Role of UHRF1 in HIV-1 transcriptional regulation

Title

Role of UHRF1 in HIV-1 transcriptional regulation

Presenter

Maryam Bendoumou

Authors

M. Bendoumou * (1), R. Verdikt (1), S. Bouchat (1), L. Nestola (1), A.O. Pasternak (2), G. Darcis (3), V. Avettand-Fenoel (4), C. Vanhulle (1), A. Ait-Ammar (1), M. Santangelo (1), E. Plant (1), V. Le Douce (5), N. Delacourt (1), A. Cicilionytè (2), C. Necsoi (6), F. Corazza (7), C. Pereira Bittencourt Passaes (8), C. Schwartz (9), M. Bizet (10), F. Fuks (10), A. Sáez-Cirión (8), C. Rouzioux (11), S. De Wit (6), B. Berkhout (2), V. Gautier (5), O. Rohr (9), C. Van Lint (1)

Institutions

(1) Université Libre de Bruxelles, Service of Molecular Virology, Gosselies, Belgium, (2) Amsterdam UMC, University of Amterdam, Amsterdam, Netherlands, the, (3) Infectious Diseases Departement, Liege University Hospital, Liège, Belgium, (4) INSERM, U 1016, Institut Cochin, Paris, France, (5) Centre for Research in Infectious Deseases, University College Dublin, Dublin, Ireland, (6) Service des Maladies Infectieuse, CHU St-Pierre, Université Libre de Bruxelles, Brussels, Belgium, (7) Laboratory of Immunology, IRISLab, CHU Brugmann, Université Libre de Bruxelles, Brussels, Belgium, (8) Institut Pasteur, Unité HIV, Inflammation et Persistance, Paris, France, (9) Université de Strasbourg, Schiltigheim, France, (10) Laboratory of Cancer Epigenetics, Faculty of Medecine, Université Libre de Bruxelles, Brussels, Belgium, (11) AP-HP, Hôpital Necker-Enfants-Malades, Service de Microbiologie Clinique, Paris, France

BACKGROUND: DNA methylation is one of the epigenetic mechanisms involved in HIV-1 latency. The latent 5'LTR methylation profile is heterogeneous in latency model cell lines and in patient cells where it increases with the duration of cART. We have previously demonstrated that 5-Azadeoxycytidine (decitabine) treatment, an inhibitor of DNA methylation, resulted in variable levels of HIV-1 reactivation in latently infected T-cell lines and in ex vivo patient cell cultures. Nevertheless, the mechanisms mediating HIV-1 latency through DNA methylation remain unclear.
METHODS: Sodium bisulfite sequencing, Electrophoretic mobility shift Assay, ChIP-qPCR, RNA interference, GFP fluorescence FACS, p24 ELISA and purification of primary cells from HIV+ patient blood.
RESULTS: Using latently infected J-Lat cell lines, displaying different integration sites, we showed that decitabine-induced reactivation of HIV-1 was accompanied by differential DNA demethylation profiles at the 5'LTR, occurring at specific CpG positions, termed DDMP (Differentially DeMethylated Positions). Interestingly, we showed that UHRF1 (Ubiquitin-like with PHD and Ring Finger domain 1) bound in vitroto several of these DDMPs through different binding modalities where DNA methylation was either non-essential, either essential, or enhancing UHRF1 binding. Moreover, since UHRF1 was originally identified as an CCAAT/enhancer binding protein (C/EBP), we were able to show in vivo recruitment of UHRF1 to four C/EBPs motifs localized in the 5'LTR independently of DNA methylation, in both cell line and primary cell models for HIV-1 latency. UHRF1 depletion through RNA interference induced an increase in HIV gene expression accompanied by global DNA and histone demethylation of the 5'LTR . We showed that UHRF1 repressed HIV-1 in absence and presence of Tat, independently of 5'LTR DNA methylation. Pharmacological inhibition of UHRF1 in PBMCs isolated from aviremic cART-treated HIV+ individuals reactivated expression of HIV-1 RNAs.
CONCLUSIONS: We demonstrate an important role in HIV-1 latency of UHRF1 which binds to multiple sites throughout the 5'LTR in a methylation-dependent and -independent manner. As UHRF1 is known to maintain heterochromatic profiles during replication, our results suggest its involvement in HIV-1 latency by maintenance of the 5'LTR methylation and other mechanisms. In this regard, UHRF1 constitutes a new therapeutic target for HIV cure strategies.

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