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1.
Nat Commun ; 12(1): 1045, 2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33594072

RESUMEN

Recurring chromosomal translocation t(10;17)(p15;q21) present in a subset of human acute myeloid leukemia (AML) patients creates an aberrant fusion gene termed ZMYND11-MBTD1 (ZM); however, its function remains undetermined. Here, we show that ZM confers primary murine hematopoietic stem/progenitor cells indefinite self-renewal capability ex vivo and causes AML in vivo. Genomics profilings reveal that ZM directly binds to and maintains high expression of pro-leukemic genes including Hoxa, Meis1, Myb, Myc and Sox4. Mechanistically, ZM recruits the NuA4/Tip60 histone acetyltransferase complex to cis-regulatory elements, sustaining an active chromatin state enriched in histone acetylation and devoid of repressive histone marks. Systematic mutagenesis of ZM demonstrates essential requirements of Tip60 interaction and an H3K36me3-binding PWWP (Pro-Trp-Trp-Pro) domain for oncogenesis. Inhibitor of histone acetylation-'reading' bromodomain proteins, which act downstream of ZM, is efficacious in treating ZM-induced AML. Collectively, this study demonstrates AML-causing effects of ZM, examines its gene-regulatory roles, and reports an attractive mechanism-guided therapeutic strategy.


Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Co-Represoras/química , Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Leucemia Mieloide Aguda/patología , Lisina Acetiltransferasa 5/metabolismo , Acetilación , Animales , Carcinogénesis , Diferenciación Celular , Proliferación Celular , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Elementos de Facilitación Genéticos/genética , Regulación Leucémica de la Expresión Génica , Genoma Humano , Células HEK293 , Células Madre Hematopoyéticas/metabolismo , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Ratones Endogámicos BALB C , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas de Fusión Oncogénica/metabolismo , Unión Proteica , Dominios Proteicos , Factores de Transcripción/metabolismo
2.
BMJ Case Rep ; 14(2)2021 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-33563691

RESUMEN

While sporadic inclusion body myositis (sIBM) is the most commonly acquired inflammatory myopathy above 50 years of age, its refractory response to conventional immunosuppressive treatments raises questions about its perplexing pathogenesis. Muscle biopsy typically reveals major histocompatibility complex I antigens and CD8+ T cell endomysial infiltrates invading non-necrotic muscle fibres early in the disease course with rimmed vacuoles, protein aggregates and amyloid inclusions later in the disease. Transactive response DNA-binding protein-43 (TDP-43), a protein implicated in transcriptional repression in neurodegenerative diseases, is also found in sIBM. C5b-9 membrane attack complex, an effector protein involved in the complement cascade of the immune response, is commonly found in dermatomyositis, but has rarely been reported in IBM. We describe a novel case of IBM with simultaneous C5b-9 and TDP-43 staining on quadriceps biopsy, raising the question of a possibility of concurrent immune-mediated inflammatory and myodegenerative pathogenesis.


Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas de Unión al ADN/metabolismo , Miositis por Cuerpos de Inclusión/diagnóstico , Miositis por Cuerpos de Inclusión/metabolismo , Anciano , Biomarcadores/sangre , Biopsia , Diagnóstico Diferencial , Humanos , Masculino , Coloración y Etiquetado
3.
Nat Commun ; 12(1): 734, 2021 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-33531470

RESUMEN

Driver genes with a mutually exclusive mutation pattern across tumor genomes are thought to have overlapping roles in tumorigenesis. In contrast, we show here that mutually exclusive prostate cancer driver alterations involving the ERG transcription factor and the ubiquitin ligase adaptor SPOP are synthetic sick. At the molecular level, the incompatible cancer pathways are driven by opposing functions in SPOP. ERG upregulates wild type SPOP to dampen androgen receptor (AR) signaling and sustain ERG activity through degradation of the bromodomain histone reader ZMYND11. Conversely, SPOP-mutant tumors stabilize ZMYND11 to repress ERG-function and enable oncogenic androgen receptor signaling. This dichotomy regulates the response to therapeutic interventions in the AR pathway. While mutant SPOP renders tumor cells susceptible to androgen deprivation therapies, ERG promotes sensitivity to high-dose androgen therapy and pharmacological inhibition of wild type SPOP. More generally, these results define a distinct class of antagonistic cancer drivers and a blueprint toward their therapeutic exploitation.


