Histone H3 Lysine 4 Dimethylation Signals the Transcriptional Competence of the Adiponectin Promoter in Preadipocytes

Abstract

Adipogenesis is regulated by a coordinated cascade of sequencespecific transcription factors and coregulators with chromatinmodifying 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 Lys 4 (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.The influence exerted by the post-translational modifications of histones over the regulation of gene expression has been extensively studied in the past few years. Numerous studies have shown a clear link between the pattern of histone modifications found at promoter regions and gene transcription, thus leading to the statement of the histone code hypothesis (1), which postulates that the pattern of histone post-translational modifications in a locus considerably extends the amount of information conveyed by the genomic code. Histone H3 and H4 hyperacetylation 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 (1-5). Unlike acetylation, histone H3 methylation can be equally associated with either transcriptional activation or repression. Methylation of the lysine residue Lys 4 of histone H3 (H3-K4) 3 correlates with activation of gene expression in most systems (2, 4 -7), whereas H3 Lys 9 (H3-K9) methylation is involved in the establishment and maintenance of silent heterochromatin regions (8). Moreover, lysine residues can be mono-, di-, or trimethylated in vivo, thus providing a further layer of complexity and exponentially increasing functional diversity (9, 10). The recent identification of LSD1, the first histone demethylase to be ch...

A chromatin perspective of adipogenesis

“…57,58,75,76,[80][81][82]97,99 At this stage, most adipogenic genes are characterized by bivalent histone marks, such as methylation of H3K27, H3K9 and H3K4. [35][36][37][38]50 Methylation of DNA is also found 120 as well as recruitment of a still inactive RNA Pol II. 50 Upon induction of differentiation, pRB phosporylation results in its inactivation and HDAC1 is dislodged from PPARg which is now free to bind CBP/p300 and a number of other coactivators.…”

“…49 Interestingly, we have previously observed a similar change in the nuclear pattern of H3K4me2 during adipogenesis. 50 In 3T3-L1 preadipocytes, H3K4me2, which was observed to be enriched in the promoters of still silent adipogenic genes such as the hormone adiponectin, was found strongly enriched along the nuclear border in inmature cells and dramatically rearranged towards the interior nucleoplasma in differentiated cells. 50 The region along the nuclear envelope is occupied by mid-to-late replicating chromatin in 3T3-L1 cells, and mostly includes silent genes.…”

“…50 In 3T3-L1 preadipocytes, H3K4me2, which was observed to be enriched in the promoters of still silent adipogenic genes such as the hormone adiponectin, was found strongly enriched along the nuclear border in inmature cells and dramatically rearranged towards the interior nucleoplasma in differentiated cells. 50 The region along the nuclear envelope is occupied by mid-to-late replicating chromatin in 3T3-L1 cells, and mostly includes silent genes. 51 Taking together all these results it is tempting to speculate that histone modifications and nuclear location collaborate in regulating transcription of adipogenic genes during adipogenesis.…”

“…Given the preponderance of HAT/HDAC interactions with adipogenic regulators, it is easy to recognize the key role that histone acetylation plays in adipogenesis. Histone acetylation increases at the promoters of adipogenic marker genes during adipocyte differentiation, correlating with their increased expression, 50 whereas a decrease in HDAC expression takes place throughout this process. 89 Furthermore, blocking HDAC activity with general inhibitors such as trichostatin A increases adipogenesis, whereas overexpression of HDAC1 impairs it.…”

“…92 Accordingly, we have observed that the promoter region of dlk1 is acetylated in 3T3-L1 fibroblasts, when the gene is highly expressed, becoming progressively deacetylated throughout differentiation as the gene is gradually silenced. 50 On the initial phases of adipogenesis, PPARg is blocked by its association with a repressive pRb-HDAC3 complex resulting in the recruitment of deacetylase activity to PPARg target promoters and their consequent repression. 81 When pRb is inactivated by phosphorylation upon induction of differentiation, this complex is dissociated and PPARg is then free to interact with HATs CBP/p300 and activate transcription.…”