195 SUPPRESSION OF SETDB1 DURING BOVINE EMBRYONIC DEVELOPMENT RESULTS IN PRE-IMPLANTATION MORTALITY AND DECREASED TRANSCRIPTION OF TRANSPOSABLE ELEMENTS

Abstract

Organization of chromatin structure by the combinatorial patterns of DNA methylation and post-translational histone modification is essential for the establishment and maintenance of proper transcriptional programs that result in the coordination of embryonic development. We previously observed that suppression of transcripts encoding SET domain, bifurcated 1 (SETDB1) using small interfering RNAs (siRNA) is embryonic lethal, with SETDB1-suppressed embryos (n = 361) arresting immediately before the blastocyst stage (blastocyst rate: Control 44.9 ± 4.9% and NULL injected 25.7 ± 6.0%). Studies in rodents indicate SETDB1 is a crucial regulator of transposable elements and that the precise epigenetic regulation of these elements is a key aspect of transcriptional programs controlling pluripotency and placentation. To better characterise the molecular basis of the observed mortality, we analysed expression of the bovine Long Interspersed Nuclear Element 1 family (LINE1) of transposable elements via quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). Mature bovine oocytes were obtained from a commercial supplier (De Soto Biosciences, Seymour, TN, USA) and IVF performed by standard laboratory protocol. Eighteen hours after IVF, cumulus cells were removed and presumptive zygotes divided into 3 different treatment groups: non-injected control (CNTL), non-targeting siRNA injected control (siNULL), and zygotes injected with siRNAs targeting SETDB1 (siSETDB1). Each embryo was injected with ~100 pL of siRNAs (10 µM) in fluorescent dextran solution. All zygotes were verified as injected by fluorescent microscopy and then cultured in Bovine Evolve (Zenith Biotech, Guilford, CT, USA) medium supplemented with 4 mg mL of BSA (Probumin, EMD Millipore, Darmstadt, Germany). Groups of embryos (15–20) from each treatment were lysed at the 4-cell, 8-cell, and morula stages, RNA extracted, and analysed by RT-qPCR using GAPDH and YWHAZ as reference genes. A two-way ANOVA and a Student's t-test were used to analyse the results from the RT-qPCR. As expected, siSETDB1-injected morulae displayed dramatic reduction in the level of Setdb1 transcripts as compared to siNULL control (96%; P < 0.05). Preliminary analysis of LINE1 transcripts at the morula stage indicated siSETDB1-injected embryos displayed a 75% reduction compared to the siNULL. Whether alteration in LINE1 regulation contributes to the developmental arrest and embryonic mortality of siSETDB1-injected embryos is under investigation.