Asunto(s)
Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Represoras/metabolismo , Regulador Transcripcional ERG/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Inmunoprecipitación , Masculino , Ratones , Ratones Desnudos , Mutación/genética , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Neoplasias de la Próstata/genética , Unión Proteica , Proteómica , Receptores Androgénicos/metabolismo , Proteínas Represoras/genética , Transducción de Señal/fisiología , Regulador Transcripcional ERG/genética , Complejos de Ubiquitina-Proteína Ligasa/genética
4.
Mol Cell ; 81(3): 426-441.e8, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33545059

RESUMEN

Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.


Asunto(s)
Replicación del ADN , Origen de Réplica , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Puntos de Control de la Fase S del Ciclo Celular , Especificidad por Sustrato , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Xenopus laevis
5.
Mol Cell ; 81(4): 644-646, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33606972

RESUMEN

In this issue of Molecular Cell, Chen et al. (2020) report the structural transition during DNA-dependent activation of DNA-PK, shedding light on the mechanism by which kinase inhibitors and auto-phosphorylation-deficient DNA-PKcs compromise non-homologous end-joining (Chen et al., 2020).


Asunto(s)
Proteínas de Unión al ADN , Proteínas Nucleares , ADN/genética , Reparación del ADN por Unión de Extremidades , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Autoantígeno Ku/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación
6.
Am J Hum Genet ; 108(2): 324-336, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33508233

RESUMEN

Human infertility is a multifactorial disease that affects 8%-12% of reproductive-aged couples worldwide. However, the genetic causes of human infertility are still poorly understood. Synaptonemal complex (SC) is a conserved tripartite structure that holds homologous chromosomes together and plays an indispensable role in the meiotic progression. Here, we identified three homozygous mutations in the SC coding gene C14orf39/SIX6OS1 in infertile individuals from different ethnic populations by whole-exome sequencing (WES). These mutations include a frameshift mutation (c.204_205del [p.His68Glnfs∗2]) from a consanguineous Pakistani family with two males suffering from non-obstructive azoospermia (NOA) and one female diagnosed with premature ovarian insufficiency (POI) as well as a nonsense mutation (c.958G>T [p.Glu320∗]) and a splicing mutation (c.1180-3C>G) in two unrelated Chinese men (individual P3907 and individual P6032, respectively) with meiotic arrest. Mutations in C14orf39 resulted in truncated proteins that retained SYCE1 binding but exhibited impaired polycomplex formation between C14ORF39 and SYCE1. Further cytological analyses of meiosis in germ cells revealed that the affected familial males with the C14orf39 frameshift mutation displayed complete asynapsis between homologous chromosomes, while the affected Chinese men carrying the nonsense or splicing mutation showed incomplete synapsis. The phenotypes of NOA and POI in affected individuals were well recapitulated by Six6os1 mutant mice carrying an analogous mutation. Collectively, our findings in humans and mice highlight the conserved role of C14ORF39/SIX6OS1 in SC assembly and indicate that the homozygous mutations in C14orf39/SIX6OS1 described here are responsible for infertility of these affected individuals, thus expanding our understanding of the genetic basis of human infertility.


Asunto(s)
Azoospermia/genética , Mutación , Insuficiencia Ovárica Primaria/genética , Adulto , Azoospermia/fisiopatología , Emparejamiento Cromosómico , Codón sin Sentido , Proteínas de Unión al ADN/metabolismo , Femenino , Homocigoto , Humanos , Masculino , Meiosis , Persona de Mediana Edad , Proteínas Nucleares/metabolismo , Linaje , Insuficiencia Ovárica Primaria/fisiopatología , Espermatocitos/metabolismo , Espermatocitos/fisiología , Complejo Sinaptonémico/genética , Complejo Sinaptonémico/metabolismo , Secuenciación Completa del Genoma
7.
Nat Commun ; 12(1): 115, 2021 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-33446654

RESUMEN

Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases.


Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/genética , Microscopía por Crioelectrón , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Ratones , Conformación Proteica en Hélice alfa , Recombinasa Rad51/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia
8.
Mol Cell ; 81(4): 845-858.e8, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33406384

RESUMEN

Mammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate.


