| Abstract |
Genome organization and high order chromatin structure are key features in the highly compartmentalized and dynamic environment of the eukaryotic cell nucleus. Recent investigations have indicated that gene positioning and chromosomal interactions (cis & trans) in the nucleus may play an important role in the regulation of gene expression. Although, interchromosomal interactions are a well-characterized phenomenon from the X-chromosome inactivation in female mammals, it is widely accepted since 2005, when detected in naive CD4+ T lymphocytes. Therefore, it was of great importance to identify and characterize protein complexes that induce and/or stabilize interchromosomal interactions in mouse CD4+ T cells. For this reason, RHS6, which is a regulatory region within the TH2 locus and was recently found to participate in interchromosomal interactions, was chosen as a candidate region for study. Moreover, we focused οn 151 bases within RHS6, which show considerable conservation between human and mouse. By utilizing DNA affinity chromatography coupled with Mass Spectrometry, we identified two specific DNA binding factors, namely SATB1 and BACH1 and mapped one phosphorylation site for each of them. The phosphorylated residues identified were S635 and S448, corresponding to proteins SATB1 and BACH1. In the frame of this study, we tried to elucidate whether serine phosphorylation, detected in each one of the two proteins was responsible for the subcellular localization of these proteins. Therefore, we made efforts for gene cloning and site-directed mutagenesis of serine residues to alanine, followed by fusion of the genes of interest (wild type and mutant) with the reporter gene of green fluorescent protein (EGFP) to an expression vector. Subsequently, we performed transfection of HEK 293 T cells with the expression vector pEGFP-c3 that expresses wild-type BACH1 protein in fusion with green fluorescent protein (EGFP), as well as with pEGFP-c3 that expresses the mutant BACH1 protein (Ser448A) in fusion with EGFP. In each case, we studied the subcellular localization of the chimeric proteins by using fluorescence microscopy and found that while the wild type BACH1 protein localized mainly in the nucleus, the mutant BACH1 exhibited mostly a cytoplasmic distribution, indicating that serine phosphorylation is a potential determinant for the nuclear localization of BACH1 protein. Despite repeated efforts for the initial cloning of the SATB1 gene, this was not possible due to missense mutations incorporated into the cDNA although a proofreading polymerase was used. However, we identified a new isoform of SATB1 containing an additional exon of about 100 bases, which resembles with a corresponding human SATB1 isoform.
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