Introduction
Results
Low cloning efficiency is thought to be caused, at least partially, by inadequate
nuclear remodeling and reprogramming of the donor nucleus. Treatment of
reconstructed zygotes with a histone deacetylase inhibitor (HDACi) after nuclear
transfer (NT) can help facilitate nuclear reprogramming by potentially opening
up the DNA architecture thus allowing proteins like RNA polymerases to gain
access to the DNA and begin transcription. Members of the hydroxamic acid
containing class I and IIa/b HDACi such as trichostatin A (TSA) and 6-(1,3dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (Scriptaid)
are potent HDACis that result in an increase in the global acetylation of histones.
It has been shown that Scriptaid does increase cloning efficiency in pigs when
compared to TSA treatment (Zhao 2009, 2010). When gene expression of Scriptaid
treated nuclear transfer blastocyst stage embryos was examined, only a few
aberrant transcripts (3 of 14 tested) returned to levels of normal in vivo
blastocysts (Whitworth et al 2011). Ono et al 2010 showed that healthy mice were
produced after treating NT zygotes with a different class I and IIa/b inhibitor,
Suberoylanilide Hydroxamic Acid (SAHA). SAHA and its hydrophobic
derivative, 4-iodo-SAHA (I-SAHA) were tested in pig and showed to have similar
blastocyst rates and slightly higher total cell numbers when compared to Scriptaid
treated NT embryos (Whitworth , manuscript in preparation). Deep sequencing
of these embryos was performed would reveal the differences in transcripts in NT
blastocysts that have been treated with HDACi. Pathway analysis revealed an up
regulation of transcripts in the lysosomal pathway in the HDACi treated
blastocyst stage embryos when compared to non-treated embryos.
Objective
Materials and Methods
NT was performed on enucleated MII oocytes using a genetically modified fetal
fibroblast cell line . After NT, oocytes were electrically fused and activated.
Presumptive zygotes were immediately cultured with HDACi, Scriptaid (500 nM),
SAHA (10 mM) or I-SAHA (1 mM) or without inhibitor (Control) for 14-16 hours.
Clones were then removed from HDACi and cultured to the blastocyst stage in PZM3
under 5% oxygen tension for 7 days. The zona pellucida were removed and four
pools of 10 NT embryos from each treatment were collected and snap frozen. RNA
was amplified using the Ovation RNA Seq System (Nugen). Libraries were prepared
using the TruSeq RNA-Seq library kit and subjected to Illumina HiSeq sequence
analysis resulting in 100 base pair reads. Reads were then analyzed by the MU
Bioinformatics Consortium and tiled to a pig custom genome described previously
(Bauer 2010). Pairwise comparisons between HDACi treated and non treated embryos
were performed in EdgeR (Empirical analysis of digital gene expression data).
Transcripts were considered significantly different with a p <0.05, FDR Q<0.05 and
>1.5 fold change. Genbank accessions from up and down regulated transcripts were
loaded in Database for Annotation, Visualization and Integrated Discovery (DAVID)
to identify enriched biological themes (Huang et al 2009).
10 μM SAHA
Table 1: Full Development of NT embryos treated with HDACi
Embryos
Number
Treatment
Transferred Recipients
858
4
1.0 μM I-SAHA
395
2
10.0 μM SAHA
0.5 μM Scriptaid
616
3
Number
Pregnant
3
2
2
Pregnancy
Rate
75%
100%
66.7%
Number
piglets
10
5
9
Table 2: Up and Down Regulated Transcripts in HDACi treated blastocyst-stage
embryos (P<0.05, Q<0.05, >1.5 fold difference from NT Control)
Treatment
SAHA
ISAHA
Scriptaid
Up
Regulated
79
82
55
Down
Regulated
81
120
66
Table 3: Up regulated lysosomal associated transcripts identified in HDACi treated
blastocyst-stage embryos (P<0.05, Q<0.05, >1.5 fold difference from NT Control)
The goal of this experiment was to determine the transcriptional profile of pig
blastocyst stage embryos that were treated with the HDACis, Scriptaid, SAHA and ISAHA following NT.
