Shared Mechanisms in Pparγ1sv and Pparγ2 Expression in 3T3-L1 Cells: Studies on Epigenetic and Positive Feedback Regulation of Pparγ during Adipogenesis

We have previously reported the identification of a novel splicing variant of the mouse peroxisome proliferator-activated receptor-γ (Pparγ), referred to as Pparγ1sv. This variant, encoding the PPARγ1 protein, is abundantly and ubiquitously expressed, playing a crucial role in adipogenesis. Pparγ1sv possesses a unique promoter and 5′ untranslated region (5′UTR), distinct from those of the canonical mouse Pparγ1 and Pparγ2 mRNAs. We observed a significant increase in DNA methylation at two CpG sites within the proximal promoter region (-733 to -76) of Pparγ1sv during adipocyte differentiation. Concurrently, chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) using antibodies against H3K4me3 and H3K27ac indicated marked elevations in both methylation and acetylation of histone H3, while the repressive histone mark H3K9me2 significantly decreased, at the transcription start sites of both Pparγ1sv and Pparγ2 following differentiation. Knocking down Pparγ1sv using specific siRNA also led to a decrease in Pparγ2 mRNA and PPARγ2 protein levels; conversely, knocking down Pparγ2 resulted in reduced Pparγ1sv mRNA and PPARγ1 protein levels, suggesting synergistic transcriptional regulation of Pparγ1sv and Pparγ2 during adipogenesis. Furthermore, our experiments utilizing the CRISPR-Cas9 system identified crucial PPARγ-binding sites within the Pparγ gene locus, underscoring their significance in adipogenesis. Based on these findings, we propose a model of positive feedback regulation for Pparγ1sv and Pparγ2 expression during the adipocyte differentiation process in 3T3-L1 cells.


Introduction
The peroxisome proliferator-activated receptors (PPARs) are key nuclear receptors that regulate the expression of genes critical for metabolic homeostasis.Among them, PPARγ, the third member of the PPAR gene subtype, is a pivotal regulator of adipogenesis [1].PPARγ modulates the transcription of essential adipocyte differentiation genes, such as Fabp4 and Cebpa, in a ligand-dependent manner [2].Two isoforms of PPARγ are known: the ubiquitously expressed PPARγ1 and the adipocyte-specific PPARγ2, with the latter being 30 amino acids longer at the N-terminus in mice [3,4].In addition to these, we have identified novel human [5] and mouse Pparγ splicing variants [6].Pparγ1sv, a mouse Pparγ splicing variant, is indispensable for adipocyte differentiation of 3T3-L1 and mouse primary cultured preadipocytes [6].Notably, Pparγ1sv is significantly upregulated during adipocyte differentiation in these cells, and its knockdown via specific siRNAs substantially inhibits adipogenesis in 3T3-L1 cells.Pparγ1sv is ubiquitously expressed, with high levels in many tissues including the placenta and embryo [6,7].Pparγ1sv-knockout (KO) mice are viable and fertile but exhibit a prolonged lower body weight during and after the weaning period [7].
Pparγ1sv possesses unique promoter and 5 ′ UTR sequences, providing distinct regulatory elements and a specific transcriptional initiation site for its expression.However, we found that the promoters of both Pparγ1sv and Pparγ2 are similarly transactivated by C/EBPβ and C/EBPδ in 3T3-L1 cells [6].Furthermore, specific knockdown of C/EBPβ resulted in the concomitant reduction of both mRNA and protein levels for Pparγ1sv and Pparγ2.These results imply that the expression of Pparγ1sv and Pparγ2 is synergistically regulated during adipogenesis.
In 3T3-L1 cells, the Pparγ2 promoter undergoes progressive demethylation of methylated CpG sites upon the induction of differentiation [8].In human colorectal cancers, specific DNA methylation in a region of the Pparγ1 promoter correlates with reduced PPARγ expression [9].In addition to DNA methylation, histone modifications also play a crucial role in the transcriptional regulation of the Pparγ gene [10].In this study, we demonstrate that the epigenetic regulation of Pparγ1sv during adipogenesis in 3T3-L1 cells predominantly involves histone modifications, with limited DNA methylation.Furthermore, we have identified PPARγ-binding sites within the Pparγ gene locus, suggesting the presence of a positive feedback mechanism regulating synergistic Pparγ1sv and Pparγ2 expression during adipocyte differentiation.
2.9.Statistical Analyses.We conducted one-way ANOVA followed by Tukey's multiple comparison test or the twotailed unpaired t-test.Differences between groups were considered significant at p < 0 05.All data were analyzed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) and are presented as the mean (SD) of the obtained values.

