Doctor of Philosophy / Biochemistry and Molecular Biophysics / Timothy P. Durrett / The membrane bound O-acyltransferase (MBOAT) family contains multi-pass membrane proteins that add fatty acids to different compounds. Despite their importance in economic activity and human health, little is known about the localization of the active site and regions important for determining substrate specificity of MBOATs. Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) is the only known MBOAT enzyme that exhibits a high preference for acetyl-CoA, the shortest possible acyl-CoA. EaDAcT catalyzes the transfer of the acetate group from acetyl-CoA to the sn-3 position of diacylglycerol to form 3-acetyl-1,2-diacyl-sn-glycerol. Our goal was to investigate the structural features and the amino acid residues that define substrate specificity of EaDAcT to provide insights into the mechanism by which MBOAT family controls substrate selection. By mapping the membrane topology of EaDAcT we obtained the first experimentally determined topology model for a plant MBOAT. The EaDAcT model contains four transmembrane domains with both the N- and C- termini oriented toward the endoplasmic reticulum lumen. The MBOAT signature region including the putative active site His-257 of the protein is embedded in the third transmembrane domain close to the interface between the membrane and the cytoplasm. In order to identify amino acid residues important for acetyltransferase activity, we isolated and characterized orthologs of EaDAcT from other acetyl-TAG producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro. Mutagenesis of conserved residues of DAcTs revealed that Ser-253, His-257 and Asp-258 are essential for enzyme activity of EaDAcT, suggesting their involvement in the enzyme catalysis. Alteration of residues unique to acetyltransferases did not alter the acyl donor specificity of EaDAcT, implying that multiple amino acids are important for substrate recognition. Together, this work identifies the structural features of EaDAcT and offers an initial view of the amino acids important for activity of the enzyme.

Although skeletal myogenesis is largely controlled by myogenic regulatory factors, epigenetic modifications have recently emerged as an essential regulatory mechanism of gene expression. Molecular regulation of stem cell differentiation is exerted through both genetic and epigenetic factors over distal enhancer regions. Understanding the mechanistic action of active or poised enhancers is therefore, imperative for the control of stem cell differentiation. Based on the genome-wide co-occurrence of different epigenetic marks in proliferating myoblasts, we have generated a chromatin state model to profile differentiation- and rexinoid-responsive histone acetylation in early myoblast differentiation. Here, we delineate the functional mode of transcription regulators during early myogenic differentiation using genome-wide chromatin state association. We define a role of transcriptional coactivator p300, when recruited by muscle master regulator MyoD, in the establishment and regulation of myogenic loci at the onset of myoblast differentiation. In addition, we reveal an enrichment of loci-specific histone acetylation at p300 associated active or poised enhancers, mainly when enlisted by MyoD. We have previously established that bexarotene, a clinically approved agonist of retinoid X receptor (RXR), promotes the specification and differentiation of skeletal muscle lineage. Hence, we investigated the genome-wide impact of rexinoids on myogenic differentiation and uncovered a new mechanism of rexinoid action, which is mediated by the nuclear receptor and largely reconciled through direct regulation of MyoD gene expression. In addition, we determined rexinoid-responsive residue-specific histone acetylation at a distinct chromatin state associated with MyoD and myogenin. Finally, through ChIP-seq and RNA-seq analyses, we have identified dystroglycan (Dag1) as a differentiation-dependent and a rexinoid-responsive model target, and we revealed a possible co-regulation of Dag1 by p300 and MyoD accompanied by enrichment of loci-specific histone acetylation. Taken together, we provide novel molecular insights into the regulation of myogenic enhancers by p300 in concert with MyoD. Furthermore, we provide novel mechanistic perceptions into the interplay between RXR signaling and chromatin states pertinent to myogenic programs in early myoblast differentiation. Our studies present a valuable insight for driving condition-specific chromatin state or enhancers pharmacologically to treat muscle-related diseases and for the identification of additional myogenic targets and molecular interactions for therapeutic development.

