IL‐2R alpha transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. To analyse the mechanisms used to activate IL‐2R alpha transcription as well as those used to block it in non‐expressing cells, we determined the protein‐DNA interactions at the IL‐2R alpha locus in three different cell types using the DMS/LMPCR genomic footprinting method. CD25/IL‐2R alpha can be efficiently induced in primary human T cells since approximately 100% express this gene when receiving an appropriate combination of mitogenic stimuli. To understand why IL‐2R alpha is not expressed in other haematopoietic cell types, we analysed BJAB B lymphoma cells which do not express the IL‐2R alpha gene and contain constitutively active nuclear NF‐kappa B. Primary fibroblasts from embryo and adult skin were selected to examine the mechanisms that may be used to keep the IL‐2R alpha gene inactive in non‐haematopoietic cells. The three main results are: (i) the stable in vivo occupancy of IL‐2R alpha kappa B element in resting T cells, most probably by constitutive NF‐kappa B p50 homodimer that could impair SRF binding to the flanking SRE/CArG box; (ii) its inducible occupancy by NF‐kappa B p50‐p65 associated with the binding of an SRE/CArG box DNA‐binding factor upon mitogenic stimulation; and (iii) a correlation between the precommitment of T cells to activation and the presence of stable preassembled protein‐DNA complexes in contrast with the bare IL‐2R alpha locus in non‐T cells.

Viral latency of human immunodeficiency virus type 1 (HIV-1) has become a major hurdle to a cure in the highly effective antiretroviral therapy (ART) era. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has successfully been demonstrated to excise or inactivate integrated HIV-1 provirus from infected cells by targeting the long terminal repeat (LTR) region. However, the guide RNAs (gRNAs) have classically avoided transcription factor binding sites (TFBSs) that are readily observed and known to be important in human promoters. Although conventionally thought unfavorable due to potential impact on human promoters, our computational pipeline identified gRNA sequences that were predicted to inactivate HIV-1 transcription by targeting the nuclear factor κB (NF-κB) binding sites (gNFKB0, gNFKB1) with a high safety profile (lack of predicted or observed human edits) and broad-spectrum activity (predicted coverage of known viral sequences). Genome-wide, unbiased identification of double strand breaks (DSBs) enabled by sequencing (GUIDE-seq) showed that the gRNAs targeting NF-κB binding sites had no detectable CRISPR-induced off-target edits in HeLa cells. 5′ LTR-driven HIV-1 transcription was significantly reduced in three HIV-1 reporter cell lines. These results demonstrate a working model to specifically target well-known TFBSs in the HIV-1 LTR that are readily observed in human promoters to reduce HIV-1 transcription with a high-level safety profile and broad-spectrum activity.

R. Costello : poste d' accueil lnsenn. P. Lecine : n.llocatrâre de recl!l'rche MESR. B. Kahn-Perlès : chmgée de 1•echerrhe au Cnrs. M. Algané : al/1) w.tmre de re cherche MESR. C. Lipcey : ingé nieur à 1'/nsenn. D. Olive : directeur de recherrhe à l'Inserm. J. 1 rn bert : chargé de 1• echerche à l'fn senn. Laboratoire d'immunologie fo nction nelle el moléculaire, Inserm U. l l9, 27, bou levard Lei-Roure, 13009 Marseille, France. Voir aussi la. mini-sy nthès e d'A. lsmil, jmge 101 7 de ce numéro.

The full length of HIV/R7/E−/GFP integrated in the J-Lat 10.6 cell line was sequenced in this study. The single copy of the integrated virus, including the breakpoints from the human chromosome to the provirus, was amplified by two separate PCRs. A 10,200-bp genome sequence was acquired, analyzed, and deposited in GenBank.