC) niche is a specialized microenvironment, where a complex and dynamic network of interactions across multiple cell types regulates HSC function. During the last years, it became progressively clearer that changes in the HSC niche are responsible for specific alterations of HSC behavior. The aging of the bone marrow (BM) microenvironment has been shown to critically contribute to the decline in HSC function over time. Interestingly, while upon aging some niche structures within the BM are degenerated and negatively affect HSC functionality, other niche cells and specific signals are preserved and essential to retaining HSC function and regenerative capacity. These new findings on the role of the aging BM niche critically depend on the implementation of new technical tools, developed thanks to transdisciplinary approaches, which bring together different scientific fields. For example, the development of specific mouse models in addition to coculture systems, new 3D-imaging tools, ossicles, and ex-vivo BM mimicking systems is highlighting the importance of new technologies to unravel the complexity of the BM niche on aging. Of note, an exponential impact in the understanding of this biological system has been recently brought by single-cell sequencing techniques, spatial transcriptomics, and implementation of artificial intelligence and deep learning approaches to data analysis and integration. This review focuses on how the aging of the BM niche affects HSCs and on the new tools to investigate the specific alterations occurring in the BM upon aging. All these new advances in the understanding of the BM niche and its regulatory function on HSCs have the potential to lead to novel therapeutical approaches to preserve HSC function upon aging and disease.
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Envejecimiento/fisiología , Médula Ósea/fisiología , Células Madre Hematopoyéticas/fisiología , Nicho de Células Madre/fisiología , Animales , Aprendizaje Profundo , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Análisis de la Célula IndividualRESUMEN
During X chromosome inactivation (XCI), the inactive X chromosome (Xi) is recruited to the nuclear lamina at the nuclear periphery. Beside X chromosome reactivation resulting in a highly penetrant aging-like hematopoietic malignancy, little is known about XCI in aged hematopoietic stem cells (HSCs). Here, we demonstrate that LaminA/C defines a distinct repressive nuclear compartment for XCI in young HSCs, and its reduction in aged HSCs correlates with an impairment in the overall control of XCI. Integrated omics analyses reveal higher variation in gene expression, global hypomethylation, and significantly increased chromatin accessibility on the X chromosome (Chr X) in aged HSCs. In summary, our data support the role of LaminA/C in the establishment of a special repressive compartment for XCI in HSCs, which is impaired upon aging.
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Senescencia Celular/genética , Células Madre Hematopoyéticas/metabolismo , Inactivación del Cromosoma X/genética , Animales , Cromatina/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Humanos , Lamina Tipo A/metabolismo , Ratones Endogámicos C57BL , Transposasas/metabolismo , Cromosoma X/genéticaRESUMEN
Histone methylases and demethylases regulate gene expression programs in hematopoiesis. The molecular function of the demethylase KDM6A in normal hematopoiesis and, in particular, for the hematopoietic stem and progenitor cell (HSPC) compartment remains only partially understood. Female but not male Kdm6a-/- HSPCs were functionally impaired in adoptive transfer experiments as well as upon proliferative stress induced by 5-fluorouracil. Loss of Kdm6a affected primarily early B cells and erythroid and myeloid progenitor cells with respect to both number and function. Global gene expression analyses revealed a shared altered gene signature in Kdm6a-/- pro-B and pre-B cells that is also present in HSPCs, supporting that altered B-cell differentiation in Kdm6a-/- animals is already initiated in HSPCs. Interestingly, loss of KDM6A did not affect the global level of methylation of H3K27, its presumed target, in hematopoietic cells. Our data indicate a critical role for KDM6A in the regulation of hematopoietic differentiation and differentiation-specific gene expression programs, with a prominent role in early B-cell differentiation that is likely independent of H3K27 methylation status.
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Diferenciación Celular , Hematopoyesis , Histona Demetilasas/metabolismo , Histonas/metabolismo , Células Precursoras de Linfocitos B/enzimología , Estrés Fisiológico , Animales , Histona Demetilasas/genética , Histonas/genética , Metilación , Ratones , Ratones NoqueadosRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which is still missing effective therapeutic strategies. Although manipulation of neuronal excitability has been tested in murine and human ALS models, it is still under debate whether neuronal activity might represent a valid target for efficient therapies. In this study, we exploited a combination of transcriptomics, proteomics, optogenetics and pharmacological approaches to investigate the activity-related pathological features of iPSC-derived C9orf72-mutant motoneurons (MN). We found that human ALSC9orf72 MN are characterized by accumulation of aberrant aggresomes, reduced expression of synaptic genes, loss of synaptic contacts and a dynamic "malactivation" of the transcription factor CREB. A similar phenotype was also found in TBK1-mutant MN and upon overexpression of poly(GA) aggregates in primary neurons, indicating a strong convergence of pathological phenotypes on synaptic dysregulation. Notably, these alterations, along with neuronal survival, could be rescued by treating ALS-related neurons with the K+ channel blockers Apamin and XE991, which, respectively, target the SK and the Kv7 channels. Thus, our study shows that restoring the activity-dependent transcriptional programme and synaptic composition exerts a neuroprotective effect on ALS disease progression.
