Abstract
We have previously reported the abundant and ubiquitous expression of a newly identified splicing variant of mouse peroxisome proliferator-activated receptor-γ (Pparγ), namely Pparγ1sv that encodes PPARγ1 protein, and plays an important role in adipogenesis. Pparγ1sv has a unique 5’UTR sequence, compared to those of mouse Pparγ1 and Pparγ2 mRNAs. This implies the presence of a novel transcriptional initiation site and promoter for Pparγ1sv. We found that DNA methylation of 42 CpG sites in the proximal promoter region (−733 to −76) of Pparγ1sv was largely unchanged five days after adipocyte differentiation, whereas chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) using antibodies against H3K4me3 and H3K27ac revealed that these modifications significantly elevated at the transcription start sites of Pparγ1sv and Pparγ2 after differentiation.
Introduction
The peroxisome proliferator-activated receptors (PPARs) function as nuclear receptors to regulate the expression of many genes involved in metabolic homeostasis. PPARγ is the third member of PPAR subtype of genes and is one of the master regulators of adipogenesis1. In a ligand-dependent manner, PPARγ regulates transcription of target genes such as Fabp4 and which are indispensable for completion of adipocyte differentiation. There are two isoforms of PPARγ; the ubiquitously expressed PPARγ1 and the adipocyte-specific PPARγ2. PPARγ2 in mice is longer than PPARγ1 by 30 amino acid residues at the N-terminus. In to Pparγ1 and Pparγ2 mRNAs, we have recently reported a novel mouse Pparγ splicing variant, Pparγ1sv, that is indispensable for adipocyte differentiation of 3T3-L1 and mouse primary cultured preadipocytes2. Pparγ1sv was significantly up-regulated during adipocyte differentiation of 3T3-L1 cells and mouse primary cultured preadipocytes, and its inhibition by specific siRNAs completely abolished the induced adipogenesis in 3T3-L1 cells. Pparγ1sv has a unique 5’UTR sequence, implying the presence of a unique transcriptional initiation site and regulatory elements for the expression. Both the Pparγ1sv and Pparγ2 promoters are transactivated by the overexpression of C/EBPβ and C/EBPδ, which are transiently expressed very early during adipocyte differentiation3, in 3T3-L1 cells. However, detailed mechanism of Pparγ1sv regulation during adipogenesis remains to be elucidated.
Methylated CpG sites on the Pparγ2 promoter are progressively demethylated upon the induction of differentiation in 3T3-L1 cells4. In humans, a specific region of the Pparγ1 promoter is methylated in colorectal cancers, which correlates with a lack of PPARγ expression5. In addition to DNA methylation of Pparγ promoters, histone modifications are involved in transcriptional regulation of Pparγ gene6. In this study, we report that epigenetic regulation of Pparγ1sv is via histone modifications, but not DNA methylation.
Experimental procedures
Cell culture and differentiation
3T3-L1 cells were obtained from JCRB Cell Bank (Osaka, Japan). Culture condition, media and method of adipocyte differentiation were described elsewhere2.
Bisulfite sequencing
Genomic DNA was isolated from 3T3-L1 preadipocytes (day 0) and differentiated adipocytes (day 5) using DNeasy Tissue kit (Qiagen). Two µg of each DNA sample was bisulfite modified using EpiTect Bisulfite kit (Qiagen). The Pparγ1sv promoter region was amplified with EpiTaq HS polymerase (Takara) using the following primers: Novelpro_UP3bis, 5′-TGTGATAGATAAGGTGATAGAGTTTGG-3′ and Novelpro_LP1bis, 5′-TCCCTTATATAAAAACAACCCAAACTA-3′. PCR fragments were cloned into the pGEM-T Easy vector (Promega) and sequenced for both strands. Bisulfite sequences from all clones were analyzed using QUMA7.
