Effect of Trichostatin A on Chromatin Remodeling, Histone Modifications, DNA Replication, and Transcriptional Activity in Cloned Mouse Embryos1

Abstract

Our group and others have found that the treatment of embryos with trichostatin A (TSA) after cloning by somatic cell nuclear transfer (SCNT) results in a significant improvement in efficiency. We believe that TSA treatment improves nuclear remodeling via histone modifications, which are important in the epigenetic regulation of gene silencing and expression. Some studies found that treatment of SCNT-generated embryos with TSA improved lysine acetylation of core histones in a manner similar to that seen in normally fertilized embryos. However, how histone methylation is modified in TSA-treated cloned embryos is not completely understood. In the present study, we found that TSA treatment caused an increase in chromosome decondensation and nuclear volume in SCNT-generated embryos similar to that in embryos produced by intracytoplasmic sperm injection. Histone acetylation increased in parallel with chromosome decondensation. This was associated with a more effective formation of DNA replication complexes in treated embryos. We also found a differential effect of TSA on the methylation of histone H3 at positions K4 and K9 in SCNT-generated embryos that could contribute to genomic reprogramming of the somatic cell nuclei. In addition, using 5-bromouridine 5'-triphosphate-labeled RNA, we showed that TSA enhanced the levels of newly synthesized RNA in 2-cell embryos. Interestingly, the amount of SCNT-generated embryos showing asymmetric expression of nascent RNA was reduced significantly in the TSA-treated group compared with the nontreated group at the 2-cell stage. We conclude that the incomplete and inaccurate genomic reprogramming of SCNT-generated embryos was improved by TSA treatment. This could enhance the reprogramming of somatic nuclei in terms of chromatin remodeling, histone modifications, DNA replication, and transcriptional activity.

Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency

“…Several reports have shown that TSA treatment of the donor cell or the reconstructed embryo can improve NT cloning efficiency, which is likely through the creation of a permissive chromatin state (Enright et al 2003, Enright et al 2005, Kishigami et al 2006, Rybouchkin et al 2006, Bui et al 2010. Recent studies have observed that donor cells with decreased H3K9me3 or H3K27me3 levels are associated with improved NT efficiency in cloned embryos (Antony et al 2013, Saini et al 2015, further suggesting that the establishment of accessible chromatin can facilitate nuclear reprogramming in cloned embryos.…”

“…Furthermore, reduction of the repressive mark H3K9me3 in donor cells also significantly increases cloning efficiency, indicating that the global reduction of the repressive histone modification is beneficial to the onset of reprogramming in cloned embryos (Antony et al 2013. In addition to treatments of donor cells, TSA-treated cloned embryos also displayed lower repressive epigenetic marks, enhanced histone acetylation, remodeling of pericentromeric heterochromatin and increased nascent RNA transcription (Kishigami et al 2006, Rybouchkin et al 2006, Maalouf et al 2009, Bui et al 2010. These results suggest that establishing an accessible chromatin state can significantly improve genomic reprogramming.…”

Derivation and application of pluripotent stem cells for regenerative medicine

“…The abnormal development of nuclear transfer embryos is always associated with defects in epigenetic reprogramming (Dean et al, 2001;Kang et al, 2001;Ohgane et al, 2001;Yamazaki et al, 2006). Treatment with histone demethylase inhibitor can increase the reprogramming efficiency (Bui et al, 2010;Dai et al, 2010;Kishigami et al, 2006;Song et al, 2014). Besides, modified culture methods showed that in vitro culture environment plays an important role in nuclear transfer reprogramming (Dai et al, 2009).…”

Epigenetic regulation in pluripotent stem cells: a key to breaking the epigenetic barrier

“…RNA interference screening of ES cells for chromatin components showed that a large set of HAT complexes to which Tip60 (TAT-interacting protein 60)/p400 contributes are ES cell development regulators, such as Gata4 and Gata6, and significantly overlap with target genes of Nanog [63,64]. On the other hand, HDAC inhibitors, such as valproic acid and trichostatin A, improve the efficiency of nuclear reprogramming by both nuclear transfer [65,66] and the transduction of pluripotency genes [67], suggesting that histone acetylation is involved in the maintenance and acquisition of pluripotency.…”