Hox and Tale Transcrıptıon Factors in Fanconı Anemıa Bone-Marrow Mesenchymal Stem Cells: Gene Expressıon and Proteın Interactıons

Abstract

HOX/TALE proteins maintain tissue identity and their altered expression is seen in many cancers. The main aim of this thesis is to evaluate whether HOX/TALE gene expression changes due to genomic instability. To answer this question, Fanconi anemia (FA) bone marrow mesenchymal stem/stromal cells (BM-MSCs) were investigated. FA is an inherited bone marrow (BM) failure disease, which shows cancer predisposition due to genomic instability. FA BM-MSCs were characterized at molecular and cellular level compared to donors. The gene expression profile was evaluated by RT-qPCR. Differentially expressed gene was further analyzed to define its function related to BM-MSCs biology, as well as, to assess its interactions with the members of FA/BRCA pathway using co-immunoprecipitation (Co-IP). PKNOX2 expression significantly decreased in FA cells compared to donors, and its expression was stimulated by rTGF-β1 treatment. When BM-MSCs, transfected with PKNOX2 expression plasmid (PKNOX2:MSC) were characterized, decrease in CD105 level and lateral motility were observed. Co-culture assays of cryopreserved CD34+HSCs on PKNOX2:MSCs displayed increase in CD34+CD38+ percentage and clonogenic capacity. Proteome analysis following Co-IP revealed that a total of 219 proteins directly interacted with PKNOX2, but FA/BRCA pathway members were not among these proteins. However, interactors of PKNOX2 interactome may regulate double-strand break repair via NHEJ, HSC differentiation and G2/M transition. In conclusion, decrease in PKNOX2 expression of FA BM-MSCs may cause cell cycle arrest, increase in NHEJ-related gene expression and trigger the formation of genomic unstable microenvironment that can promote hematopoietic defects seen in patients.