Fatty acid amides are an emerging family of bioactive lipids that consists of N-acylethanolamines, N-acylarylalkylamides, N-acylglycines, N-acyl amino acids, N-monoacylpolyamides, and primary fatty acid amides. Short chain fatty acid amides are products of inactivated biogenic amines such as dopamine, histamine, octopamine, and serotonin, whereas long chain fatty acid amides have been implicated in a number of physiological process such as the perception and inhibition of chronic pain through binding to their specific receptors. The most famous; therefore, the most studied long chain fatty acid amide is anandamide or also known as N-arachidonylethanolamine. The biosynthesis of anandamide is well defined; however, other long-chain fatty acid amides, such as the N-acyldopamines, N-acylserotonins, N-acylglycines, N-acyl amino acids, and primary fatty acid amides have remained elusive to date. Understanding the complete biosynthetic pathway for these cell signaling lipids, may yield new exciting molecular targets for human health and disease. Discovery of the long-chain fatty acid amide biosynthetic enzymes has proven to be challenging due to the low biologic abundance of the respective metabolites found in organisms, the interconnection of the pathways, and expense of using mammalian cells and/or organisms. This led to the transition of studying these metabolites and their respective biosynthetic enzymes in Drosophila melanogaster. D. melanogaster is an ideal system to study fatty acid amide biosynthesis because the respective metabolites have been identified, the cost of maintaining the organism is relatively low, and genetic manipulation (RNAi) is universally available. This dissertation is dedicated to defining enzymes involved in D. melanogaster N-acylarylalkyamide biosynthesis. The biologically relevant long-chain N-acylarylalkylamides are comprised of long-chain N-acyldopamines and N-acylserotonins. Very little is known for how these potent cell signaling lipids are biosynthesized in the cell. One possible route is the N -acylation of the respective biogenic amine by an N-acyltransferase enzyme. An enzyme known to catalyze this chemistry is arylalkylamine N-acetyltransferase (AANAT), which catalyzes the formation of N-acetylarylalkylamides from acetyl CoA and the corresponding arylalkylamide. The N-acetylation of biogenic amines is a critical step in Drosophila melanogaster for the inactivation of amine neurotransmitters, sclerotization of the cuticle, and to serve as the penultimate intermediate in the biosynthesis of melatonin. Two AANAT(L) enzymes has been previously evaluated in D. melanogaster and six other putative AANATL enzymes have identified in the fly genome. One AANAT is expressed as two biologically relevant isoforms, AANAT variant A (AANATA) and AANAT variant B (AANATB), where AANATA differs from AANATB by the truncation of 35 amino acids on the N-terminus. The other AANATL enzyme to be previously studied is AANATL2, which was found to catalyze the formation of N-acetyltryptamine from acetyl CoA and tryptamine. Herein, we expressed six AANAT(L) enzymes (AANATA and AANATB, AANATL2, AANATL3, AANATL7, and AANATL8) and sought to define the acyl-CoA and amine substrates for each enzyme. To accomplish this, we developed an activity based screening assay to define acyl-CoA and amine substrates for AANATL2, AANATL3, AANATL7, and AANATL8. Following this work, we defined the acyl-CoA and amine substrate specificity for AANATA, AANATL2, AANATL3, and AANATL7. We have identified acetyl CoA and arylalkylamines as substrates for AANATA, AANATL2, and AANATL3; whereas AANATL7 acetylates histamine and arylalkylamines. AANATL2 was additionally shown to catalyze the formation of long-chain N-acyldopamines and N-acylserotonins. Following these important set of results, we solved the kinetic mechanism for AANATA, AANATL2, and AANATL7 in which these enzymes were shown to catalyze the formation of N-acylarylalkylamides by an ordered sequential mechanism where the acyl-CoA substrate binds first followed by the corresponding amine substrate. Finally, we evaluated the function of structural amino acids on regulating catalysis, structural features of substrates that effect binding and/or catalysis, and generated data leading to a proposed chemical mechanism by means of pH-activity profiles and site-directed mutagenesis of prospective catalytic residues.

