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1.
Blood ; 142(4): 336-351, 2023 07 27.
Artículo en Inglés | MEDLINE | ID: mdl-36947815

RESUMEN

Structural variants (SVs) involving enhancer hijacking can rewire chromatin topologies to cause oncogene activation in human cancers, including hematologic malignancies; however, because of the lack of tools to assess their effects on gene regulation and chromatin organization, the molecular determinants for the functional output of enhancer hijacking remain poorly understood. Here, we developed a multimodal approach to integrate genome sequencing, chromosome conformation, chromatin state, and transcriptomic alteration for quantitative analysis of transcriptional effects and structural reorganization imposed by SVs in leukemic genomes. We identified known and new pathogenic SVs, including recurrent t(5;14) translocations that cause the hijacking of BCL11B enhancers for the allele-specific activation of TLX3 in a subtype of pediatric leukemia. Epigenetic perturbation of SV-hijacked BCL11B enhancers impairs TLX3 transcription, which are required for the growth of t(5;14) leukemia cells. By CRISPR engineering of patient-derived t(5;14) in isogenic leukemia cells, we uncovered a new mechanism whereby the transcriptional output of SV-induced BCL11B enhancer hijacking is dependent on the loss of DNA hypermethylation at the TLX3 promoter. Our results highlight the importance of the cooperation between genetic alteration and permissive chromatin as a critical determinant of SV-mediated oncogene activation, with implications for understanding aberrant gene transcription after epigenetic therapies in patients with leukemia. Hence, leveraging the interdependency of genetic alteration on chromatin variation may provide new opportunities to reprogram gene regulation as targeted interventions in human disease.


Asunto(s)
Cromatina , Leucemia , Humanos , Niño , Cromatina/genética , Elementos de Facilitación Genéticos , Cromosomas/metabolismo , Factores de Transcripción/genética , Leucemia/genética , Proteínas Supresoras de Tumor/genética , Proteínas Represoras/genética
2.
Br J Haematol ; 205(2): 607-612, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38385580

RESUMEN

T/myeloid mixed phenotype acute leukaemia (MPAL) is a rare aggressive acute leukaemia with poorly understood pathogenesis. Herein, we report two cases of T/myeloid MPAL harbouring BCL11B-associated structural variants that activate TLX3 (TLX3::BCL11B-TLX3-activation) by genome sequencing and transcriptomic analyses. Both patients were young males with extramedullary involvement. Cooperative gene alterations characteristic of T/myeloid MPAL and T-lymphoblastic leukaemia (T-ALL) were detected. Both patients achieved initial remission following lineage-matched ALL-based therapy with one patient requiring a lineage-switched myeloid-based therapy. Our study is the first to demonstrate the clinicopathological and genomic features of TLX3::BCL11B-TLX3-activated T/myeloid MPAL and provide insights into leukaemogenesis.


Asunto(s)
Proteínas Represoras , Humanos , Masculino , Proteínas Represoras/genética , Adulto , Proteínas de Fusión Oncogénica/genética , Leucemia Bifenotípica Aguda/genética , Leucemia Bifenotípica Aguda/patología , Proteínas Supresoras de Tumor/genética , Proteínas de Homeodominio/genética
3.
Proc Natl Acad Sci U S A ; 116(37): 18455-18465, 2019 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-31451669

RESUMEN

The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. To uncover the molecular mechanisms underlying neonatal heart regeneration, we compared the transcriptomes and epigenomes of regenerative and nonregenerative mouse hearts over a 7-d time period following myocardial infarction injury. By integrating gene expression profiles with histone marks associated with active or repressed chromatin, we identified transcriptional programs underlying neonatal heart regeneration, and the blockade to regeneration in later life. Our results reveal a unique immune response in regenerative hearts and a retained embryonic cardiogenic gene program that is active during neonatal heart regeneration. Among the unique immune factors and embryonic genes associated with cardiac regeneration, we identified Ccl24, which encodes a cytokine, and Igf2bp3, which encodes an RNA-binding protein, as previously unrecognized regulators of cardiomyocyte proliferation. Our data provide insights into the molecular basis of neonatal heart regeneration and identify genes that can be modulated to promote heart regeneration.


