Intracellular HIV-1 Tat regulator induces epigenetic changes in the DNA methylation landscape.[EPIC]

The HIV regulatory protein Tat enhances viral transcription but also modifies host gene expression, affecting cell functions like cell cycle and apoptosis. Cohort studies indicate that, despite virological suppression, people with HIV (PWH) are at increased risk of comorbidities linked to chronic inflammation, accelerated immune-aging and cellular senescence, sometimes associated with abnormal genomic methylation patterns. In this study, we analyzed whether Tat influences DNA methylation and subsequently impact the transcriptional signature, contributing to inflammation and accelerated aging. To this aim, Jurkat cells were transfected with full-length Tat (Tat101), Tat’s first exon (Tat72), or an empty vector (TetOFF) and DNA methylation changes were assessed. Differentially expressed genes (DEG) were identified via RNA-seq. Results showed that Tat101 expression resulted in major hyper- and hypomethylations changes at individual CpG sites resulting in a slightly global DNA hypermethylation. Methylation changes at gene promoters and bodies resulted in altered gene expression, specifically regulating gene transcription in 5.1% of DEG in Tat101 expressing cells. In contrast Tat72 had a minimal impact on this epigenetic process. The observed differentially methylated and expressed genes were involved in inflammatory responses, lipid antigen presentation, and apoptosis, and may constitute a key epigenetic mechanism contributing to HIV pathogenesis and chronic inflammation. We aimed to profile the epigenetic changes at DNA methylation level induced by HIV protein Tat with an InfiniumEPIC Methylation array to study the effects of Tat in gene regulation. Jurkat TetOFF cell line from Clontech was stably transfected with a pTRE2hyg plasmid encoding full-length HIV-Tat cDNA (101aa; Tat101), a pTRE2hyg plasmid encoding only the first exon of HIV-Tat (72aa; Tat72), or an empty pTRE2hyg vector as a control (TetOFF). In this model, the administration of 1 μg/ml doxycycline to the culture medium for 48h repress Tat expression. A total of 400.000 Jurkat cells per well with the different HIV-Tat constructs or the empty vector control (3 biological replicates) were grown in the presence or absence of 1 μg/ml doxycycline for 2 days. Cell pellets were then obtained and frozen. Genomic DNA was isolated and used for bisulfite conversion. Subsequently, all the samples were assayed with the Infinium MethylationEPIC BeadChip microarray v1.0 (Illumina) in a HISCanSQ Analysis System (Illumina).Beta values were obtained with minfi and differentially methylated regions were calculated between groups using MissMethyl R package.