The Retrovirus HTLV-1 Inserts an Ectopic CTCF-Binding Site Into the Human Genome
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The retrovirus HTLV-1 inserts an ectopic CTCF-binding site into the human genome Yorifumi Satoua,b,c,d,e,1, Paola Miyazatob,c,d, Ko Ishiharac,e, Hiroko Yaguchia, Anat Melameda, Michi Miuraa, Asami Fukudab,c,d, Kisato Nosakaf, Takehisa Watanabeg,2, Aileen G. Rowana, Mitsuyoshi Nakaoe,g, and Charles R. M. Banghama,1 aSection of Virology, Division of Infectious Diseases, Imperial College, London W2 1PG, United Kingdom; bCentre for AIDS Research, Kumamoto University, Kumamoto 860-0811, Japan; cPriority Organization for Innovation and Excellence, Kumamoto University, Kumamoto 860-0811, Japan; dInternational Research Centre for Medical Science, Kumamoto University, Kumamoto 860-0811, Japan; eCore Research for Evolutionary Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan; fCancer Centre, Kumamoto University Hospital, Kumamoto University, Kumamoto 860-0811, Japan; and gDepartment of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan Edited by Robert C. Gallo, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, and approved February 1, 2016 (received for review December 4, 2014) Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes One mechanism used by HTLV-1 to suppress transcription of the malignant and inflammatory diseases in ∼10% of infected people. plus strand is methylation of the 5′ LTR, whereas there is little DNA 4 5 A typical host has between 10 and 10 clones of HTLV-1–infected methylation in the 3′ LTR(5).TheDNAmethylationissharply T lymphocytes, each clone distinguished by the genomic integration reduced at the middle of the provirus (6). This observation raised the HBZ site of the single-copy HTLV-1 provirus. The HTLV-1 bZIP ( )factor question of whether there is a regulatory mechanism that divides the gene is constitutively expressed from the minus strand of the provirus, methylated 5′ part from the unmethylated 3′ part of the provirus, whereas plus-strand expression, required for viral propagation to un- HBZ infected cells, is suppressed or intermittent in vivo, allowing escape perhaps to allow the constitutive expression of the gene that from host immune surveillance. It remains unknown what regulates appears to be required for clonal persistence of HTLV-1 (3, 7). this pattern of proviral transcription and latency. Here, we show that A chromatin insulator is a DNA region that separates tran- CTCF, a key regulator of chromatin structure and function, binds to scriptionally active and inactive regions by binding to certain pro- the provirus at a sharp border in epigenetic modifications in the pX teins. The best-characterized insulator-binding protein in higher region of the HTLV-1 provirus in T cells naturally infected with HTLV-1. eukaryotes is CTCF, an 11 zinc-finger protein highly conserved CTCF is a zinc-finger protein that binds to an insulator region in ge- from flies to humans (8), which binds to tens of thousands of sites in nomic DNA and plays a fundamental role in controlling higher order the human genome and regulates chromatin structure, transcrip- chromatin structure and gene expression in vertebrate cells. We show tional activation, repression, silencing, imprinting, and alternative that CTCF bound to HTLV-1 acts as an enhancer blocker, regulates splicing (9). We therefore set out to test the hypothesis that CTCF HTLV-1 mRNA splicing, and forms long-distance interactions with binds to the HTLV-1 provirus at an epigenetic border and regulates flanking host chromatin. CTCF-binding sites (CTCF-BSs) have been propagated throughout the genome by transposons in certain pri- proviral transcription. mate lineages, but CTCF binding has not previously been described in present-day exogenous retroviruses. The presence of an ectopic Significance CTCF-BS introduced by the retrovirus in tens of thousands of geno- mic locations has the potential to cause widespread abnormalities The retrovirus human T-lymphotropic virus type 1 (HTLV-1) in host cell chromatin structure and gene expression. causes inflammatory and malignant diseases in humans. To maintain latency and avoid immune detection in vivo, HTLV-1 retrovirus | latency | epigenetics | HTLV-1 | CTCF minimizes expression of genes on the plus-strand of the in- tegrated provirus but allows constitutive expression of the etroviruses integrate a dsDNA copy of their genome, the minus-strand gene, which maintains clonal persistence. It is not Rprovirus, into the genome of the cell they infect. Human understood how this gene expression is regulated. We show T-lymphotropic virus type 1 (HTLV-1) is an exogenous retrovirus, that CTCF, a master regulator of chromatin structure and gene widespread in the tropics. Most infected people are asymptomatic expression, binds to HTLV-1, forms loops between the provirus carriers, but ∼10% develop a malignant or inflammatory disease. and host genome, and alters expression of proviral and