Transforming growth factor β (TGF-β) is a member of a large family of cytokines that regulate many crucial events in cells, including proliferation, differentiation, migration and apoptosis. Deregulated TGF-β signaling is associated with various forms of cancers and developmental disorders. TGF-β binds to a receptor complex at the surface of cells and activates a signaling cascade involving specific intracellular signaling proteins, known as Smads. Following receptor activation, the Smads are activated by phosphorylation and translocate to the nucleus, where they activate or repress the expression of specific genes. Posttranslational modifications regulate the function of proteins in a number of ways, including their activity, stability, localization, and/or interactions with other proteins. These modifications are important to modulate the strength and specificity of cellular signal transduction. Smad7, an important negative modulator of TGF-β signaling, has been shown to be acetylated by the acetyltransferase p300. My aim was to further explore the involvement of protein acetylation in TGF-β-dependent signaling. We could show that the acetylation of Smad7 is a reversible process. Interestingly, earlier work had shown that the acetylation of Smad7 prevented its degradation. In agreement with this observation, we found that the ubiquitylation and degradation of Smad7 was increased following cotransfection with HDAC1, a protein deacetylase. Based on our observations, we propose a model in which the stability of Smad7 is controlled by the balance between its acetylation, deacetylation and ubiquitylation. In a separate study, we found that also Smad2 and Smad3 are acetylated by p300/CBP and P/CAF upon TGF-β stimulation. Moreover, we found that the acetylation of the short isoform of Smad2 promoted its DNA binding activity, resulting in an increased transcriptional activity. Our results suggest that the increased DNA binding in response to acetylation is due to a conformational change in Smad2.

Post translational modifications in histone proteins are transmissible changes that are not coded for in the DNA sequence itself but have a significant affect in the control of gene expression. Eukaryotic transcription is a regulated process, and acetylation plays a major role in this regulation. Deranged equilibrium of histone acetylation can lead to alteration in chromatin structure and transcriptional dysregulation of genes that are involved in the control of proliferation, cell-cycle progression, differentiation and or apoptosis. Evidence shows that high glucose conditions mimicking diabetes can increase histone acetylation and augment the inflammatory gene expression. Recent advances also highlight the involvement of altered histone acetylation in gastrointestinal carcinogenesis or hyperacetylation in amelioration of experimental colitis. However, the role of histone acetylation under obesity conditions is not yet known. Therefore in the present study, western blot analysis in the liver of Zucker obese versus lean rats was performed to determine the pattern and level of H3 and H4 acetylation (both in nuclear and homogenate fractions) at specific lysine (K) in pathological state of hepatic steatosis The same technique was also applied in the liver of obese rats fed higher amounts of vitamin B6 (OH) versus those fed normal amounts of vitamin B6 (ON) to assess if hyper-acetylation can be a protective response to hepatic steatosis. In both experimental models, it was also of interest to elucidate the expression of anti- and pro- apoptotic factor Bcl-2 and Bax in respect to histone acetylation. It was observed that, in liver homogenate fractions in control animals (LC/OC), there was a higher level of histone H3 acetylation at (K9, K14) and H4 acetylation at K5 in the obese animals. In contrast, the nuclear level of H3 and H4 acetylation at the same lysine residues was considerably higher in the lean and lower in the obese animals. Obese animals contained lower liver preneoplastic lesions as well as liver weight as a result of higher amounts of vitamin B6, had significantly higher H3 acetylation at K9 and K14 and H4 acetylation at K5, in both homogenate and nuclear fractions. However, histone acetylation was not detected for histone H4 at lysine 12 (K12) in either control group (LC/OC) or obese with different B6 diet group (OH/ON). Nevertheless, global histone H3 and H4 acetylation in both homogenate and nuclear fractions, was slightly higher in the lean rats and obese rats fed higher amounts of B6. By using the western blot technique, the level of anti- and pro- apoptotic Bcl-2 and Bax were also evaluated. The moderately higher level expression of anti-apoptotic Bcl2 protein was found in lean animals, whereas the expression of pro-apoptotic Bax was significantly higher in obese animals. Furthermore, anti-apoptotic Bcl2 protein expression was slightly higher in the obese rats fed normal amounts of B6 diet; but, pro-apoptotic Bax was higher in the obese rats fed higher amounts of vitamin B6. This is the first study which shows that hyperacetylation of histones in liver nuclei can be correlated with amelioration of hepatic steatotis. Histone acetylation and B6 rich diet might be involved in the regulation of biological availability of key apoptotic proteins, which, in turn, can possibly modify the severity of the disease.

