Recently, it is demonstrate that the invertebrates have a immune memory, called Immune priming (IP). It was partially studied that the IP is mainly regulated by epigenetic modification. Here, to understand the IP on antimicrobial peptides (AMPs) production, we investigated larval mortality and time-dependent expression patterns of AMP genes in T. molitor larvae challenged with E. coli (two-times injection with a one-month interval). Interestingly, the results indicate that the higher and faster expression levels of most AMP genes were detected compared to the non-primed T. molitor larvae. Our results may used to improve the understanding of mechanisms of invertebrate immune memory.

α-solanine is toxic to human health by disturbing digestive and central nervous systems. However, little information has been focused on investigated with respect to α-solanine influence in mammal oocyte maturation and quality. In this study, we investigated the effects of α-solanine on oocyte maturation, quality and possible molecular mechanisms in a pig model. Porcine Cumulus-oocyte complexes (COCs) were treated with increasing concentration (0, 1, 10, 20, 50 μM) of α-solanine subjected to further in vitro maturation culture. The result showed that α-solanine significantly inhibited cumulus cells expansion and increased oocyte death rates when the concentration of α-solanine more than 10 μM. After cell cycle and cytoskeleton analysis, the results showed that α-solanine (10 μM) disturbed meiotic resumption, increased abnormal spindle formation and cortical granules (CGs) distribution rates when compared with the untreated group. α-solanine (10 μM) triggered autophagy by increasing the expression of autophagy-related genes (LC3, ATG7, LAMP2) and accumulation of LC3-specific puncta (an autophagy maker). TUNEL staining assay showed that α-solanine significantly increased apoptosis in porcine oocytes confirmed by up-regulated the levels of BAX and CAPS3 genes. Further study revealed that exposure α-solanine (10 μM) to porcine oocytes induced ROS generation, reduced mitochondrial membrane potential. In addition, our results suggested that α-solanine (10 μM) significantly increased the levels of H3K36me3 and H3K27me3 in porcine oocytes. Taken together, these data indicated that α-solanine toxic impaired oocyte maturation and quality by inhibited cumulus cells expansion, increased abnormal spindle and CGs distribution rates, triggered autophagy/apoptosis occur, accumulated ROS, decreased mitochondrial membrane potential, and changed epigenetic modifications.

A viral histone H4 is encoded in a polydnavirus called Cotesia plutellae bracovirus (CpBV), which is symbiotic to an endoparasitoid wasp, C. plutellae. Compared to general histone H4, the viral H4 possesses an extra N-terminal tail containing 38 amino acid residues, which has been presumed to control host gene expression in an epigenetic mode. This study addressed the mutational analysis of extra N-terminal amino acid residues of a viral histone H4 and their epigenetic control efficacy. Mutational analysis was performed by serially deleting each of the nine amino acid residues from N-terminal tail of a viral histone H4. Transient expression of each truncated mutants (K1M-K19) in diamondback moth, Plutella xylostella, was performed by microinjection of a recombinant expression vector and confirmed by RT-PCR. Under transient expression, we analysed the effect of these mutations on target gene, transferin. Interestingly, we found that truncated mutants (K1M-K15) did not inhibit the expression of target gene but mutations thereafter (K6M-K9M) significantly alter its expression. As expected these truncated mutants (K1M-K5M) also inhibit hemocyte nodule formation and development of Plutella xylostella. This suggest that lysine residue (K6) in the N-terminal tail is very crucial for the epigenetic control efficacy of viral histone H4.

A viral histone H4 (=CpBV-H4) is encoded in a polydnavirus, Cotesia plutellae bracovirus, and symbiotically associated with an endoparasitoid wasp, C. plutellae. It has an extended N-terminal tail consisting of 38 amino acid residues, compared to the host H4 and this extended N-terminal tail has been postulated to play a crucial role in an epigenetic control of gene expression. The (SSH) suppression subtractive hybridization analysis was analyzed in transcriptome by short-read sequencing technology. The SSH analysis provided several target and nontarget genes of a viral histone H4. In this study, we analyzed the effect CpBV-H4 on the expression of two target genes serpins and histone lysine N-methyl transferase. Transient expression of CpBV-H4 by microinjecting recombinant expression vector to non parasitized larvae of Plutella xylostella showed that it was expressed up to 70 h. Under this transient expression condition, we analyzed the effect of CpBV-H4 on the expression of target genes by RT-PCR at different time points. Interestingly, the CpBV-H4 significantly inhibited the expression of target genes after 44 h, while the truncated CpBV-H4 deleting the N-terminal tail did not show the inhibitory activity.