Methylation at Two CpG Sites in the Upstream Region of the Pparγ1sv
Transcription Initiation Site Is Enhanced during Adipocyte Differentiation.Pparγ1sv exhibited a remarkable upregulation as early as day 3 following the induction of adipogenesis in 3T3-L1 cells [6].To explore the regulatory mechanisms of Pparγ1sv transcription, we investigated the DNA methylation of CpG sites within the promoter region of Pparγ1sv.The methylation status of 42 CpG sites located within the upstream region spanning -733 to -76 nucleotides (nt) relative to the transcription start site (TSS) located in exon C (Figure 1(a), indicated by a wave line) was analyzed using bisulfite genomic sequencing.A comparison of the methylation percentages before (day 0) and after (day 5) differentiation in 3T3-L1 cells showed that two CpG sites, at positions -643 and -638 from TSS (Figure 1(b), indicated by dot lines), were significantly methylated in differentiated cells compared to undifferentiated cells (p < 0 05).However, the remaining 40 CpG sites did no undergo notable methylation or demethylation during the process of adipocyte differentiation.

Histone H3 Methylation and Acetylation at the Upstream Region of Pparγ1sv
Transcription Start Site Are Elevated during Adipocyte Differentiation.Increases in histone modification levels at the Pparγ promoter have been associated with adipocyte differentiation [12,13].We examined the trimethylation of histone H3 lysine 4 (H3K4me3), acetylation of histone H3 lysine 27 (H3K27ac), monomethylation of histone H4 lysine 20 (H4K20me1), and dimethylation of histone H3 lysine 9 (H3K9me2) at the Pparγ promoters (Figure 2(a)).Previous report indicated that H3K4me3 and H3K27ac levels were elevated in the region spanning the TSSs [14].For H4K20me1, an increase was observed in the downstream regions of the transcription start sites for both Pparγ1 and Pparγ2 [12].In our study, ChIP-qPCR with specific primers revealed remarkable increases in H3K4me3 and H3K27ac modifications at the upstream regions of the TSSs for Pparγ1sv and Pparγ2 on day 5 of adipocyte differentiation (Figure 2(b)), indicating active transcription of these genes.This was further supported by substantial demethylation of H3K9me2, a repressive histone marks, at the promoter regions of Pparγ1sv and 3

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Pparγ2.Meanwhile, the levels of H4K20me1, associated with DNA damage repair or DNA replication, remained largely unchanged.

Synergistic Regulation of Gene Expression between
Pparγ1sv and Pparγ2.We next investigated whether the specific knockdown of either Pparγ1sv or Pparγ2 would  ).An asterisk ( * ) denotes a significant difference (p < 0 05) compared to the siControl group, as determined by one-way ANOVA with Tukey's multiple comparison test.ns: not significant.

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influence the expression of the other.Using a siRNA (siγ1sv22) we designed (Figure 3(a)), which specifically targets Pparγ1sv transcripts in 3T3-L1 cells [6] (Figure 3(b)), we observed a concurrent decrease in Pparγ2 mRNA levels (Figure 3(c)) during adipocyte differentiation, as measured by quantitative PCR (qPCR).Similarly, a siRNA specific to Pparγ2 (siγ2_8) led to a significant reduction in Pparγ1sv levels (Figure 3(b)).We then assessed whether the protein levels of PPARγ1 and PPARγ2, which are derived from Pparγ1sv and Pparγ2, respectively, were influenced by treatment with their specific siRNAs.Indeed, knocking down Pparγ1sv with siγ1sv22 significantly reduced the levels of both PPARγ1 and PPARγ2 proteins on day 1 (Figures 3(d), 3(e), and 3(f)); similarly, silencing Pparγ2 with siγ2_8 led to decreased levels of both PPARγ1 and PPARγ2 proteins (Figures 3(d), 3(e), and 3(f)), whereas C/ EBPβ (p30), the major isoform of C/EBPβ [6] that is transiently expressed at the early stages of adipocyte differentiation [3], was similarly upregulated in cells transduced with siControl, siγ1sv22, and siγ2_8 (Figures 3(d) and 3(g)), indicating that the induction of adipogenesis was effectively achieved.These findings imply the existence of synergistic regulatory mechanisms for the gene expression of Pparγ1sv and Pparγ2 and their protein products, PPARγ1 and PPARγ2, during adipocyte differentiation.Initially, we analyzed NCBI GEO datasets for a chromatin immunoprecipitation (ChIP) sequencing study with an anti-PPARγ antibody [15].Based on this analysis, we found three potential PPARγ-binding sites (sites 1-3) (Figure 4(a)).The association of CREB-binding protein (CBP) with sites 2 and 3, and C/EBPα with site 3, supports the notion that these are possible binding sites of transcription regulators.We validated the notable binding of PPARγ protein to all three sites during adipocyte differentiation using a ChIP-qPCR assay with an anti-PPARγ antibody (Figure 4(b)).To further demonstrate the significance of these sites in adipocyte differentiation and the regulation of Pparγ expression, we established heterozygous KO-3T3-L1 cells lacking each of three binding sites using CRISPR-Cas9 system with the targeting vectors (Figure 5(a)).During adipocyte differentiation, the accumulation of intracellular triglyceride (TG) was significantly reduced in the sites 1 and 2 KO clones, while the effect was less pronounced in the site 3 KO clones (Figure 5(b)), suggesting that sites 1 and 2 are critical for adipogenesis.Consistent with TG accumulation patterns, Pparγ1sv and Pparγ2 mRNA levels were largely suppressed in the sites 1 and 2 KO clones on day 9 of differentiation, whereas levels in the site 3 KO clones were comparable with those in control cells (Figure 5(c)).

Discussion
Previous studies have demonstrated that in murine 3T3-L1 cells, methylated CpG sites on the Pparγ2 promoter undergo progressive demethylation during adipogenesis [8].Similarly, study on human colorectal cancer has revealed an inverse correlation between methylation of specific regions in the Pparγ1 promoter and PPARγ expression [9].However, in our studies, DNA methylation levels of the Pparγ1sv promoter remained largely unchanged during adipogenesis in 3T3-L1 cells (Figure 1(b)).This suggests that DNA methylation may not play a significant role in the epigenetic regulation of Pparγ1sv expression.Instead, our findings indicate that Pparγ1sv and Pparγ2 transcripts are likely regulated through a common mechanism involving histone modifications.ChIP-qPCR with antibodies against H3K4me3 and H3K27ac showed significant elevations in these histone modifications at the regions close to transcription start sites of both Pparγ1sv and Pparγ2 upon differentiation (Figure 2(b)).Although H3K4me3 and H3K27ac levels also increased at the Pparγ1 promoter, the extent was comparatively smaller, which may be indicative of a limited enhancement of Pparγ1 mRNA expression during adipogenesis in 3T3-L1 cells.
Positive feedback mechanisms play vital regulatory roles in the expression of genes required for proper cellular differentiation, proliferation, and metabolic homeostasis [16].Previous studies have suggested the involvement of PPARγ-binding sites within the Pparγ gene locus [13,15] and feedback regulation of Pparγ expression by PPARγ protein in adipogenesis [10,12].These studies primarily focused on Pparγ2 expression, a key regulator of adipogenesis.In this study, we revealed that the specific knockdown of either Pparγ1sv or Pparγ2 leads to the reciprocal downregulation of each transcript (Figures 3(b) and 3(c)) and their protein products (Figures 3(d), 3(e), and 3(f)).Furthermore, we demonstrated that two out of three identified PPARγ-binding sites within the Pparγ gene locus are critical for both adipocyte differentiation (Figure 5(b)) and Pparγ expression (Figure 5(c)) in 3T3-L1 cells.In contrast, while PPARγ binding was observed at site 3, this site does not appear to be significantly involved in Pparγ expression or the subsequent adipocyte differentiation.This could be due to site 3 being located considerably downstream of the TSSs (Figure 4(a)).Therefore, the binding of PPARγ to site 3 might be involved in the regulation of other gene expressions by PPARγ, but this aspect will be clarified in future studies.
In conclusion, we have shown that the promoter regions of two splicing variants, Pparγ1sv and Pparγ2, undergo similar histone modifications during adipogenesis.Furthermore, the expression of these variants mutually influences each other's levels, potentially regulated by the binding of their protein products, PPARγ1 and PPARγ2, to shared regions in the Pparγ gene.Given that these sites are consistent between 3T3-L1 cells and mouse primary adipocytes [17], we propose that our findings can be applied to in vivo studies to gain a more comprehensive understanding of Pparγ expression regulation during adipogenesis.

