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Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription.

ABSTRACT: Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy.

SUBMITTER: Tosoni E

PROVIDER: S-EPMC4605322 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription.

Tosoni Elena E Frasson Ilaria I Scalabrin Matteo M Perrone Rosalba R Butovskaya Elena E Nadai Matteo M Palù Giorgio G Fabris Dan D Richter Sara N SN

Nucleic acids research 20150909 18

Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex ...[more]

PMID: 26354862

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Project description:DNA secondary structures are important for fundamental genome functions such as transcription and replication1. The G-quadruplex (G4) structural motif has been linked to gene regulation2,3 and genome instability4,5 and may be important to cancer development and other diseases6-8. Recently, ~700,000 discrete G4s have been observed in naked human single-stranded genomic DNA using G4-seq, a high-throughput sequencing technique that detects structural features in vitro.9 It is of vital importance to investigate G4 structures within an endogenous chromatin context, which until now remained elusive10,11. Herein, we address this via the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We identified ~10,000 endogenous G4 structures and show that G4s are predominantly seen in regulatory, nucleosome-depleted, chromatin regions. G4s were enriched in the promoters and 5âUTR regions of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as MYC. Reorganization of the chromatin landscape using a histone deacetylase inhibitor, resulted in de novo G4 formation in new and more prominent regulatory, nucleosome-depleted regions associated with increased transcriptional output. Our findings suggest a striking relationship between promoter nucleosome-depleted regions, G4 formation and elevated transcriptional activity. Comparison between normal human epidermal keratinocytes and their immortalized counterparts revealed a ï¾7-fold greater G4 abundance in immortalized cells, of which 80 % were found in regulatory, nucleosome-depleted regions common to both cell types. Consequently, cells exhibiting more G4s displayed significantly increased transcriptional output and were more sensitive to growth inhibition by a small molecule G4 ligand. Overall, our results provide new mechanistic insights into where and when DNA adopts G4 structure in vivo. Our findings show for the first time that regulatory, nucleosome-depleted chromatin and transcriptional states predominantly shape the endogenous G4 DNA landscape. Two cell lines, treated with entinostat or untreated, analyzed to detect gene expression differences, presence of G-Qudruplexes and chromatin state. Each combination of conditions replicated in duplicates or triplicates.

Project description:BACKGROUND:The human immunodeficiency virus (HIV) cell reservoir is currently a main obstacle towards complete eradication of the virus. This infected pool is refractory to anti-viral therapy and harbors integrated proviruses that are transcriptionally repressed but replication competent. As transcription silencing is key for establishing the HIV reservoir, significant efforts have been made to understand the mechanism that regulate HIV gene transcription, and the role of the elongation machinery in promoting this step. However, while the role of the super elongation complex (SEC) in enhancing transcription activation of HIV is well established, the function of SEC in modulating viral latency is less defined and its cell partners are yet to be identified. RESULTS:In this study we identify fused in sarcoma (FUS) as a partner of AFF4 in cells. FUS inhibits the activation of HIV transcription by AFF4 and ELL2, and silences overall HIV gene transcription. Concordantly, depletion of FUS elevates the occupancy of AFF4 and Cdk9 on the viral promoter and activates HIV gene transcription. Live cell imaging demonstrates that FUS co-localizes with AFF4 within nuclear punctuated condensates, which are disrupted upon treating cells with aliphatic alcohol. In HIV infected cells, knockout of FUS delays the gradual entry of HIV into latency, and similarly promotes viral activation in a T cell latency model that is treated with JQ1. Finally, effects of FUS on HIV gene transcription are also exhibited genome wide, where FUS mainly occupies gene promoters at transcription starting sites, while its knockdown leads to an increase in AFF4 and Cdk9 occupancy on gene promoters of FUS affected genes. CONCLUSIONS:Towards eliminating the HIV infected reservoir, understanding the mechanisms by which the virus persists in the face of therapy is important. Our observations show that FUS regulates both HIV and global gene transcription and modulates viral latency, thus can potentially serve as a target for future therapy that sets to reactivate HIV from its latent state.

Dataset Information

Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription.

Publications

Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription.

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