Post translational modifications in histone proteins are transmissible changes that are not coded for in the DNA sequence itself but have a significant affect in the control of gene expression. Eukaryotic transcription is a regulated process, and acetylation plays a major role in this regulation. Deranged equilibrium of histone acetylation can lead to alteration in chromatin structure and transcriptional dysregulation of genes that are involved in the control of proliferation, cell-cycle progression, differentiation and or apoptosis. Evidence shows that high glucose conditions mimicking diabetes can increase histone acetylation and augment the inflammatory gene expression. Recent advances also highlight the involvement of altered histone acetylation in gastrointestinal carcinogenesis or hyperacetylation in amelioration of experimental colitis. However, the role of histone acetylation under obesity conditions is not yet known. Therefore in the present study, western blot analysis in the liver of Zucker obese versus lean rats was performed to determine the pattern and level of H3 and H4 acetylation (both in nuclear and homogenate fractions) at specific lysine (K) in pathological state of hepatic steatosis The same technique was also applied in the liver of obese rats fed higher amounts of vitamin B6 (OH) versus those fed normal amounts of vitamin B6 (ON) to assess if hyper-acetylation can be a protective response to hepatic steatosis. In both experimental models, it was also of interest to elucidate the expression of anti- and pro- apoptotic factor Bcl-2 and Bax in respect to histone acetylation. It was observed that, in liver homogenate fractions in control animals (LC/OC), there was a higher level of histone H3 acetylation at (K9, K14) and H4 acetylation at K5 in the obese animals. In contrast, the nuclear level of H3 and H4 acetylation at the same lysine residues was considerably higher in the lean and lower in the obese animals. Obese animals contained lower liver preneoplastic lesions as well as liver weight as a result of higher amounts of vitamin B6, had significantly higher H3 acetylation at K9 and K14 and H4 acetylation at K5, in both homogenate and nuclear fractions. However, histone acetylation was not detected for histone H4 at lysine 12 (K12) in either control group (LC/OC) or obese with different B6 diet group (OH/ON). Nevertheless, global histone H3 and H4 acetylation in both homogenate and nuclear fractions, was slightly higher in the lean rats and obese rats fed higher amounts of B6. By using the western blot technique, the level of anti- and pro- apoptotic Bcl-2 and Bax were also evaluated. The moderately higher level expression of anti-apoptotic Bcl2 protein was found in lean animals, whereas the expression of pro-apoptotic Bax was significantly higher in obese animals. Furthermore, anti-apoptotic Bcl2 protein expression was slightly higher in the obese rats fed normal amounts of B6 diet; but, pro-apoptotic Bax was higher in the obese rats fed higher amounts of vitamin B6. This is the first study which shows that hyperacetylation of histones in liver nuclei can be correlated with amelioration of hepatic steatotis. Histone acetylation and B6 rich diet might be involved in the regulation of biological availability of key apoptotic proteins, which, in turn, can possibly modify the severity of the disease.

