Dynamics of HOX gene expression and regulation in adipocyte development

HOX proteins are homeodomain-containing transcription factors that play a central role in development. We have applied genome-wide approaches to develop time-dependent profile of differentially expressed genes in early and mature adipocytes. The list of differentially expressed HOX genes were developed by analyzing the microarray datasets of murine adipocyte samples at different time points of development. Since these datasets were obtained from Gene Expression Omnibus (GEO), we were able to find a new HOX gene, HOXC13 in adipogenesis. To investigate whether these members of the homeobox gene family are expressed and regulated in preadipocytes or mature adipocytes, RNA was isolated from 3T3-L1 preadipocyte cells at different time point's through-out the preadipocyte and adipocyte state. A reverse transcriptase-polymerase chain reaction strategy was applied for the analysis of gene expression. We have observed that HOXA5 and HOXC13 were differentially expressed in preadipocytes and HOXD4 and HOXD8 in mature adipocytes. To understand this difference in expression pattern, we have considered to investigate the role of the major regulators of adipogenesis in HOX gene regulation. Since Retinoic acid receptor (RAR) was reported previously as a regulator of Hox genes, we chose the combination of Peroxisome proliferator-activated receptor gamma (PPARγ) and Retinoic X receptor (RXR) which are modulated by the presence of RAR. To provide a detailed analysis of retinoic acid (RA) and/or PPARγ induced transcriptional and epigenetic changes within the homeotic clusters of mouse fibroblast cells (3T3-L1), we have performed a promoter mapping of HOX genes and observed an enriched binding site for PPARγ and RXR in their promoter regions. We further confirmed this PPARγ and RXR binding to HOX gene promoters by re-analyzing the anti-PPARγ/anti-RXR ChIP-Seq data. Based on the results, we modulated the PPARγ expression at the transcriptional and translational levels by using 5 different pharmacological molecules (TSA, GW9662, ATRA, FH535, and Pioglitazone) to elucidate their effect on the HOX gene transcription. These pharmacological molecules had a direct or indirect regulatory effect on the PPARγ activity. We observed that PPARγ suppression alone is enough for the upregulation of HOXA5 and HOXD4 genes. In addition, HOXD8 regulation was mediated by RAR activation in mature adipocytes but the regulation of HOXC13 gene expression was not clear. We suggest that it might be partially mediated through suppressing PPARγ activation. Further insights are required to provide a mechanistic detail about HOX gene regulation through PPARγ. In this study, we have reported a time-dependent expression analysis of HOXA5, HOXD4, HOXD8, and HOXC13 in preadipocytes and mature adipocytes. Also, we have suggested PPARγ/RAR dependent regulation for these genes during adipogenesis.