Identification of Differentially Methylated Regions (DMR) in the genome of bovine endometrial epithelial cells (bEEC)challenged by E.Coli LPS and its effect on transcription

• Lipopolysaccharide (LPS) from Gram-negative bacteria induces the activation of pro-inflammatory pathways in the endometrium and such reactions potentially affect fertility and more specifically the implantation process. Gene expression studies based on RNA sequencing [Guo et al., Epiconcept Conference 2015, Abstract O-23, 34p] have shown that more than 2000 genes were differentially expressed (DEGs) in bEEC exposed to LPS. This study aimed to identify epigenetic correlates of these DEGs by detecting Differentially Methylated Regions (DMRs) in same cells. bEEC were exposed in vitro to 0, 2, and 8 μg/ml LPS for 24 hours. DNA libraries were prepared at The Babraham Institute, UK for reduced representation bisulfite sequencing (RRBS) using MspI digestion followed by end-repair/A-tailing and 5mC adaptor ligation and bisulfite conversion plus PCR. Sequences were analyzed using BS-Seeker2 for alignment of reads and MethylKit v0.9.5 for finding significant DMRs. AliBaba2 was used to predict potential transcription factor binding sites (TFBS) at DMRs using the TRANSFAC database. Analysis of RRBS data revealed 78 DMRs with q-value<0.05 between controls and 2 μg/ml LPS samples (34 hyper- and 44 hypo-methylated) and 109 DMRs between controls and 8 μg/ml LPS samples (47 hyper- and 62 hypo-methylated). 17 common DMRs (in 2 μg/ml and 8μg/ml LPS groups vs. controls) were identified. A reciprocal relationship was found between methylation and gene expression data for a sub-set of loci known to be key regulators of endometrial function. For example, LIF (Leukemia Inhibitory Factor) gene was found to contain a hypo-methylated DMR and its expression was up-regulated. Predicted binding sites in this DMR have 100% conservation and gene synteny in the human genome. These analyses allow the identification of regions harbouring candidates for key regulatory elements of endometrial function, thus contributing to the understanding of LPS-induced deregulation that may impact implantation.

Ämnesord

LANTBRUKSVETENSKAPER  -- Bioteknologi med applikationer på växter och djur -- Genetik och förädling inom lantbruksvetenskap (hsv//swe)

AGRICULTURAL SCIENCES  -- Agricultural Biotechnology -- Genetics and Breeding in Agricultural Sciences (hsv//eng)

NATURVETENSKAP  -- Biologi -- Bioinformatik och systembiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Bioinformatics and Systems Biology (hsv//eng)

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