Key determinants of target DNA recognition by retroviral intasomes

BACKGROUND: Retroviral integration favors weakly conserved palindrome sequences at the sites of viral DNA joining and generates a short (4–6 bp) duplication of host DNA flanking the provirus. We previously determined two key parameters that underlie the target DNA preference for prototype foamy virus (PFV) and human immunodeficiency virus type 1 (HIV-1) integration: flexible pyrimidine (Y)/purine (R) dinucleotide steps at the centers of the integration sites, and base contacts with specific integrase residues, such as Ala188 in PFV integrase and Ser119 in HIV-1 integrase. Here we examined the dinucleotide preference profiles of a range of retroviruses and correlated these findings with respect to length of target site duplication (TSD). RESULTS: Integration datasets covering six viral genera and the three lengths of TSD were accessed from the literature or generated in this work. All viruses exhibited significant enrichments of flexible YR and/or selection against rigid RY dinucleotide steps at the centers of integration sites, and the magnitude of this enrichment inversely correlated with TSD length. The DNA sequence environments of in vivo-generated HIV-1 and PFV sites were consistent with integration into nucleosomes, however, the local sequence preferences were largely independent of target DNA chromatinization. Integration sites derived from cells infected with the gammaretrovirus reticuloendotheliosis virus strain A (Rev-A), which yields a 5 bp TSD, revealed the targeting of global chromatin features most similar to those of Moloney murine leukemia virus, which yields a 4 bp duplication. In vitro assays revealed that Rev-A integrase interacts with and is catalytically stimulated by cellular bromodomain containing 4 protein. CONCLUSIONS: Retroviral integrases have likely evolved to bend target DNA to fit scissile phosphodiester bonds into two active sites for integration, and viruses that cut target DNA with a 6 bp stagger may not need to bend DNA as sharply as viruses that cleave with 4 bp or 5 bp staggers. For PFV and HIV-1, the selection of signature bases and central flexibility at sites of integration is largely independent of chromatin structure. Furthermore, global Rev-A integration is likely directed to chromatin features by bromodomain and extraterminal domain proteins. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12977-015-0167-3) contains supplementary material, which is available to authorized users

Hsmar1 transposition is sensitive to the topology of the transposon donor and the target

Hsmar1 is a member of the Tc1-mariner superfamily of DNA transposons. These elements mobilize within the genome of their host by a cut-and-paste mechanism. We have exploited the in vitro reaction provided by Hsmar1 to investigate the effect of DNA supercoiling on transposon integration. We found that the topology of both the transposon and the target affect integration. Relaxed transposons have an integration defect that can be partially restored in the presence of elevated levels of negatively supercoiled target DNA. Negatively supercoiled DNA is a better target than nicked or positively supercoiled DNA, suggesting that underwinding of the DNA helix promotes target interactions. Like other Tc1-mariner elements, Hsmar1 integrates into 5′-TA dinucleotides. The direct vicinity of the target TA provides little sequence specificity for target interactions. However, transposition within a plasmid substrate was not random and some TA dinucleotides were targeted preferentially. The distribution of intramolecular target sites was not affected by DNA topology

BF Integrase Genes of HIV-1 Circulating in São Paulo, Brazil, with a Recurrent Recombination Region

Although some studies have shown diversity in HIV integrase (IN) genes, none has focused particularly on the gene evolving in epidemics in the context of recombination. The IN gene in 157 HIV-1 integrase inhibitor-naïve patients from the São Paulo State, Brazil, were sequenced tallying 128 of subtype B (23 of which were found in non-B genomes), 17 of subtype F (8 of which were found in recombinant genomes), 11 integrases were BF recombinants, and 1 from subtype C. Crucially, we found that 4 BF recombinant viruses shared a recurrent recombination breakpoint region between positions 4900 and 4924 (relative to the HXB2) that includes 2 gRNA loops, where the RT may stutter. Since these recombinants had independent phylogenetic origin, we argue that these results suggest a possible recombination hotspot not observed so far in BF CRF in particular, or in any other HIV-1 CRF in general. Additionally, 40% of the drug-naïve and 45% of the drug-treated patients had at least 1 raltegravir (RAL) or elvitegravir (EVG) resistance-associated amino acid change, but no major resistance mutations were found, in line with other studies. Importantly, V151I was the most common minor resistance mutation among B, F, and BF IN genes. Most codon sites of the IN genes had higher rates of synonymous substitutions (dS) indicative of a strong negative selection. Nevertheless, several codon sites mainly in the subtype B were found under positive selection. Consequently, we observed a higher genetic diversity in the B portions of the mosaics, possibly due to the more recent introduction of subtype F on top of an ongoing subtype B epidemics and a fast spread of subtype F alleles among the B population