Asunto(s)
Composición de Base , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Ratones , Ratones Mutantes , Células Madre Embrionarias de Ratones/citología , Mutación , Neuronas/citología , Factores de Transcripción/genética , Regulación hacia Arriba , Dedos de Zinc
9.
Nat Commun ; 12(1): 73, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397987

RESUMEN

In the male germ cells of placental mammals, 26-30-nt-long PIWI-interacting RNAs (piRNAs) emerge when spermatocytes enter the pachytene phase of meiosis. In mice, pachytene piRNAs derive from ~100 discrete autosomal loci that produce canonical RNA polymerase II transcripts. These piRNA clusters bear 5' caps and 3' poly(A) tails, and often contain introns that are removed before nuclear export and processing into piRNAs. What marks pachytene piRNA clusters to produce piRNAs, and what confines their expression to the germline? We report that an unusually long first exon (≥ 10 kb) or a long, unspliced transcript correlates with germline-specific transcription and piRNA production. Our integrative analysis of transcriptome, piRNA, and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA clusters. Furthermore, a highly methylated promoter, often containing a low or intermediate level of CG dinucleotides, correlates with germline expression and somatic silencing of pachytene piRNA clusters. Pachytene piRNA precursor transcripts bind THOC1 and THOC2, THO complex subunits known to promote transcriptional elongation and mRNA nuclear export. Together, these features may explain why the major sources of pachytene piRNA clusters specifically generate these unique small RNAs in the male germline of placental mammals.


Asunto(s)
Epigénesis Genética , Exones/genética , Mamíferos/genética , Fase Paquiteno/genética , ARN Interferente Pequeño/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Acetilación , Animales , Metilación de ADN/genética , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Histonas/metabolismo , Intrones/genética , Masculino , Ratones Endogámicos C57BL , Proteínas Nucleares/metabolismo , Especificidad de Órganos/genética , Regiones Promotoras Genéticas/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/genética , Testículo/metabolismo , Transcripción Genética
10.
Nat Commun ; 12(1): 585, 2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33500419

RESUMEN

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.


Asunto(s)
Síndrome de Bloom/genética , Replicación del ADN , ADN de Cadena Simple/metabolismo , RecQ Helicasas/metabolismo , Proteína de Replicación A/metabolismo , Secuencias de Aminoácidos/genética , Sistemas CRISPR-Cas/genética , Daño del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Mitosis/genética , Mutación , Unión Proteica/genética , Dominios Proteicos/genética , RecQ Helicasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reparación del ADN por Recombinación/genética
11.
Science ; 371(6524)2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33384352

RESUMEN

Tissue homeostasis is perturbed in a diversity of inflammatory pathologies. These changes can elicit endoplasmic reticulum (ER) stress, protein misfolding, and cell death. ER stress triggers the unfolded protein response (UPR), which can promote recovery of ER proteostasis and cell survival or trigger programmed cell death. Here, we leveraged single-cell RNA sequencing to define dynamic transcriptional states associated with the adaptive versus terminal UPR in the mouse intestinal epithelium. We integrated these transcriptional programs with genome-scale CRISPR screening to dissect the UPR pathway functionally. We identified QRICH1 as a key effector of the PERK-eIF2α axis of the UPR. QRICH1 controlled a transcriptional program associated with translation and secretory networks that were specifically up-regulated in inflammatory pathologies. Thus, QRICH1 dictates cell fate in response to pathological ER stress.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Estrés del Retículo Endoplásmico/genética , Regulación de la Expresión Génica , Inflamación/metabolismo , Proteostasis/genética , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada/genética , Animales , Apoptosis , Células Cultivadas , Proteínas de Unión al ADN/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Inflamación/genética , Inflamación/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Organoides , RNA-Seq , Análisis de la Célula Individual , Factores de Transcripción/genética , Transcripción Genética , eIF-2 Quinasa/metabolismo
12.
Adv Exp Med Biol ; 1281: 201-217, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33433877

RESUMEN

Frontotemporal lobar degeneration with TPD-43-immunoreactive pathology (FTLD-TDP) is subclassified based on the type and cortical laminar distribution of neuronal inclusions. The relevance of these pathological subtypes is supported by the presence of relatively specific clinical and genetic correlations. Recent evidence suggests that the different patterns of pathology are a reflection of biochemical differences in the pathological TDP-43 species, each of which is influenced by differing genetic factors. As a result, patient FTLD-TDP subtype may be an important factor to consider when developing biomarkers and targeted therapies for frontotemporal dementia. In this chapter, we first describe the pathological features, clinical and genetic correlations of the currently recognized FTLD-TDP subtypes. We then discuss a number of novel patterns of TDP-43 pathology. Finally, we provide an overview of what is currently known about the biochemical basis of the different FTLD-TDP subtypes and how this may explain the observed phenotypic and pathological heterogeneity.