Epigenetic status of the genome of a donor nucleus has an important effect on the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). In our previous study has results showed that the donor cells treated with 5-aza-2’- deoxyctidine (5-aza-dC, DNA methylation inhibitors) and Trichostatin A (TSA, histone deacetylase inhibitors) could improve the development of porcine nuclear transfer embryos in vitro. In this study we want to investigate why these two drugs treatment with the donor cell can improve the cloning efficiency, whether they can alter the epigenetic status of the genome of the donor nucleus. This study included 6 groups: control group, the donor cell (porcine fetal fibroblast cell) with no treatment; 2.5 nM 5-aza-dC group, the donor cells treated with 2.5 nM 5-aza-dC for 1h; 5-aza-dC group, the donor cells treated with 5 nM 5-aza-dC for 1h; TSA group, the donor cells treated with 50 nM TSA for 1h; 2.5 nM 5-aza-dC+TSA group, the donor cells treated with 2.5 nM 5-aza-dC for 1h and subsequently treated with 50 nM TSA for another 1h; 5-aza-dC+TSA group, the donor cells treated with 5 nM 5-aza-dC and 50 nM TSA together for 1h. The first experiment detected the DNA methylation status in the different groups. After treatment with these two drugs, the DNA methylation level of the donor cells decreased, however there is no significant difference among the groups. This result indicated that the donor cell treatment with 5-aza-dC and TSA can partially alter the DNA methylation status of the donor cells. The second experiment checked the histone acetylation level of the donor cells treated with these two drugs by western blot. TSA, 2.5 nM 5-aza-dC+TSA, 5 nM 5-aza-aC+TSA, these three groups can significantly improve the hisone acetylation level compared with control and 5-aza-dC groups, there is no significant difference among these three groups. The results of this study suggest that the donor cells treated with 5-aza-dC and TSA can partially decrease DNA methylation and can significantly improve the histone acetylation level of the donor cells, these alterations of the epigenetic modification maybe can improve the clonging efficiency.

Testes‐derived unipotent male germ‐line stem (GS) cells can acquire multipotency under appropriate culture conditions to become mGS cells which can contribute to all three germ‐layers. This study was designed to investigate the epigenetic characteristics of mGS cells derived from adult mouse testes (maGS cells). The GS cells were isolated from 4 6 week DBA mouse and were cultured in Dulbecco’s modified Eagle Medium supplemented with 15% (v/v) fetal bovine serum, 1,000 U/ml LIF, 4 ng/ml GDNF at 37℃ in an humidified atmosphere of 5% CO2 in air to derive the maGS cells. The multipotency of maGS cells were verified by morphological and gene expression analyses, teratoma formation upon transplantation into nude mouse and in vitro differentiation ability. Bisulfite genomic sequencing revealed that GS cells had androgenetic DNA methylation pattern at the Igf2‐H19, Gnas‐Nespas , and Dlk1‐Dio3 imprinted gene clusters which changed to hemi‐zygotic embryonic stem (ES)‐cell like pattern in the maGS cells. Western blot analysis, using modification‐ and residue‐specific antibodies, revealed that both maGS and ES cells had similar level of histone di‐methylation at 4th and 27th lysine residue of histone 3 (H3K4me2 and H3K27me2) which represent “bivalent domain” for regulating self‐renewal and differentiation of mouse ES cells. Both maGS and ES cells also shared similar hisone modification for H3K9me2, H3K79me2, H3K9ac and H3K18ac. However, maGS cells had higher level of H3K- 36me2 and H3S10p. These data suggest that maGS and ES cells share several epigenetic characteristics but they also have their own unique epigenetic marks that may be useful as a molecular marker for their identification.

Oct4 and Nanog are well-known transcription factors related with self renewal of embryonic stem cell. In low-dose of Nanog, transcription of oct4 is increased; however, oct4 is down-regulated upon high-dose of Nanog. There is a negative feedback loop between oct4 and Nanog. To identify this regulation, we generated 4 nested sets for mouse oct4 promoter. Luciferase activities of oct4 were declined upon high-dose Nanog in all constructs. The declined effects of oct4 upon high-dose Nanog were moderated with DNMT and HDAC inhibitors (5-AZA-cytidine and trichostatin A) in 3 constructs (1867, 1346, 754). But, one construct (2179) was only sensitive to TSA. Taken together, these effects were also represented in semi-quantitative RT-PCR and Western blotting data. These data suggest that negative regulation of oct4 gene upon high-dose Nanog would be accomplished by DNMT and HDAC. Further, it will be studied whether these constraining molecules bind to CR1-4 region of oct4 promoter upon low- and high-dose of Nanog.

