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1.
Nat Commun ; 10(1): 487, 2019 01 30.
Artículo en Inglés | MEDLINE | ID: mdl-30700703

RESUMEN

Mammalian transcription factors (TFs) differ broadly in their nuclear mobility and sequence-specific/non-specific DNA binding. How these properties affect their ability to occupy specific genomic sites and modify the epigenetic landscape is unclear. The association of TFs with mitotic chromosomes observed by fluorescence microscopy is largely mediated by non-specific DNA interactions and differs broadly between TFs. Here we combine quantitative measurements of mitotic chromosome binding (MCB) of 501 TFs, TF mobility measurements by fluorescence recovery after photobleaching, single molecule imaging of DNA binding, and mapping of TF binding and chromatin accessibility. TFs associating to mitotic chromosomes are enriched in DNA-rich compartments in interphase and display slower mobility in interphase and mitosis. Remarkably, MCB correlates with relative TF on-rates and genome-wide specific site occupancy, but not with TF residence times. This suggests that non-specific DNA binding properties of TFs regulate their search efficiency and occupancy of specific genomic sites.


Asunto(s)
Cromatina/metabolismo , Cromosomas/metabolismo , Interfase/fisiología , Mitosis/fisiología , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Cromosomas/genética , ADN/genética , ADN/metabolismo , Humanos , Interfase/genética , Mitosis/genética , Unión Proteica , Factores de Transcripción/genética
2.
FEBS Lett ; 592(6): 878-887, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-28862742

RESUMEN

During mitosis, gene transcription stops, and the bulk of DNA-binding proteins are excluded from condensed chromosomes. While most gene-specific transcription factors are largely evicted from mitotic chromosomes, a subset remains bound to specific and non-specific DNA sites. Here, we review the current knowledge on the mechanisms leading to the retention of a subset of transcription factors on mitotic chromosomes and discuss the implications in gene expression regulation and their potential as an epigenetic mechanism controlling stem cell self-renewal and differentiation.


Asunto(s)
Cromosomas Humanos/metabolismo , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética/fisiología , Mitosis/fisiología , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Animales , Humanos , Células Madre/citología
3.
Genes Dev ; 30(22): 2538-2550, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27920086

RESUMEN

Mitotic bookmarking transcription factors remain bound to chromosomes during mitosis and were proposed to regulate phenotypic maintenance of stem and progenitor cells at the mitosis-to-G1 (M-G1) transition. However, mitotic bookmarking remains largely unexplored in most stem cell types, and its functional relevance for cell fate decisions remains unclear. Here we screened for mitotic chromosome binding within the pluripotency network of embryonic stem (ES) cells and show that SOX2 and OCT4 remain bound to mitotic chromatin through their respective DNA-binding domains. Dynamic characterization using photobleaching-based methods and single-molecule imaging revealed quantitatively similar specific DNA interactions, but different nonspecific DNA interactions, of SOX2 and OCT4 with mitotic chromatin. Using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) to assess the genome-wide distribution of SOX2 on mitotic chromatin, we demonstrate the bookmarking activity of SOX2 on a small set of genes. Finally, we investigated the function of SOX2 mitotic bookmarking in cell fate decisions and show that its absence at the M-G1 transition impairs pluripotency maintenance and abrogates its ability to induce neuroectodermal differentiation but does not affect reprogramming efficiency toward induced pluripotent stem cells. Our study demonstrates the mitotic bookmarking property of SOX2 and reveals its functional importance in pluripotency maintenance and ES cell differentiation.


Asunto(s)
Diferenciación Celular/genética , Mitosis/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Animales , Reprogramación Celular/genética , Cromatina/metabolismo , Células Madre Embrionarias , Fase G1 , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Células 3T3 NIH , Placa Neural/citología , Placa Neural/fisiología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Unión Proteica
4.
J Bacteriol ; 197(23): 3686-97, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26369580

RESUMEN

UNLABELLED: Mycobacterium tuberculosis possesses a thick and highly hydrophobic cell wall principally composed of a mycolyl-arabinogalactan-peptidoglycan complex, which is critical for survival and virulence. DprE1 is a well-characterized component of decaprenyl-phospho-ribose epimerase, which produces decaprenyl-phospho-arabinose (DPA) for the biosynthesis of mycobacterial arabinans. Upstream of dprE1 lies rv3789, which encodes a short transmembrane protein of the GtrA family, whose members are often involved in the synthesis of cell surface polysaccharides. We demonstrate that rv3789 and dprE1 are cotranscribed from a common transcription start site situated 64 bp upstream of rv3789. Topology mapping revealed four transmembrane domains in Rv3789 and a cytoplasmic C terminus consistent with structural models built using analysis of sequence coevolution. To investigate its role, we generated an unmarked rv3789 deletion mutant in M. tuberculosis. The mutant was characterized by impaired growth and abnormal cell morphology, since the cells were shorter and more swollen than wild-type cells. This phenotype likely stems from the decreased incorporation of arabinan into arabinogalactan and was accompanied by an accumulation of DPA. A role for Rv3789 in arabinan biosynthesis was further supported by its interaction with the priming arabinosyltransferase AftA, as demonstrated by a two-hybrid approach. Taken together, the data suggest that Rv3789 does not act as a DPA flippase but, rather, recruits AftA for arabinogalactan biosynthesis. IMPORTANCE: Upstream of the essential dprE1 gene, encoding a key enzyme of the decaprenyl phospho-arabinose (DPA) pathway, lies rv3789, coding for a short transmembrane protein of unknown function. In this study, we demonstrated that rv3789 and dprE1 are cotranscribed from a common transcription start site located 64 bp upstream of rv3789 in M. tuberculosis. Furthermore, the deletion of rv3789 led to a reduction in arabinan content and to an accumulation of DPA, confirming that Rv3789 plays a role in arabinan biosynthesis. Topology mapping, structural modeling, and protein interaction studies suggest that Rv3789 acts as an anchor protein recruiting AftA, the first arabinosyl transferase. This investigation provides deeper insight into the mechanism of arabinan biosynthesis in mycobacteria.


Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Arabinosa/metabolismo , Proteínas Bacterianas/metabolismo , Galactanos/metabolismo , Mycobacterium tuberculosis/enzimología , Aldehído Oxidorreductasas/química , Aldehído Oxidorreductasas/genética , Secuencia de Aminoácidos , Arabinosa/análogos & derivados , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Alineación de Secuencia , Terpenos/metabolismo
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