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1.
Genes Dev ; 38(1-2): 70-94, 2024 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-38316520

RESUMEN

Since genome instability can drive cancer initiation and progression, cells have evolved highly effective and ubiquitous DNA damage response (DDR) programs. However, some cells (for example, in skin) are normally exposed to high levels of DNA-damaging agents. Whether such high-risk cells possess lineage-specific mechanisms that tailor DNA repair to the tissue remains largely unknown. Using melanoma as a model, we show here that the microphthalmia-associated transcription factor MITF, a lineage addition oncogene that coordinates many aspects of melanocyte and melanoma biology, plays a nontranscriptional role in shaping the DDR. On exposure to DNA-damaging agents, MITF is phosphorylated at S325, and its interactome is dramatically remodeled; most transcription cofactors dissociate, and instead MITF interacts with the MRE11-RAD50-NBS1 (MRN) complex. Consequently, cells with high MITF levels accumulate stalled replication forks and display defects in homologous recombination-mediated repair associated with impaired MRN recruitment to DNA damage. In agreement with this, high MITF levels are associated with increased single-nucleotide and copy number variant burdens in melanoma. Significantly, the SUMOylation-defective MITF-E318K melanoma predisposition mutation recapitulates the effects of DNA-PKcs-phosphorylated MITF. Our data suggest that a nontranscriptional function of a lineage-restricted transcription factor contributes to a tissue-specialized modulation of the DDR that can impact cancer initiation.


Asunto(s)
Melanoma , Humanos , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Daño del ADN , Inestabilidad Genómica/genética , ADN
2.
Genes Dev ; 36(11-12): 664-683, 2022 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-35710139

RESUMEN

Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESSs) and ossifying fibromyxoid tumors (OFMTs), leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a coactivator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a megacomplex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically associating domain including part of the HOXD cluster. This is linked to aberrant gene expression-most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1-implicated with a PRC2 component in the most frequent translocation in ESSs, JAZF1-SUZ12-is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating outcomes similar to those of EPC1-PHF1 Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas use the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes, leading to mislocalization of histone marks and aberrant Polycomb target gene expression.


Asunto(s)
Neoplasias Endometriales , Sarcoma Estromático Endometrial , Sarcoma , Cromatina , Proteínas de Unión al ADN/metabolismo , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Femenino , Histonas/metabolismo , Humanos , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Sarcoma/genética , Sarcoma Estromático Endometrial/genética , Sarcoma Estromático Endometrial/metabolismo , Sarcoma Estromático Endometrial/patología , Translocación Genética/genética
3.
Genes Dev ; 35(23-24): 1657-1677, 2021 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-34819350

RESUMEN

Senescence shapes embryonic development, plays a key role in aging, and is a critical barrier to cancer initiation, yet how senescence is regulated remains incompletely understood. TBX2 is an antisenescence T-box family transcription repressor implicated in embryonic development and cancer. However, the repertoire of TBX2 target genes, its cooperating partners, and how TBX2 promotes proliferation and senescence bypass are poorly understood. Here, using melanoma as a model, we show that TBX2 lies downstream from PI3K signaling and that TBX2 binds and is required for expression of E2F1, a key antisenescence cell cycle regulator. Remarkably, TBX2 binding in vivo is associated with CACGTG E-boxes, present in genes down-regulated by TBX2 depletion, more frequently than the consensus T-element DNA binding motif that is restricted to Tbx2 repressed genes. TBX2 is revealed to interact with a wide range of transcription factors and cofactors, including key components of the BCOR/PRC1.1 complex that are recruited by TBX2 to the E2F1 locus. Our results provide key insights into how PI3K signaling modulates TBX2 function in cancer to drive proliferation.


Asunto(s)
Melanoma , Proteínas de Dominio T Box , Expresión Génica , Humanos , Melanoma/genética , Melanoma/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismo
4.
Mol Cell ; 79(3): 472-487.e10, 2020 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-32531202

RESUMEN

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genoma , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Procesamiento Proteico-Postraduccional , Neoplasias Cutáneas/genética , Acetilación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular Tumoral , Secuencia Conservada , Elementos de Facilitación Genéticos , Femenino , Xenoinjertos , Humanos , Masculino , Melanocitos/metabolismo , Melanocitos/patología , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Desnudos , Factor de Transcripción Asociado a Microftalmía/química , Factor de Transcripción Asociado a Microftalmía/metabolismo , Motivos de Nucleótidos , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Pez Cebra
5.
Cell ; 149(4): 936-48, 2012 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-22579291

