Role of the histone demethylase PHF2 during early neurogenesis

Abstract

During neural development, signaling molecules and networks of expressed transcription factors cooperate to control cellular fate. Chromatin acting factors are essential players in cell proliferation and differentiation events. PHF2 is a histone demethylase, previously associated with cancer and autism spectrum disorder (ASD) but despite its clinical relevance its function during neural development remains unclear. PHF2 belongs to the KDM7 family of demethylases. It harbors an N-terminal plant homeodomain, (PHD) a domain that was shown to be associated with methylated lysine residues and an enzymatically active Jumonji-C (JmjC) domain responsible for their demethylase activity towards H3K9me and H3K9me2. PHF2 has been proposed to be a transcriptional coactivator, although, its genomic localization still remains unclear. To address the function of this protein during early neurogenesis constitutes the major goal of this Ph.D. thesis. To do so, we are using in vitro (cortical neural stem cells, NSC) and in vivo (the chick embryo neural tube) models. We demonstrated that this histone demethylase binds mainly promoter regions along the genome and more specifically cell cycle gene promoters facilitating their transcription in NSCs. PHF2 was shown to regulate genes involved in G1-S transition (E2f2/3/7/8, Cdc7, Cdc25a, Cdk4, Mcm3/4/8), DNA replication (Orc1/2/6, Pcna), mitosis (Cdk1, Smc2/3/4, Aurkb, Topo2a), as well as chromatin activity (Cenpa, Kdm1b, Hat1, Parp1, Prmt5). In addition, we demontrated that this demethylase colocalizes with H3K4me2/3 marks, partially colocalizes with E2F1 and E2F4 transcription factors and mediates H3K9 demethylation at global and promoter levels. Moreover, PHF2 binds to the centromeric and pericentromeric regions and requiring its catalytic activity suppresses unprogrammed transcription from satellite repeats. In that way, PHF2 allows neural stem cells proliferation and preserves heterochromatin integrity during progenitor expansion. We also showed that PHF2 interacts with heterochromatic components such as the histone methyltransferase SUV39H1 and the HP1-binding protein 3. Furthermore, PHF2 fine-tunes H3K9 methylation levels, ensuring genome stability and chromatin homeostasis, as we observed that its lack of function leads to γH2Ax and R-loops accumulation in NSCs. Interestingly, we demonstrated that PHF2 is essential for neural progenitor self- renewal in the chick embryo neural tube and for neural subpopulation specification. To conclude, this work helps to move forward our understanding of the multiple crosstalks between epigenetics and developmental programs.

Durante el desarrollo neural, los factores reguladores de la actividad de la cromatina son esenciales en el control de la proliferación y diferenciación de las células. PHF2 es una desmetilasa de histonas, asociada a cáncer y a trastornos del espectro autista (ASD) cuya función en el desarrollo neural no ha sido estudiada. PHF2 pertenece a la familia de desmetilasas KDM7. Contiene un dominio PHD, capaz de unirse a residuos de lysinas metiladas y un dominio Jumonji-C (JmjC) enzimáticamente activo, responsable de la actividad desmetilasa hacía las marcas H3K9me y H3K9me2. PHF2 ha sido propuesta como un activador transcripcional pero su localización genómica no ha sido descrita. El objetivo principal de esta tesis doctoral es abordar la función de esta desmetilasa de histonas durante la neurogénesis temprana. Para ello, hemos usado modelos in vitro (progenitores neurales de córtex, NSCs) e in vivo (el tubo neural del embrión de pollo). Nuestros resultados han permitido identificar los lugares de unión de PHF2 en el genoma, esencialmente promotores y más específicamente promotores de genes de ciclo celular facilitando su transcripción. Además, esta desmetilasa colocaliza con las marcas H3K4me2/3, media desmetilación de las marcas H3K9 a nivel global y en promotores. Los experimentos realizados han demostrado que PHF2 se une a las regiones centromérica y pericentromérica y así mantiene niveles bajos de la transcripción de las secuencias repetidas. De esta forma preserva la integridad de la heterocromatina, asegurando la estabilidad genómica y la homeostasis de la cromatina. Estos resultados han sido validados en nuestro modelo in vivo; en el tubo neural, hemos demostrado que PHF2 es esencial para la proliferación y el mantenimiento de la pluripotencia de los progenitores, así como para la especificación de los diferentes tipos de las poblaciones neurales. En resumen, el trabajo presentado ayuda a avanzar en el conocimiento de las múltiples interacciones moleculares entre los programas de desarrollo y la epigenética.