Research
Regulation of gene expression can be accomplished “in cis ” by several regulatory elements (promoters, enhancers, locus control regions, silencers, boundary elements, etc.) exerting their action on physically linked genes on the same chromosome . During my postdoctoral studies in the lab of Dr Richard A. Flavell ( Yale Medical School ), I extended my studies on gene regulation utilizing an in vivo immunological system, namely the differentiation of CD4 + helper T cells in T(helper)H1 and TH2 cells. The TH2-specific cytokine-encoding genes Il4 , Il5 and Il13 are located on mouse chromosome 11 covering a genomic region of 120 kilobases. These three cytokine-encoding genes are coordinately expressed in a TH2 cell by the action of a locus control region (LCR) present in the same locus.
Two levels of a poised chromatin configuration in the TH2 cytokine gene locus.
A “ trans” regulation aspect of gene expression has also recently been brought to light by studies of the interactions and function of regulatory elements located on a chromosome different from the one that carries the regulated gene. Such regulatory elements include enhancers or locus control regions that are shown to be able to regulate the expression not only of cis linked genes but also of genes located on different chromosomes, in trans .
Following the characterization of the multiple intrachromosomal interactions in the TH2 locus and the identification of the mode of action of an LCR in the coordinate regulation of multiple genes, motivated by some interesting initial observations, we decided to test the possibility of physical interaction between the TH2 LCR and alternatively expressed genes located on different chromosomes. The subnuclear localization of chromatin is not random, and specific genetic loci or whole chromosomes reside at specific locations within the nucleus, restricted within limited and specific boundaries, called ‘territories'. DNA sequences within a chromosome are packed with chromatin, organized in distinct, repetitive “domains” of euchromatin and heterochromatin with specific, functional localization in the nucleus. In some cases, large chromosomal loops containing active genes extend outside the defined chromosomal territories. As cellular differentiation advances, changes in transcriptional activity are often coupled with changes in subnuclear localization of chromosomes.
We reported that the Ifn g gene, located on mouse chromosome 10 physically interacts with the TH2 locus, located on mouse chromosome 11, and specifically, with a particular DNase I hypersensitive site of the TH2 LCR, among other contact sites. The interactions detected were notably strong in naive non differentiated CD4 + T cells and were subsequently greatly reduced after the differentiation of naive T cells to effector TH1 or TH2 cells. In contrast, non-T-cell types do not exhibit these interactions. Concomitant with the loss of these interchromosomal interactions, in TH1 cells the Ifn g gene region instead strengthens intrachromosomal interactions with a regulatory element located downstream of the Ifn g gene.
Co-localization of the Ifn g and TH2 loci as revealed by FISH.
Our study showed for the first time that there is direct interchromosomal interaction between two loci that are expressed as mutually exclusive alternatives in two different cell types. The co-localization of the Ifn ? locus with the TH2 locus was established with the 3C technique and confirmed with Fluorescence In Situ Hybridization experiments. We suggested that there are dynamic intra- and interchromosomal interactions between specific genetic loci regulating their transcriptional activation or repression. We also suggested that one of the functions of the LCRs, in addition to regulating the expression of adjacent genes in cis , is to participate in developmentally regulated interchromosomal interactions of related genes (acting in trans ).
There seems to be a cell type-specific dynamic network of interactions between alternating chromatin partners whereby interchromosomal interactions are apparently lost in favor of intrachromosomal ones upon gene activation. Thus, we provided an example of eukaryotic genes located on separate chromosomes; nevertheless associated physically in the nucleus via interactions that may be functional regarding the establishment of coordinated gene expression. It is now obvious that interchromosomal association of coordinately regulated genes is a common trait of gene organization and gene expression mechanisms of higher organisms.
Research Activities
Main Objective
The main objective of this laboratory is to apply a combination of biocomputing, molecular, biochemical, imaging and genetic approaches in order to identify and characterize the protein complexes that generate and/or maintain interchromosomal interactions in T cells. The main project is defined in five stages aiming at the isolation, purification and evaluation of the isolated protein complexes for their potency to regulate gene expression via interchromosomal interactions as well as to define nuclear structures. The project should provide substantial information on how the genome is shaped as a whole and how the nuclear structure patterns in the context of distinct subnuclear microenvironments regulate global gene expression (3D epigenetics). Identification of the proteins involved could lead to the future development of small interfering molecules aiming specifically at the inactivation of these proteins; thus developing an intervening tool for twisting the expression profile of certain loci and ultimately manipulating/reprogramming the immune system or modifying the expression of translocated loci in cancer cells.
