DNA methylation alters transcriptional rates of differentially expressed genes and contributes to pathophysiology in mice fed a high fat diet
Authors: Robert M O&#x27;Doherty
Liora S Katz
Lucy Li
David G Peters
Ian Sipula
Pili Zhang
Carmen Argmann
Jun Zhu
Tianjiao Chu
Donald K Scott
Patricia A Shaw
Kimberly D Bunce
Benjamin S Mantell
N Dedousis
Journal: Molecular Metabolism (2017)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: DNA methylation; High fat diet; Liver; Phlda1; RNA-seq
Affiliations: Diabetes, Obesity and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA., Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, USA; Center for Fetal Medicine, Magee-Women's Research Institute, Pittsburgh, PA, USA., Division of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA., Department of Genetics and Genomic Sciences and Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA., Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, USA; Center for Fetal Medicine, Magee-Women's Research Institute, Pittsburgh, PA, USA. Electronic address: ., Diabetes, Obesity and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. Electronic address: .

Abstract:  Objective: Overnutrition can alter gene expression patterns through epigenetic mechanisms that may persist through generations. However, it is less clear if overnutrition, for example a high fat diet, modifies epigenetic control of gene expression in adults, or by what molecular mechanisms, or if such mechanisms contribute to the pathology of the metabolic syndrome. Here we test the hypothesis that a high fat diet alters hepatic DNA methylation, transcription and gene expression patterns, and explore the contribution of such changes to the pathophysiology of obesity. Methods: RNA-seq and targeted high-throughput bisulfite DNA sequencing were used to undertake a systematic analysis of the hepatic response to a high fat diet. RT-PCR, chromatin immunoprecipitation and in vivo knockdown of an identified driver gene, Phlda1, were used to validate the results. Results: A high fat diet resulted in the hypermethylation and decreased transcription and expression of Phlda1 and several other genes. A subnetwork of genes associated with Phlda1 was identified from an existing Bayesian gene network that contained numerous hepatic regulatory genes involved in lipid and body weight homeostasis. Hepatic-specific depletion of Phlda1 in mice decreased expression of the genes in the subnetwork, and led to increased oil droplet size in standard chow-fed mice, an early indicator of steatosis, validating the contribution of this gene to the phenotype. Conclusions: We conclude that a high fat diet alters the epigenetics and transcriptional activity of key hepatic genes controlling lipid homeostasis, contributing to the pathophysiology of obesity. 

Retracted: No

PubPeer comments:
 In this article authors performed Methylseq and RNAseq analyses of control and high fat fed mouse liver and found Phlda1 as the gene methylated and reduced mRNA expressed in HFD mouse liver. We have listed some of the issues found with the data shown in this article. Fig 3B-C: In the ChIP assay data shown in Fig 3B, the data plotted was the background corrected ChIP signal for RNA Pol II total, phospho-Ser 5 RNA Pol II and phospho-Ser2 RNA Pol II show huge variation among the biological replicates. However, ratio of Phospho-Ser5 total RNA Pol II shown in Fig 3C very little variation, as if the samples with little variation were picked to plot in this graph (Fig 3C). If they would have considered all the values, the difference between SD and HFD would not be statistically significant. Also, ratio of Phospho-Ser5 total RNA Pol II is incorrect. For example, Phospho-Ser5 total RNA Pol II at the promoter for SD would be 0.5 1= 0.5 but it is shown in Fig. 3B as 1.8. Also authors havenu2019t considered the stochastic dynamics of RNA Pol II phosphorylation dephosphorylation and the effect of HFD on this process. They say Phospho-Ser5 dephosphorylated quickly, isnu2019t that could be the reason for lower ChIP signal. If they had considered the Phospho-Ser2 total RNA Pol II ratio, they would not see the lower Phospho-Ser2 total RNA Pol II ratio in HFD compared to SD. Fig 5B and C: The micrographs of oil Red O stained mouse liver sections there is not much difference between control and Phlda1 shRNA treated mouse liver. But they chose a highly biased method of measuring TAG accumulation by manually determining the lipid particle size to demonstrate the anticipated result. They should have co-stained the liver sections with hematoxylin and oil red O so the readers can get an idea of cell number and magnification of the displayed micrographs. ,