Asunto(s)
Animales Recién Nacidos/fisiología , Corazón/fisiología , Código de Histonas/fisiología , Regeneración/fisiología , Transcriptoma/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Lesiones Cardíacas/genética , Lesiones Cardíacas/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Infarto del Miocardio/genética , Regeneración/genética , Transcriptoma/genética
4.
Exp Dermatol ; 30(11): 1711-1716, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34036652

RESUMEN

BACKGROUND: Human papillomavirus (HPV) infection is known to promote the development of mucosal squamous cell carcinoma (mSCC), including pathologically high-grade lesions, but its role in cutaneous squamous cell carcinoma (cuSCC) remains unclear, particularly in lesions that are considered high risk. OBJECTIVE: We aimed to determine whether enhanced HPV transcriptional activity can be detected in high-risk cuSCC samples compared with low-grade SCC samples or normal skin. METHODS: We performed RNA sequencing of cuSCC across 23 risk-stratified skin lesions. A subset of samples was tested for the presence of HPV DNA. High-quality, non-human reads from each sample group were used for viral analysis using Microbiome Coverage Profiler. RESULTS: None of the samples analysed had detectable expression of HPV RNA, while 64% of samples tested positive for HPV DNA. All samples were found to have expression of human endogenous retrovirus, and multiple samples showed expression of other viruses. CONCLUSIONS: Viral and prophage gene expression can be monitored in cuSCC or normal skin biopsies, yet no sample in our study showed evidence of active HPV gene expression despite evidence of HPV genome presence. This suggests HPV transcription does not play a role in differentiating high-risk cuSCCs from low-risk cuSCCs or normal skin.


Asunto(s)
Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/virología , Expresión Génica , Papillomaviridae/genética , Infecciones por Papillomavirus/complicaciones , Infecciones por Papillomavirus/patología , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/virología , Anciano , Biopsia , Sondas de ADN de HPV , Femenino , Humanos , Masculino , Medición de Riesgo
5.
PLoS Pathog ; 13(7): e1006496, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28704551

RESUMEN

Successful host defense against pathogens requires innate immune recognition of the correct pathogen associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs) to trigger the appropriate gene program tailored to the pathogen. While many PRR pathways contribute to the innate immune response to specific pathogens, the relative importance of each pathway for the complete transcriptional program elicited has not been examined in detail. Herein, we used RNA-sequencing with wildtype and mutant macrophages to delineate the innate immune pathways contributing to the early transcriptional response to Staphylococcus aureus, a ubiquitous microorganism that can activate a wide variety of PRRs. Unexpectedly, two PRR pathways-the Toll-like receptor (TLR) and Stimulator of Interferon Gene (STING) pathways-were identified as dominant regulators of approximately 95% of the genes that were potently induced within the first four hours of macrophage infection with live S. aureus. TLR signaling predominantly activated a pro-inflammatory program while STING signaling activated an antiviral/type I interferon response with live but not killed S. aureus. This STING response was largely dependent on the cytosolic DNA sensor cyclic guanosine-adenosine synthase (cGAS). Using a cutaneous infection model, we found that the TLR and STING pathways played opposite roles in host defense to S. aureus. TLR signaling was required for host defense, with its absence reducing interleukin (IL)-1ß production and neutrophil recruitment, resulting in increased bacterial growth. In contrast, absence of STING signaling had the opposite effect, enhancing the ability to restrict the infection. These results provide novel insights into the complex interplay of innate immune signaling pathways triggered by S. aureus and uncover opposing roles of TLR and STING in cutaneous host defense to S. aureus.


Asunto(s)
Citosol/inmunología , Proteínas de la Membrana/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/fisiología , Receptores Toll-Like/inmunología , Animales , Citosol/microbiología , ADN , ADN Bacteriano/genética , ADN Bacteriano/inmunología , Femenino , Humanos , Inmunidad Innata , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Transducción de Señal , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Receptores Toll-Like/genética
6.
Methods Mol Biol ; 2599: 69-97, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36427144

RESUMEN

The spatiotemporal control of tissue-specific gene expression is coordinated by cis-regulatory elements (CREs) and associated trans-acting factors. Despite major advances in genome-wide annotation of candidate CREs, the in situ regulatory composition of the vast majority of CREs remain unknown. To address this challenge, we developed the CRISPR affinity purification in situ of regulatory elements (CAPTURE) toolbox that employs an in vivo biotinylated nuclease-deficient Cas9 (dCas9) protein and programmable single-guide RNAs (sgRNAs) to identify CRE-associated macromolecular complexes and chromatin looping. In this chapter, we provide a detailed protocol for implementing the latest iteration of the CRISPR-based CAPTURE methods to interrogate the molecular composition of locus-specific chromatin complexes and configuration in a mammalian genome.


