USP51 deubiquitylates H2AK13,15ub and regulates DNA damage response

Dynamic regulation of RNF168-mediated ubiquitylation of histone H2A Lys13,15 (H2AK13,15ub) at DNA double-strand breaks (DSBs) is crucial for preventing aberrant DNA repair and maintaining genome stability. However, it remains unclear which deubiquitylating enzyme (DUB) removes H2AK13,15ub. Here we show that USP51, a previously uncharacterized DUB, deubiquitylates H2AK13,15ub and regulates DNA damage response. USP51 depletion results in increased spontaneous DNA damage foci and elevated levels of H2AK15ub and impairs DNA damage response. USP51 overexpression suppresses the formation of ionizing radiation-induced 53BP1 and BRCA1 but not RNF168 foci, suggesting that USP51 functions downstream from RNF168 in DNA damage response. In vitro, USP51 binds to H2A–H2B directly and deubiquitylates H2AK13,15ub. In cells, USP51 is recruited to chromatin after DNA damage and regulates the dynamic assembly/disassembly of 53BP1 and BRCA1 foci. These results show that USP51 is the DUB for H2AK13,15ub and regulates DNA damage response

Distinct versus overlapping functions of MDC1 and 53BP1 in DNA damage response and tumorigenesis

The importance of the DNA damage response (DDR) pathway in development, genomic stability, and tumor suppression is well recognized. Although 53BP1 and MDC1 have been recently identified as critical upstream mediators in the cellular response to DNA double-strand breaks, their relative hierarchy in the ataxia telangiectasia mutated (ATM) signaling cascade remains controversial. To investigate the divergent and potentially overlapping functions of MDC1 and 53BP1 in the ATM response pathway, we generated mice deficient for both genes. Unexpectedly, the loss of both MDC1 and 53BP1 neither significantly increases the severity of defects in DDR nor increases tumor incidence compared with the loss of MDC1 alone. We additionally show that MDC1 regulates 53BP1 foci formation and phosphorylation in response to DNA damage. These results suggest that MDC1 functions as an upstream regulator of 53BP1 in the DDR pathway and in tumor suppression

A c-Myc–SIRT1 feedback loop regulates cell growth and transformation

The protein deacetylase SIRT1 has been implicated in a variety of cellular functions, including development, cellular stress responses, and metabolism. Increasing evidence suggests that similar to its counterpart, Sir2, in yeast, Caenorhabditis elegans, and Drosophila melanogaster, SIRT1 may function to regulate life span in mammals. However, SIRT1's role in cancer is unclear. During our investigation of SIRT1, we found that c-Myc binds to the SIRT1 promoter and induces SIRT1 expression. However, SIRT1 interacts with and deacetylates c-Myc, resulting in decreased c-Myc stability. As a consequence, c-Myc's transformational capability is compromised in the presence of SIRT1. Overall, our experiments identify a c-Myc–SIRT1 feedback loop in the regulation of c-Myc activity and cellular transformation, supporting/suggesting a role of SIRT1 in tumor suppression

The Long Non-Coding RNA GAS5 Cooperates with the Eukaryotic Translation Initiation Factor 4E to Regulate c-Myc Translation

<div><p>Long noncoding RNAs (lncRNAs) are important regulators of transcription; however, their involvement in protein translation is not well known. Here we explored whether the lncRNA GAS5 is associated with translation initiation machinery and regulates translation. GAS5 was enriched with eukaryotic translation initiation factor-4E (eIF4E) in an RNA-immunoprecipitation assay using lymphoma cell lines. We identified two RNA binding motifs within eIF4E protein and the deletion of each motif inhibited the binding of GAS5 with eIF4E. To confirm the role of GAS5 in translation regulation, GAS5 siRNA and <i>in vitro</i> transcribed GAS5 RNA were used to knock down or overexpress GAS5, respectively. GAS5 siRNA had no effect on global protein translation but did specifically increase c-Myc protein level without an effect on c-Myc mRNA. The mechanism of this increase in c-Myc protein was enhanced association of c-Myc mRNA with the polysome without any effect on protein stability. In contrast, overexpression of <i>in vitro</i> transcribed GAS5 RNA suppressed c-Myc protein without affecting c-Myc mRNA. Interestingly, GAS5 was found to be bound with c-Myc mRNA, suggesting that GAS5 regulates c-Myc translation through lncRNA-mRNA interaction. Our findings have uncovered a role of GAS5 lncRNA in translation regulation through its interactions with eIF4E and c-Myc mRNA.</p></div

