Major research Interests
Our goal is to understand gene transcription mechanisms that control important cell processes and further exploit this towards designing and testing novel biological or chemical tools for monitoring or treatment. The main current directions are:
- Transcriptional regulation of Major Histocompatibility class II (MHC II) genes. Studies on enhanceosome dynamics in vivo, mechanisms of transcriptional memory and maintenance of antigen presenting ability through the cell cycle.
- Analysis of the epigenetic state of the cell following employment of appropriate inhibitors and development of new biomedical applications
- Development of novel fluorescent tools and technologies: This is a collaborative interdisciplinary approach to improve biological and technological tools and know how for in vivo real time imaging of cellular processes.
I. Transcriptional regulation of Major Histocompatibility class II (MHC II) genes.
MHC II gene products control antigen presentation in the thymus and the periphery and thus determine immune response to foreign or auto-antigens. Expression of MHC II genes is primarily transcriptional and of critical importance for determining immune regulation.
Expression of MHC II depends on a promoter proximal regulatory region (MHC II enhancer) and factors that interact to form a cooperative DNA binding complex (MHC II enhanceosome) on the enhancer. The enhanceosome in turn recruits a gene specific transactivator called CIITA that initiates transcription. Contrary to the ubiquitously expressed enhanceosomal subunits, CIITA is expressed developmentally in B lymphocyte? and dendritic cells or by cytokines (primarily Interferon ?) in other cell types. We employ state of the art molecular biology approaches and novel genetically encoded fluorophores to dissect the molecular mechanisms that control MHC II transcription with emphasis in:
- The interplay between DNA binding factors of the enhanceosome and the transactivator CIITA. The mechanism of transcriptional control by CIITA.
- Arginine methylation and enhanceosome function: Following our original observation that histone methytransferase (MTs) inhibitors block MHC II expression, we have determined that enhanceosome components are methylated by arginine MTs. We are identifying and then mutagenizing methylation targeted arginines to study their effect on enhanceosome assembly and function.
- The in vivo assembly and dynamics of enhanceosome studied by fluorescent fusions of some ennhanceosomal subunits and live microscopy (nucleocytoplasmic trafficking, interactions, co localisation and recovery after photobleaching, FRET, bimolecular fluorescent complementation etc). These studies will improve understanding of the molecular hierarchy and dynamics of MHC II enhanceosome assembly and its function in the living cell.
Fluorescence Recovery after Photobleaching ( FRAP) analysis showing intracellular dynamics of enhnceosome factors under specific conditions.
II. Epigenetic control of gene expression
In collaboration with Dr. Kretsovali, IMBB-FoRTH, we studied the effects of the Histone Deacetylase inhibitor Trichostatin A (TSA) on the MHC II expression. The cell type specific and transcriptome- wide events that correlate with its transcriptional effects are currently analysed.
Through a collaborative program (with Dr. Kretsovali, IMBB and Giannis, Un. of Leipzig) we have characterized and demonstrated the efficacy of new chemicals to inhibit HAT enzyme activity in vivo and in vitro. These studies provide new insight into important epigenetic processes and help to develop new biological tools for dissecting molecular processes or potential biomedical applications.
III. Molecular imaging
This is a recently established collaborative, interdisciplinary approach that aims at the integration of new information and technologies to advance real time in vivo imaging and produce valuable tools and technological platforms.This activity is part of a major consortium of various European groups and companies working in diverse aspects of physics, engineering, analysis and fluorescent tools coordinated by Dr. E. Economou (FoRTH-light propagation physics). This Core collaboration involves Dr K. Mamalaki (IMBB-FoRTH, fluorescent gene expression in transgenic models) Dr. J.Ripoll (IESL-FoRTH, physics engineering and applications of fluorescent tomography development of) and Dr. K. Lukyanov (Institute of Bioorganic, Moscow Russia, fluorescent proteins). Our aim is to develop and use novel fluorescent tools and technology (novel spectral properties, photoactivatable fluorescent molecules, spectral shift timers in combination with confocal microscopy or molecular tomography) for in vivo real time imaging of cellular processes.