RESUMEN

Lysine acetylation is a dynamic posttranslational modification with a well-defined role in regulating histones. The impact of acetylation on other cellular functions remains relatively uncharacterized. We explored the budding yeast acetylome with a functional genomics approach, assessing the effects of gene overexpression in the absence of lysine deacetylases (KDACs). We generated a network of 463 synthetic dosage lethal (SDL) interactions involving class I and II KDACs, revealing many cellular pathways regulated by different KDACs. A biochemical survey of genes interacting with the KDAC RPD3 identified 72 proteins acetylated in vivo. In-depth analysis of one of these proteins, Swi4, revealed a role for acetylation in G1-specific gene expression. Acetylation of Swi4 regulates interaction with its partner Swi6, both components of the SBF transcription factor. This study expands our view of the yeast acetylome, demonstrates the utility of functional genomic screens for exploring enzymatic pathways, and provides functional information that can be mined for future studies.


Asunto(s)
Genómica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetilación , Secuencia de Aminoácidos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Datos de Secuencia Molecular , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo
6.
Cell ; 149(1): 214-31, 2012 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-22464331

RESUMEN

Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resolution crystal structures, covering all BRD families. Comprehensive crossfamily structural analysis identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-containing peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family.


Asunto(s)
Histonas/química , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Acetilación , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Genoma Humano , Histonas/metabolismo , Humanos , Lisina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Dominios y Motivos de Interacción de Proteínas , Proteoma/análisis
7.
Nature ; 595(7865): 120-124, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34079125

RESUMEN

Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy1 and biochemical fractionation coupled with mass spectrometry2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells5-7. Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.


Asunto(s)
Biotinilación , Compartimento Celular , Transporte de Proteínas , Proteoma/análisis , Proteoma/química , Células Cultivadas , Conjuntos de Datos como Asunto , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Espectrometría de Masas , Mitocondrias/química , Mitocondrias/metabolismo , Orgánulos/química , Orgánulos/metabolismo , Proteoma/metabolismo , Reproducibilidad de los Resultados
8.
Mol Cell ; 73(3): 621-638.e17, 2019 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-30554943

RESUMEN

Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.


Asunto(s)
Antineoplásicos/farmacología , Azepinas/farmacología , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Unión al ARN/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Antineoplásicos/química , Azepinas/química , Proteínas de Ciclo Celular , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Células K562 , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Triazoles/química
9.
Genes Dev ; 33(5-6): 310-332, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30804224

RESUMEN

Whether cell types exposed to a high level of environmental insults possess cell type-specific prosurvival mechanisms or enhanced DNA damage repair capacity is not well understood. BRN2 is a tissue-restricted POU domain transcription factor implicated in neural development and several cancers. In melanoma, BRN2 plays a key role in promoting invasion and regulating proliferation. Here we found, surprisingly, that rather than interacting with transcription cofactors, BRN2 is instead associated with DNA damage response proteins and directly binds PARP1 and Ku70/Ku80. Rapid PARP1-dependent BRN2 association with sites of DNA damage facilitates recruitment of Ku80 and reprograms DNA damage repair by promoting Ku-dependent nonhomologous end-joining (NHEJ) at the expense of homologous recombination. BRN2 also suppresses an apoptosis-associated gene expression program to protect against UVB-, chemotherapy- and vemurafenib-induced apoptosis. Remarkably, BRN2 expression also correlates with a high single-nucleotide variation prevalence in human melanomas. By promoting error-prone DNA damage repair via NHEJ and suppressing apoptosis of damaged cells, our results suggest that BRN2 contributes to the generation of melanomas with a high mutation burden. Our findings highlight a novel role for a key transcription factor in reprogramming DNA damage repair and suggest that BRN2 may impact the response to DNA-damaging agents in BRN2-expressing cancers.