Polycomb CBX7 Directly Controls Trimethylation of Histone H3 at Lysine 9 at the p16 Locus

BACKGROUND: H3K9 trimethylation (H3K9me3) and binding of PcG repressor complex-1 (PRC1) may play crucial roles in the epigenetic silencing of the p16 gene. However, the mechanism of the initiation of this trimethylation is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we found that upregulating the expression of PRC1 component Cbx7 in gastric cancer cell lines MGC803 and BGC823 led to significantly suppress the expression of genes within the p16-Arf-p15 locus. H3K9me3 formation was observed at the p16 promoter and Regulatory Domain (RD). CBX7 and SUV39H2 binding to these regions were also detectable in the CBX7-stably upregulated cells. CBX7-SUV39H2 complexes were observed within nucleus in bimolecular fluorescence complementation assay (BiFC). Mutations of the chromodomain or deletion of Pc-box abolished the CBX7-binding and H3K9me3 formation, and thus partially repressed the function of CBX7. SiRNA-knockdown of Suv39h2 blocked the repressive effect of CBX7 on p16 transcription. Moreover, we found that expression of CBX7 in gastric carcinoma tissues with p16 methylation was significantly lower than that in their corresponding normal tissues, which showed a negative correlation with transcription of p16 in gastric mucosa. CONCLUSION/SIGNIFICANCE: These results demonstrated for the first time, to our knowledge, that CBX7 could initiate H3K9me3 formation at the p16 promoter

LEDGF gene silencing impairs the tumorigenicity of prostate cancer DU145 cells by abating the expression of Hsp27 and activation of the Akt/ERK signaling pathway

Lens epithelium-derived growth factor (LEDGF) maintains survival pathways by augmenting the transcription of stress-response genes such as small heat-shock protein 27. Recently, aberrant expression of LEDGF was found in prostate cancer (PC). Herein, we showed that LEDGF overexpression upregulated Hsp27 in PC cells, DU145, PC-3 and LNCaP and promoted antiapoptotic pathways in PCs. We found that these cells had higher abundance of Hsp27, which was correlated with the levels of LEDGF expression. Transactivation assay in DU145 cells revealed that transactivation of Hsp27 was related to the magnitude of LEDGF expression. Silencing of LEDGF in DU145 cells abrogated Hsp27 expression and inhibited stimulated cell proliferation, invasiveness and migration. These cells were arrested in S and G2 phase, and failed to accumulate cyclin B1, and showed increased apoptosis. Furthermore, LEDGF-depleted DU145 cells displayed elevated Bax and cleaved caspase 9 expression and reduced levels of Bcl2, Bcl-XL. The activated survival pathway(s), ERK1/2 and Akt, were selectively decreased in these cells, which characteristically have lower tumorigenicity. Conversely, the depleted cells, when re-overexpressed with LEDGF or Hsp27, regained tumorigenic properties. Collectively, results reveal the involvement of LEDGF-mediated elevated expression of Hsp27-dependent survival pathway(s) in PC. Our findings suggest new lines of investigation aimed at developing therapies by targeting LEDGF or its aberrant expression-associated stimulated antiapoptotic pathway(s)

LEDGF/p75 Proteins with Alternative Chromatin Tethers Are Functional HIV-1 Cofactors

LEDGF/p75 can tether over-expressed lentiviral integrase proteins to chromatin but how this underlies its integration cofactor role for these retroviruses is unclear. While a single integrase binding domain (IBD) binds integrase, a complex N-terminal domain ensemble (NDE) interacts with unknown chromatin ligands. Whether integration requires chromatin tethering per se, specific NDE-chromatin ligand interactions or other emergent properties of LEDGF/p75 has been elusive. Here we replaced the NDE with strongly divergent chromatin-binding modules. The chimeras rescued integrase tethering and HIV-1 integration in LEDGF/p75-deficient cells. Furthermore, chromatin ligands could reside inside or outside the nucleosome core, and could be protein or DNA. Remarkably, a short Kaposi's sarcoma virus peptide that binds the histone 2A/B dimer converted GFP-IBD from an integration blocker to an integration cofactor that rescues over two logs of infectivity. NDE mutants were corroborative. Chromatin tethering per se is a basic HIV-1 requirement and this rather than engagement of particular chromatin ligands is important for the LEDGF/p75 cofactor mechanism

