Identification of Apoptosis-Related Genes Regulated by the PLZF Protein.
Authors: Bernardo Maria Victoria , Yelo Estefania , Gimeno Lourdes , Majado Maria Juliana , Alvarez-Lopez Maria Rocio , Parrado Antonio 
Published: April 2020

Abstract
The PLZF gene was identified in acute promyelocytic leukemia (APL, AML-M3). PLZF expression is altered in chronic lymphocytic leukemia (CLL). PLZF is a transcriptional repressor involved in the control of cell proliferation, differentiation and survival. Overexpression of PLZF induces cell cycle arrest and growth suppression, whereas alteration of its normal function due to the presence of the reciprocal fusion proteins, PLZF-RARA and RARA-PLZF, resulting from the t(11;17) traslocation, is associated to the development of APL. We have established stable inducible PLZF expressor clones from different hematopoietic cell lines, using the tet-off system. PLZF induced inhibition of cell growth in T lymphocytic Jurkat cells, but not in erythroid K562 cells, or B lymphocytic DG75 cells. Growth suppression in Jurkat cells was dependent on its levels of expression, since it was observed only in the highest PLZF expressor clones. Moreover, it was inversely proportional to the initial density of the cultures, suggesting a dependence on exogenous mitogens. Deletion of the BTB/POZ domain abrogated growth suppression. Cell cycle and apoptosis analysis suggest that cell death may be the primary and predominant mechanism responsible for growth suppression in Jurkat cells. Since PLZF may mediate its biological effects by transcriptional control, we conducted a search of potential transcriptional targets by microarray analysis (CodeLink, human whole genome, 55K). Biological duplicates of a high PLZF expressor Jurkat clone, cultured at low cell density (25.000 cells per ml) in the presence and absence of doxycycline, were analysed at 24, 48, 72, and 96 hours. As a result, we have identified around 70 genes, involved in diverse cellular functions, whose expression was significantly modulated by PLZF. Some of them, which have been implicated in cell death, were verified using real time RT-PCR: TERT, which has been described as anti-apoptotic, was repressed; conversely, TP53INP1, ID1 and ID3, which have been described as pro-apoptotic, were induced. The identification of these genes is consistent with the pro-apoptotic phenotype observed for PLZF, suggesting that they could be major mediators of PLZF function. Other genes confirmed by real time RT-PCR included ZNF496 and IGLL1, significantly down-regulated, and MYCN, TBL1X, and TRB2, significantly up-regulated, respectively. None of these targets were significantly modulated when cultures were established at high cell density (250.000 cells per ml). At this cell density, PLZF expression did not induce growth suppression or apoptosis, suggesting that PLZF may integrate external signals before initiating specific genetic programmes that lead to growth arrest and cell death. These findings could contribute to elucidate the mechanism by which PLZF induces apoptosis, and to assess the relevance that structural alterations or abnormal expression of PLZF, respectively, may have in APL and CLL.