Asunto(s)
Apoptosis , Reparación del ADN por Unión de Extremidades/genética , Proteínas de Homeodominio/metabolismo , Melanoma/genética , Melanoma/fisiopatología , Mutación/genética , Factores del Dominio POU/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Humanos , Autoantígeno Ku/metabolismo , Factores del Dominio POU/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Unión Proteica , Dominios Proteicos , Transporte de Proteínas
10.
Mol Psychiatry ; 2024 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-38514804

RESUMEN

Bridging Integrator 1 (BIN1) is the second most important Alzheimer's disease (AD) risk gene, but its physiological roles in neurons and its contribution to brain pathology remain largely elusive. In this work, we show that BIN1 plays a critical role in the regulation of calcium homeostasis, electrical activity, and gene expression of glutamatergic neurons. Using single-cell RNA-sequencing on cerebral organoids generated from isogenic BIN1 wild type (WT), heterozygous (HET) and homozygous knockout (KO) human-induced pluripotent stem cells (hiPSCs), we show that BIN1 is mainly expressed by oligodendrocytes and glutamatergic neurons, like in the human brain. Both BIN1 HET and KO cerebral organoids show specific transcriptional alterations, mainly associated with ion transport and synapses in glutamatergic neurons. We then demonstrate that BIN1 cell-autonomously regulates gene expression in glutamatergic neurons by using a novel protocol to generate pure culture of hiPSC-derived induced neurons (hiNs). Using this system, we also show that BIN1 plays a key role in the regulation of neuronal calcium transients and electrical activity via its interaction with the L-type voltage-gated calcium channel Cav1.2. BIN1 KO hiNs show reduced activity-dependent internalization and higher Cav1.2 expression compared to WT hiNs. Pharmacological blocking of this channel with clinically relevant doses of nifedipine, a calcium channel blocker, partly rescues electrical and gene expression alterations in BIN1 KO glutamatergic neurons. Further, we show that transcriptional alterations in BIN1 KO hiNs that affect biological processes related to calcium homeostasis are also present in glutamatergic neurons of the human brain at late stages of AD pathology. Together, these findings suggest that BIN1-dependent alterations in neuronal properties could contribute to AD pathophysiology and that treatment with low doses of clinically approved calcium blockers should be considered as an option to slow disease-onset and progression.

11.
Mol Cell ; 65(1): 78-90, 2017 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-27916662

RESUMEN

During DNA replication, proliferating cell nuclear antigen (PCNA) adopts a ring-shaped structure to promote processive DNA synthesis, acting as a sliding clamp for polymerases. Known posttranslational modifications function at the outer surface of the PCNA ring to favor DNA damage bypass. Here, we demonstrate that acetylation of lysine residues at the inner surface of PCNA is induced by DNA lesions. We show that cohesin acetyltransferase Eco1 targets lysine 20 at the sliding surface of the PCNA ring in vitro and in vivo in response to DNA damage. Mimicking constitutive acetylation stimulates homologous recombination and robustly suppresses the DNA damage sensitivity of mutations in damage tolerance pathways. In comparison to the unmodified trimer, structural differences are observed at the interface between protomers in the crystal structure of the PCNA-K20ac ring. Thus, acetylation regulates PCNA sliding on DNA in the presence of DNA damage, favoring homologous recombination linked to sister-chromatid cohesion.


Asunto(s)
Acetiltransferasas/metabolismo , Cromátides , Cromosomas Fúngicos , Daño del ADN , Inestabilidad Genómica , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Procesamiento Proteico-Postraduccional , Reparación del ADN por Recombinación , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Acetilación , Acetiltransferasas/química , Acetiltransferasas/genética , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Genotipo , Humanos , Lisina , Modelos Moleculares , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fenotipo , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/genética , Conformación Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relación Estructura-Actividad
12.
Curr Issues Mol Biol ; 46(1): 934-947, 2024 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-38275674

RESUMEN

The present study investigated the association of genetic predisposition for white matter hyperintensities (WMHs) with incident amnestic mild cognitive impairment (aMCI) or Alzheimer's disease (AD), as well as whether such an association was influenced by age, sex, and cognitive reserve. Overall, 537 individuals without aMCI or dementia at baseline were included. Among them, 62 individuals developed aMCI/AD at follow up. Genetic propensity to WMH was estimated using a polygenic risk score for WMHs (PRS WMH). The association of PRS WMH with aMCI/AD incidence was examined using COX models. A higher PRS WMH was associated with a 47.2% higher aMCI/AD incidence (p = 0.015) in the fully adjusted model. Subgroup analyses showed significant results in the older age group, in which individuals with a higher genetic predisposition for WMHs had a 3.4-fold higher risk for developing aMCI/AD at follow up (p < 0.001), as well as in the lower cognitive reserve (CR, proxied by education years) group, in which individuals with a higher genetic predisposition for WMHs had an over 2-fold higher risk (p = 0.013). Genetic predisposition for WMHs was associated with aMCI/AD incidence, particularly in the group of participants with a low CR. Thus, CR might be a modifier in the relationship between genetic predisposition for WMHs and incident aMCI/AD.

