Epigenetic regulation is a mechanism by which heritable changes in gene expression are controlled by chromatin status rather than primary DNA sequence. Changes in chromatin structure affect accessibility of DNA elements to the transcriptional machinery and thus affect transcription activity of the gene. A key event in this process is reversible modification of core histones, which is catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDs, HDAs, or HDACs). In general, histone deacetylation is related to transcriptional gene silencing, whereas acetylation is associated with gene activation.To study the role of histone deacetylase in plant gene regulation and development, we generated constitutive antisense histone deacetylase 1 (CASH) transgenic plants. AtHD1 is a homolog of RPD3 protein, a global transcriptional regulator in yeast. Expression of the antisense AtHD1 caused dramatic reduction in endogenous AtHD1 transcription, resulting in accumulation of acetylated histones. Down-regulation of histone deacetylation caused a variety of growth and developmental abnormalities and ectopic expression of tissue-specific genes. However, changes in genomic DNA methylation were not detected in repetitive DNA sequences in the transgenic plants.We also identified a T-DNA insertion line in exon 2 of AtHD1 gene (athd1-t1), resulting in a null allele at the locus. The complete inhibition of the AtHD1 expression induced growth and developmental defects similar to those of CASH transgenic plants. The phenotypic abnormalities were heritable across the generations in the mutants. When the athd1-t1/athd1-t1 plants were crossed to wild-type plants, the mutant phenotype was corrected in the F1 hybrids, which correlated with the AtHD1 expression and reduction of histone H4 Lys12 acetylation. Microarray analysis was applied to determine genome-wide changes in transcriptional profiles in the athd1-t1 mutant. Approximately 6.7% (1,753) of the genes were differentially expressed in leaves between the wild-type (Ws) and the athd1-t1 mutant, whereas 4.8% (1,263) of the genes were up- or down-regulated in flower buds of the mutant. These affected genes were randomly distributed across five chromosomes of Arabidopsis and represented a wide range of biological functions. Chromatin immunoprecipitation assays indicated that the activation for a subset of genes was directly associated with changes in acetylation profiles.

Epigenetic regulation is a mechanism by which heritable changes in gene expression are controlled by chromatin status rather than primary DNA sequence. Changes in chromatin structure affect accessibility of DNA elements to the transcriptional machinery and thus affect transcription activity of the gene. A key event in this process is reversible modification of core histones, which is catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDs, HDAs, or HDACs). In general, histone deacetylation is related to transcriptional gene silencing, whereas acetylation is associated with gene activation.To study the role of histone deacetylase in plant gene regulation and development, we generated constitutive antisense histone deacetylase 1 (CASH) transgenic plants. AtHD1 is a homolog of RPD3 protein, a global transcriptional regulator in yeast. Expression of the antisense AtHD1 caused dramatic reduction in endogenous AtHD1 transcription, resulting in accumulation of acetylated histones. Down-regulation of histone deacetylation caused a variety of growth and developmental abnormalities and ectopic expression of tissue-specific genes. However, changes in genomic DNA methylation were not detected in repetitive DNA sequences in the transgenic plants.We also identified a T-DNA insertion line in exon 2 of AtHD1 gene (athd1-t1), resulting in a null allele at the locus. The complete inhibition of the AtHD1 expression induced growth and developmental defects similar to those of CASH transgenic plants. The phenotypic abnormalities were heritable across the generations in the mutants. When the athd1-t1/athd1-t1 plants were crossed to wild-type plants, the mutant phenotype was corrected in the F1 hybrids, which correlated with the AtHD1 expression and reduction of histone H4 Lys12 acetylation. Microarray analysis was applied to determine genome-wide changes in transcriptional profiles in the athd1-t1 mutant. Approximately 6.7% (1,753) of the genes were differentially expressed in leaves between the wild-type (Ws) and the athd1-t1 mutant, whereas 4.8% (1,263) of the genes were up- or down-regulated in flower buds of the mutant. These affected genes were randomly distributed across five chromosomes of Arabidopsis and represented a wide range of biological functions. Chromatin immunoprecipitation assays indicated that the activation for a subset of genes was directly associated with changes in acetylation profiles.