Cloning or somatic cell nuclear transfer (SCNT) using adult somatic cell to derive cloned embryos is a promising new technology with potential applications in both agriculture and regenerative medicine. Mammalian embryos derived by nuclear transfer are capable of development to the blastocyst stage with a relatively high efficiency of 30～ 50%. However, in full-time development, usually only 2% of NT embryos can result in live births due to abnormalities in placenta formation. In SCNT embryos, the donor cell nucleus is epigenetically reprogrammed by oocyte cytoplasm during development. Incomplete reprogramming of the donor cell genome is considered a major reason for low cloning efficiency. Aberrant epigenetic modifications include DNA methylation, histone modification and X-chromosome-inactivation. Due to a lack of basic knowledge regarding the embryos following nuclear transfer, the success rate of cloning is low. Therefore, elucidation of the molecular mechanism of SCNT embryo development will be of great value for further research. MicroRNAs (microRNA) are single-strand RNA molecules of about 19 23 nucleotides in length, which regulate gene expression by imperfect base pairing with target mRNA, subsequently guiding mRNA cleavage or translational repression. Since the first discovery and functional annotation in 1993 of the small RNA, lin-4 and let-7, which are involved in developmental timing and gene regulation during C. elegans larval development, microRNAs have received scientific attention. Now hundreds of microRNAs have been identified in various multicellular organisms, and many microRNAs have been shown to be evolutionarily conserved. The roles proposed for this novel class of tiny RNA molecules are diverse. They are likely to be involved in developmental timing, differentiation, cell proliferation, signaling pathways, apoptosis, metabolism, heterochromatin formation, genome rearrangement, brain development and carcinogenesis. Currently (2006- present) we are working to determine the role of microRNAs on the epigenetic regulation of fertilized and cloned embryo development. The general hypothesis of our research is that genetic and epigenetic factors regulate the development of preimplantation mammalian embryos, and aberrant modulations in cloned embryos are causes of abnormal development and low success rate of cloned embryos.

The vast majority of embryo generated by Assisted Reproductive Technologies (ART) do not result in a live offspring and a multiple birth is the single biggest health risk associated with human fertility treatment, and the used of frozen embryos increased for medical or personal reasons. However, practical and ethical reasons might hamper study of human embryos. Therefore, animal models are necessary to elucidate the molecular and morphological changes during development. In the serial experiments, we employed mouse embryos and a Cdx-inducible ES cell system that transdifferentiates into TS cells. We found aberrant gene expression profiles including apoptosis associated (Bcl2), lineage formation related genes (Cdx-2, Tcfap2c, Oct4, and Nanog), and/or mitochondrial DNA replication related genes (mt-cox-1, mt-cox-2, Polg, Polg2, Tfam) in mouse embryos that showed developmentally retardation between morula to blastocyst transition or post implantation development after embryo transfer to surrogate mothers, compared to control embryos. To determine direct interaction between knockdown genes via siRNA approach and putative down-stream genes involved in blastocyst formation and further development, we carried out qPCR and Chip assay in either mouse embryos or the ES cells. qPCR and Chip assay results showed target gene directly bound to promoter regions of down-regulated genes in TS cells. In conclusion, we suggested that an increased understanding of epigenetic regulation of early embryonic development through animal models may ultimately lead to better methodologies for selecting more competent embryos and and/or protocols for augmenting embryos viability.

The epigenetic therapy of cancers is emerging as an effective and valuable approach to both chemotherapy and the chemoprevention of cancer. The utilization of epigenetic targets that include histone methyltransferase (HMTase), Histone deacetylatase, and DNA methyltransferase, are emerging as key therapeutic targets. SET containing proteins such as the HMTase Setd1b has been found significantly amplified in cancerous cells. In order to shed some light on the histone methyl transferase family, we cloned the Setd1b gene from Mus musculus and build a collection of vectors for recombinant protein expression in E.coli that will pave the way for further structural biology studies. We prospect the role of the Setd1b pathway in cancer therapy and detail its unique value for designing novel anti-cancer epigenetic-drugs.

The objective of this study was to analyzed pattern of proteins expression abnormally in cloned bovine placenta. TIMP-2 protein whose function is related to extracellular matrix degradation and tissue remodeling processes was one of differentially up-regulated proteins in SCNT placenta. And one of down-regulated protein in SCNT placenta was identified as vimentin protein that is presumed to stabilize the architecture of the cytoplasm. The expression patterns of these proteins were validated by Western blotting. To evaluate how regulatory loci of TIMP-2 and vimentin genes was programmed reprogramming in cloned placenta, the status of DNA methylation in the promoter region of TIMP-2 and vimentin genes was analyzed by sodium Bisulfite mapping. The DNA methylation results showed that there was not difference in methylation pattern of TIMP-2 and vimentin loci between cloned and normal placenta. Histone H3 acetylation state of the nucleosome was analyzed in the cloned placental and normal placenta by Western blotting. A small portion of the protein lysates were subjected to Western blotting with the antibodies against anti acetyl-Histone H3. Overall histone H3 acetylation state of SCNT placenta was significantly higher than those of normal placenta cells. It is postulated that cloned placenta at the end of gestation seems to be unusual in function and morphology of placenta via improper expression of TIMP-2 and vimentin by abnormal acetylation states of cloned genome.