KRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins) are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential ...cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences.
To investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression.
We found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes.
Together, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression.
The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, ...N6-methyladenosine (m
A) and 5-methylcytosine (m
C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections. Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m
A and m
C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m
A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m
C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile. Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.
While analyses of cell populations provide averaged information about viral infections, single-cell analyses offer individual consideration, thereby revealing a broad spectrum of diversity as well as ...identifying extreme phenotypes that can be exploited to further understand the complex virus-host interplay. Single-cell technologies applied in the context of human immunodeficiency virus (HIV) infection proved to be valuable tools to help uncover specific biomarkers as well as novel candidate players in virus-host interactions. This review aims at providing an updated overview of single-cell analyses in the field of HIV and acquired knowledge on HIV infection, latency, and host response. Although HIV is a pioneering example, similar single-cell approaches have proven to be valuable for elucidating the behavior and virus-host interplay in a range of other viruses.
HIV DNA integration is favored in active genes, but the underlying mechanism is unclear. Cellular lens epithelium-derived growth factor (LEDGF/p75) binds both chromosomal DNA and HIV integrase, and ...might therefore direct integration by a tethering interaction. We analyzed HIV integration in cells depleted for LEDGF/p75, and found that integration was (i) less frequent in transcription units, (ii) less frequent in genes regulated by LEDGF/p75 and (iii) more frequent in GC-rich DNA. LEDGF is thus the first example of a cellular protein controlling the location of HIV integration in human cells.
Retroviral vectors are often used to introduce therapeutic sequences into patients' cells. In recent years, gene therapy with retroviral vectors has had impressive therapeutic successes, but has also ...resulted in three cases of leukaemia caused by insertional mutagenesis, which has focused attention on the molecular determinants of retroviral-integration target-site selection. Here, we review retroviral DNA integration, with emphasis on recent genome-wide studies of targeting and on the status of efforts to modulate target-site selection.
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