Abstract
USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression. We mutated the USP16 gene in a high differentiation clone of the acute monocytic leukemia cell line THP-1 using the CRISPR-Cas9 system and generated four homozygous knockout clones. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. Three clones showed sustained expression of the progenitor cell marker MYB, indicating that differentiation had not completely blocked proliferation in these clones. Network analysis of transcriptomic differences among wild type, heterozygotes and homozygotes showed clusters of genes that were up- or down-regulated after differentiation in all cell lines. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including SRPRB, encoding an interaction partner of USP16. There was also apparent loss of interferon signaling. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases (USP12, BAP1, and MYSM1). However, three homozygotes failed to fully induce USP3 during differentiation. Other network clusters showed effects prior to or after differentiation in the homozygous clones. Thus the removal of USP16 affected the transcriptome of the cells, although all these lines were able to survive, which suggests that the functions attributed to USP16 may be redundant. Our analysis indicates that the leukemic line can adapt to the extreme selection pressure applied by the loss of USP16, and the harsh conditions of the gene editing and selection protocol, through different compensatory pathways. Similar selection pressures occur during the evolution of a cancer in vivo, and our results can be seen as a case study in leukemic cell adaptation. USP16 has been considered a target for cancer chemotherapy, but our results suggest that treatment would select for escape mutants that are resistant to USP16 inhibitors.
Highlights
The development of a tumorigenic lineage from healthy cells is usually associated with a wide range of genetic changes, including point mutations, small and large deletions and insertions and chromosomal rearrangements (Vogelstein et al, 2013)
We report the knockout of USP16 in four homozygous cell lines derived from the high differentiation clone of THP-1
The day, the membrane was washed six times for 5 min in PBS-T, and secondary antibodies were diluted at 1:2000 in 5% milk powder in PBS-T, and the membrane immersed rotating for 1 h at room temperature
Summary
The development of a tumorigenic lineage from healthy cells is usually associated with a wide range of genetic changes, including point mutations, small and large deletions and insertions (indels) and chromosomal rearrangements (Vogelstein et al, 2013). The net result of either change was to reduce the cell’s ability to repair DNA damage, either because the initial ubiquitination of H2A at the site of the break was suppressed (over-expression of USP16) or because the ubiquitin could not be removed after the repair (down-regulation) (Zhang et al, 2014). A triplicated USP16 gene had decreased DNA damage response (Zhang et al, 2014) These results show that USP16 has three functions: cell cycle progression (Xu et al, 2013), DNA damage repair (Zhang et al, 2014) and gene activation by removal of H2A ubiquitin (Yang et al, 2014). We report the creation of a loss of function mutation in the USP16 gene using CRISPR-Cas technology in the THP-1 human acute monocytic leukemia (AML) cell line. We observed heterogeneity amongst the homozygous knockout lines and examined their transcriptomic profiles to understand whether these cells have evolved mechanisms to compensate for the impact of the USP16 mutation
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