No description available.

Breast cancer is the most common malignancy and a major cause of cancer-related death among Canadian women. Although treatment of primary breast tumours is highly successful through surgery, metastatic breast cancer is difficult to treat. Cancer progression and metastasis require the accumulation of numerous genetic and epigenetic alterations. Normal cells that acquire such alterations can transform into cancer cells, resulting in primary tumour formation. Primary tumours are a heterogeneous population, containing cells of various metastatic potentials. Cells that acquire a high potential for metastasis can spread to secondary locations. Our model system consists of two subpopulations, with different metastatic potential, derived from the same rat mammary adenocarcinoma. Using this model, a differentially expressed novel gene, termed MS-1, was discovered. Due to significant expression of this gene in the poorly metastatic subpopulation and lack of expression in the highly metastatic subpopulation, MS-1 may have involvement in metastasis suppression. Several breast cancer metastasis suppressor genes have been identified on the basis that they are down-regulated during the progression of metastasis. Epigenetic mechanisms, such as DNA methylation, account for loss of expression in several of these genes. Hypermethylation of CpG islands within gene promoters results in deacetylation of histone proteins and produces a compact chromatin structure that is unfavourable for transcription. A CpG island spans the 5 untranslated region, exon 1 and part of intron 1 of the MS-1 gene. Our data reveals aberrant methylation patterns of this CpG island in our model. Also, MS-1 expression appears to be partially induced by both DNA methylation and histone deacetylation inhibitors. Following a screen of several cancer cell lines of various metastatic potential, it appears that the presence of DNA methylation in the CpG island of MS-1 correlates with the lack of MS-1 expression. Therefore, these results suggest that MS-1 may be silenced in cells of high metastatic potential through epigenetic mechanisms.

Breast cancer is the most common malignancy and a major cause of cancer-related death among Canadian women. Although treatment of primary breast tumours is highly successful through surgery, metastatic breast cancer is difficult to treat. Cancer progression and metastasis require the accumulation of numerous genetic and epigenetic alterations. Normal cells that acquire such alterations can transform into cancer cells, resulting in primary tumour formation. Primary tumours are a heterogeneous population, containing cells of various metastatic potentials. Cells that acquire a high potential for metastasis can spread to secondary locations. Our model system consists of two subpopulations, with different metastatic potential, derived from the same rat mammary adenocarcinoma. Using this model, a differentially expressed novel gene, termed MS-1, was discovered. Due to significant expression of this gene in the poorly metastatic subpopulation and lack of expression in the highly metastatic subpopulation, MS-1 may have involvement in metastasis suppression. Several breast cancer metastasis suppressor genes have been identified on the basis that they are down-regulated during the progression of metastasis. Epigenetic mechanisms, such as DNA methylation, account for loss of expression in several of these genes. Hypermethylation of CpG islands within gene promoters results in deacetylation of histone proteins and produces a compact chromatin structure that is unfavourable for transcription. A CpG island spans the 5 untranslated region, exon 1 and part of intron 1 of the MS-1 gene. Our data reveals aberrant methylation patterns of this CpG island in our model. Also, MS-1 expression appears to be partially induced by both DNA methylation and histone deacetylation inhibitors. Following a screen of several cancer cell lines of various metastatic potential, it appears that the presence of DNA methylation in the CpG island of MS-1 correlates with the lack of MS-1 expression. Therefore, these results suggest that MS-1 may be silenced in cells of high metastatic potential through epigenetic mechanisms.