13.
Mol Psychiatry ; 28(7): 2716-2727, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37131074

RESUMEN

Alzheimer's disease (AD) is considered to have a large genetic component. Our knowledge of this component has progressed over the last 10 years, thanks notably to the advent of genome-wide association studies and the establishment of large consortia that make it possible to analyze hundreds of thousands of cases and controls. The characterization of dozens of chromosomal regions associated with the risk of developing AD and (in some loci) the causal genes responsible for the observed disease signal has confirmed the involvement of major pathophysiological pathways (such as amyloid precursor protein metabolism) and opened up new perspectives (such as the central role of microglia and inflammation). Furthermore, large-scale sequencing projects are starting to reveal the major impact of rare variants - even in genes like APOE - on the AD risk. This increasingly comprehensive knowledge is now being disseminated through translational research; in particular, the development of genetic risk/polygenic risk scores is helping to identify the subpopulations more at risk or less at risk of developing AD. Although it is difficult to assess the efforts still needed to comprehensively characterize the genetic component of AD, several lines of research can be improved or initiated. Ultimately, genetics (in combination with other biomarkers) might help to redefine the boundaries and relationships between various neurodegenerative diseases.


Asunto(s)
Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/genética , Estudio de Asociación del Genoma Completo , Factores de Riesgo , Biomarcadores , Apolipoproteínas E/genética
14.
Mol Cell ; 62(3): 409-421, 2016 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-27153538

RESUMEN

The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here we identified MBTD1 as a stable subunit of the complex, and we reveal that, via a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double-strand breaks by homologous recombination. It was previously suggested that TIP60-dependent acetylation of H4 regulates binding of the non-homologous end joining factor 53BP1, which engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub. We find that the TIP60 complex regulates association of 53BP1 partly by competing for H4K20me2 and by regulating H2AK15ub. Ubiquitylation of H2AK15 by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex to regulate 53BP1-dependent repair through competitive bivalent binding and modification of chromatin.


Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Acetilación , Sitios de Unión , Unión Competitiva , Sistemas CRISPR-Cas , Proteínas Cromosómicas no Histona/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Histona Acetiltransferasas/genética , Histonas/genética , Humanos , Células K562 , Lisina Acetiltransferasa 5 , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Transfección , Proteína 1 de Unión al Supresor Tumoral P53/genética , Ubiquitinación
15.
Mol Cell Proteomics ; 21(7): 100253, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35636729

RESUMEN

MRG15/MORF4L1 is a highly conserved protein in eukaryotes that contains a chromodomain (CHD) recognizing methylation of lysine 36 on histone H3 (H3K36me3) in chromatin. Intriguingly, it has been reported in the literature to interact with several different factors involved in chromatin modifications, gene regulation, alternative mRNA splicing, and DNA repair by homologous recombination. To get a complete and reliable picture of associations in physiological conditions, we used genome editing and tandem affinity purification to analyze the stable native interactome of human MRG15, its paralog MRGX/MORF4L2 that lacks the CHD, and MRGBP (MRG-binding protein) in isogenic K562 cells. We found stable interchangeable association of MRG15 and MRGX with the NuA4/TIP60 histone acetyltransferase/chromatin remodeler, Sin3B histone deacetylase/demethylase, ASH1L histone methyltransferase, and PALB2-BRCA2 DNA repair protein complexes. These associations were further confirmed and analyzed by CRISPR tagging of endogenous proteins and comparison of expressed isoforms. Importantly, based on structural information, point mutations could be introduced that specifically disrupt MRG15 association with some complexes but not others. Most interestingly, we also identified a new abundant native complex formed by MRG15/X-MRGBP-BRD8-EP400NL (EP400 N-terminal like) that is functionally similar to the yeast TINTIN (Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes) complex. Our results show that EP400NL, being homologous to the N-terminal region of NuA4/TIP60 subunit EP400, creates TINTIN by competing for BRD8 association. Functional genomics indicate that human TINTIN plays a role in transcription of specific genes. This is most likely linked to the H4ac-binding bromodomain of BRD8 along the H3K36me3-binding CHD of MRG15 on the coding region of transcribed genes. Taken together, our data provide a complete detailed picture of human MRG proteins-associated protein complexes, which are essential to understand and correlate their diverse biological functions in chromatin-based nuclear processes.


