SIRT3 deregulation is linked to mitochondrial dysfunction in Alzheimer's disease

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Despite decades of study, effective treatments for AD are lacking. Mitochondrial dysfunction has been closely linked to the pathogenesis of AD, but the relationship between mitochondrial pathology and neuronal damage is poorly understood. Sirtuins (SIRT, silent mating type information regulation 2 homolog in yeast) are NAD-dependent histone deacetylases involved in aging and longevity. The objective of this study was to investigate the relationship between SIRT3 and mitochondrial function and neuronal activity in AD. SIRT3 mRNA and protein levels were significantly decreased in AD cerebral cortex, and Ac-p53 K320 was significantly increased in AD mitochondria. SIRT3 prevented p53-induced mitochondrial dysfunction and neuronal damage in a deacetylase activity-dependent manner. Notably, mitochondrially targeted p53 (mito-p53) directly reduced mitochondria DNA-encoded ND2 and ND4 gene expression resulting in increased reactive oxygen species (ROS) and reduced mitochondrial oxygen consumption. ND2 and ND4 gene expressions were significantly decreased in patients with AD. p53-ChIP analysis verified the presence of p53-binding elements in the human mitochondrial genome and increased p53 occupancy of mitochondrial DNA in AD. SIRT3 overexpression restored the expression of ND2 and ND4 and improved mitochondrial oxygen consumption by repressing mito-p53 activity. Our results indicate that SIRT3 dysfunction leads to p53-mediated mitochondrial and neuronal damage in AD. Therapeutic modulation of SIRT3 activity may ameliorate mitochondrial pathology and neurodegeneration in AD.ope

Sp3 and Sp4 can repress transcription by competing with Sp1 for the core cis-elements on the human ADH5/FDH minimal promoter

The human alcohol dehydrogenase 5 gene (also known as the formaldehyde dehydrogenase gene, ADH5/FDH) has a GC-rich promoter with many sites at which transcription factors bind. A minimal promoter extending from -34 base pairs (bp) to +61 bp directs high levels of transcription in several different cells, consistent with the ubiquitous expression of the gene. Nearly the entire minimal promoter can be bound by Sp1. We analyzed the transcriptional regulation of ADH5/FDH by members of the Sp1 multigene family. Two core cis-elements (-22 bp to +22 bp) had the highest affinity for Sp1. Mutagenesis revealed that these cis-elements are critical for transcriptional activation. The zinc-finger domains of Sp3 and Sp4 also bind selectively to the core cis-elements. In Drosophila SL2 cells, which lack endogenous Sp1, the minimal promoter cannot drive transcription. Introduction of Sp1 activated transcription over 50-fold, suggesting that Sp1 is critical in the initiation of transcription. Neither Sp3 nor Sp4 was able to activate transcription in those cells, and transcriptional activation by Sp1 was repressed by Sp3 or Sp4. These data suggest that Sp3 and Sp4 can repress transcription by competing with Sp1 for binding to the core cis-elements. The content of Sp1, Sp3, and Sp4 in different cells may be critical factors regulating transcription of the ADH5/FDH gene.ope

Expression patterns of alpha-synuclein in human hematopoietic cells and in Drosophila at different developmental stages.

Alpha-synuclein, a presynaptic protein of the central nervous system, has been implicated in the synaptic events such as neuronal plasticity during development and learning, and neuronal degeneration under pathological conditions. As an effort to understand the biological function of alpha-synuclein, we examined the expression patterns of alpha-synuclein in various human hematopoietic cells, and in Drosophila at different developmental stages. The alpha-synuclein was ubiquitously expressed in all the tested hematopoietic cells including T cells, B cells, NK cells, and monocytes, as well as in the lymphoma cell lines, Jurkat and K562. A potential alpha-synuclein homologue was also expressed in Drosophila, and its expression appeared to be temporally and spatially regulated during development. Our data suggest that alpha-synuclein may function in invertebrates as well as in vertebrates and its function may not be restricted to the neuron.ope

A unique histone deacetylase inhibitor alters microRNA expression and signal transduction in chemoresistant ovarian cancer cells

