A functional study of UBE2G1 in murine and human cell lines

Recent data collected by the laboratory of Hartmut Geiger show that mice transplanted with haematopoietic stem cells (HSCs) overexpressing (OE) the Ubiquitin E2 conjugating enzyme G1 (UBE2G1) showed an MDS/AML-like phenotype upon transplantation. The phenotype is characterised by cytopenia, myeloid differentiation bias, and cell dysplasia. The role of UBE2G1 in the insurgence of an MDS/AML-like disease remains to be investigated. Revealing what effect UBE2G1 OE has on cell’s basic functions is the first step toward an understanding of the mechanism behind the development of MDS. This could provide useful information for establishing an in vivo model to study the disease. In this study, three AML-derived cell lines (32D, THP1, and MOLM13) were used to generate OE and knock down (KD) models to study the effects of UBE2G1 perturbation on a molecular level. The OE and KD of the UBE2G1 gene were obtained by lentiviral vector transduction. Gene expression was assessed by RT-PCR and protein levels were quantified by immunoblot in 3T3 cells, confirming that an overall 6.5-fold increase of UBE2G1 protein levels in the OE and an 84% decrease of UBE2G1 levels in the KD. Additional bands were visible at higher molecular weights compatible with the addition of a light-weighted protein. The intensity of the additional bands was drastically decreased in reducing conditions, while both endogenous UBE2G1 and the fusion protein UBE2G1-GFP bands were enriched suggesting that the additional proteins may have been the result of UBEG1 bound to Ubiquitin (Ub) on its catalytic cysteine. This result also shows identical behaviour between the endogenous UBE2G1 and the UBE2G1-GFP fusion protein, confirming unaltered functionality. Quantification of proteins ubiquitinated on Lysine (Lys) 48 showed unaltered levels in both 32D OE and KD cell lines. The cyclin-dependent kinase inhibitor 1 (p21) was observed to be transcriptionally increased when UBE2G1 was knocked down in HCT116p53-/- cells, and the increase affected protein levels accordingly. KD and OE of UBE2G1 led to increased p21 levels up to 2-fold in 32D cells, as previously reported, possibly as an intrinsic mechanism in response to protein imbalance. The effect of p21 increase was then assessed in two functional assays to measure the impact of these alterations on cell cycle and cell growth. Flow cytometric analysis of cell cycle in 32D, THP1, and MOLM13 cell lines did not show any increase in G1 or G2 populations upon UBE2G1 OE, and the growth rate was unaltered in the OE, but significantly decreased in the KD in 32D cells suggesting that UBE2G1 may play a role in the regulation of the cell cycle and consequently in the growth rate.