Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors

Hosseini et al. BMC Genomics (2016) 17:16 DOI 10.1186/s12864-015-2264-z RESEARCH ARTICLE Open Access Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors Sayyed Morteza Hosseini1,2, Isabelle Dufort2, Julie Nieminen2, Fariba Moulavi1, Hamid Reza Ghanaei1, Mahdi Hajian1, Farnoosh Jafarpour1, Mohsen Forouzanfar1, Hamid Gourbai2, Abdol Hossein Shahverdi2, Mohammad Hossein Nasr-Esfahani1,2* and Marc-André Sirard3* Abstract Background: The limited duration and compromised efficiency of oocyte-mediated reprogramming, which occurs during the early hours following somatic cell nuclear transfer (SCNT), may significantly interfere with epigenetic reprogramming, contributing to the high incidence of ill/fatal transcriptional phenotypes and physiological anomalies occurring later during pre- and post-implantation events. A potent histone deacetylase inhibitor, trichostatin A (TSA), was used to understand the effects of assisted epigenetic modifications on transcriptional profiles of SCNT blastocysts and to identify specific or categories of genes affected. Results: TSA improved the yield and quality of in vitro embryo development compared to control (CTR-NT). Significance analysis of microarray results revealed that of 37,238 targeted gene transcripts represented on the microarray slide, a relatively small number of genes were differentially expressed in CTR-NT (1592 = 4.3 %) and TSA-NT (1907 = 5.1 %) compared to IVF embryos. For both SCNT groups, the majority of downregulated and more than half of upregulated genes were common and as much as 15 % of all deregulated transcripts were located on chromosome X. Correspondence analysis clustered CTR-NT and IVF transcriptomes close together regardless of the embryo production method, whereas TSA changed SCNT transcriptome to a very clearly separated cluster. Ontological classification of deregulated genes using IPA uncovered a variety of functional categories similarly affected in both SCNT groups with a preponderance of genes required for biological processes. Examination of genes involved in different canonical pathways revealed that the WNT and FGF pathways were similarly affected in both SCNT groups. Although TSA markedly changed epigenetic reprogramming of donor cells (DNA-methylation, H3K9 acetylation), reconstituted oocytes (5mC, 5hmC), and blastocysts (DNA-methylation, H3K9 acetylation), these changes did not recapitulate parallel marked changes in chromatin remodeling, and nascent mRNA and OCT4-EGFP expression of TSA-NT vs. CRT-NT embryos. (Continued on next page) * Correspondence: ; Marc-Andre.Sirard@ fsaa.ulaval.ca 1 Department of Reproduction and Development, Reproductive Biomedicine Centre, Royan Institute for Biotechnology, ACECR, Isfahan, Iran 3 Centre de Recherche en Biologie de la Reproduction, Faculté des Sciences de l’Agriculture et de l’Alimentation, Département des Sciences Animales, Pavillon INAF, Université Laval, Québec, QC G1V 0A6, Canada Full list of author information is available at the end of the article © 2016 Hosseini et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hosseini et al. BMC Genomics (2016) 17:16 Page 2 of 21 (Continued from previous page) Conclusions: The results obtained suggest that despite the extensive reprogramming of donor cells that occurred by the blastocyst stage, SCNT-specific errors are of a non-random nature in bovine and are not responsive to epigenetic modifications by TSA. Keywords: Somatic cell nuclear transfer, Trichostatin A, Transcriptome, Bovine Background Early embryonic development in mammals begins in transcriptional silence [1] with an oocyte-mediated transcriptional reprogramming of parental gametes occurring during an across-the-board process of “erase-and-rebuild” [2]. In this process, the parental transcription programs are erased long before (maternal) or soon after (paternal) fertilization to generate a relatively naïve zygotic chromatin upon which the transcription program of a new life cycle is rebuilt de novo after activation of the zygotic genome [2, 3]. Any perturbation in this process will result in ill or fatal transcriptional phenotypes of the resultant embryos [4]. The very few viable clones obtained at the end of a typical cloning experiment underscore that substantial differences exist between transcriptional reprogramming of somatic cell nuclei and gametes [5]. Six microarray studies have been carried out to explore the transcriptomic profiles of cloned (SCNT) vs. fertilized (IVF) bovine blastocysts [4, 6–10] and all reported extensive transcriptional reprogramming of the donor cells by the blastocyst stage with few genes, even as low as twenty [8], being deregulated in SCNT embryos. Conversely, numerous studies have reported abnormal patterns of DNA-methylation and histone acetylation/methylation of bovine clones [11–14]. These studies concluded that bovine SCNT embryos suffer from genome-wide hypermethylation, associated with elevated heterochromatic histone methylation (H3K9me2) and H3K9 acetylation in the trophectoderm layer. Therefore, a typical SCNT embryo may be transcriptionally close to (euchromatin), but epigenetically far from (heterochromatin), normal embryos, demonstrating that epigenetic but not expression barriers limit reprogramming efficiency [15]. Accordingly, it would be interesting to determine how small transcriptional aberrations translate into broad epigenetic anomalies or vice versa. Somatic cell chromatin is compact and static, due to the tight association of chromatin with heterochromatin binding proteins and of histones with chromatin. Proper erasure of these epigenotype markers is essential for chromatin remodeling and pluripotency, as restricting the exchange of these chromatin factors makes the chromatin inaccessible to oocyte reprogramming factors [13]. The short duration and compromised efficiency of the erasure process is considered to be the main cause of the transcriptomic and epigenomic anomalies of cloned embryos [16]. To circumvent this problem, assisted epigenetic modification of somatic cells, before and/or soon after SCNT, with DNA-methyltransferase (DNMTi) and histone deacetylase (HDACi) inhibitors has been recently used to assist the endogenous epigenome-modifying machinery and enhance the speed and extent (...truncated)