Previously, we demonstrated potent antineoplastic activity of a distinctive histone deacetylase inhibitor (HDACI), AR42, against chemoresistant CP70 ovarian cancer cells in vitro and in vivo. Here, in follow-up to that work, we explored AR42 global mechanisms-of-action by examining drug-associated, genome-wide microRNA and mRNA expression profiles, which differed from those of the well-studied HDACI vorinostat. Expression of microRNA genes in negative correlation with their "target" coding gene (mRNA) transcripts, and transcription factor genes with expression positively correlated with coding genes having their cognate binding sites, were identified and subjected to gene ontology analyses. Those evaluations showed AR42 gene expression patterns to negatively correlate with Wnt signaling (> 18-fold induction of SFRP1), the epithelial-to-mesenchymal transition (40% decreased ATF1), and cell cycle progression (33-fold increased 14-3-3σ). By contrast, AR42 transcriptome alterations correlated positively with extrinsic ("death receptor") apoptosis (> 2.3-fold upregulated DAPK) and favorable ovarian cancer histopathology and prognosis. Inhibition of Wnt signaling was experimentally validated by: (1) > 2.6-fold reduced Wnt reporter activity; and (2) 36% reduction in nuclear, activated β-catenin. Likely AR42 induction of multiple (type I or type II autophagic) cell death cascades was further supported by 57% decreased reliance upon reactive oxygen, increased mitochondrial membrane disruption, and caspase independence, as compared with vorinostat. Taken together, we demonstrate distinct antineoplastic pathway alterations, in aggressive ovarian cancer cells, following treatment with a promising HDACI, AR42. These combined computational and experimental approaches may also represent a straightforward means for mechanistic studies of other promising antineoplastics, and/or the identification of agents that may complement epigenetic therapies.ope

Zeste maintains repression of Ubx transgenes: support for a new model of Polycomb repression

During late embryogenesis, the expression domains of homeotic genes are maintained by two groups of ubiquitously expressed regulators: the Polycomb repressors and the Trithorax activators. It is not known how the activities of the two maintenance systems are initially targeted to the correct genes. Zeste and GAGA are sequence-specific DNA-binding proteins previously shown to be Trithorax group activators of the homeotic gene Ultrabithorax (Ubx). We demonstrate that Zeste and GAGA DNA-binding sites at the proximal promoter are also required to maintain, but not to initiate, repression of Ubx. Furthermore, the repression mediated by Zeste DNA-binding site is abolished in zeste null embryos. These data imply that Zeste and probably GAGA mediate Polycomb repression. We present a model in which the dual transcriptional activities of Zeste and GAGA are an essential component of the mechanism that chooses which maintenance system is to be targeted to a given promoter.ope

Activation of dynamin I gene expression by Sp1 and Sp3 is required for neuronal differentiation of N1E-115 cells.

Dynamin I is a key molecule required for the recycling of synaptic vesicles in neurons, and it has been known that dynamin I gene expression is induced during neuronal differentiation. Our previous studies established that neuronal restriction of dynamin I gene expression is controlled by Sp1 and nuclear factor-κB-like element-1. Here, using a series of deletion constructs and site-directed mutation, we found that transcription of dynamin I gene during neuronal differentiation of N1E-115 cells is controlled primarily by the Sp1 element located between −13 to −4 bp of the dynamin I promoter. Gel shift analysis demonstrated that in addition to Sp1, Sp3 could interact with this Sp1 element. The requirement for Sp family transcription factors in dynamin I gene expression was confirmed by using mithramycin, an inhibitor of Sp1/Sp3 binding. Mithramycin repressed dynamin I gene expression and resulted in blocking of neuronal differentiation of N1E-115 cells. The localization of the dynamin I protein was also restricted in the peripheral region of the nucleus by the mithramycin treatment. Thus, all of our results suggest that induction of dynamin I gene expression during N1E-115 cell differentiation is modulated by Sp1/Sp3 interactions with the dynamin I promoter, and its expression is important for neuronal differentiation of the N1E-115 cells.ope

Proto-oncoprotein Zbtb7c and SIRT1 repression: implications in high-fat diet-induced and age-dependent obesity

Zbtb7c is a proto-oncoprotein that controls the cell cycle and glucose, glutamate, and lipid metabolism. Zbtb7c expression is increased in the liver and white adipose tissues of aging or high-fat diet-fed mice. Knockout or knockdown of Zbtb7c gene expression inhibits the adipocyte differentiation of 3T3-L1 cells and decreases adipose tissue mass in aging mice. We found that Zbtb7c was a potent transcriptional repressor of SIRT1 and that SIRT1 was derepressed in various tissues of Zbtb7c-KO mice. Mechanistically, Zbtb7c interacted with p53 and bound to the proximal promoter p53RE1 and p53RE2 to repress the SIRT1 gene, in which p53RE2 was particularly critical. Zbtb7c induced p53 to interact with the corepressor mSin3A-HADC1 complex at p53RE. By repressing the SIRT1 gene, Zbtb7c increased the acetylation of Pgc-1α and Pparγ, which resulted in repression or activation of Pgc-1α or Pparγ target genes involved in lipid metabolism. Our study provides a molecular target that can overexpress SIRT1 protein in the liver, pancreas, and adipose tissues, which can be beneficial in the treatment of diabetes, obesity, longevity, etc.ope