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In order to improve the synthetic route for diazonium perfluoroalkyl benzenesulfonylimide (PFSI) zwitterionic monomers, N-deacetylation of the coupling product was proposed to replace the reduction of aromatic amine intermediates. A series of hydrolysis methods, such as acid and base catalyzed refluxing, were explored for the N-deacetylation to obtain the PFSI aromatic amine. Factors such as temperature, concentration of acid/base and the time needed for the reaction to take place were investigated in an attempt to optimize the reaction condition. The basic hydrolysis was preferred since it was expected to carry out the N-deacetylation and debromination in one batch reaction. N-deacetylation in base at high concentrations was successful, however, side reaction of the perfluorovinyl ether occurred. It was discovered that the best N-deacetylation method is to reflux/sonicate the coupling product with acid in methanol for six hours. The intermediates and purified products were characterized with 1HNMR, 19FNMR, GC-MS and IR.

As an important posttranslational modification, protein acetylation plays critical roles in many biological processes such as gene transcription, DNA damage repair, apoptosis and metabolism. The acetylation occurs on the ε-amino group of specific lysine residues, and is catalyzed by histone acetyltransferases (HATs). In cellular contexts, HATs are found to target hundreds and thousands of substrates including histone and nonhistone proteins. Lysine acetylation changes the microenvironment of protein and may potentially alter protein activity and protein-protein interaction. The goal of this dissertation project is to investigate the impact of lysine acetylation on the catalysis of MYST HATs, and to establish the strategy for labeling substrates of the MYST HATs at cellular level. To understand the regulatory mechanism of MYST HATs, a detailed study was carried out to investigate the active site lysine acetylation of two MYST HATs (MOF and Tip60). Autoradiography and immunoblotting data shows that mutation of active site lysine differentially affects the enzyme autoacetylation activity and the cognate substrate acetylation activity. In addition, deacetylated MOF and Tip60 were prepared by using the nonspecific lysine deacetylase Sirt1. Kinetic study demonstrated that the acetylation of the active site lysine on MYST HATs marginally modulates the HAT catalysis. This work provides new insights into the regulatory mechanism of MYST catalysis. In the second part of my work, we designed and synthesized a series of Ac-CoA analogs conjugated with alkynyl or azido functional groups. Meanwhile, the active site of the MOF was engineered to expand the cofactor binding capability. Fluorescence screening was carried out to characterize the enzyme activity to Ac-CoA analogs. MOF-I317A with all analogs and MOF-I317A/H273A–5HYCoA were identified and further applied in the labeling of the cognate histone H4 protein and HAT substrates in 293T cell lysate. Visualizing of the labeled substrate was achieved using the alkynyl or azido-tagged fluorescent reporters through the copper-catalyzed azide−alkyne cycloaddition. As expected, the histone H4 protein was successfully labeled by the active enzyme-cofactor pairs. More intriguingly, multiple protein bands in cell lysate were labeled and observed. This work provides a new versatile strategy in exploring the substrates of MYST HATs at the proteomic level.