ChIP-qPCR analyses
3T3-L1 cells cultured in 10 cm dishes were fixed with 1% formaldehyde at around 25°C for 10 min, after which fixation was halted by addition of glycine solution. Immunoprecipitated protein/DNA complexes were prepared using the Magna ChIP A kit (Millipore) following manufacture’s instructions. Anti-acetyl-histone H3 (Lys27) and anti-monomethyl-histone H4 (Lys20) antibodies were purchased from Millipore. Anti-trimethyl-histone H3 (Lys4) antibody was purchased from Wako Pure Chemical Industries. ChIP samples were analyzed by qPCR with gene-specific primers as follows: NVpro_ChIP_UP1, 5′-GTCGGAGGGTGGGGAGGAGGATG-3′ and NVpro_ChIP_LP1, 5′-CCCAATCCCAAGCCATAAAGCAC-3′ for Pparγ1sv promoter; NVpro_ChIP_UP3, 5′-GGAGCAAGGCGGCCAGGTAACCA-3′ and NVpro_ChIP_LP3, 5′-GGCGGGTGCTGTGCGTCGGTGAG-3′ for Pparγ1 promoter; g2pro_ChIP_UP2, 5′-GCCTTTATTCTGTCAACTATTCCTTTT-3′ and g2pro_ChIP_LP2, 5′-AGTATTTATCTTTGGTTGAAACTCCTA-3′ for Pparγ2 promoter.
Results
Pparγ1sv was remarkably up-regulated as early as day 3 upon induction of adipogenesis in 3T3-L1 cells2. To address how transcription of Pparγ1sv is regulated, we investigated the DNA methylation of CpG sites in the 658 bp upstream region of the Pparγ1sv transcription initiation site. The methylation status of 42 CpG sites in the −733/−76 of the Pparγ1sv promoter region (Fig. 1A, wave line) were analyzed by bisulfite genomic sequencing. Comparison of the methylation percentage before (day 0) and after (day 5) differentiation of 3T3-L1 cells revealed that 2 CpG sites at position −643 and −638 (Fig. 1B, two arrowheads) of differentiated 3T3-L1 were more frequently methylated than those of undifferentiated cells (P<0.05). However, the other 40 CpG sites were not significantly methylated or demethylated during adipocyte differentiation.
Increase in levels of histone modification at the Pparγ promoter have been associated with adipocyte differentiation8, 9. We examined the trimethylation of histone H3 lysine 4 (H3K4me3), acetylation of histone H3 lysine 27 (H3K27ac), and monomethylation of histone H4 lysine 20 (H4K20me1) at the Pparγ1sv promoter (Fig. 2A). We found that H3K4me3 and H3K27ac are elevated in the region spanning the transcription start sites10. H4K20me1 increases in the downstream regions of transcription start sites for both Pparγ1 and Pparγ28. ChIP-qPCR using specific primers revealed remarkable increases in modifications by H3K4me3 and H3K27ac around the transcription start sites of Pparγ1sv and Pparγ2 at day 5 of adipocyte differentiation (Fig. 2B). In contrast, H4K20me1 levels at the promoter regions of Pparγ1sv and Pparγ2 were unchanged.
Discussion
Previous studies have found that methylated CpG sites on the Pparγ2 promoter get demethylated progressively upon adipogenesis in murine 3T3-L14. Also, as mentioned before, studies on human colorectal cancer have shown an inverse correlation between methylation of specific regions of Pparγ1 promoter and PPARγ expression5. In our studies, DNA methylation levels of the Pparγ1sv promoter were essentially unchanged during adipogenesis in 3T3-L1 cells (Fig. 1B). Therefore, epigenetic regulation by DNA methylation may not contribute to the regulation of Pparγ1sv expression. Instead, it is likely that Pparγ1sv and Pparγ2 transcripts share a common regulatory mechanism by histone modifications. ChIP-qPCR using antibodies against H3K4me3 and H3K27ac revealed that these histone modifications significantly elevated at the transcription start sites of Pparγ1sv and Pparγ2 by the induction of differentiation (Fig. 2B). H3K4me3 and H3K27ac levels on the Pparγ1 promoter also increased but to a smaller extent relative to those of Pparγ1sv and Pparγ2, which may be indicative of a limited enhancement of Pparγ1 mRNA expression during adipogenesis in 3T3-L1 cells.
Declaration of competing financial interests (CFI)
The authors declare they have no actual or potential competing financial interests.
Grant Information
This study was supported by JSPS KAKENHI (https://www.jsps.go.jp/english/index.html) Grant number 25460299, 26461367, and 17K09866.
Acknowledgements
We are grateful to Ms. Sawako Sato (Saitama Medical University) for her technical assistances. We would like to thank Editage (www.editage.jp) for English language editing.