HTLV-1 bZIP factor impairs DNA mismatch repair system

Abstract

Adult T-cell leukemia (ATL) is a peripheral T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). Microsatellite instability (MSI) has been observed in ATL cells. Although MSI results from impaired mismatch repair (MMR) pathway, no null mutations in the genes encoding MMR factors are detectable in ATL cells. Thus, it is unclear whether or not impairment of MMR causes the MSI in ATL cells. HTLV-1 bZIP factor (HBZ) protein interacts with numerous host transcription factors and significantly contributes to disease pathogenesis and progression. Here we investigated the effect of HBZ on MMR in normal cells. The ectopic expression of HBZ in MMR-proficient cells induced MSI, and also suppressed the expression of several MMR factors. We then hypothesized that the HBZ compromises MMR by interfering with a transcription factor, nuclear respiratory factor 1 (NRF-1), and identified the consensus NRF-1 binding site at the promoter of the gene encoding MutS homologue 2 (MSH2), an essential MMR factor. The luciferase reporter assay revealed that NRF-1 overexpression enhanced MSH2 promoter activity, while co-expression of HBZ reversed this enhancement. These results supported the idea that HBZ suppresses the transcription of MSH2 by inhibiting NRF-1. Our data demonstrate that HBZ causes impaired MMR, and may imply a novel oncogenesis driven by HTLV-1.

Keywords: ATL; DNA mismatch Repair; HBZ; HTLV-1; MSI; NRF-1.

Fig. 1.

Transduction of HBZ into MMR-proficient cells induces microsatellite alterations (A) RT-PCR of the indicated genes in TK6 cells stably transduced with HBZ (TK6-HBZ) or empty vector (TK6-EV). (B) Electropherograms for eight loci of microsatellites (D10S190, D10S191, D18S21, BAT40, BAT25, BAT26, D2S123 and D5S346) in TK6-EV and TK6-HBZ cells. The arrows indicate microsatellite alterations. (C, D) TK6-EV and TK6-HBZ cells were treated with the indicated concentration of CCNU (C) or MMC (D) for 48h. Cell viability was determined by MTS assays and expressed as relative to vehicle-treated controls (n = 3, mean ± SD). *p < 0.05, **p < 0.01 (unpaired Student’s t-test).

Fig. 2.

Transduction of HBZ into MMR-proficient cells suppresses the expression of several MMR factors (A) mRNA expression of indicated genes measured by qRT-PCR in TK6-EV and TK6-HBZ cells (n = 4, mean ± SEM). *p < 0.05, **p < 0.01 (unpaired Student’s t-test). (B) Immunoblotting of the indicated proteins in TK6-EV and TK6-HBZ cells.

Fig. 3.

Expression of MMR genes in primary acute type ATL samples. mRNA expression levels of the indicated genes in human CD4⁺ T lymphocytes from the peripheral blood of healthy volunteers (n = 5) and primary acute type ATL cells (n = 7), obtained from the microarray dataset in the Gene Expression Omnibus database, accession number GSE43017 (mean ± SD). *p < 0.05 (unpaired Welch’s t-test).

Fig. 4.

HBZ suppresses transcription of MSH2 via the transcription factor NRF-1 (A, B) UCSC genome browser view of the NRF-1 binding site in the MSH2 promoter. (A) Tracks show signals of NRF-1 ChIP-seq in the promoter region of MSH2 from the ENCODE project. (B) Sequences of the signal peak are shown. Blue bars represent the degree of sequence conservation across different vertebrates from the phastCons program. The black-lined region represents the NRF-1 binding motif predicted using Factorbook. (C) Activity of MSH2 promoter constructs truncated at different positions upstream of the transcription start site (TSS). Sequence upstream of the TSS of MSH2, and the NRF-1 binding motif predicted using Factorbook are shown. Each MSH2-Luc was transfected into HEK293T cells together with Renilla-Luc. MSH2-luciferase activity was assayed as relative to Renilla-Luc (n = 3, mean ± SEM). (D) Effects of NRF-1 expression on MSH2 promoter activity. MSH2-Luc and Renilla-Luc were transfected into HEK293T cells, with or without vectors expressing NRF-1 (0.05 μg or 0.2 μg) (n = 3, mean ± SEM). (E) Effects of HBZ on MSH2 promoter activity. MSH2-Luc and Renilla-Luc were transfected into HEK293T cells, with or without vectors expressing HBZ (0.4 μg or 0.6 μg) and NRF-1 (0.0125 or 0.025 μg) (n = 3, mean ± SEM). (F) Immunoblotting of the indicated proteins in TK6-HBZ cells infected with lentivirus expressing FLAG-tagged NRF-1 or the corresponding empty vector (EV). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (unpaired Student’s t-test).