0.5 μM Scriptaid
The analysis identified 121 differentially expressed transcripts between NT and Scriptaid,
160 between NT and SAHA and 120 between NT and I-SAHA. Genbank accessions from up
and down regulated transcripts were loaded in Database for Annotation, Visualization and
Integrated Discovery (DAVID) to identify enriched biological themes. All three HDACi
treatments yielded the highest enrichment for transcripts within the KEGG pathway,
lysosome, including up regulation of Cathepsins A and K, Table 3..
1 μM I-SAHA
References
Bauer BK, Isom SC, Spate LD, Whitworth KM, Spollen WG, Blake SM, Springer GK, Murphy CN, Prather RS. 2010. Transcriptional profiling by deep
sequencing identifies differences in mRNA transcript abundance in in vivo-derived versus in vitro-cultured porcine blastocyst stage embryos. Biology of
reproduction 83(5):791-798.
Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc.
2009;4(1):44-57.
Ono T, Li C, Mizutani E, Terashita Y, Yamagata K, Wakayama T. 2010. Inhibition of Class IIb Histone Deacetylase Significantly Improves Cloning
Efficiency in Mice. Biol Reprod.
Zhao J, Hao Y, Ross JW, Spate LD, Walters EM, Samuel MS, Rieke A, Murphy CN, Prather RS. 2010. Histone deacetylase inhibitors improve in vitro and
in vivo developmental competence of somatic cell nuclear transfer porcine embryos. Cloning Stem Cells 12(1):75-83.
Zhao J, Ross JW, Hao Y, Spate LD, Walters EM, Samuel MS, Rieke A, Murphy CN, Prather RS. 2009. Significant improvement in cloning efficiency of an
inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer. Biol Reprod 81(3):525-530.
Whitworth KM, Zhao J, Spate LD, Li R, Prather RS. 2011. Scriptaid corrects gene expression of a few aberrantly reprogrammed transcripts in nuclear
transfer pig blastocyst stage embryos. Cell Reprogram 13(3):191-204.
Gene
Name
CTSK
CTSA
HEXA
HEXB
LGMN
Fold
Fold
Fold
Change Change Change GenBank
SAHA
ISAHA Scriptaid Accession
3.03
4.13
3.45 NM_214302
1.63
1.6 no change NM_001243629
2.16
2.12
1 NM_001123221.1
1.91
1.67
1.84 NM_213921.1
2.48
2.17
2.27 XM_001927082.4
SMPD1
no change
Annotation
Sus scrofa cathepsin K (CTSK), mRNA
Sus scrofa cathepsin A (CTSA), mRNA
Sus scrofa hexosaminidase A (alpha polypeptide) (HEXA), mRNA
Sus scrofa hexosaminidase B (beta polypeptide) (HEXB), mRNA
PREDICTED: Sus scrofa legumain (LGMN), mRNA
PREDICTED: Sus scrofa sphingomyelin phosphodiesterase 1,
3.57 no change XM_003482522.1 acid lysosomal (SMPD1), mRNA
Summary
HDACi inhibitor treated blastocyst stage embryos have an increase in expression of
transcripts associated with the KEGG pathway, lysosome. Interestingly, treated
embryos have increase d gene expression of acid hyrdrolases, CTSK and LGMN and an
increase in glycosidases, HEXA and HEXB. The increase in CTSA would provide
greater stability to the glycosidase, GLB, indicating an increased ability of need for
protein turnover within these NT embryos.
Travel to this meeting was funded by Douglas D. Randall Young Scientists Development Fund
and Food for the 21st Century which are both dedicated to the growth of young scientists.
The authors would like to acknowledge NSRRC Project Director Dr. Eric Walters for his
assistance with this project