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Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Fármacos Neuroprotectores , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Animales , Humanos , Ratones , Neuronas MotorasRESUMEN
Cdc42 organizes cellular polarity and directs the formation of cellular structures in many organisms. By locating Cdc24, the source of active Cdc42, to the growing front of the yeast cell, the scaffold protein Bem1, is instrumental in shaping the cellular gradient of Cdc42. This gradient instructs bud formation, bud growth, or cytokinesis through the actions of a diverse set of effector proteins. To address how Bem1 participates in these transformations, we systematically tracked its protein interactions during one cell cycle to define the ensemble of Bem1 interaction states for each cell cycle stage. Mutants of Bem1 that interact with only a discrete subset of the interaction partners allowed to assign specific functions to different interaction states and identified the determinants for their cellular distributions. The analysis characterizes Bem1 as a cell cycle-specific shuttle that distributes active Cdc42 from its source to its effectors. It further suggests that Bem1 might convert the PAKs Cla4 and Ste20 into their active conformations.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/fisiología , Secuencia de Aminoácidos/genética , Ciclo Celular , Proteínas de Ciclo Celular/química , División Celular , Polaridad Celular , Factores de Intercambio de Guanina Nucleótido/metabolismo , Unión Proteica/fisiología , Proteínas Serina-Treonina Quinasas/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomycetales/metabolismo , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/fisiología , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/genética , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae/metabolismoRESUMEN
Ph+ acute lymphoblastic leukemia (ALL) is characterized by the expression of an oncogenic fusion kinase termed BCR-ABL1. Here, we show that interleukin 7 receptor (IL7R) interacts with the chemokine receptor CXCR4 to recruit BCR-ABL1 and JAK kinases in close proximity. Treatment with BCR-ABL1 kinase inhibitors results in elevated expression of IL7R which enables the survival of transformed cells when IL7 was added together with the kinase inhibitors. Importantly, treatment with anti-IL7R antibodies prevents leukemia development in xenotransplantation models using patient-derived Ph+ ALL cells. Our results suggest that the association between IL7R and CXCR4 serves as molecular platform for BCR-ABL1-induced transformation and development of Ph+ ALL. Targeting this platform with anti-IL7R antibody eliminates Ph+ ALL cells including those with resistance to commonly used ABL1 kinase inhibitors. Thus, anti-IL7R antibodies may provide alternative treatment options for ALL in general and may suppress incurable drug-resistant leukemia forms.
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Proteínas de Fusión bcr-abl/metabolismo , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores CXCR4/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/efectos de los fármacos , Femenino , Proteína Forkhead Box O1/metabolismo , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-7/farmacología , Subunidad alfa del Receptor de Interleucina-7/antagonistas & inhibidores , Subunidad alfa del Receptor de Interleucina-7/genética , Ratones , Ratones Mutantes , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Receptores CXCR4/genética , Transducción de Señal/efectos de los fármacosRESUMEN
With ageing, intrinsic haematopoietic stem cell (HSC) activity decreases, resulting in impaired tissue homeostasis, reduced engraftment following transplantation and increased susceptibility to diseases. However, whether ageing also affects the HSC niche, and thereby impairs its capacity to support HSC function, is still widely debated. Here, by using in-vivo long-term label-retention assays we demonstrate that aged label-retaining HSCs, which are, in old mice, the most quiescent HSC subpopulation with the highest regenerative capacity and cellular polarity, reside predominantly in perisinusoidal niches. Furthermore, we demonstrate that sinusoidal niches are uniquely preserved in shape, morphology and number on ageing. Finally, we show that myeloablative chemotherapy can selectively disrupt aged sinusoidal niches in the long term, which is linked to the lack of recovery of endothelial Jag2 at sinusoids. Overall, our data characterize the functional alterations of the aged HSC niche and unveil that perisinusoidal niches are uniquely preserved and thereby protect HSCs from ageing.