Analysis of Differential Expression of TG2 Variants During Treatment with Retinoic Acid Highlights a New 3'-Terminal Transcript and Putatively Involved Proteins

In the last few years great attention has been aimed at understanding the role of alternative splicing and differential expression of variant isoforms especially as hallmarks of cancer, in cell death and in neurodegenerative processes. In this context it was reported that Transglutaminase 2 expression is associated with tumor status, progression and response to applied therapies, and that distinct isoforms of the enzyme are detected in several neurodegenerative diseases. In this work we have analyzed the modulation of TGM2 variants and other gene transcripts in hematologic and neurogenic cell lines after treatment with retinoic acid, a well known differentiating agent also used in combined therapies for glioblastoma and acute leukaemia. Our data indicate that treatment of acute progranulocytic leukaemia HL-60 cells with ATRA for variable time intervals (16-80hs) increased the expression of the truncated and alternatively-spliced variants and induced also a short 3’-terminal transcript associated with up-regulation of TGM2, along with the lncRNA included in the gene. This new 3’-terminal transcript is constituted by the sequence corresponding to the last 3 exons of the gene and is strongly modulated by ATRA in HL-60 cells. We scrutinized their expression in the early stages after treatment (0-8h) of HL-60 comparing the results with those obtained in erythroleukemia K562 cells. These latter cells have mutational inactivation of the p53 and hypomethylation of the promoter, thus supporting a basal level of transglutaminase expression and undergoing apoptosis upon administration of ATRA. In contrast to what happens in HL-60, the short 3’-terminal transcript was detected also in untreated K562 cells, and likely decreasing upon ATRA administration. We have extended the analysis to gene expression profiling for TGM2 in other cell lines responsive to retinoic acids: acute promyelocytic leukemia NB4 cells and glioblastoma GBM cells, both tumors arising from astrocytes. In both cases we observed accumulation of TGM2 transcriptional variants associated with an increase of the lncRNA and the short 3’-terminal transcript. To attempt to identify the transcriptional factors that could be involved in the differential expression of these transcripts we analyzed ChIP-seq experiments from the ENCODE database performed on neuroblastoma SK-N-SH cell line untreated and treated with retinoic acid, focusing attention on TGM2 gene promoter and intronic regions recognized by transcription or DNA-binding factors. The results paired to that differentially expressed in some of the cell lines used in this study could be helpful to shed light on transcriptional ATRA-modulation of TGM2 isoforms, where lncRNA seems to play a role as enhancer RNA. It will be interesting to further explore whether the overexpression of a specific transcript is also followed by concomitant increase of the alternative enzyme variant(s). These expression studies about TGM2 variants in tumour cells could represent a valuable tool to investigate their association with the progression and invasiveness of the disease and be also a prognostic factor for effectiveness or resistance to chemotherapeutic agents. A deeper understanding of the factors involved in gene modulation of TGM2 variants could be helpful to develop innovative approaches for gene therapy.