Prostate cancer (PCa) is one of the most prevelant cancers in males in many countries, increasing in frequency with age. PCa incidence and mortality rates are not evenly distributed worldwide. Family history is an established risk factor for prostate cancer and families demonstrating autosomal dominant or X-linked transmission of susceptibility have been observed. Although an increasing number of candidate genes or hereditary prostate cancer susceptibility have been identified, only 5 to 10 percent of prostate cancer cases in the population may arise from major susceptibility genes. A few risk factors for PCa development are advanced age, an intact androgen metabolism, ethnicity, and genetic background. Other genetic factors, in combination with possible environmental risk factors for prostate cancer, may have greater public health importance. Genetic polymorphisms that may be associated with prostate cancer risk are much more common in the population than are high-penetrance cancer susceptibility genes. In this study, the effect of N-acetyltransferase 2 (NAT2) and Glutathione S-transferases (GSTM1 and GSTT1) were investigated, since polymorphism in these genes may alter their enzymatic activity and, therefore, their capacity to biotransform xenobiotic compounds. In order to evaluate the potential association between NAT2 , GSTM1 and GSTT1 genotypes and prostate cancer risk, a hospital based case control study was carried out in a Turkish population consisting of 30 histologically confirmed incident prostate cancer cases and 67 control subjects with no present or previous history of cancer. The GSTM1 and GSTT1 genotypes showed no significant differences between case and control groups, with respect to their frequencies and it was observed that GSTM1 null genotype was more common in cases with a 60% frequency. Even though the frequency of slow NAT2 acetylator genotype was 80% in cases and 50,7% in controls NAT2 rapid acetylator showed no association with prostate cancer statistically. These results suggested that GSTM1 null genotype is a susceptibility factor for prostate cancer, particularly in the presence of NAT2 slow acetylator genotype with no significance. Further studies with a larger size are required to confirm the presence and significance of this relationship.

Huntington’s disease (HD) is caused by expression of the huntingtin gene containing an expanded CAG repeat. N-terminal mutant huntingtin protein (N-mHtt) accumulates in the nucleus and impairs transcription of a subset of genes through incorporation into transcriptional complexes or sequestration of proteins away from the promoter. CREB-binding protein (CBP) is a transcriptional co-activator and acetyltransferase (AT) that binds to N-mHtt. We hypothesized that overexpressing CBP fragments that lack a promoter association domain would block N-mHtt-mediated transcriptional dysregulation and toxicity. We found that overexpressing full-length CBP or CBP fragments did not reverse transcriptional dysregulation, but did decrease toxicity in a cell model of HD. Overexpressing fragments of CBP containing the AT domain increased toxicity in wild-type cells, while overexpressing a fragment lacking this domain had no effect. We conclude that excess AT activity was detrimental in wild-type cells, while overexpressing CBP or CBP fragments was protective in HD cells.

Le gène suppresseur de tumeur LKB1 code une protéine sérine/thréonine kinase qui régule le métabolisme et la polarité cellulaires. LKB1 exerce une partie de ses fonctions biologiques en phosphorylant et en activant les 14 kinases appartenant à la famille des protéines kinases activées par l'AMP (AMPK). Le membre éponyme de cette famille, AMPK, agit comme un senseur nutritionnel essentiel dans la cellule. La recherche que j'ai conduite au cours de ma thèse a porté sur le mode de régulation de LKB1. L'holoenzyme LKB1, un hétérotrimère comprenant deux autres protéines appelées STRAD et MO25, est dotée d'une activité catalytique constitutive. Mon travail a permis de montrer que la lysine 48 de LKB1 est acétylée par l´acétyltransférase GCN5. Par des approches biochimiques et des techniques d'imagerie, j'ai montré que l'acétylation de LKB1 par GCN5 favorise sa localisation nucléaire, la fraction non-acétylée étant localisée à la fois dans le cytoplasme et le noyau. GCN5 promeut également l´export cytoplasmique de LKB1 de