host Adult T-cell leukemia (ATL) is a leukemia of HTLV-1–infected genes. Because a typical HTLV-1–infected host carries >104 in- + CD4 T cells. ATL cells frequently contain chromosomal abnor- fected T-cell clones, each containing a provirus integrated in a malities, and the disease is refractory to conventional chemo- different genomic site, CTCF binding gives HTLV-1 the potential therapy. HTLV-1–associated myelopathy/tropical spastic paraparesis to cause widespread abnormalities in the human genome. (HAM/TSP) is a chronic inflammatory disease of the spinal cord. HTLV-1 spreads within the individual both by cell-to-cell trans- Author contributions: Y.S., K.N., M.N., and C.R.M.B. designed research; Y.S., P.M., K.I., mission and by clonal proliferation of infected cells: HTLV-1 gene H.Y., A.M., M.M., A.F., T.W., and A.G.R. performed research; Y.S., P.M., K.I., H.Y., A.M., M.M., M.N., and C.R.M.B. analyzed data; K.N. made clinical diagnoses; and Y.S. and products induce proliferation and enhance survival of infected cells C.R.M.B. wrote the paper. (1, 2). In addition to the viral genes that encode enzymes and The authors declare no conflict of interest. structural proteins, HTLV-1 encodes several regulatory and acces- This article is a PNAS Direct Submission. sory genes in the pX region, between the env gene and the 3′ long HBZ Data deposition: ChIP-sequencing data reported in this paper have been deposited in the terminal repeat (LTR). The gene is constitutively expressed DNA Data Bank of Japan (accession no. DRA004162). from the minus strand of the integrated provirus (3), whereas plus- 1To whom correspondence may be addressed. Email: [email protected] or strand expression, required for viral propagation to uninfected [email protected]. cells, is suppressed or intermittently expressed in vivo, allowing 2Present address: Department of Gastroenterology and Hepatology, Graduate School of escape from host immune surveillance (2, 4). It is unknown how Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan. HTLV-1 maintains this chromatin state and strand-selective This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. transcription. 1073/pnas.1423199113/-/DCSupplemental. 3054–3059 | PNAS | March 15, 2016 | vol. 113 | no. 11 www.pnas.org/cgi/doi/10.1073/pnas.1423199113 Downloaded by guest on October 2, 2021 Results transcribed gene bodies, increasing toward the 3′ end of the gene Epigenetic Border in the HTLV-1 Provirus. We used chromatin im- (10). These observations, together with previous reports (5, 6), munoprecipitation (ChIP) assays to identify epigenetic marks over suggested the presence of a regulatory mechanism that establishes the HTLV-1 provirus (Fig. 1A), including H3K36me3 and H3K9Ac, the distinctive pattern of respective epigenetic modifications in the each associated with actively transcribed genes; H3K4me3, char- 5′ and 3′ portions of the HTLV-1 provirus. acteristic of enhancer and promoter regions; and H2A.Z, a histone variant frequently observed in promoter and enhancer regions (10). The Host Insulator-Binding Protein CTCF Binds to the HTLV-1 Provirus The results showed a sharp border in each of these epigenetic in Vivo. Insulator elements demarcate the boundary between tran- modifications in the pX region of the provirus in both a non- scriptionally active (euchromatic) and inactive (heterochromatic) malignant primary T-cell clone (Fig. 1B) and an ATL cell line (Fig. regions of the genome (11) and prevent enhancers from activating 1C). The epigenetic border was more sharply defined in the ATL inappropriate promoters. We therefore tested the hypothesis that cell line, in which the spliced HBZ transcript (sHBZ) is transcribed CTCF binds to HTLV-1 at the observed boundary in epigenetic but plus-strand transcription is significantly suppressed (Fig. S1), modifications (Fig. 1). First, we searched for the consensus CTCF than in nonmalignant T-cell clones, in which both tax, a plus-strand DNA-binding motif (9) (Fig. S4 A–C) in the HTLV-1 genome and transcript, and sHBZ, a minus-strand transcript, are actively tran- identified several candidate binding sites (Fig. 2A); the site in pX scribed. The same pattern was also observed in three other HTLV- showed the highest similarity to the consensus sequence. CTCF 1–infected T-cell lines (Fig. S2 A–C) and in peripheral blood ChIP assays revealed CTCF binding in pX [viral CTCF-binding site mononuclear cells (PBMCs) freshly isolated from patients with (vCTCF-BS)], but not in the other proviral regions tested (Fig. 2 B ATL (Fig. S3 A–F). In addition, a border was observed in DNA and C); similar results were obtained in fresh PBMCs from patients methylation, consistent with previous observations (6) (Fig. 1 D and with either HAM/TSP (Fig. 2D)orATL(Fig.2E), demonstrating E). The results are summarized schematically in Fig. 1F.Theob- that CTCF binds to the pX region of HTLV-1 in natural infection served pattern of epigenetic modifications was consistent with the in vivo.