PPAR-γ enrichment sites in gyk and ob have differential changes in H3K4 methylation status after treatment with rosiglitazone in 3T3-L1 adipocytes

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 649-677-Adipocyte Biology
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-673
Aniket Sidhaye1, Viviane P Lima*2 and Fredric Edward Wondisford3
1Johns Hopkins Univ, Baltimore, MD, 2Johns Hopkins Univ. School of Medicine, Baltimore, MD, 3Johns Hopkins Med Instit, Baltimore, MD
Leptin is secreted by adipocytes in proportion to fat mass and is the product of the ob gene. Through its interaction with leptin receptors in hypothalamic appetite control centers, it forms an important part of the network regulating whole body energy expenditure.  While it is known that ligands of PPAR-γ produce changes in histone acetylation on genes such as glycerol kinase (gyk), there is little information on the role of other post-translational histone modifications. Further, PPAR-γ ligands down-regulate obexpression but there is no model to explain how this occurs.

Methods: We used the 3T3-L1 adipocyte cell system to perform all studies.  ChIP-qPCR was performed to determine enrichment of histone H3 modifications K4-dimethyl (H3K4m2), K4-trimethyl (H3K4m3), K9-dimethyl (H3K9m2). PCR primer sets were designed to tile across the oblocus, such that amplicons were ~100bp in size and separated by ~500bp since well defined PPAR response elements (PPRE) are unknown. In the case of gyk locus, we designed 1 primer set near the described PPREs and 1 primer set ~4kb proximal to that site as a control region where we did not expect to find enrichment of PPAR-γ or of histone marks associated with transcriptional activation.

Results: We found strong enrichment of H3K4m2 and –m3, near the reported gyk PPRE (gyk -1893) but not in a region proximal (gyk -6085).   On the ob locus, we found enrichment of H3K4m2 ~ but not m3 ~ 16kb proximal to the reported transcriptional start site (TSS). Interestingly, there was only background enrichment of H3K4m2 near the ob TSS.  After treatment with rosiglitazone (1uM) for 6 hours, we found that at gyk -1893  H3K4m2 decreased but H3K4m3 was increased.  In contrast, at ob -16kb H3K4m2 decreased and H3K4m3 remained low.

Conclusion: Based on the above data we hypothesize that there may be differential recruitment of histone modifying enzymes in response to rosiglitazone at gyk vs. ob response elements.  Further work is focused on identifying which histone demethylases or histone methyltransferases are involved in this regulation.

Nothing to Disclose: AS, VPL, FEW

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm