65 Identification of chromatin state related to H3K27 acetylation in pre-implantation bovine embryos by cleavage under targets and tagmentation analysis
A. M. Fonseca Junior A , J. Ispada A , E. C. dos Santos B , C. B. de Lima B , P. K. Fontes A , J. V. Alcantara da Silva A , P. J. Ross C and M. P. Milazzotto AA Federal University of ABC, Santo André, SP, Brazil
B Laval University, Laval, QC, Canada
C University of California, Davis, Davis, CA, USA
Reproduction, Fertility and Development 35(2) 159-159 https://doi.org/10.1071/RDv35n2Ab65
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
The metabolic landscape of the early embryo is known for having an intrinsic correspondence with its quality and viability attributes, besides being an important element capable of influencing the molecular and epigenetic status of the conceptus. We have previously demonstrated that the modulation of the pyruvate metabolism was capable of altering the profile of H3K27ac and the transcriptional status of bovine blastocysts. In this work, we performed a cleavage under targets and tagmentation (CUT&Tag) analysis, with the aim of investigating the interactions between H3K27ac and DNA altered by the modulation of pyruvate metabolism. For this, in vitro-produced bovine embryos were cultured from Day 5 in two experimental groups (synthetic oviducal fluid with amino acids [SOFaa] + 4% bovine serum albumin): Control (no additional supplementation), or in the presence of 2 mM dichloroacetate (DCA), a pyruvate to acetyl-CoA conversion stimulator. Blastocysts were collected on Day 7 and the inner cell masses were submitted to the CUT&Tag analysis. Briefly, cells were immobilised on concanavalin A-coated magnetic beads and permeabilised, incubated with a primary antibody specific for H3K27ac, and followed by incubation with a secondary antibody. The cells were then incubated with assembled transposomes (protein A fused to the Tn5 transposase enzyme). DNA cleavage and library preparation were managed in one single step and the resulting fragments were sequenced. To compare DCA to control, three experimental replicates of each group were sequenced in NextSeq PE40 high output. Data were pre-processed and analysed by CLC genomics workbench 22.0.2 (QIAGEN®). After quality control and duplicate removal, the number of reads for control group had a mean of 12.607.455 and for DCA 8.588.631 reads, with a 96% mean of high quality reads. Nearby gene information annotation revealed 588 peaks in control (376 genes) and 751 in DCA (481 genes) considering P ≤ 0,05. From those, 506 were common in both groups, while 40 were exclusively found in control and 174 in DCA. Enrichment of H3K27ac in DCA group was found, among others, in ALDOA (peak shape score = 1,75), subunit of a glycolytic enzyme that catalyses the conversion of fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate as well as CYTB (peak shape score = 20,54), which plays a key role in the mitochondrial complex III, which performs one step in the oxidative phosphorylation, corroborating the findings of our previous transcriptomic data of this model. These initial findings corroborate the thoroughgoing character of the metaboloepigenetics concept in the pre-implantation bovine embryo. A robust integrative analysis with our previous metabolic and molecular data is now necessary to better elucidate the mechanisms by which the metabolism alters the epigenetic profile of pre-implantation embryos.
This research was supported by FAPESP 2019/22025-1.