manière HAT-indépendente et régule son niveau d´expression. Afin de préciser la contribution de cette acétylation à la fonction in vivo de LKB1, j'ai utilisé le modèle expérimental de la crête neurale (CN) chez le poulet. En effet, j'ai été impliquée au cours de ma thèse dans une étude issue du laboratoire, qui a établi que l'activité de LKB1 est requise pour la délamination, la migration polarisée et la survie des cellules de la CN céphalique. Ces dernières contribuent à la formation de la majorité du squelette cranio-facial des vertébrés. Le signal LKB1 dans ces cellules est relayé par l'AMPK et la kinase ROCK et converge sur le moteur moléculaire dépendant de l'actine, la Myosine II. A l'aide du même modèle expérimental, j'ai montré que GCN5 est exprimé dans les cellules de la CN au cours de l'embryogenèse et que l'interaction fonctionnelle entre LKB1 et GCN5 est nécessaire à l'activité de LKB1 au cours de l'ontogénie des cellules de la CN céphalique et donc de la formation de la tête. / The tumor suppressor gene LKB1 encodes a serine/threonine kinase which regulates the cellular metabolism and polarity. Its biological activity is partly exerted through the phosphorylation and activation of 14 kinases which belong to the AMP-activated protein kinases (AMPK). The eponym member of this family acts as an essential nutritional sensor in the cell. The research that I conducted during my PhD focused on the regulation of LKB1. The LKB1 holoenzyme is a constitutively active heterotrimer comprising two other proteins called STRAD and MO25. My PhD project shows that LKB1 is acetylated on the lysine 48 residue by the acetyltransferase GCN5. Using biochemical approaches and cell imaging, I have shown that the acetylation of LKB1 by GCN5 favors its nuclear localization, while the non-acetylated fraction is localized in both the nucleus and the cytoplasm. GCN5 also promotes the cytoplasmic export of LKB1 in an HAT-independent manner and regulates its expression levels. In order to investigate the contribution of this acetylation to the functions of LKB1 in vivo, I have used the experimental model of the neural crest (NC) in chick embryos. Indeed, during my PhD, I have contributed to a study, initiated by my host laboratory, in which we show that LKB1 is required for the delamination, polarized migration and survival of neural crest cells (NCCs) which contribute to the formation of most craniofacial structures in vertebrates. LKB1 signaling is mediated by AMPK and the ROCK kinase and converges towards the actin-dependent molecular motor, Myosin II. Using the same experimental model, I have shown that GCN5 is expressed in NCCs during embryogenesis and that the functional interaction between GCN5 and LKB1 is essential for the activity of LKB1 in the cephalic NCCs ontogenesis and head formation.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy P. Durrett / Many Euonymus species express an acetyltransferase enzyme in their seeds which catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol (DAG) producing unusual acetyl-1,2-diacyl-sn-glycerols (acetyl-TAG). The presence of the sn-3 acetate group gives acetyl-TAG with unique physical properties over regular triacylglycerol (TAG) found in vegetable oils. The useful characteristics of acetyl-TAG oil offer advantages for its use as emulsifiers, lubricants, and 'drop-in' biofuels. One enzyme, Euonymus alatus diacylglycerol acetyltransferase (EaDAcT), responsible for acetyl-TAG synthesis in nature was previously isolated from the seeds of Euonymus alatus (burning bush) and expressed in the oilseed crop Camelina sativa. Expression of EaDAcT successfully led to production of high levels of acetyl-TAG in camelina seeds. To further increase acetyl-TAG accumulation in transgenic camelina seeds, multiple strategies were examined in this study. Expression of a new acetyltransferase enzyme (EfDAcT) isolated from the seeds of Euonymus fortunei, which was previously shown to possess higher in vitro activity and in vivo acetyl-TAG levels compared to EaDAcT, increased acetyl-TAG accumulation by 20 mol%. Suppression of the endogenous competing enzyme DGAT1 further enhanced acetyl-TAG accumulation to 90 mol% in selected transgenic line. Studying the regulation of EfDAcT transcript, protein, and acetyl-TAG levels during seed development further provided new insights on the factors limiting acetyl-TAG accumulation.