Asunto(s)
Demencia Frontotemporal , Degeneración Lobar Frontotemporal , Enfermedad de Pick , Encéfalo/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Degeneración Lobar Frontotemporal/genética , Humanos , Cuerpos de Inclusión
13.
Adv Exp Med Biol ; 1281: 243-267, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33433879

RESUMEN

Following the discovery of TDP-43 and FUS involvement in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD), the major challenge in the field has been to understand their physiological functions, both in normal and disease conditions. The hope is that this knowledge will improve our understanding of disease and lead to the development of effective therapeutic options. Initially, the focus has been directed at characterizing the role of these proteins in the control of RNA metabolism, because the main function of TDP-43 and FUS is to bind coding and noncoding RNAs to regulate their life cycle within cells. As a result, we now have an in-depth picture of the alterations that occur in RNA metabolism following their aggregation in various ALS/FTLD models and, to a somewhat lesser extent, in patients' brains. In parallel, progress has been made with regard to understanding how aggregation of these proteins occurs in neurons, how it can spread in different brain regions, and how these changes affect various metabolic cellular pathways to result in neuronal death. The aim of this chapter will be to provide a general overview of the trending topics in TDP-43 and FUS investigations and to highlight what might represent the most promising avenues of research in the years to come.


Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Degeneración Lobar Frontotemporal , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Demencia Frontotemporal/genética , Degeneración Lobar Frontotemporal/genética , Humanos , Proteína FUS de Unión a ARN/genética
14.
Nat Commun ; 12(1): 99, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397934

RESUMEN

CD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling. Here we use single cell RNA sequencing (scRNA-seq) to classify mouse thymocyte selection intermediates by coreceptor gene expression. In the unperturbed thymus, Cd4+Cd8a- selection intermediates appear before Cd4-Cd8a+ selection intermediates, but the timing of these subsets is flexible according to the strength of TCR signals. Our data show that selection intermediates discriminate MHC class prior to the loss of coreceptor expression and suggest a model where signal strength informs the timing of coreceptor gene activity and ultimately CD4/CD8 lineage choice.


Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Timo/citología , Timo/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Citocinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad/metabolismo , Activación de Linfocitos/genética , Ratones Endogámicos C57BL , Análisis de Componente Principal , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo
15.
mBio ; 12(1)2021 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-33402539

RESUMEN

Bacteriocins are natural antimicrobial peptides produced by bacteria to kill closely related competitors. The opportunistic pathogen Streptococcus gallolyticus subsp. gallolyticus was recently shown to outcompete commensal enterococci of the murine microbiota under tumoral conditions thanks to the production of a two-peptide bacteriocin named gallocin. Here, we identified four genes involved in the regulatory control of gallocin in S. gallolyticus subsp. gallolyticus UCN34 that encode a histidine kinase/response regulator two-component system (BlpH/BlpR), a secreted peptide (GSP [gallocin-stimulating peptide]), and a putative regulator of unknown function (BlpS). While BlpR is a typical 243-amino-acid (aa) response regulator possessing a phospho-receiver domain and a LytTR DNA-binding domain, BlpS is a 108-aa protein containing only a LytTR domain. Our results showed that the secreted peptide GSP activates the dedicated two-component system BlpH/BlpR to induce gallocin transcription. A genome-wide transcriptome analysis indicates that this regulatory system (GSP-BlpH/BlpR) is specific for bacteriocin production. Importantly, as opposed to BlpR, BlpS was shown to repress gallocin gene transcription. A conserved operator DNA sequence of 30 bp was found in all promoter regions regulated by BlpR and BlpS. Electrophoretic mobility shift assays (EMSA) and footprint assays showed direct and specific binding of BlpS and BlpR to various regulated promoter regions in a dose-dependent manner on this conserved sequence. Gallocin expression appears to be tightly controlled in S. gallolyticus subsp. gallolyticus by quorum sensing and antagonistic activity of 2 LytTR-containing proteins. Competition experiments in gut microbiota medium and 5% CO2 to mimic intestinal conditions demonstrate that gallocin is functional under these in vivo-like conditions.IMPORTANCE Streptococcus gallolyticus subsp. gallolyticus, formerly known as Streptococcus bovis biotype I, is an opportunistic pathogen causing septicemia and endocarditis in the elderly often associated with asymptomatic colonic neoplasia. Recent studies indicate that S. gallolyticus subsp. gallolyticus is both a driver and a passenger of colorectal cancer. We previously showed that S. gallolyticus subsp. gallolyticus produces a bacteriocin, termed gallocin, enabling colonization of the colon under tumoral conditions by outcompeting commensal members of the murine microbiota such as Enterococcus faecalis Here, we identified and extensively characterized a four-component system that regulates gallocin production. Gallocin gene transcription is activated by a secreted peptide pheromone (GSP) and a two-component signal transduction system composed of a transmembrane histidine kinase receptor (BlpH) and a cytosolic response regulator (BlpR). Finally, a DNA-binding protein (BlpS) was found to repress gallocin genes transcription, likely by antagonizing BlpR. Understanding gallocin regulation is crucial to prevent S. gallolyticus subsp. gallolyticus colon colonization under tumoral conditions.


Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriocinas/genética , Regulación Bacteriana de la Expresión Génica , Streptococcus gallolyticus/genética , Streptococcus gallolyticus/metabolismo , Proteínas de Unión al ADN/metabolismo , Microbioma Gastrointestinal , Perfilación de la Expresión Génica , Genes Bacterianos/genética , Genoma Bacteriano , Histidina Quinasa/genética , Histidina Quinasa/metabolismo , Percepción de Quorum , Infecciones Estreptocócicas/microbiología , Transcriptoma
16.
Neurology ; 96(6): e916-e925, 2021 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-33441454

RESUMEN

OBJECTIVE: To determine whether memory is preserved longitudinally in primary progressive aphasia (PPA) associated with Alzheimer disease (AD) and to identify potential factors that maintain memory despite underlying neurofibrillary degeneration of mediotemporal memory areas. METHODS: Longitudinal memory assessment was done in 17 patients with PPA with autopsy or biomarker evidence of AD (PPA-AD) and 14 patients with amnestic dementia of the Alzheimer type with AD at autopsy (DAT-AD). RESULTS: In PPA-AD, episodic memory, tested with nonverbal items, was preserved at the initial testing and showed no decline at retesting 2.35 ± 0.78 years later, at which time symptoms had been present for 6.26 ± 2.21 years. In contrast, language functions declined significantly during the same period. In DAT-AD, both verbal memory and language declined with equal severity. Although imaging showed asymmetric left-sided mediotemporal atrophy in PPA-AD, autopsy revealed bilateral hippocampo-entorhinal neurofibrillary degeneration at Braak stages V and VI. Compared to DAT-AD, however, the PPA-AD group had lower incidence of APOE ε4 and of mediotemporal TAR DNA-binding protein 43 (TDP-43) pathology. CONCLUSIONS: Memory preservation in PPA is not just an incidental finding at onset but a core feature that persists for years despite the hippocampo-entorhinal AD neuropathology that is as severe as that of DAT-AD. Asymmetry of mediotemporal atrophy and a lesser impact of APOE ε4 and of TDP-43 on the integrity of memory circuitry may constitute some of the factors underlying this resilience. Our results also suggest that current controversies on memory in PPA-AD reflect inconsistencies in the diagnosis of logopenic PPA, the clinical variant most frequently associated with AD. CLINICALTRIALSGOV IDENTIFIER: NCT00537004 and NCT03371706.


Asunto(s)
Enfermedad de Alzheimer , Amnesia , Afasia Progresiva Primaria , Corteza Entorrinal/patología , Hipocampo/patología , Anciano , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Amnesia/patología , Amnesia/fisiopatología , Afasia Progresiva Primaria/patología , Afasia Progresiva Primaria/fisiopatología , Apolipoproteína E4/genética , Atrofia , Autopsia , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Femenino , Humanos , Estudios Longitudinales , Masculino , Memoria Episódica , Persona de Mediana Edad , Ovillos Neurofibrilares/patología , Índice de Severidad de la Enfermedad
17.
Nat Commun ; 12(1): 448, 2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33469008

RESUMEN

In self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors.


Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Proteínas de Unión al ADN/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Queratinocitos/fisiología , Repitelización/genética , Células Madre/fisiología , Factores de Transcripción/metabolismo , Sistemas CRISPR-Cas/genética , Diferenciación Celular/genética , Autorrenovación de las Células/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Intrones/genética , Poliadenilación/genética , Cultivo Primario de Células , Factores de Transcripción/genética
18.
Nat Commun ; 12(1): 647, 2021 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-33510144

RESUMEN

Ferroptosis is a type of iron-dependent regulated cell death, representing an emerging disease-modulatory mechanism. Transcription factors play multiple roles in ferroptosis, although the key regulator for ferroptosis in iron metabolism remains elusive. Using NanoString technology, we identify NUPR1, a stress-inducible transcription factor, as a driver of ferroptosis resistance. Mechanistically, NUPR1-mediated LCN2 expression blocks ferroptotic cell death through diminishing iron accumulation and subsequent oxidative damage. Consequently, LCN2 depletion mimics NUPR1 deficiency with respect to ferroptosis induction, whereas transfection-enforced re-expression of LCN2 restores resistance to ferroptosis in NUPR1-deficient cells. Pharmacological or genetic blockade of the NUPR1-LCN2 pathway (using NUPR1 shRNA, LCN2 shRNA, pancreas-specific Lcn2 conditional knockout mice, or the small molecule ZZW-115) increases the activity of the ferroptosis inducer erastin and worsens pancreatitis, in suitable mouse models. These findings suggest a link between NUPR1-regulated iron metabolism and ferroptosis susceptibility.


Asunto(s)
Proteínas de Unión al ADN/genética , Ferroptosis/genética , Regulación Neoplásica de la Expresión Génica , Hierro/metabolismo , Lipocalina 2/genética , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Humanos , Estimación de Kaplan-Meier , Lipocalina 2/metabolismo , Ratones Noqueados , Ratones Desnudos , Ratones Transgénicos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapia , Piperazinas/farmacología , Tratamiento con ARN de Interferencia/métodos , Transducción de Señal/genética , Tiazinas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos
19.
Viruses ; 13(1)2021 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-33435393

RESUMEN

The bacteriophage T4 early gene product MotB binds tightly but nonspecifically to DNA, copurifies with the host Nucleoid Associated Protein (NAP) H-NS in the presence of DNA and improves T4 fitness. However, the T4 transcriptome is not significantly affected by a motB knockdown. Here we have investigated the phylogeny of MotB and its predicted domains, how MotB and H-NS together interact with DNA, and how heterologous overexpression of motB impacts host gene expression. We find that motB is highly conserved among Tevenvirinae. Although the MotB sequence has no homology to proteins of known function, predicted structure homology searches suggest that MotB is composed of an N-terminal Kyprides-Onzonis-Woese (KOW) motif and a C-terminal DNA-binding domain of oligonucleotide/oligosaccharide (OB)-fold; either of which could provide MotB's ability to bind DNA. DNase I footprinting demonstrates that MotB dramatically alters the interaction of H-NS with DNA in vitro. RNA-seq analyses indicate that expression of plasmid-borne motB up-regulates 75 host genes; no host genes are down-regulated. Approximately 1/3 of the up-regulated genes have previously been shown to be part of the H-NS regulon. Our results indicate that MotB provides a conserved function for Tevenvirinae and suggest a model in which MotB functions to alter the host transcriptome, possibly by changing the association of H-NS with the host DNA, which then leads to conditions that are more favorable for infection.


Asunto(s)
Bacterias/metabolismo , Bacterias/virología , Proteínas Bacterianas/metabolismo , Bacteriófago T4/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Genes Virales , Interacciones Huésped-Patógeno , Proteínas Bacterianas/química , Secuencia de Bases , Proteínas de Unión al ADN/química , Filogenia , Fagos T/genética
20.
Mol Cell ; 81(3): 514-529.e6, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33385327

RESUMEN

Termination of RNA polymerase II (RNAPII) transcription in metazoans relies largely on the cleavage and polyadenylation (CPA) and integrator (INT) complexes originally found to act at the ends of protein-coding and small nuclear RNA (snRNA) genes, respectively. Here, we monitor CPA- and INT-dependent termination activities genome-wide, including at thousands of previously unannotated transcription units (TUs), producing unstable RNA. We verify the global activity of CPA occurring at pA sites indiscriminately of their positioning relative to the TU promoter. We also identify a global activity of INT, which is largely sequence-independent and restricted to a ~3-kb promoter-proximal region. Our analyses suggest two functions of genome-wide INT activity: it dampens transcriptional output from weak promoters, and it provides quality control of RNAPII complexes that are unfavorably configured for transcriptional elongation. We suggest that the function of INT in stable snRNA production is an exception from its general cellular role, the attenuation of non-productive transcription.


Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/biosíntesis , Terminación de la Transcripción Genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Poliadenilación , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , ARN Nuclear Pequeño/genética
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