Figure 1 :
Figure 1: DNA methylation of Pparγ1sv promoter during adipogenesis.(a) The wave line represents the region analyzed by bisulfite sequencing.Arrows indicate the transcription start sites of three Pparγ transcripts.(b) Percentages of DNA methylation of 42 CpG islands within the 658 bp region of the Pparγ1sv promoter were determined through bisulfite sequencing of 90 clones at day 0 and 88 clones at day 5. Two dot lines indicate CpG sites that exhibited significant DNA methylation during adipocyte differentiation (day 5) compared to undifferentiated cells (p < 0 05).

Figure 2 :Figure 3 :
Figure 2: Histone modifications at the Pparγ1sv promoter.The promoter regions of Pparγ1, Pparγ1sv, and Pparγ2 were analyzed for H3K4me3, H3K27ac, H4K20me1, and H3K9me2 modifications using ChIP-qPCR with specific primers.(a) Arrows indicate positions of transcription start sites of the three transcription variants.The gray bars mark the analyzed promoter regions, with distances from each transcription start site indicated in nucleotides.(b) Enrichment of the Pparγ1sv promoter region by ChIP using antibodies specific to H3K4me3, H3K27ac, H4K20me1, or H3K9me2 on days 0 and 5 of adipocyte differentiation.The values represent means (SD) from three separate experiments.An asterisk ( * ) indicates p < 0 05 by the two-tailed unpaired t-test for day 0 vs. day 5.

Figure 4 :
Figure 4: Potential binding sites of PPARγ protein in the Pparγ gene locus.(a) Identified binding sites of PPARγ, CREB-binding protein (CBP),and C/EBPα in the Pparγ gene locus, as indicated by the densities from ChIP-seq data (NCBI GSE20752).Sites 1-3 are candidate PPARγbinding sites, while g2 UL2 is a non-PPARγ-binding site.(b) ChIP-qPCR assay with anti-PPARγ antibody and normal mouse IgG was performed to evaluate the binding of PPARγ protein to candidate sites 1-3 at days 0, 2, and 6 of 3T3-L1 adipocyte differentiation.Values are expressed as mean (SD) (n = 4).An asterisk ( * ) indicates p < 0 05 by the two-tailed unpaired t-test for day 0 vs. day 6.

Figure 5 :
Figure 5: PPARγ-binding sites 1 and 2 are crucial to the expression of Pparγ1sv, Pparγ2, and adipogenesis in 3T3-L1 cells.(a) Knockout (KO) strategy for PPARγ-binding sites 1-3 using the CRISPR-Cas9 with targeting vectors.(b) Relative triglyceride content (TG) in 3T3-L1 control cells and sites 1-3 KO clones was evaluated on days 0 and 6 of adipocyte differentiation.Values are shown as mean (SD) (n = 4).An asterisk ( * ) indicates a significant difference (p < 0 05) compared to control cells on day 6.ns: not significant.(c) Relative Pparγ1sv and Pparγ2 mRNA levels in control and sites 1-3 KO clones on day 6 of adipocyte differentiation.Values were obtained by averaging the results from two independent experiments.
or adjacent to the Pparγ gene locus.
Sites within the Pparγ Gene Locus Regulate Pparγ1sv and Pparγ2 Expression and Adipogenesis.To elucidate the synergistic gene regulatory mechanisms between Pparγ1sv and Pparγ2, we explored potential cis-