Rewired chromatin structure and epigenetic gene dysregulation during HTLV-1 infection to leukemogenesis

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) broadly impacts host genes, affecting the infected cell population and inducing the development of a disease with a poor prognosis, adult T-cell leukemia-lymphoma (ATL). This study aimed to provide a comprehensive epigenomic characterization of the infected cell population and evaluated the transcriptome and chromatin structures of peripheral blood cells in HTLV-1-infected individuals using RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin sequencing (ATAC-seq). The infected cells showed significant changes in gene expression patterns from the polyclonal stage and before ATL onset while demonstrating similarities to tumor-forming ATL cells. These similarities were a result of large-scale open chromatin changes, supporting the independent early formation of epigenomic aberrations as an underlying mechanism for later clonal propagation. This study also demonstrated that HTLV-1 Tax directly affects the host chromatin structure, thereby developing fundamental epigenomic characteristics. Several Tax target genes, including the RASGRP3-ERK pathway, were recognized, indicating an impact on signaling pathways. This genome-wide variability in chromatin structural property is a novel feature of HTLV-1 infection and may contribute to pathogenic mechanisms. In addition, it has crucial implications for better understanding the impact of HTLV-1 on the host genome and identifying novel therapeutic targets.

Keywords: ATL; HTLV‐1; chromatin; epigenetics; gene expression.

FIGURE 1

Transcriptome and epigenome analyses for HTLV‐1‐infected cells. (A) Workflow illustrates the collection and processing of fresh peripheral blood samples and the following multilayered experimental platform. (B) Bar graph shows the cell population size (%) based on HAS‐Flow. Line graph represents PVL (%) before sorting. (C) Beeswarm plots show proviral load (%) and expression levels of tax/rex, HBZ, and miR‐31 in each cell fraction (P, n = 16; D, n = 16; N, n = 22) after cell sorting (*P < 0.05; **P < 10⁻⁸). (D) Immunoblot shows Tax protein level in the representative cell fraction after cell sorting. (E, F) Hierarchical clustering (E) and non‐hierarchical t‐SNE plot (F) based on the expression of all genes (22,074 genes). Diagnosis, phenotype, and proviral load are indicated by colors.

FIGURE 2

Dysregulation of transcriptome in HTLV‐1‐infected cells. (A) Box plots show expression levels [log₂(TPM + 0.01)] of functionally important genes (*P < 0.05, **P < 10⁻⁸ vs. AC_P). (B) Venn diagram depicts overlapped dysregulated genes (P < 0.05) in AC_D, iATL_N, and aATL_N compared to uninfected “P” fraction. (C) Bar graph shows enriched gene ontology terms with one‐sided Fisher's exact P values (−log₁₀) for commonly dysregulated genes in ATL cells and HTLV‐1‐infected cells in AC (P < 0.05). (D) Dot plot shows log2 fold changes in the expression of “Cell division” genes in HTLV‐1‐infected cells and ATL cells compared to uninfected T‐cells. (E) Non‐hierarchical dimensional reduction using t‐SNE based on the expression of H3K27me3 target 644 genes. (F) Box plots show expression levels [log₂(TPM + 0.01)] of upregulated 1146 genes (left, FC ≥2 in aATL) and downregulated 3116 genes (right, FC ≤ −2 in aATL) in each indicated subgroup based on phenotype and PVL. PVL “L” represents a low group (PVL 0.96%–6.83%). “H” represents a high group (PVL ≥14.2%). (*P < 0.05; **P < 10⁻⁸). (G) Box plots show expression levels of upregulated and downregulated genes in each indicated subgroup based on clonal expansion. “‐” represents a polyclonal group (median top clone size 2.32%; 1.14%–6.81%, n = 8). “+” represents a clonal group (median top clone size 54.3%; 26.8%–92.4%, n = 9). (*P < 0.05; **P < 10⁻⁸).

FIGURE 3

Characteristics of chromatin accessibility in HTLV‐1‐infected cells and ATL cells. (A) Bar graph shows numbers of chromatin structural abnormalities per Mbp common in four aATL cases. (B, C) Heatmaps depict ATAC peak values of differential regions (left, open chromatin in aATL; right, closed chromatin in aATL) in ATL and AC compared to normal T‐cells. (D) Bar graph shows enriched gene ontology terms for commonly open chromatin genes in ATL cells and HTLV‐1‐infected cells. (E) Scatter plots show ATAC‐seq changes in aATL_N (x axis) and iATL_N or AC_D (y axis) compared to normal T‐cells in promoter regions with different chromatin accessibility. (F) Scatter plots show ATAC‐seq changes in promoter regions (x axis) and gene expression changes (y axis) in iATL_N and AC_D compared to normal T‐cells. (G) Representative IGV tracks for RNA‐seq and ATAC‐seq in normal T‐cells, AC HTLV‐1 infected cells, and ATL cells. The numbers on the right of each track represent RNA‐seq TPM values.

FIGURE 4

Chromatin localization and epigenetic influence of HTLV‐1 Tax. (A) Heatmaps show Tax and H3K27ac ChIP‐seq values centered on H3K27ac clusters (20‐kb windows) in C91/PL cells. (B) Heatmaps depict clustered ChIP‐seq peak values of Tax, RelA, and RelB within H3K27ac‐positive sites. (C) Venn diagram depicts overlapped ChIP‐seq and ATAC‐seq peaks in open chromatin region in aATL. (D) Heatmaps depict enriched gene ontology terms for commonly associated genes with Tax, NF‐κB, and open chromatin. (E) Scatter plot shows ATAC‐seq changes in promoter regions (x axis) and gene expression changes based on RNA‐seq (y axis) in Tax‐cell compared to normal T‐cells. (F) Heatmaps depict clustered ATAC‐seq peak values and RNA‐seq TPM as fold‐changes in Tax‐cell and aATL cells compared to normal T‐cells. (G) Representative IGV tracks for ChIP‐seq and ATAC‐seq. (H) Bar graph shows enriched gene ontology terms for overexpressed genes in Tax‐cell.

FIGURE 5

RASGRP3 as a Tax target gene. (A) Dot plot shows Tax ChIP‐seq values. Functionally important genes are annotated. (B) IGV tracks for ChIP‐seq and ATAC‐seq in RASGRP3 locus. (C) Box plot shows expression levels of RASGRP3. (D) Immunoblots show protein levels of RASGRP3 and β‐actin in Tax⁺ (C91/PL) and Tax⁻ (TL‐Om1, ATN‐1) cell lines treated with indicated dose of NF‐κB inhibitor DHMEQ. (E) Immunoblots show protein levels of RASGRP3 and p‐ERK in RASGRP3‐tarnsduced Jurkat cells and parental cells. (F) HTLV‐1‐infected (C91/PL) and ATL‐derived (TL‐Om1, ATN‐1, MT‐1) cell lines were transduced with lentiviruses expressing shRASGRP3. Graph shows results of Venus‐competitive assay. Data are representative of two independent experiments. (G) p‐ERK staining of PBMCs in acute ATL cases (n = 3) gated on CD4⁺ cell populations. (H) Schematic diagram showing Tax‐dependent and subsequent sustained chromatin regulation.