RESUMEN
Epigenetic regulation by histone modification can activate or repress transcription through changes in chromatin dynamics and regulates development and the response to environmental signals in both animals and plants. Chromatin immunoprecipitation (ChIP) is an indispensable tool to identify histones with specific post-translational modifications. The lack of a ChIP technique for macroalgae has hindered understanding of the role of histone modification in the expression of genes in this organism. In this study, a ChIP method with several modifications, based on existing protocols for plant cells, has been developed for the red macroalga, Neopyropia yezoensis, that consists of a heterogeneous alternation of macroscopic leaf-like gametophytes and microscopic filamentous sporophytes. ChIP method coupled with qPCR enables the identification of a histone mark in generation-specific genes from N. yezoensis. The results indicate that acetylation of histone H3 at lysine 9 in the 5' flanking and coding regions from generation-specific genes was maintained at relatively high levels, even in generation-repressed gene expression. The use of this ChIP method will contribute significantly to identify the epigenetic regulatory mechanisms through histone modifications that control a variety of biological processes in red macroalgae.
Asunto(s)
Rhodophyta , Algas Marinas , Animales , Histonas/genética , Histonas/metabolismo , Código de Histonas , Epigénesis Genética , Procesamiento Proteico-Postraduccional , Inmunoprecipitación de Cromatina/métodos , Rhodophyta/genética , Rhodophyta/metabolismo , Algas Marinas/genética , Algas Marinas/metabolismoRESUMEN
Forkhead box protein O1 (FoxO1) is a transcription factor and a critical regulator of angiogenesis. Various environmental stimuli, including growth factors, nutrients, shear stress, oxidative stress and hypoxia, affect FoxO1 subcellular localization and strongly influence its transcriptional activity; however, FoxO1-localization patterns in endothelial cells (ECs) during development have not been clarified in vivo. Here, we reported that FoxO1 expression was observed in three layers of angiogenic vessels in developing mouse retinas and that among these layers, the front layer showed high levels of FoxO1 expression in the nuclei of most tip ECs. Because tip ECs migrate toward the avascular hypoxic area, we focused on hypoxia as a major stimulus regulating FoxO1 subcellular localization in tip cells. In cultured ECs, FoxO1 accumulated into the nucleus under hypoxic conditions, with hypoxia also inducing expression of tip-cell-specific genes, including endothelial-specific molecule 1 (ESM1), which was suppressed by FoxO1 knockdown. Additionally, in murine models, EC-specific FoxO1 deletion resulted in reduced ESM1 expression and suppressed tip-cell migration during angiogenesis. These findings indicated roles for FoxO1 in tip-cell migration and that its transcriptional activity is regulated by hypoxia.