Investigation of HJURP functions in the acquisition of stemness in glioblastoma lines

Abstract

Glioblastomas (GBMs) are the most frequent and aggressive tumors of the central nervous system. This is because in most cases there is a high rate of relapses that, in a short time, allow the formation of a new tumor mass. This is due to the high resistance and proliferative capacity of tumor cells, which has been associated with the presence of large numbers of tumor stem cells (CTTs) in GBMs. These cells have the potential of unlimited self-renewal, asymmetric division capacity, proliferation and differentiation, and resistance to genotoxic treatments, which together are called potential stemness. Previous data from our group have shown that HJURP (Holliday Junction-Recognizing Protein) has increased expression in GBMs and that their high levels correlate with poorer prognosis in patients. We have also shown that the strain of GBM U87MG gains proliferative capacity when stably transformed with a construct that allows overexpression of this protein. HJURP acts as a histone chaperone and is responsible for the incorporation of CENP-A (Centromeric Protein A) into centromeric chromatin. CENP-A is a variant of canonical histone H3 that promotes a loosening of chromatin in the central region of the centromere, restricting the formation of heterochromatin at this site. Recent results from our laboratory have shown that overexpression of HJURP also promotes a decrease in the number of HP1 foci (Heterochromatin Protein 1). HP1 binds in regions of compacted chromatin contributing to maintain it in this state, therefore it is a marker of gene repression. Thus, we hypothesized that overexpression of HJURP could promote a global loosening of chromatin by allowing silenced genes to be reexpressed. Thus, in this work we intend to investigate whether the overexpression of HJURP would be re-connecting genetic programs associated with the potential stemmness of GBM cells. For this, we intend to standardize oncoesphere generation models from GBM strains and to evaluate the potential stemmness (proliferation, self-renewal and differentiation) of the cells comparing parental and superexpressor lines of HJURP. We also intend to perform chromatin immunoprecipitation assays, followed by sequencing (ChiP-Seq), to identify genes with higher HJURP occupancy in superexpressor cells. Finally, target genes will be chosen for silencing with shRNAs and evaluation of the effect caused on the maintenance of potential stemness.