Transcriptional Activity of Sp1 Is Regulated by Molecular Interactions between the Zinc Finger DNA Binding Domain and the Inhibitory Domain with Corepressors, and This Interaction Is Modulated by MEK

Sp1 activates the transcription of many cellular and viral genes with the GC-box in either the proximal promoter or the enhancer. Sp1 is composed of several functional domains, such as the inhibitory domain (ID), two serine/threonine-rich domains, two glutamine-rich domains, three C2H2-type zinc finger DNA binding domains (ZFDBD), and a C-terminal D domain. The ZDDBD is the most highly conserved domain among the Sp-family transcription factors and plays a critical role in GC-box recognition. In this study, we investigated the protein-protein interactions occurring at the Sp1ZFDBD and the Sp1ID, and the molecular mechanisms controlling the interaction. Our results found that Sp1ZFDBD and Sp1ID repressed transcription once they were targeted to the proximal promoter of the pGal4 UAS reporter fusion gene system, suggesting molecular interaction with the repressor molecules. Indeed, mammalian two-hybrid assays, GST fusion protein pull-down assays, and co-immunoprecipitation assays showed that Sp1ZFDBD and Sp1ID are able to interact with corepressor proteins such as SMRT, NcoR, and BCoR. The molecular interactions appear to be regulated by MAP kinase/Erk kinase kinase (MEK). The molecular interactions between Sp1ID and the corepressor might explain the role of Sp1 as a repressor under certain circumstances. The siRNA-induced degradation of the corepressors resulted in an up-regulation of Sp1-dependent transcription. The cellular context of the corepressors and the regulation of molecular interaction between corepressors and Sp1ZFDBD or Sp1ID might be important in controlling Sp1 activity.ope

Interleukin 6 gene promoter polymorphism is not associated with Kawasaki disease

We examined the IL-6 gene promoter and detected several interesting promoter polymorphisms: GGGCTG insertion at +162 bp and G deletion at +168 bp positions (M1), A to G substitution at -594 bp (M2) of the reported IL-6 promoter sequence. Other rare variations were also observed at several positions: -583 bp (T insertion), -507 bp (C insertion), -71 bp (T deletion), +17 bp (C insertion), and +121 bp (GC insertion). Although Kawasaki disease (KD) patients demonstrate a drastic increase in serum interleukin-6 (IL-6) during the acute phase that parallels the duration of fever, there were no significant differences in the nucleotide sequence between the KD patients and normal control group. By transient transfection with IL-6 gene promoter-luciferase fusion plasmids into CV-1 cells, we tested the functional significances of the polymorphisms. Mutations at +162 bp, +168 bp and -594 bp significantly decreased luciferase expression (P < 0.05), suggesting the promoter elements flanking the mutated nucleotides are important in transcriptional activation.ope

KR-POK interacts with p53 and represses its ability to activate transcription of p21WAF1/CDKN1A.

Transcriptional regulation by p53 is thought to play a role in its ability to suppress tumorigenesis. However, there remain gaps in understanding about how p53 regulates transcription and how disrupting this function may promote cancer. Here we report a role in these processes for the kidney cancer-related gene KR-POK (ZBTB7C), a POZ domain and Krüppel-like zinc finger transcription factor that we found to physically interact with p53. Murine embryonic fibroblasts isolated from genetically deficient mice (Kr-pok(-/-) MEFs) exhibited a proliferative defect relative to wild-type mouse embryonic fibroblasts (MEF). The zinc finger domain of Kr-pok interacted directly with the DNA binding and oligomerization domains of p53. This interaction was essential for Kr-pok to bind the distal promoter region of the CDKN1A gene, an important p53 target gene encoding the cell-cycle regulator p21WAF1, and to inhibit p53-mediated transcriptional activation of CDKN1A. Kr-pok also interacted with the transcriptional corepressors NCoR and BCoR, acting to repress histone H3 and H4 deacetylation at the proximal promoter region of the CDKN1A gene. Importantly, Kr-pok(-/-) MEFs displayed an enhancement in CDKN1A transactivation by p53 during the DNA damage response, without any parallel changes in transcription of either the p53 or Kr-pok genes themselves. Furthermore, Kr-pok promoted cell proliferation in vitro and in vivo, and its expression was increased in more than 50% of the malignant human kidney cancer cases analyzed. Together, our findings define KR-POK as a transcriptional repressor with a pro-oncogenic role that relies upon binding to p53 and inhibition of its transactivation function.ope