Asunto(s)
Factores de Transcripción , Cromatina/metabolismo , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Humanos , Nucleosomas/metabolismo , Factores de Transcripción/metabolismo
16.
Alzheimers Dement ; 20(2): 1298-1308, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-37985413

RESUMEN

INTRODUCTION: Genome-wide association studies (GWAS) are fundamental for identifying loci associated with diseases. However, they require replication in other ethnicities. METHODS: We performed GWAS on sporadic Alzheimer's disease (AD) including 539 patients and 854 controls from Argentina and Chile. We combined our results with those from the European Alzheimer and Dementia Biobank (EADB) in a meta-analysis and tested their genetic risk score (GRS) performance in this admixed population. RESULTS: We detected apolipoprotein E ε4 as the single genome-wide significant signal (odds ratio  = 2.93 [2.37-3.63], P = 2.6 × 10-23 ). The meta-analysis with EADB summary statistics revealed four new loci reaching GWAS significance. Functional annotations of these loci implicated endosome/lysosomal function. Finally, the AD-GRS presented a similar performance in these populations, despite the score diminished when the Native American ancestry rose. DISCUSSION: We report the first GWAS on AD in a population from South America. It shows shared genetics modulating AD risk between the European and these admixed populations. HIGHLIGHTS: This is the first genome-wide association study on Alzheimer's disease (AD) in a population sample from Argentina and Chile. Trans-ethnic meta-analysis reveals four new loci involving lysosomal function in AD. This is the first independent replication for TREM2L, IGH-gene-cluster, and ADAM17 loci. A genetic risk score (GRS) developed in Europeans performed well in this population. The higher the Native American ancestry the lower the GRS values.


Asunto(s)
Enfermedad de Alzheimer , Azidas , Estudio de Asociación del Genoma Completo , Humanos , Chile , Enfermedad de Alzheimer/genética , Predisposición Genética a la Enfermedad/genética , Polimorfismo de Nucleótido Simple/genética
17.
J Proteome Res ; 22(1): 78-90, 2023 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-36484504

RESUMEN

Bromodomains (BRDs) are a family of evolutionarily conserved domains that are the main readers of acetylated lysine (Kac) residues on proteins. Recently, numerous BRD-containing proteins have been proven essential for transcriptional regulation in numerous contexts. This is exemplified by the multi-subunit mSWI/SNF chromatin remodeling complexes, which incorporate up to 10 BRDs within five distinct subunits, allowing for extensive integration of Kac signaling to inform transcriptional regulation. As dysregulated transcription promotes oncogenesis, we sought to characterize how BRD-containing subunits contribute molecularly to mSWI/SNF functions. By combining genome editing, functional proteomics, and cellular biology, we found that loss of any single BRD-containing mSWI/SNF subunit altered but did not fully disrupt the various mSWI/SNF complexes. In addition, we report that the downregulation of BRD7 is common in invasive lobular carcinoma and modulates the interactome of its homologue, BRD9. We show that these alterations exacerbate sensitivities to inhibitors targeting epigenetic regulators─notably, inhibitors targeting the BRDs of non-mSWI/SNF proteins. Our results highlight the interconnections between distinct mSWI/SNF complexes and their far-reaching impacts on transcriptional regulation in human health and disease. The mass spectrometry data generated have been deposited to MassIVE and ProteomeXchange and assigned the identifiers MSV000089357, MSV000089362, and PXD033572.


Asunto(s)
Proteínas Cromosómicas no Histona , Factores de Transcripción , Humanos , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica , Dominios Proteicos
18.
Neurobiol Dis ; 182: 106140, 2023 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-37120095

