Lens epithelium-derived growth factor (LEDGF)/p75 is a cellular cofactor for HIV-1 DNA integration. It is well established that the simultaneous binding of LEDGF/p75 to chromatin and to HIV-1 integrase is required for its cofactor activity. However, the exact molecular mechanism of LEDGF/p75 in HIV-1 integration is not yet completely understood. Our hypothesis is that evolutionarily conserved regions in LEDGF/p75 exposed to solvent and harboring posttranslational modifications may be involved in its HIV-1 cofactor activity. Therefore, a panel of LEDGF/p75 deletion mutants targeting these protein regions were evaluated for their HIV-1 cofactor activity, chromatin binding, integrase interaction, and integrase-to-chromatin-tethering activity by using different cellular and biochemical approaches. The deletion of amino acids 267 to 281 reduced the cofactor activity of LEDGF/p75 to levels observed for chromatin-binding-defective mutants. This region contains a serine cluster (residues 271, 273, and 275) recurrently found to be phosphorylated in both human and mouse cells. Importantly, the conversion of these Ser residues to Ala was sufficient to impair the ability of LEDGF/p75 to mediate HIV-1 DNA integration, although these mutations did not alter chromatin binding, integrase binding, or the integrase-to-chromatin-tethering capability of LEDGF/p75. These results clearly indicated that serine residues 271, 273, and 275 influence the HIV-1 cofactor activity of integrase-to-chromatin-tetheringcompetent LEDGF/p75.

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Lens epithelium-derived growth factor (LEDGF) proteins, p75 and p52, are transcriptional co-activators that connect sequence-specific activators to the basal transcription machinery. We have found that these proteins are post translationally modified by the Small Ubiquitin-like Modifier (SUMO)-1 and SUMO-3. Three SUMOylation sites, K75, K250 and K254, were mapped in the shared N-terminal region of these molecules, while a fourth site, K364, was identified in the C-terminal part exclusive of LEDGF/p75. The N-terminal SUMO targets are located in evolutionarily conserved charge-rich regions that lack resemblance to the described consensus SUMOylation motif, whereas the C-terminal SUMO target is solvent-exposed and situated in a typical consensus motif. SUMOylation did not affect the cellular localization of LEDGF proteins and was not necessary for their chromatin-binding ability, nor did it affect this activity. However, lysine to arginine mutations of the identified SUMO acceptor sites drastically inhibited LEDGF SUMOylation, extended the half-life of LEDGF/p75 and significantly increased its transcriptional activity on the Heat shock protein 27 (Hsp27) promoter, indicating a negative effect of SUMOylation on the transcriptional activity of LEDGF/p75. Considering that SUMOylation is known to negatively affect the transcriptional activity of all transcription factors known to transactivate Hsp27 expression, these findings support the paradigm establishing SUMOylation as a global neutralizer of cellular processes upregulated upon cellular stress.

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Lysine demethylase 2A (KDM2A) functions in transcription as a demethylase of lysine 36 on histone H3. Herein, we characterise a role for KDM2A in the DNA damage response in which KDM2A stimulates conjugation of ubiquitin to 53BP1. Impaired KDM2A-mediated ubiquitination negatively affects the recruitment of 53BP1 to DSBs. Notably, we show that KDM2A itself is recruited to DSBs in a process that depends on its demethylase activity and zinc finger domain. Moreover, we show that KDM2A plays an important role in ensuring genomic stability upon DNA damage. Depletion of KDM2A or disruption of its zinc finger domain results in the accumulation of micronuclei following ionizing radiation (IR) treatment. In addition, IR-treated cells depleted of KDM2A display premature exit from the G2/M checkpoint. Interestingly, loss of the zinc finger domain also resulted in 53BP1 focal distribution in condensed mitotic chromosomes. Overall, our data indicates that KDM2A plays an important role in modulating the recruitment of 53BP1 to DNA breaks and is crucial for the preservation of genome integrity following DNA damage.

BackgroundChromatin binding plays a central role in the molecular mechanism of LEDGF/p75 in HIV-1 DNA integration. Conflicting results have been reported in regards to the relevance of the LEDGF/p75 chromatin binding element PWWP domain in its HIV-1 cofactor activity.ResultsHere we present evidence that re-expression of a LEDGF/p75 mutant lacking the PWWP domain (ΔPWWP) rescued HIV-1 infection in cells verified to express background levels of endogenous LEDGF/p75 that do not support efficient HIV-1 infection. The HIV-1 cofactor activity of LEDGF/p75 ΔPWWP was similar to that of LEDGF/p75 wild type (WT). A possible molecular explanation for the nonessential role of PWWP domain in the HIV-1 cofactor activity of LEDGF/p75 comes from the fact that coexpression of HIV-1 integrase significantly restored the impaired chromatin binding activity of LEDGF/p75 ΔPWWP. However, integrase failed to promote chromatin binding of a non-chromatin bound LEDGF/p75 mutant that lacks both the PWWP domain and the AT hook motifs (ΔPWWP/AT) and that exhibits negligible HIV-1 cofactor activity. The effect of integrase on the chromatin binding of LEDGF/p75 requires the direct interaction of these two proteins. An HIV-1 integrase mutant, unable to interact with LEDGF/p75, failed to enhance chromatin binding, whereas integrase wild type did not increase the chromatin binding strength of a LEDGF/p75 mutant lacking the integrase binding domain (ΔIBD).ConclusionsOur data reveal that the PWWP domain of LEDGF/p75 is not essential for its HIV-1 cofactor activity, possibly due to an integrase-mediated increase of the chromatin binding strength of this LEDGF/p75 mutant.

Processing of unintegrated linear HIV-1 cDNA by the host DNA repair system results in its degradation and/or circularization. As a consequence, deficient viral cDNA integration generally leads to an increase in the levels of HIV-1 cDNA circles containing one or two long terminal repeats (LTRs). Intriguingly, impaired HIV-1 integration in LEDGF/p75-deficient cells does not result in a correspondent increase in viral cDNA circles. We postulate that increased degradation of unintegrated linear viral cDNA in cells lacking the lens epithelium-derived growth factor (LEDGF/p75) account for this inconsistency. To evaluate this hypothesis, we characterized the nucleotide sequence spanning 2-LTR junctions isolated from LEDGF/p75-deficient and control cells. LEDGF/p75 deficiency resulted in a significant increase in the frequency of 2-LTRs harboring large deletions. Of note, these deletions were dependent on the 3′ processing activity of integrase and were not originated by aberrant reverse transcription. Our findings suggest a novel role of LEDGF/p75 in protecting the unintegrated 3′ processed linear HIV-1 cDNA from exonucleolytic degradation.

Glutathione-S-transferase (GST) is a family of enzymes involved in the detoxification of toxic and carcinogenic compounds. In the present study, the effect of thioacetamide (TA), a hepatotoxic and hepatocarcinogenic compound, on the activity and expression of GST of Wistar female rats was tested. Animals were treated with a single dose of TA (250 mg/kg) for 12, 24, 48 and 72 hours. GST activity toward the broad substrate 1-chloro-2,4-dinitrobenzene was enhanced by TA. The protein level of the GST classes alpha and mu as well as the mRNA level of several GST subunits were also positively affected by the TA treatment. Female Wistar rats of the same age but from two other different colonies had their GST activity either inhibited or not affected by TA. The basal mRNA level of class alpha and class mu GST was also tested in female Wistar rats obtained from five different sources. Differences in the basal level of class alpha mRNA were observed in rats from at least three different sources, while class mu mRNA level was distinct in two groups of animals.