The HDAC Inhibitor LAQ824 Enhances Epigenetic Reprogramming and In Vitro Development of Porcine SCNT Embryos

Abstract

Background/Aims: Hypoacetylation caused by aberrant epigenetic nuclear reprogramming results in low efficiency of mammalian somatic cell nuclear transfer (SCNT). Many epigenetic remodeling drugs have been used in attempts to improve in vitro development of porcine SCNT embryos. In this study, we examined the effects of LAQ824, a structurally novel histone acetylase inhibitor, on the nuclear reprogramming and in vitro development of porcine SCNT embryos. Methods: LAQ824 treatment was supplemented during the culture of SCNT embryos. The reprogramming levels were measured by immunofluorescence and quantified by image J software. Relative expression levels of 18 genes were analyzed by quantitative real-time PCR. Results: 100 nM LAQ824 treatment of post-activation SCNT embryos for 24 h significantly improved the subsequent blastocyst formation rate. The LAQ824 treatment enhanced histone 3 lysine 9 (H3K9) levels, histone 4 lysine 12 (H4K12) levels, and reduced global DNA methylation levels as well as anti-5-methylcytosine (5-mC) at the pseudo-pronuclear and 2-cell stages. Furthermore, LAQ824 treatment positively regulated the mRNA expression of genes for histone acetylation (HAT1, HDAC1, 2, 3, and 6), DNA methylation (DNMT1, 3a and 3b), development (Pou5f1, Nanog, Sox2, and GLUT1) and apoptosis (Bax, Bcl2, Caspase 3 and Bak) in blastocysts. Conclusion: Optimum exposure (100 nM for 24 h) to LAQ824 post-activation improved the in vitro development of porcine SCNT embryos by enhancing levels of H3K9 and H4K12, reducing 5-mC, and regulating gene expression.

Down-Regulation of H3K4me3 by MM-102 Facilitates Epigenetic Reprogramming of Porcine Somatic Cell Nuclear Transfer Embryos

“…The expression of pluripotency genes is crucial for the development of preimplantation embryos, but SCNT embryos usually have defects in this respect that limits reprogramming efficiency [37, 38]_ENREF_42. LAQ824, a HDAC inhibitor, could improve porcine SCNT embryos development by increasing levels of H3K9 and H4K12, decreasing the level of 5mC, and positively regulating the expression patterns of genes related to pluripotency and apoptosis [39]. In our study, MM-102 treatment significantly improved developmental rates at each stage and the total number of blastocyst cells.…”

Enhancement of epigenetic reprogramming status of porcine cloned embryos with zebularine, a DNA methyltransferase inhibitor

“…Indirect immunofluorescence staining was done as described in our previous studies with minor modifications (Jin, Lee, Taweechaipaisankul, Kim, & Lee, ). Briefly, a total of 20–25 embryos at various stages (pseudo‐pronuclear; PNC, 2‐cell, and 4‐cell) were washed three times in 1% polyvinyl alcohol (PVA; w/v) solution, then fixed in 4% paraformaldehyde (PFA; w/v) in 1% PVA solution.…”

“…Then, amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, TX) was used to synthesize the complementary DNA (cDNA) according to the manufacturer's protocol (all cDNAs were prepared by reverse transcription of 200 ng total RNA in a reaction volume of 20 μl). The primer sequences for all genes were synthesized according to our previous studies (Jin et al, ) and are listed in Table . The RT‐qPCR mix of each gene (20 µl) consisted of 1 µl cDNA, 0.4 µl (10 pmol/µl) forward primer, 0.4 µl (10 pmol/µl) reverse primer, 10 µl SYBR Premix Ex Taq (TaKaRa, Otsu, Japan) and 8.2 µl of DEPC.…”

Expression of pluripotency‐related genes is highly dependent on trichostatin A‐assisted epigenomic modulation of porcine mesenchymal stem cells analysed for apoptosis and subsequently used for generating cloned embryos

“…On the one hand, those epigenetic modifiers represent not only the subclass of highly specific extrinsic HMT inhibitors (HMTi) such as G9A (H3K9) HMTi, whose pivotal member is diazepin‐quinazolin‐amine derivative termed BIX‐01294 (Cao et al, ; Huang et al, ), but also the subclass of ectopic non‐specific DNMT inhibitors, whose most important members are: (a) 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) (Huan, Wang, et al, ; Huan, Wu, et al, ; Huan et al, , ; Ning et al, ); and (b) zebularine (a nucleoside analog of cytidine; Diao et al, ). On the other hand, they represent the subclass of ectopic non‐selective HDAC inhibitors (HDACi), whose main members are (a) TSA (Huan, Wang, et al, ; Huan, Wu, et al, ; Huan et al, ; Opiela et al, ; Samiec et al, ); (b) scriptaid (SCPT) (Liang, Zhao, Choi, Kim, & Cui, ; Xu et al, ; Zhang et al, ); (c) oxamflatin (Hou et al, ; Mao et al, ); (d) sodium butyrate (NaBu) (Kumar et al, ; Liu et al, ); (e) m ‐carboxycinnamic acid bis hydroxamide (CBHA) (Song et al, ); (f) panobinostat, also known as LBH589 (Jin et al, ); (g) abexinostat, also termed PCI‐24781 (Jin et al, ); (h) quisinostat, also called JNJ‐26481585 (Jin, Guo, et al, ); and (i) dacinostat, also named as LAQ824 (Jin, Lee, Taweechaipaisankul, Kim, & Lee, ). The initiation of chromatin decondensation and gene transcriptional activity is triggered both via highly specific and transient inactivation of G9A (H3K9) HMTs by BIX‐01294 (Cao et al, ; Huang et al, ) and via non‐specifically blocking the biocatalytic activity of either DNMTs by 5‐aza‐dC and zebularine (Diao et al, ; Saini et al, ) or HDACs by TSA, SCPT, oxamflatin, NaBu, CBHA, panobinostat, abexinostat, quisinostat and dacinostat (Jin, Lee, et al, ; Jin et al, , ; Jin, Guo, et al, ; Kumar et al, ; Song et al, ; Zhang et al, ).…”

“…Such epigenomic modulation of nuclear donor cells, nuclear recipient cells and/or SCNT‐derived embryos appears to enhance the reprogrammability of donor cell nuclei that have been transferred into a host cytoplasm of enucleated oocytes. As a consequence, they undergo the faithful recapitulation of the scenario inevitable for switching off somatic cell‐inherited epigenetic memory and accompanying cessation of their tissue‐specific gene expression, followed by correct switching on the epigenetically dependent program of transcriptional activity, characteristic for preimplantation development of cloned embryos (Huan, Wang, et al, ; Huan, Wu, et al, ; Huang et al, ; Jin, Lee, et al, ; Samiec & Skrzyszowska, ; Wang et al, ).…”