<p>Reversible acetylation has emerged as an important post-translational modification that rivals phosphorylation in regulating chromatin structure and gene expression. Acetylation of histone is associated with transcriptional activation while deacetylation is linked to transcriptional repression. Moreover, histone deacetylase inhibitors induce growth arrest, differentiation and apoptosis of cancer cells and therefore appear to be promising anti-tumor agents. While transcriptional deregulation is thought to be the main mechanism underlying their therapeutic effects, the exact mechanisms and targets by which HDAC inhibitors, which are mostly non-specific, achieve their anti-tumor effects remain poorly understood. In other words, it is not known which and how each HDAC members are involved in supporting tumor growth.</p><p>In this thesis, I have showed that HDAC6, a cytoplasmic localized and cytoskeleton-associated deacetylase, is required for efficient oncogenic transformation and tumor formation. I have found that HDAC6 expression is induced upon oncogenic Ras-induced transformation in both human somatic cells and murine fibroblasts. Conversely, murine fibroblasts deficient in HDAC6 are more resistant to both oncogenic Ras and ErbB2-dependent transformation, indicating a critical role for HDAC6 in oncogene-induced transformation. Supporting this hypothesis, inactivation of HDAC6 in several human cancer cell lines effectively impairs anchorage-independent growth <em>in vitro</em> and their ability to form tumors in immunocompromised mice. I have demonstrated that the impairment of anchorage independent growth in HDAC6 deficient cells is associated with increased anoikis and mechanistically a defect in activation of the AKT and ERK kinase cascades. Additionally, <em>HDAC6 </em>null mice are more resistant to two-stage chemical carcinogen-induced skin tumors. Finally, I have demonstrated that the tumor-promoting effect of HDAC6 is probably mediated through the molecular chaperon Hsp90. While Hsp90 is known to be deacetylated by HDAC6 and has been implicated in stabilizing Raf-1 and ErbB2, I have found that suppression of HDAC6 impairs the stability of Raf-1 and the association between Hsp90 and ErbB2.</p><p>In conclusion, my work provides the first experimental evidence that of all the HDAC members, the cytoplasmic deacetylase HDAC6 is required for efficient oncogenic transformation, indicating that reversible acetylation plays a critical role in regulating cellular functions of non-histone non-nuclear cytoplasmic proteins that contributes to malignant transformation. Furthermore, this work identifies HDAC6 as an important component underlying the anti-tumor effects of HDAC inhibitors.</p> / Dissertation

Trimethylation of histone H3 lysine 9 (H3K9me3) and DNA methylation mark heterochromatin, contributing to gene silencing and normal cellular functions. My research investigated the control of H3K9me3 and DNA methylation in the filamentous fungus Neurospora crassa. The H3K9 methyltransferase complex, DCDC, consists of DIM-5, DIM-7, DIM-9, DDB1, and CUL4. Each component of DCDC is required for H3K9me3. The DIM-9/DDB1/CUL4 subunits are reminiscent of known cullin E3 ubiquitin ligases. I showed that core features of CUL4-based E3 ubiquitin ligases are not required for H3K9me3 and DNA methylation in Neurospora. H3K9me3 is bound by heterochromatin protein 1 (HP1) to recruit the DIM-2 DNA methyltransferase and the HCHC histone deacetylase complex. HCHC consists of HP1, CDP-2, HDA-1, and CHAP. Both HP1 and CDP-2 harbor conserved chromodomains that bind H3K9me3, and CHAP contains two putative AT-hook domains that bind A:T-rich DNA. To test the contributions of these domains to HCHC function, I deleted the chromodomains of HP1 and CDP-2. Deletion of the HP1 chromodomain resulted in a reduction of DNA methylation, which was not exacerbated by deletion of the CDP-2 chromodomain. A strain with deletions of chap and the HP1 chromodomain showed a DNA methylation phenotype comparable to the loss of the HDA-1 catalytic subunit. These findings support a model in which recognition of H3K9me3 and A:T-rich DNA by HP1 and CHAP, respectively, are required for proper HCHC function. To examine the relationships between H3K9me3, DNA methylation, and histone acetylation, I utilized in vivo protein tethering of core heterochromatin components. The requirement of DIM-7 for native heterochromatin, previously implicated in localizing the H3K9 methyltransferase DIM-5, was not bypassed by DIM-5 tethering, indicating that DIM-7 has additional roles within the DCDC. Artificial localization of the HCHC histone deacetylase, by tethering HP1 or HDA-1, resulted in induction of H3K9me3, DNA methylation, and gene silencing, but silencing did not require H3K9me3 or DNA methylation. HCHC-mediated establishment of H3K9me3 was not required for de novo heterochromatin formation at native heterochromatic loci suggesting a role in heterochromatin spreading. Together, this work implicates HDA-1 activity as a key driver of heterochromatin spreading and silencing. This dissertation includes previously published co-authored material.