RESUMEN

The rare A673T variant was the first variant found within the amyloid precursor protein (APP) gene conferring protection against Alzheimer's disease (AD). Thereafter, different studies have discovered that the carriers of the APP A673T variant show reduced levels of amyloid beta (Aß) in the plasma and better cognitive performance at high age. Here, we analyzed cerebrospinal fluid (CSF) and plasma of APP A673T carriers and control individuals using a mass spectrometry-based proteomics approach to identify differentially regulated targets in an unbiased manner. Furthermore, the APP A673T variant was introduced into 2D and 3D neuronal cell culture models together with the pathogenic APP Swedish and London mutations. Consequently, we now report for the first time the protective effects of the APP A673T variant against AD-related alterations in the CSF, plasma, and brain biopsy samples from the frontal cortex. The CSF levels of soluble APPß (sAPPß) and Aß42 were significantly decreased on average 9-26% among three APP A673T carriers as compared to three well-matched controls not carrying the protective variant. Consistent with these CSF findings, immunohistochemical assessment of cortical biopsy samples from the same APP A673T carriers did not reveal Aß, phospho-tau, or p62 pathologies. We identified differentially regulated targets involved in protein phosphorylation, inflammation, and mitochondrial function in the CSF and plasma samples of APP A673T carriers. Some of the identified targets showed inverse levels in AD brain tissue with respect to increased AD-associated neurofibrillary pathology. In 2D and 3D neuronal cell culture models expressing APP with the Swedish and London mutations, the introduction of the APP A673T variant resulted in lower sAPPß levels. Concomitantly, the levels of sAPPα were increased, while decreased levels of CTFß and Aß42 were detected in some of these models. Our findings emphasize the important role of APP-derived peptides in the pathogenesis of AD and demonstrate the effectiveness of the protective APP A673T variant to shift APP processing towards the non-amyloidogenic pathway in vitro even in the presence of two pathogenic mutations.


Asunto(s)
Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Enfermedad de Alzheimer/líquido cefalorraquídeo , Péptidos beta-Amiloides/metabolismo , Heterocigoto , Encéfalo/metabolismo
19.
Development ; 147(19)2020 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-32907847

RESUMEN

Pattern formation is influenced by transcriptional regulation as well as by morphogenetic mechanisms that shape organ primordia, although factors that link these processes remain under-appreciated. Here we show that, apart from their established transcriptional roles in pattern formation, IRX3/5 help to shape the limb bud primordium by promoting the separation and intercalation of dividing mesodermal cells. Surprisingly, IRX3/5 are required for appropriate cell cycle progression and chromatid segregation during mitosis, possibly in a nontranscriptional manner. IRX3/5 associate with, promote the abundance of, and share overlapping functions with co-regulators of cell division such as the cohesin subunits SMC1, SMC3, NIPBL and CUX1. The findings imply that IRX3/5 coordinate early limb bud morphogenesis with skeletal pattern formation.


Asunto(s)
Cromátides/metabolismo , Proteínas de Homeodominio/metabolismo , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Factores de Transcripción/metabolismo , Animales , Western Blotting , Segregación Cromosómica/genética , Segregación Cromosómica/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Células HEK293 , Proteínas de Homeodominio/genética , Humanos , Inmunoprecipitación , Espectrometría de Masas , Ratones , Mitosis/genética , Mitosis/fisiología , Embarazo , RNA-Seq , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genética
20.
Chembiochem ; 24(10): e202300143, 2023 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-37042352

RESUMEN

Ring-closure is a key step in current pyrimidine anabolism and one may wonder whether cyclisation reactions could be promoted in the geochemical context at the origins of life, i. e. with the help of minerals. Various prebiotic minerals were tested in this work, including silica, carbonates, microporous minerals. In particular, the role of zinc ions supported on minerals was investigated in view of its presence in the catalytic site of cyclic amidohydrolase enzymes. Based on in situ (TGA: ThermoGravimetric Analysis, ATR-IR: Attenuated Total Reflectance-InfraRed) and ex situ (1 H NMR- Nuclear Magnetic Resonance) characterisations, we identified the products of thermal activation of NCA (N-carbamoyl-aspartic acid) in wetting-and-drying scenarios on the surface of minerals. NCA can cyclize extensively only on some surfaces, with the predominant product being 5-carboxymethylhydantoin (Hy) rather than dihydroorotate (DHO), while there is a competition with hydrolysis on others. Replacing the enzymes with heterogeneous catalysts also works with other reactions catalysed by enzymes of the cyclic amidohydrolases family. The role of the hydrophilicity/hydrophobicity of minerals as well as the regioselectivity of the cyclisation (5-carboxymethylhydantoin versus dihydroorotate) are examined.


Asunto(s)
Amidohidrolasas , Ácido Aspártico , Hidantoínas , Minerales , Origen de la Vida , Minerales/síntesis química , Minerales/química , Dominio Catalítico , Zinc/química , Amidohidrolasas/química , Ciclización , Ácido Aspártico/química , Hidantoínas/química
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