HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells

Background The persistence of the latent HIV-1 reservoir is a major obstacle to curing HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2). Methods We investigated HIV-1 promoter activity after integration into specific sites in BACH2 in Jurkat T-cells. The HIV-1-based vector LTatCL[M] contains two fluorophores: (1) Cerulean, which reports the activity of the HIV-1 promoter and (2) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean+/mCherry+) and inactive (Cerulean–/mCherry+) HIV-1 promoters were characterised. Results Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2, the HIV-1 promoter was gradually silenced as reflected by the decrease in Cerulean expression over a period of 162 days. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. Conclusion Successful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity.

HIV-1 promoter is gradually silenced when integrated into BACH2

The persistence of the latent HIV-1 reservoir is a major obstacle to cure HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2). We investigated HIV-1 promoter activity after integration into specific sites in BACH2. The HIV-1-based vector LTatCL[M] contains two fluorophores: 1.) Cerulean, which reports the activity of the HIV-1 promoter, and 2.) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean+/mCherry+) and inactive (Cerulean−/mCherry+) HIV-1 promoters were characterized. Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2, active HIV-1 promoters were gradually silenced as reflected by decrease in Cerulean expression over a period of 162 days in culture. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. Our results show that the HIV-1 promoter is silenced when integrated into BACH2 without impairing BACH2 mRNA and protein expression. This might contribute to HIV-1 persistence, enabling infected T-cells to complete differentiation into a memory phenotype, persist, and clonally expand over time.

Three-Way Translocations and Insertions Are the Molecular Basis for Complex MLL Rearrangements.

The human MLL gene on 11q23 is frequently involved in chromosomal rearrangements including almost all human chromosomes. These rearrangements are associated with high-risk acute leukemias. Today more than 60 MLL fusion partner genes which represent the MLL recombinome have been characterized at the molecular level. Interestingly, more than 20% of all MLL translocations are highly complex. Here we distinguish between two types of complex rearrangements: Type I: A complex rearrangement is a genetic prerequisite due to the transcriptional orientation of the involved genes, for example t(10;11) with MLL and AF10, respectively; Type II: A complex rearrangement is not prerequisite, although identified in leukemia patients. More than 500 MLL rearrangements have been analyzed at the Diagnostic Center of Acute Leukemia so far and here, we present data on 60 complex translocations that were resolved at the molecular level. Three-way chromosomal translocations, insertions and combinations thereof are the molecular basis for most of these complex MLL rearrangements which in most cases create 3 or more fusions. However, type I is mainly based on insertions and type II is based on 3 way translocations. Within these complex rearrangements, the 5′ part of the MLL gene is in general fused in frame to one of the most frequent partner genes. But the 3′ part of the MLL gene is fused in 36 cases (60%) to a novel gene such as, for example, APBB1IP or CMAH. We defined this heterogeneous group of novel translocation partner genes as “reciprocal MLL fusion genes” as they had not been identified as fusion partners to the 5′ part of the MLL gene before. These fusions are only in frame in four cases; the remaining fusions are out of frame or the genes have an opposite transcriptional orientation. I.e. these genes show genetic haplo-insufficiency and thus may also cooperate in promoting leukemogenesis. For the remaining 40% the 3′ part of the MLL gene is fused to a chromosomal area which does not encode any gene. In many complex aberrations additional genes like for example RREB1 or OS9 are fused mostly out of frame to other genes or non-coding chromosomal areas of these complex rearrangements. These genes are also haplo-insufficient and may also contribute to the leukemogenic process. These results demonstrate that LDI-PCR is a potent tool to identify highly complex and cryptic rearrangements as both MLL fusion alleles are analyzed at the same time and reveal immediately if a balanced or more complex rearrangement is present. Whether the pathobiology of type II complex rearrangements is different in comparison to their not complex counterparts has still to be proven. Supported in part by grants Ma 1876/7-1, /8-1 and /9-1 from the DFG, grant N1KR-S12T13 from the BMBF and grant 102362 from the Deutsche Krebshilfe.