Browsing by Subject "histone"
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Item Characterization and Analysis of the Bovine Epigenome during Preimplantation Embryo Development In Vitro(2012-10-19) Williamson, Gayle LingerDuring early mammalian embryogenesis, the embryonic genome undergoes critical reprogramming events that include changes in both DNA methylation and histone modifications necessary to control chromatin structure and thus, gene expression. Improper reprogramming of the epigenome during this window of development can lead to a vast number of imprinting anomalies, which are increased in children and livestock conceived in vitro. In the bovine, which closely resembles human preimplantation development, epigenetic changes occur from fertilization through the blastocyst stages. In particular, and concurrent with embryonic genome activation (EGA), de novo DNA methylation begins at the 8-cell stage. In order to explore the roles of histone-modifying enzymes during this crucial period of development, we characterized the transcript expression of several enzymes key enzymes across in vitro bovine preimplantation development using quantitative real-time PCR. Two of the 7 genes analyzed (Suz12 and Lsh) exhibited notable increases at the 8-16 cell stages, with basal levels observed both before and after this. These increases coincided with both EGA and de novo DNA methylation. We further explored their roles in bovine preimplantation embryos by knocking down expression via the use of gene-specific targeting siRNAs. Independent suppression of either Suz12 or Lsh via cytoplasmic microinjection of targeting siRNAs resulted in lower development rates (p < 0.0001), and poorer embryo quality of the morulas and blastocysts that survived. In addition, Suz12 suppression led to reductions in both H3K27 (p < 0.0001) and H3K9 (p = 0.07) trimethylation, and an increase in DNA methylation levels (p < 0.0001), as compared to the null-injected controls. Lsh suppression did not change H3K27, but led to a reduction in H3K9 trimethylation (p = 0.006) and an increase in DNA methylation (p < 0.0001). Clearly our data demonstrate that these epigenetic modifiers play a critical role in formation of the embryonic epigenome, but further research would be necessary in order to fully characterize gene activities during this developmental window.Item Characterization and expression of histone deacetylase 1 (athd1) in Arabidopsis thaliana(Texas A&M University, 2005-08-29) Fong, Man KimThe reversible process of histone acetylation and deacetylation is an important mechanism of epigenetic regulation in the control of gene expression and chromatin structure. In general, histone acetylation is related to gene activation, whereas histone deacetylation is associated with transcriptional gene silencing and maintenance of heterochromatin. A large number of histone deacetylases (HDACs), the enzymes that catalyze the reaction of histone deacetylation, have been identified in plants and other eukaryotes, and they were found to play crucial roles in plant growth and development. In Arabidopsis thaliana, histone deacetylase 1 (AtHD1) is a homolog of Saccharomyces cerevisiae Rpd3 that is a global transcriptional regulator. Downregulation of AtHD1 in transgenic Arabidopsis results in histone hyperacetylation and induces a variety of phenotypic and developmental defects, suggesting that AtHD1 is also a global regulator of many physiological and developmental processes. To characterize the expression pattern and distribution of AtHD1 in cells, the subcellular location of AtHD1 was determined by monitoring the expression of an AtHD1-GFP fusion protein in a transient expression assay and in transgenic Arabidopsis.The results show that AtHD1 is localized in the nucleus and appears to be excluded from the nucleolus. The histone deacetylase activity of AtHD1 was studied in an in vitro assay using radiolabeled histone peptides as a substrate. Recombinant AtHD1 produced by bacteria demonstrated a moderate but significant HDAC activity, whereas that produced by the baculovirus expression system did not have activity. This suggests that AtHD1 may require other cofactors or association with other proteins, rather than post-translational modifications, in order to have full HDAC activity. To study the possible interactions of AtHD1 with other proteins, a recombinant AtHD1 protein with two units of c-myc epitope fused to its C-terminus was expressed in transgenic Arabidopsis. We attempted to isolate proteins interacting with AtHD1 by co-immunoprecipitation (Co-IP). However, in the first few trials of Co-IP, a lot of contaminating proteins were present in the eluent along with the recombinant AtHD1-cmyc protein. Improvements in the experimental conditions are required for further investigation.