GENE REGULATION LAB
Dimitris Kardassis, Ph.D., Head
Our lab investigates the mechanisms by which the expression of eukaryotic genes is regulated at the level of transcription. Eukaryotic transcription is a complicated process that involves numerous proteins including chromatin modification enzymes, basal transcription factors, transcriptional activators and repressors and co-regulatory proteins. Deregulated expression of physiologically important genes such as the human apolipoprotein genes or other genes involved in the biogenesis and catabolism of HDL leads to disease such as atherosclerosis. Understanding the structure and function of transcription factors is also crucial for developing drugs for the treatment of certain diseases. The field of steroid hormones provides some of the best examples of disease treatment or prevention which is based on the knowledge of the structure and function of transcription factors, in this case the hormone nuclear receptors. Gene regulation is a core component of all major signal transduction pathways which are associated with distinct gene expression profiles depending on the cellular context. This is accomplished via the activation of specific transcription factors that activate or repress gene networks in response to the stimulus. One of the best examples is the Transforming Growth Factor ß signaling pathway which influences the expression of hundreds of genes via the function of Smad proteins in the nucleus. Mutations in Smads or other components of the TGF ß pathway are frequently found in patients with different forms of cancer.
1. T GF ß /Smad signaling and regulation of gene expression (D. Kardassis, P. Papakosta, E. Vasilaki , P. Fotakis, N. Skourti-Stathaki)
Transforming Growth Factor ß (TGF ß ) is a pleiotropic cytokine that controls several key biological processes including cell proliferation, differentiation, apoptosis and extracellular matrix production. TGF ß signals via heteromeric complexes of type I and type II serine/threonine kinase receptors and via intracellular effectors called Smads. Following their activation by TGF ß , type I receptors phosphorylate a subset of the Smad family called R-Smads (Smad2 and Smad3) causing their heterooligomerization with a common Smad (Smad4) and their translocation to the nucleus where they activate the transcription of target genes in cooperation with DNA binding cofactors and coactivators. Deregulation of TGF ß signaling either by mutations in key signaling components or by negative interference with other signaling pathways is a common characteristic in many types of cancer (pancreatic, colon, breast cancer).
We are studying the structure-function relationship in Smad proteins and the characterization of tumorigenic mutations. By using a large panel of Smad mutants generated in our laboratory, we are investigating the structural requirements for Smad oligomerization, nuclear translocation and transcriptional activation. We are investigating the role of linker phosphorylation and of small C-terminal phosphatases SCP1-3 in Smad functions in the nucleus. We are characterizing the mechanisms of transcriptional activation by Smad proteins by analyzing the promoter regions of novel TGF ß target genes and the interactions of Smads with cofactors. We have initiated this effort by focusing on the promoter of the small GTPase RhoB gene which is involved in the TGF ß -induced cytoskeleton reorganization (in collaboration with Dr Christos Stournaras, U. of Crete Medical School and Dr Antonis Fotsis, University of Ioannina ).
2. Transcriptional regulation of the human apolipoprotein genes (D. Kardassis, V. Nikolaidou-Neokosmidou, I. Mosialou, In collaboration with Dr Vassilis I. Zannis))
a) Transcriptional regulation of the genes of the apoA-I/apoC-III/apoA-IV gene cluster: The role of hormone nuclear receptors.
Using in vitro and in vivo approaches, we hare investigating the mechanisms that govern the regulation of transcription of the genes of the apoA-I/C-III/A-IV gene cluster. Our emphasis is on the role of orphan and ligand-dependent nuclear receptors such as the hepatocyte nuclear factor 4 (HNF-4) and heterodimers of Retinoid X receptor (RXR) that bind to multiple sites on the promoters of all three genes as well as on the common enhancer. More recently, we are using chromatin immunoprecipitation assays to monitor in vivo the recruitment of the above nuclear receptors and of the ubiquitous transcription factor Sp1 to the above promoters and the common enhancer. We are studying the effect of pro- and inflammatory cytokines (TNF a , TGF ß ) on the transcriptional activity of nuclear receptors and the expression of genes of the apoA-I/C-III/A-IV gene cluster. Our studies have revealed a positive and negative cross talk between nuclear receptors and key components of these two signalling pathways. We are also studying mechanisms of coordinated regulation of the genes of the apoE/C-I/C-IV/C-II gene cluster, with emphasis on the role of Hepatic Control Regions (HCRs) and of hormone nuclear receptors (RXR, T3R, FXR) that bind to the promoters of all genes and to the HCRs.
b) Transcriptional regulation of a novel human apolipoprotein (apoM) that is important for HDL metabolism
Apolipoprotein M ( apoM ) is a 26-kDa protein of the lipocalin superfamily which is expressed exclusively in the liver and the kidneys. In the plasma, apoM is found mainly in the HDL fraction. Although the precise function of apoM is unknown , it seems to play an important role in HDL metabolism as it is required for the formation of pre ß HDL particles. Silencing of apoM gene expression in mice via siRNA led to the decrease in plasma HDL-C levels whereas overexpression of apoM in LDLR KO mice inhibited atheromatic plaque formation. We are using in vitro and in vivo approaches to study the regulation of human apoM gene expression. Our preliminary studies have revealed that hepatocyte nuclear factor 1 and hormone nuclear receptors including RXR and LXR are important regulators of the apoM promoter in hepatic cell cultures.
c) Transcriptional regulation of the human apoE gene in macrophages and in the brain (In collaboration with Anca Gafencu, Institute of Cell Biology “N. Simionescu”, Bucharest).
