Recruitment of lysine demethylase 2A to DNA double strand breaks and its interaction with 53BP1 ensures genome stability.

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.

LEDGF/p75 Deficiency Increases Deletions at the HIV-1 cDNA Ends.

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.

SUMOylation negatively modulates target gene occupancy of the KDM5B, a histone lysine demethylase.

The histone lysine demethylase KDM5B plays key roles in gene repression by demethylating trimethylated lysine 4 of histone H3 (H3K4me3), a modification commonly found at the promoter region of actively transcribed genes. KDM5B is known to regulate the expression of genes involved in cell cycle progression; however, little is known about the post-translational modifications that regulate KDM5B. Herein, we report that KDM5B is SUMOylated at lysine residues 242 and 278 and that the ectopic expression of the hPC2 SUMO E3 ligase enhances this SUMOylation. Interestingly, the levels of KDM5B and its SUMOylated forms are regulated during the cell cycle. KDM5B is modulated by RNF4, an E3 ubiquitin ligase that targets SUMO-modified proteins to proteasomal degradation. Digital gene expression analyses showed that cells expressing the SUMOylation-deficient KDM5B harbor repressed mRNA expression profiles of cell cycle and DNA repair genes. Chromatin immunoprecipitations confirmed some of these genes as KDM5B targets, as they displayed reduced H3K4me3 levels in cells ectopically expressing KDM5B. We propose that SUMOylation by hPC2 regulates the activity of KDM5B.

Poly(ADP-ribose) polymerase 1 promotes transcriptional repression of integrated retroviruses.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a cellular enzyme with a fundamental role in DNA repair and the regulation of chromatin structure, processes involved in the cellular response to retroviral DNA integration. However, the function of PARP-1 in retroviral DNA integration is controversial, probably due to the functional redundancy of the PARP family in mammalian cells. We evaluated the function of PARP-1 in retroviral infection using the chicken B lymphoblastoid cell line DT40. These cells lack significant PARP-1 functional redundancy and efficiently support the postentry early events of the mammalian-retrovirus replication cycle. We observed that DT40 PARP-1(-/-) cells were 9- and 6-fold more susceptible to infection by human immunodeficiency virus type 1 (HIV-1)- and murine leukemia virus (MLV)-derived viral vectors, respectively, than cells expressing PARP-1. Production of avian Rous-associated virus type 1 was also impaired by PARP-1. However, the susceptibilities of these cell lines to infection by the nonretrovirus vesicular stomatitis virus were indistinguishable. Real-time PCR analysis of the HIV-1 life cycle demonstrated that PARP-1 did not impair reverse transcription, nuclear import of the preintegration complex, or viral DNA integration, suggesting that PARP-1 regulates a postintegration step. In support of this hypothesis, pharmacological inhibition of the epigenetic mechanism of transcriptional silencing increased retroviral expression in PARP-1-expressing cells, suppressing the differences observed. Further analysis of the implicated molecular mechanism indicated that PARP-1-mediated retroviral silencing requires the C-terminal region, but not the enzymatic activity, of the protein. In sum, our data indicate a novel role of PARP-1 in the transcriptional repression of integrated retroviruses.

SUMOylation of the lens epithelium-derived growth factor/p75 attenuates its transcriptional activity on the heat shock protein 27 promoter.

Lens epithelium-derived growth factor (LEDGF) proteins p75 and p52 are transcriptional coactivators that connect sequence-specific activators to the basal transcription machinery. We have found that these proteins are posttranslationally modified by SUMO (small ubiquitin-like modifier)-1 and SUMO-3. Three SUMOylation sites, K75, K250, and K254, were mapped on 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 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 heat shock protein 27 expression, these findings support the paradigm establishing SUMOylation as a global neutralizer of cellular processes upregulated upon cellular stress.

Implication of serine residues 271, 273, and 275 in the human immunodeficiency virus type 1 cofactor activity of lens epithelium-derived growth factor/p75.

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-tethering-competent LEDGF/p75.