Asunto(s)
Cromatina , Escarabajos , Animales , Cromatina/genética , Cromatografía de Afinidad , Proteína 9 Asociada a CRISPR , Endonucleasas , Mamíferos
7.
Sci Adv ; 9(13): eadg1123, 2023 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-37000871

RESUMEN

Biomolecular condensates participate in the regulation of gene transcription, yet the relationship between nuclear condensation and transcriptional activation remains elusive. Here, we devised a biotinylated CRISPR-dCas9-based optogenetic method, light-activated macromolecular phase separation (LAMPS), to enable inducible formation, affinity purification, and multiomic dissection of nuclear condensates at the targeted genomic loci. LAMPS-induced condensation at enhancers and promoters activates endogenous gene transcription by chromatin reconfiguration, causing increased chromatin accessibility and de novo formation of long-range chromosomal loops. Proteomic profiling of light-induced condensates by dCas9-mediated affinity purification uncovers multivalent interaction-dependent remodeling of macromolecular composition, resulting in the selective enrichment of transcriptional coactivators and chromatin structure proteins. Our findings support a model whereby the formation of nuclear condensates at native genomic loci reconfigures chromatin architecture and multiprotein assemblies to modulate gene transcription. Hence, LAMPS facilitates mechanistic interrogation of the relationship between nuclear condensation, genome structure, and gene transcription in living cells.


Asunto(s)
Cromatina , Proteómica , Cromatina/genética , Núcleo Celular/genética , Factores de Transcripción/genética , Genoma
8.
J Clin Invest ; 133(7)2023 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-36809258

RESUMEN

Although certain human genetic variants are conspicuously loss of function, decoding the impact of many variants is challenging. Previously, we described a patient with leukemia predisposition syndrome (GATA2 deficiency) with a germline GATA2 variant that inserts 9 amino acids between the 2 zinc fingers (9aa-Ins). Here, we conducted mechanistic analyses using genomic technologies and a genetic rescue system with Gata2 enhancer-mutant hematopoietic progenitor cells to compare how GATA2 and 9aa-Ins function genome-wide. Despite nuclear localization, 9aa-Ins was severely defective in occupying and remodeling chromatin and regulating transcription. Variation of the inter-zinc finger spacer length revealed that insertions were more deleterious to activation than repression. GATA2 deficiency generated a lineage-diverting gene expression program and a hematopoiesis-disrupting signaling network in progenitors with reduced granulocyte-macrophage colony-stimulating factor (GM-CSF) and elevated IL-6 signaling. As insufficient GM-CSF signaling caused pulmonary alveolar proteinosis and excessive IL-6 signaling promoted bone marrow failure and GATA2 deficiency patient phenotypes, these results provide insight into mechanisms underlying GATA2-linked pathologies.


Asunto(s)
Deficiencia GATA2 , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Humanos , Deficiencia GATA2/genética , Interleucina-6/genética , Hematopoyesis/genética , Expresión Génica , Dedos de Zinc/genética , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo
9.
Nat Commun ; 12(1): 6241, 2021 10 29.
Artículo en Inglés | MEDLINE | ID: mdl-34716321

RESUMEN

Precise control of gene expression during differentiation relies on the interplay of chromatin and nuclear structure. Despite an established contribution of nuclear membrane proteins to developmental gene regulation, little is known regarding the role of inner nuclear proteins. Here we demonstrate that loss of the nuclear scaffolding protein Matrin-3 (Matr3) in erythroid cells leads to morphological and gene expression changes characteristic of accelerated maturation, as well as broad alterations in chromatin organization similar to those accompanying differentiation. Matr3 protein interacts with CTCF and the cohesin complex, and its loss perturbs their occupancy at a subset of sites. Destabilization of CTCF and cohesin binding correlates with altered transcription and accelerated differentiation. This association is conserved in embryonic stem cells. Our findings indicate Matr3 negatively affects cell fate transitions and demonstrate that a critical inner nuclear protein impacts occupancy of architectural factors, culminating in broad effects on chromatin organization and cell differentiation.


