Dynamic Changes of Chromatin Structure and Transcriptome By Transient Expression of HTLV-1 Tax in ATL Cells

Abstract

Adult T-cell leukemia-lymphoma (ATL) is a fatal malignancy of CD4+ T cells, which is caused by human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 encodes two oncogenic viral factors, Tax and HTLV-1 bZIP factor (HBZ) in the sense and antisense of the provirus respectively. HBZ is constitutively expressed in infected cells and plays critical roles for cell proliferation. Tax potently promotes viral replication and activates various oncogenic signaling pathways in HTLV-1 infected cells; however, Tax expression is generally suppressed in infected cells owing to its high immunogenicity. Therefore, the dynamics of Tax expression and contribution of oncogenic transformation by Tax in HTLV-1 infected cells have not been well elucidated. Recently, we have reported that Tax is transiently expressed in a small subpopulation of ATL cells, which induces drastic changes in the host transcriptional profile (Mahgoub M., et al. Proc Natl Acad Sci USA. 2018). Moreover, the single cell analysis showed that expression of anti-apoptotic and NF-kB related genes were upregulated in Tax-expressing cells compared to Tax-non-expressing cells. HBZ expression was increased in Tax-non-expressing cells than Tax-expressing cells, suggesting that HBZ may regulate cell proliferation in a different time phase from Tax. On the other hand, precise mechanisms for regulation of host gene expression by transient Tax expression are not well known. In this study, we analyzed structural change of host chromatin accompanied by transient Tax expression to clarify the transcriptional dynamics of host genes in ATL cells.ATL cell lines (MT-1 and KK-1), which express EGFP under the control of Tax, were used to sort Tax-expressing and -non-expressing cells by a cell sorter, and each population was subjected to H3K27ac ChIP-seq and ATAC-seq analyses. DNA libraries were quantified and sequenced on Illumina NextSeq 500 (single-end) for ChIP-seq, and on Illumina Hiseq 4000 (paired-end) for ATAC-seq, respectively.H3K27ac ChIP-seq data showed that the peaks correlated with Tax expression were observed in many genetic loci including NF-kB related genes. Among Tax-expressing MT-1 cells, H3K27ac were highly enriched super-enhancers, such as IL2RA, TRAF3, TNFRSF8, PHF13 loci, compared to Tax-non-expressing cells. There were more H3K27ac-marked active enhancers in Tax-expressing cells than Tax-non-expressing cells across the entire region, suggesting that global structural change may occur in Tax-expressing cells.Using the data of ATAC-seq, pathway analyses were performed by GREAT. This analysis revealed that the pathways associated with immune activation, such as Toll-like receptor signaling, TNFR2 signaling, IL-2 signaling, and Th1/Th2 differentiation pathways were significantly enriched in Tax-expressing cells. Analysis of genome-wide distribution of motifs in open chromatin region, were carried out with HOMER, and result showed that motifs of NF-kB, and AP-1 family (e.g., JunB, Fra1, Fra2) were significantly enriched in Tax-expressing cells compared to Tax-non-expressing cells, which is consistent with the results of pathway analyses of RNA-seq. The genome-wide motif footprinting analysis was performed using Protein interaction quantitation (PIQ) methods. It was found that motifs of NF-kB, and AP-1 family (e.g., JunB, Fra1, Fra2) were occupied in large number of regions and showed higher DNA accessibility in Tax-expressing cells. In contrast, motif of CTCF was likely occupied in large number of regions and exhibit higher DNA accessibility in Tax-non-expressing cells. Importantly, mRNA levels of NF-kB and AP-1 transcription factors were significantly higher in Tax-expressing than Tax-non-expressing cells.These findings suggest that structural changes of NF-kB and AP-1 family recognition sites, and activation of pathways related to these factors could trigger global transcriptional changes in host genome by transient Tax expression, which is the critical event for persistent infection of HTLV-1 and leukemogenesis of ATL. DisclosuresMatsuoka:Bristol Myers Squibb: Research Funding.