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“Biochemical isolation and functional characterization of the protein complexes that regulate interchromosomal interactions”

Final Report Summary - INTERPRO (“Biochemical isolation and functional characterization of the protein complexes that regulate interchromosomal interactions”)

Charalampos G. Spilianakis
Scope: “Biochemically identify and functionally characterize protein complexes that regulate interchromosomal interactions”

Regulation of gene expression can be accomplished “in cis” by several regulatory elements exerting their action on physically linked genes on the same chromosome. A “trans” regulation aspect of gene expression has also recently been brought to light by the study of the interactions and the 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.

In diploid organisms, the two alleles that exist for each genetic locus typically function independently. However, there are several puzzling circumstances in which they do not; instead, they can in someway sense the presence, in trans, of homologous alleles and “respond” by altering the gene expression pattern. A feature shared among trans allelic phenomena is that their mechanisms are sensitive to, and depend on the epigenetic status of the target-chromatin regions. A thorough understanding of how the mammalian genome is organized and functions will require that the mechanisms governing such phenomena are characterized and studied in detail and vice versa.

Similarly to the aforementioned trans-allelic phenomena, we have shown, utilizing the CD4+ T cell differentiation system that the association of regulatory elements on one chromosome can determine the transcriptional activation of genes on another chromosome, affecting the differentiation program of the cell and the implicated cellular functions. A new parameter is added to the already complex field of epigenetics. Epigenetic regulation of gene expression will, from now on, integrate the effects of the three dimensional organization of the genome in the cell nucleus and the mechanisms that sustain or change it over time.

The main aim of our work focused on identifying and characterizing the factors involved in regulating interchromosomal interactions with the goal to propose a new epigenetic mechanism for gene regulation in multiple systems.

The focus of our efforts can summarize in the following objectives:
Aim-I: Identify the DNA elements involved in interchromosomal interactions and trans-allelic transcriptional regulation.
Aim-II: Identify the protein complexes that generate/maintain interchromosomal associations.
Aim-III: Evaluate the protein complexes involved in interchromosomal associations that regulate gene transcription.
Aim-IV: In vivo evaluation and functional characterization of the proteins that regulate gene expression via interchromosomal interactions.

The work we have performed so far for the completion of each specific Aim is as following:
Aim-1: We did identify the DNA elements involved in the interchromosomal interactions of the TH2 locus with the LT/TNF and the IFNγ loci. This element is characterized as a conserved DNAse I hypersensitive site of the TH2 LCR.
Aim-2: By means of mass spectrometry analysis we have identified two proteins bound in the aforementioned conserved DNA element and include the SATB1 and the BACH1 proteins.
Aim-3: We did characterize the expression profile of these two proteins in the cell types of the immune system and also found the DNA elements where these two proteins are bound in the gene loci under study.
Aim-4: We have acquired genetically modified mice that either lack completely the BACH1 protein and mice that the SATB1 protein is conditionally eliminated in CD4 cells.

We found that the SATB1 and BACH1 proteins maintain a protein network within the CD4 cell nucleus whereupon chromatin is organized and cytokine gene loci are poised but inactive for transcription.

Our effort for the identification of the protein complexes involved in driving long range interactions in T cells led in two different groups of proteins: Factors of general importance and use, in maintaining the genome shape and involved in chromosome movements and a second group of tissue and cell specific factors that drive the specificity of binding of the general factors in specific loci.

The mechanistic insight gained from our performed work can be used in the future to study the deregulation of gene expression in disease models. Chronic Myelogenous Leukemia (CML) is marked by the presence of a distinct cytogenetic abnormality that results from a translocation between two chromosomes, known as the Philadelphia chromosome. Also in murine plasmacytomas (MPCs) deregulation of the myc transcript is achieved by chromosomal translocation that juxtaposes the c-myc/Pvt-1 locus with one of the immunoglobulin (Ig) loci, IgH, Ig or Igλ. We propose that the developmentally regulated colocalization of multiple loci such as Bcr and Abl in the cell nucleus might result in this kind of malignancies. Future analysis of interchromosomal interactions between candidate loci may shed light in the higher order regulation of expression in a cell nucleus.