Apolipoprotein E (apoE) is a key protein component of plasma lipoproteins and promotes the clearance of excess cholesterol from the plasma by binding to several members of the LDL receptor family. ApoE deficiency in animal models and in human patients with type III hyperlipoproteinemia is associated with premature atherosclerosis. We are concentrating our efforts on the identification and characterization of the regulatory elements responsible for the basal level of apoE expression in the healthy state and for the modulation of its expression in pathological states. The experiments are focused on two different cell types involved in two distinct major diseases in which inflammation takes an important counterpart: macrophages, one of the key cell types involved in atherogenesis and astrocytes, one of the major cell types implicated in Alzheimer's Disease (AD).
3. Transcriptional regulation of genes ABCA1 and SR-BI involved in the biogenesis and metabolism of HDL (D. Kardassis, E. Thymiakou, S. Mavridou)
a) Transcriptional regulation of the gene encoding the lipid transporter ABCA1:
Clinical and epidemiological studies have revealed an inverse correlation between plasma HDL cholesterol and the risk of coronary heart disease. The biogenesis of HDL requires functional interactions of lipid-free apoA-I with the lipid transporter ABCA1. Specific mutations in ABCA1 such as in patients with Tangier disease prevent the formation of HDL. In contrast, overexpression of ABCA1 is associated with elevated plasma HDL levels, decreased plasma LDL levels and protection from atherosclerosis.
Our aim is to understand the molecular mechanisms that control the hepatic and peripheral expression of the human ABCA1 gene and the ultimate goal is to utilize this knowledge to develop novel approaches for the correction of abnormal HDL levels. Specifically, we are characterizing important regulatory regions present in the upstream ABCA1 promoter as well as in the promoter of the first intron which drives transcription starting from exon 2. We have recently revealed the role of Sp1 in the regulation of ABCA1 gene expression by oxysterols via the Liver X Receptors (LXRs). Using adenovirus-mediated gene transfer, we are studying the role of FOXA1 (HNF-3 ß ) and Sterol Regulatory Element Binding Proteins (SREBPs) in the regulation of ABCA1 in the liver. Finally, we are studying the role of AP1 family members and of AP1 sites in the first intron in the mechanism of ABCA1 gene induction during macrophage differentiation.
b) Transcriptional regulation of the gene encoding the HDL receptor SR-BI:
Scavenger Receptor calss B type I (SR-BI) is a membrane protein that is expressed at high levels in the liver and the stereoidogenic tissues. It binds to HDL and mediates the selective cholesterol ester uptake thus contributing significantly to HDL metabolism in the plasma. SR-BI also facilitates the uptake of cholesterol in adrenal gland to be used for the synthesis of steroid hormones. SR-BI deficiency in animals is associated with premature atherosclerosis . We are studying: a) the regulatory elements and factors that are implicated in the control of SR-BI gene expression in hepatic, adrenal and ovary cells; b) the effect of glucocorticoids on SR-BI gene expression in adrenal cells; c) the effect of corticotrophin releasing factor (CRF) on SR-BI gene expression in adrenal cells (in collaboration with Dr Christos Tsatsanis, University of Crete Medical School). We are also using adenovirus-mediated gene transfer in order to delineate the role of transcription factors Small Heterodimer Partner (SHP), glucocorticoid receptor (GR) and cyclic AMP responsive element binding protein (CREB) on SR-BI gene regulation in mice.
4. Structure-function analysis of humal apolipoprotein genes and prospects for gene therapy of dyslipidemias (D. Kardassis, K. Tzavlaki, in collaboration with Dr Vassilis Zannis)
We are investigating the structure-function relationship in human apolipoproteins A-I (apoA-I) and A-IV (apoA-IV) that are constituents of High Density Lipoprotein (HDL) with the aim to understand their functions and to develop novel tools for the gene therapy of dyslipidemias. We are employing recombinant adenoviruses in order to transfer the wild type and mutant apolipoproteins into animals. We are developing novel methods to purify HDL particles from the plasma of the adenovirus-infected mice based on tagged apolipoproteins.
Vassilis I. Zannis, University of Crete and Boston University Medical Center
European Commission , fp 6 program “ HDLomics ”