Asunto(s)
Cromatina/química , Leucemia Eritroblástica Aguda/patología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/fisiología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias/fisiología , Células Eritroides/patología , Leucemia Eritroblástica Aguda/metabolismo , Ratones Noqueados , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas de Unión al ARN/genética , Cohesinas
10.
Nat Genet ; 53(5): 672-682, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33833453

RESUMEN

Transposable elements or transposons are major players in genetic variability and genome evolution. Aberrant activation of long interspersed element-1 (LINE-1 or L1) retrotransposons is common in human cancers, yet their tumor-type-specific functions are poorly characterized. We identified MPHOSPH8/MPP8, a component of the human silencing hub (HUSH) complex, as an acute myeloid leukemia (AML)-selective dependency by epigenetic regulator-focused CRISPR screening. Although MPP8 is dispensable for steady-state hematopoiesis, MPP8 loss inhibits AML development by reactivating L1s to induce the DNA damage response and cell cycle exit. Activation of endogenous or ectopic L1s mimics the phenotype of MPP8 loss, whereas blocking retrotransposition abrogates MPP8-deficiency-induced phenotypes. Expression of AML oncogenic mutations promotes L1 suppression, and enhanced L1 silencing is associated with poor prognosis in human AML. Hence, while retrotransposons are commonly recognized for their cancer-promoting functions, we describe a tumor-suppressive role for L1 retrotransposons in myeloid leukemia.


Asunto(s)
Silenciador del Gen , Leucemia Mieloide/genética , Elementos de Nucleótido Esparcido Largo/genética , Animales , Sistemas CRISPR-Cas/genética , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Epigénesis Genética , Regulación Leucémica de la Expresión Génica , Genoma Humano , Inestabilidad Genómica , Hematopoyesis/genética , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoproteínas/genética
11.
Genome Biol ; 21(1): 59, 2020 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-32138752

RESUMEN

The spatiotemporal control of 3D genome is fundamental for gene regulation, yet it remains challenging to profile high-resolution chromatin structure at cis-regulatory elements (CREs). Using C-terminally biotinylated dCas9, endogenous biotin ligases, and pooled sgRNAs, we describe the dCas9-based CAPTURE method for multiplexed analysis of locus-specific chromatin interactions. The redesigned system allows for quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling comparisons across CREs within and between gene clusters. Multiplexed analyses of the spatiotemporal configuration of erythroid super-enhancers and promoter-centric interactions reveal organizational principles of genome structure and function.


Asunto(s)
Proteína 9 Asociada a CRISPR , Cromatina/química , Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Biotinilación , Diferenciación Celular/genética , Células Eritroides , Sitios Genéticos , Región de Control de Posición , Globinas beta/genética
12.
Cell Stem Cell ; 25(1): 69-86.e5, 2019 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-31080136

RESUMEN

The cardiogenic transcription factors (TFs) Mef2c, Gata4, and Tbx5 can directly reprogram fibroblasts to induced cardiac-like myocytes (iCLMs), presenting a potential source of cells for cardiac repair. While activity of these TFs is enhanced by Hand2 and Akt1, their genomic targets and interactions during reprogramming are not well studied. We performed genome-wide analyses of cardiogenic TF binding and enhancer profiling during cardiac reprogramming. We found that these TFs synergistically activate enhancers highlighted by Mef2c binding sites and that Hand2 and Akt1 coordinately recruit other TFs to enhancer elements. Intriguingly, these enhancer landscapes collectively resemble patterns of enhancer activation during embryonic cardiogenesis. We further constructed a cardiac reprogramming gene regulatory network and found repression of EGFR signaling pathway genes. Consistently, chemical inhibition of EGFR signaling augmented reprogramming. Thus, by defining epigenetic landscapes these findings reveal synergistic transcriptional activation across a broad landscape of cardiac enhancers and key signaling pathways that govern iCLM reprogramming.