Explore the biochemical and genetic mechanisms underlying interchromosomal interactions.
Since T helper cell differentiation is the first system in which the existence and significance of interchromosomal interactions was demonstrated, it will serve as a model system in future studies to identify general protein factor(s) or protein complexes responsible for the generation and/or maintenance of such interactions, common for different gene systems. The isolated protein candidates will be characterized in the attempt to identify functional protein domains. Immunohistochemical analysis will explore the presence and function of these protein networks in T and other cell types. Chip on chip technology will be performed for the ultimate identification of the genetic loci, participating in a nuclear gene network on which this/these protein(s) bind and function, regulating their transcriptional expression in a coordinate manner. This approach will lead in the identification of complexes with specific chromatin remodeling activity, acting at a long distance, rather than on promoter and enhancer elements.
Another open and interesting question I would like to address utilizing the T helper cell differentiation system, taking advantage of the substantially useful knowledge and experience accumulated over the past three years of research, is whether the interchromosomal interactions identified in terminally differentiated cells are already formed in earlier developmental stages and if that is true, can one conclude that stem cells or precursors of certain cell types are predetermined for the cell fate they will adopt later as terminally differentiated effectors? In other words, can one claim that the TH2 LCR regulates developmentally the positioning of the TH2 locus and other target loci, to the so-called transcriptional factories or other compartments of the nucleus that facilitate gene expression and how early in development does this take place? Analyzing cell populations sorted from distinct developmental stages will be useful in answering these questions, utilizing the 3C technology in conjunction with FISH analysis. Alternatively, one can generate and analyze genetically modified animals with disrupted interchromosomal interactions between two loci in order to relate specific chromatin conformation states to certain developmental or differentiation stages and respective patterns of expression.
Extension of the analysis to other systems (TLR and genes deregulated in diseases).
In the near future I would like to focus the study of interchromosomal interactions on loci implicated in disease. As a first step, analysis of physiologic cells utilizing the 3C technique in conjunction with FISH analysis will provide the reference status regarding physical interactions and colocalization/proximity status for the loci of interest. The aim of my future studies will be an attempt to generalize the phenomenon of interchromosomal interactions, address the scope and significance of its normal operation and hopefully point out an obvious common link for diverse experimental models.
Among other ideas I would like to explore whether this phenomenon plays a role in the expression of the different members of the Toll like receptor (TLR) family, which is restricted to certain cell types and activated via different signals, in diverse each time combinations. Members of this family are located on different chromosomes and no hint has come to light so far, regarding that common regulatory element, which drives the coordinate expression of specific gene clusters. The above project is of low risk and can be completed in a relatively short period of time since I have a good command of the two major techniques (3C, FISH) necessary to perform the proposed study. The use of genetically modified mice for several members of the TLR family, already available in the lab of my current supervisor, will be of great contribution to such a study, in the context of collaboration.
Development of the 4C technology.
In the course of experimental work, in order to identify, isolate and characterize novel, specific activating/repressive elements crucial for the expression of different gene families, an advanced 3C technique will be employed; namely the 3C combined with another technique known as Inverse PCR (I-PCR). I-PCR was developed in Drosophila, commonly applied for the characterization of the integration sites of transposable elements but its use can be extended to the identification of novel genomic interactors for specific, known target loci. This approach will be followed for cloning additional regulatory elements for the loci of interest mentioned above, such as different members of the TLR family. Following application of the 3C technique for the isolation of novel genomic fragment interactors for the target sequence of interest, I-PCR will be performed for amplification of the unknown fragments and subsequent further analysis will involve cloning, sequencing and in silico studies, taking advantage of biocomputing resources and methodologies. The data will be verified with 3C and FISH analysis. Genetically manipulated mice with deletion of the identified regulatory elements could be generated to further advance this work and analysis of the phenotype of the mutants will address the physiological significance of such regulatory elements in vivo .