GAS5 suppresses c-Myc expression at protein level.

<p>(<b>A</b>) GAS5 was knocked down by siRNA in HEK-293T cells, and global protein translation was measured by [³H]-leucine incorporation assay. Bars represent mean ±SD from 3 replicates for Q-PCR and 6 replicates for [³H]-leucine incorporation assay. The experiments were performed two times. (<b>B</b>) pRF and pRF-SL plasmid were transfected into HEK-293T cells and cap-dependent protein translation efficiency was evaluated by luciferase assay. Bars represent mean ±SD from 4 replicates. The data were repeated three times. (<b>C</b>) The protein level of c-Myc (P = 0.007), Mcl1 (P = 0.88), survivin (P = 0.47) and Bcl2 (P = 0.47) was assessed by western blot (n = 3) after the cells were treated with GAS5 or control siRNA. (<b>D</b>) The mRNA level of c-Myc (P = 0.10), Mcl1 (P = 0.75), survivin (P = 0.31) and Bcl2 (P = 0.19) was quantified by Q-PCR after the cells were treated with GAS5 or control siRNA. Bars represent mean ±SD from 3 replicates. The experiment was repeated three times. (<b>E</b>) The protein level of c-Myc (P = 0.005) was assessed by western blot (n = 3) after the cells were transfected with <i>in vitro</i> transcribed GAS5 RNA. (<b>F-G</b>) The mRNA level of c-Myc (P = 0.37) (<b>F</b>) and GAS5 (<b>G</b>) was quantified by Q-PCR after the cells were transfected with <i>in vitro</i> transcribed GAS5 RNA. Bars represent mean ±SD from 3 replicates. The experiment was performed three times.</p

A schematic diagram of the GAS5 and c-Myc translation regulation.

<p>A schematic diagram of the GAS5 and c-Myc translation regulation.</p

GAS5 binds to eIF4E through RNA binding motifs.

<p>(<b>A</b>) RNA binding motifs, which are italic and bold, in the eIF4E protein were predicted with 2 web-based tools, BindN and PPRInt. The motif, W56, W102 and E103 for m7G binding is bold and underlined. N-terminally located sequence is motif-1 and the C-terminal one is motif-2. (<b>B-C</b>) GAS5 RNA was detected by RT-PCR after RNA-IP assay using HA antibody in the cells transfected with (<b>B</b>) RNA binding deletion mutants (HA-eIF4E^Del1, HA-eIF4E^Del2 and HA-eIF4E^Del1&2) and (<b>C</b>) cap binding mutant (HA-eIF4E^{cap mutant}). GAPDH was used as a control.</p

GAS5 interacts with eIF4E.

<p>(<b>A</b>) GAS5 mRNA was detected by RT-PCR after RNA-IP using eIF4E and IgG antibodies. The RNA-IP was repeated for two times with similar results. (<b>B</b>) The polysome and non-polysome fractions, as shown by the profile of the absorbance at 254 nm, were separated by sucrose gradient centrifugation in HEK-293T cells. The experiments were repeated three times. (<b>C</b>) The abundance of GAS5 lncRNA in the polysome and non-polysome fractions was measured by Q-PCR and normalized with total GAS5 mRNA in all the fractions. Bars represent mean ±SD from 3 replicates. Experiment was repeated three times, and a representative experiment is shown. (<b>D</b>) Protein levels of eIF4E and eIF4G in the polysome and non-polysome fractions were detected by western blot.</p