Abstract

Adipogenesis is regulated by a coordinated cascade of sequence-specific transcription factors and coregulators with chromatin-modifying activities that are between them responsible for the establishment of the gene expression pattern of mature adipocytes. Here we examine the histone H3 post-translational modifications occurring at the promoters of key adipogenic genes during adipocyte differentiation. We show that the promoters of apM1, glut4, gpd1, and leptin are enriched in dimethylated histone H3 Lys4 (H3-K4) in 3T3-L1 fibroblasts, where none of these genes are yet expressed. A detailed study of the apM1 locus shows that H3-K4 dimethylation is restricted to the promoter region in undifferentiated cells and associates with RNA polymerase II (pol II) loading. The beginning of apM1 transcription at the early stages of adipogenesis coincides with promoter H3 hyperacetylation and H3-K4 trimethylation. At the coding region, H3 acetylation and dimethylation, as well as pol II binding, are found in cells at later stages of differentiation, when apM1 transcription reaches its maximal peak. This same pattern of histone modifications is detected in mouse primary preadipocytes and adipocytes but not in a related fibroblast cell line that is not committed to an adipocyte fate. Inhibition of H3-K4 methylation by treatment of 3T3-L1 cells with methylthioadenosine results in decreased apM1 gene expression as well as decreased adipogenesis. Taken together, our data indicate that H3-K4 dimethylation and pol II binding to the promoter of key adipogenic genes are distinguishing marks of cells that have undergone determination to a preadipocyte stage.

Highlights

  • Acetylation in promoter regions is closely correlated with gene activation in organisms ranging from yeast to mammals, and transcriptionally active euchromatin regions are highly enriched in acetylated histones [1,2,3,4,5]

  • Dimethylation of Histone H3-K4 Is the First Modification Observed at the Promoters of Adipogenic Genes during Adipocyte Differentiation—The mouse 3T3-L1 cell line is frequently used as a model to study adipocyte differentiation

  • Recent studies have shown that the post-translational modifications of histones play a key role in regulating the programmatically determined gene expression patterns arising during differentiation of several cell types

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Summary

The abbreviations used are

H3-K4, histone H3 lysine 4; H3-K9, histone H3 lysine 9; H3-K9/K14, histone H3 lysine 9/14; Pol II, RNA polymerase II; PPAR, peroxisome proliferator-activated receptor; IBMX, 3-isobutyl-1-methyl-xanthine; MTA, 5Ј-methylthioadenosine; ChIP, chromatin immunoprecipitation; CREB, cAMP-response element-binding protein; PBS, phosphate-buffered saline; RT, reverse transcription; PIPES, 1,4-piperazinediethanesulfonic acid; D0 –D9, day 1–9, respectively; AdoMet, S-adenosyl-L-methione. The same pattern can be extended to other adipogenic genes such as glut or gpd (glycerol-3-phosphate dehydrogenase)

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