Psip1/Ledgf p52 Binds Methylated Histone H3K36 and Splicing Factors and Contributes to the Regulation of Alternative Splicing

Increasing evidence suggests that chromatin modifications have important roles in modulating constitutive or alternative splicing. Here we demonstrate that the PWWP domain of the chromatin-associated protein Psip1/Ledgf can specifically recognize tri-methylated H3K36 and that, like this histone modification, the Psip1 short (p52) isoform is enriched at active genes. We show that the p52, but not the long (p75), isoform of Psip1 co-localizes and interacts with Srsf1 and other proteins involved in mRNA processing. The level of H3K36me3 associated Srsf1 is reduced in Psip1 mutant cells and alternative splicing of specific genes is affected. Moreover, we show altered Srsf1 distribution around the alternatively spliced exons of these genes in Psip1 null cells. We propose that Psip1/p52, through its binding to both chromatin and splicing factors, might act to modulate splicing

Whole Genome Resequencing Reveals Natural Target Site Preferences of Transposable Elements in Drosophila melanogaster

Transposable elements are mobile DNA sequences that integrate into host genomes using diverse mechanisms with varying degrees of target site specificity. While the target site preferences of some engineered transposable elements are well studied, the natural target preferences of most transposable elements are poorly characterized. Using population genomic resequencing data from 166 strains of Drosophila melanogaster, we identified over 8,000 new insertion sites not present in the reference genome sequence that we used to decode the natural target preferences of 22 families of transposable element in this species. We found that terminal inverted repeat transposon and long terminal repeat retrotransposon families present clade-specific target site duplications and target site sequence motifs. Additionally, we found that the sequence motifs at transposable element target sites are always palindromes that extend beyond the target site duplication. Our results demonstrate the utility of population genomics data for high-throughput inference of transposable element targeting preferences in the wild and establish general rules for terminal inverted repeat transposon and long terminal repeat retrotransposon target site selection in eukaryotic genomes

A Novel Zinc Finger Protein Zfp277 Mediates Transcriptional Repression of the Ink4a/Arf Locus through Polycomb Repressive Complex 1

Polycomb group (PcG) proteins play a crucial role in cellular senescence as key transcriptional regulators of the Ink4a/Arf tumor suppressor gene locus. However, how PcG complexes target and contribute to stable gene silencing of the Ink4a/Arf locus remains little understood.We examined the function of Zinc finger domain-containing protein 277 (Zfp277), a novel zinc finger protein that interacts with the PcG protein Bmi1. Zfp277 binds to the Ink4a/Arf locus in a Bmi1-independent manner and interacts with polycomb repressor complex (PRC) 1 through direct interaction with Bmi1. Loss of Zfp277 in mouse embryonic fibroblasts (MEFs) caused dissociation of PcG proteins from the Ink4a/Arf locus, resulting in premature senescence associated with derepressed p16(Ink4a) and p19(Arf) expression. Levels of both Zfp277 and PcG proteins inversely correlated with those of reactive oxygen species (ROS) in senescing MEFs, but the treatment of Zfp277(-/-) MEFs with an antioxidant restored the binding of PRC2 but not PRC1 to the Ink4a/Arf locus. Notably, forced expression of Bmi1 in Zfp277(-/-) MEFs did not restore the binding of Bmi1 to the Ink4a/Arf locus and failed to bypass cellular senescence. A Zfp277 mutant that could not bind Bmi1 did not rescue Zfp277(-/-) MEFs from premature senescence.Our findings implicate Zfp277 in the transcriptional regulation of the Ink4a/Arf locus and suggest that the interaction of Zfp277 with Bmi1 is essential for the recruitment of PRC1 to the Ink4a/Arf locus. Our findings also highlight dynamic regulation of both Zfp277 and PcG proteins by the oxidative stress pathways