Asunto(s)
Receptores ErbB/metabolismo , Fibroblastos/fisiología , Miocitos Cardíacos/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Cultivadas , Reprogramación Celular , Receptores ErbB/genética , Factor de Transcripción GATA4/genética , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo , Factores de Transcripción MEF2/genética , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Proteínas de Dominio T Box/genética
13.
Stem Cell Reports ; 11(4): 869-882, 2018 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-30197120

RESUMEN

Understanding the cellular properties controlling neural stem and progenitor cell (NSPC) fate choice will improve their therapeutic potential. The electrophysiological measure whole-cell membrane capacitance reflects fate bias in the neural lineage but the cellular properties underlying membrane capacitance are poorly understood. We tested the hypothesis that cell surface carbohydrates contribute to NSPC membrane capacitance and fate. We found NSPCs differing in fate potential express distinct patterns of glycosylation enzymes. Screening several glycosylation pathways revealed that the one forming highly branched N-glycans differs between neurogenic and astrogenic populations of cells in vitro and in vivo. Enhancing highly branched N-glycans on NSPCs significantly increases membrane capacitance and leads to the generation of more astrocytes at the expense of neurons with no effect on cell size, viability, or proliferation. These data identify the N-glycan branching pathway as a significant regulator of membrane capacitance and fate choice in the neural lineage.


Asunto(s)
Linaje de la Célula , Membrana Celular/metabolismo , Fenómenos Electrofisiológicos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Polisacáridos/metabolismo , Acetilglucosamina/metabolismo , Animales , Astrocitos/citología , Encéfalo/citología , Diferenciación Celular , Proliferación Celular , Tamaño de la Célula , Supervivencia Celular , Fucosa/metabolismo , Regulación de la Expresión Génica , Glicosilación , Ratones , Ácido N-Acetilneuramínico/metabolismo , Neurogénesis , Nicho de Células Madre
14.
Acta Biomater ; 43: 122-138, 2016 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-27475528

RESUMEN

UNLABELLED: Human neural stem/progenitor cells (hNSPCs) are good candidates for treating central nervous system (CNS) trauma since they secrete beneficial trophic factors and differentiate into mature CNS cells; however, many cells die after transplantation. This cell death can be ameliorated by inclusion of a biomaterial scaffold, making identification of optimal scaffolds for hNSPCs a critical research focus. We investigated the properties of fibrin-based scaffolds and their effects on hNSPCs and found that fibrin generated from salmon fibrinogen and thrombin stimulates greater hNSPC proliferation than mammalian fibrin. Fibrin scaffolds degrade over the course of a few days in vivo, so we sought to develop a novel scaffold that would retain the beneficial properties of fibrin but degrade more slowly to provide longer support for hNSPCs. We found combination scaffolds of salmon fibrin with interpenetrating networks (IPNs) of hyaluronic acid (HA) with and without laminin polymerize more effectively than fibrin alone and generate compliant hydrogels matching the physical properties of brain tissue. Furthermore, combination scaffolds support hNSPC proliferation and differentiation while significantly attenuating the cell-mediated degradation seen with fibrin alone. HNSPCs express two fibrinogen-binding integrins, αVß1 and α5ß1, and several laminin binding integrins (α7ß1, α6ß1, α3ß1) that can mediate interaction with the scaffold. Lastly, to test the ability of scaffolds to support vascularization, we analyzed human cord blood-derived endothelial cells alone and in co-culture with hNSPCs and found enhanced vessel formation and complexity in co-cultures within combination scaffolds. Overall, combination scaffolds of fibrin, HA, and laminin are excellent biomaterials for hNSPCs. STATEMENT OF SIGNIFICANCE: Interest has increased recently in the development of biomaterials as neural stem cell transplantation scaffolds to treat central nervous system (CNS) injury since scaffolds improve survival and integration of transplanted cells. We report here on a novel combination scaffold composed of fibrin, hyaluronic acid, and laminin to support human neural stem/progenitor cell (hNSPC) function. This combined biomaterial scaffold has appropriate physical properties for hNSPCs and the CNS, supports hNSPC proliferation and differentiation, and attenuates rapid cell-mediated scaffold degradation. The hNSPCs and scaffold components synergistically encourage new vessel formation from human endothelial cells. This work marks the first report of a combination scaffold supporting human neural and vascular cells to encourage vasculogenesis, and sets a benchmark for biomaterials to treat CNS injury.


Asunto(s)
Vasos Sanguíneos/fisiología , Fibrina/farmacología , Ácido Hialurónico/farmacología , Laminina/farmacología , Células-Madre Neurales/citología , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Vasos Sanguíneos/efectos de los fármacos , Bovinos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Integrinas/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Polimerizacion/efectos de los fármacos , Salmón
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