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Affinity purification of HERC2 complex and Mass Spectrometry; Sister chromatid exchange (SCE) assays.

BLM and WRN are RecQ DNA helicasesessential for genomic stability. Here, we demonstrate that HERC2, a HECT E3 ligase, is critical for their functions to suppress G-quadruplex (G4) DNA. HERC2 interacted with BLM, WRN, and replication protein A (RPA) complexes during the S-phase of the cell cycle. Depletion of HERC2 dissociated RPA from BLM and WRN complexes and significantly increased G4 formation. Triple depletion revealed that HERC2 has an epistatic relationship with BLM and WRN in their G4-suppressing function. In vitro, HERC2 released RPA onto single-stranded DNA (ssDNA) rather than anchoring onto RPA-coated ssDNA. CRISPR/Cas9-mediated deletion of the catalytic ubiquitin-binding site of HERC2 inhibited ubiquitination of RPA2, caused RPA accumulation in the helicase complexes, and increased G4, indicating an essential role for E3 activity in the suppression of G4. Both depletion of HERC2 and inactivation of E3 sensitized cells to the G4-interacting compounds telomestatin and pyridostatin. Overall, these results indicate that HERC2 is a master regulator of G4 suppression that affects the sensitivity of cells to G4 stabilizers. Given that HERC2 expression is frequently reduced in many types of cancers, G4 accumulation as a result of HERC2 deficiency may provide a therapeutic target for G4 stabilizers.Significance: HERC2 is revealed as a master regulator of G-quadruplex, a DNA secondary structure that triggers genomic instability and may serve as a potential molecular target in cancer therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6371/F1.large.jpg. Cancer Res; 78(22); 6371–85. ©2018 AACR.

S1. Analyses of HERC2 complex immunoprecipitated with a newly generated antibody; S2. HERC2 regulates BLM-RPA complex formation; S3. Effect of HERC2 depletion on BLM-RPA and WRN-RPA complex formation was reproduced with a different shRNA; S4. Detection of nuclear G4 foci in cells in different conditions; S5. HERC2 suppresses SCE; S6. Specificity of the HERC2 antibody for the immunostain; S7. Analyses of HERC2 interacting proteins in HERC2Î"E3/Î"E3 HCT116 cells; S8. Inhibition of HERC2 E3 activity increases SCE; S9. Proliferation of HERC-perturbed cell lines; S10. HERC2 is downregulated in various human cancers; S11. Possible model for the role of HERC2 on G4 unwinding.

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Supplementary Figure 1 from BRCA1 Ubiquitinates RPB8 in Response to DNA Damage

Supplementary Figure 6 from Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

The breast cancer suppressor BRCA1 forms a stable heterodimeric E3 ubiquitin ligase with BARD1. Each protein controls the abundance and stability of the other, and loss of the interaction leads to BRCA1 degradation. Here, we show that HERC2, a protein recently implicated in DNA damage repair, targets BARD1-uncoupled BRCA1 for degradation. HERC2 shuttles between the nucleus and the cytoplasm. Its COOH-terminal HECT-containing domain interacts with an NH2-terminal degron domain in BRCA1. HERC2 ubiquitinates BRCA1; this reaction depends on Cys4762 of HERC2, the catalytic ubiquitin binding site, and the degron of BRCA1. The HERC2-BRCA1 interaction is maximal during the S phase of the cell cycle and rapidly diminishes as cells enter G2-M, inversely correlated with the steady-state level of BRCA1. Significantly, HERC2 depletion antagonizes the effects of BARD1 depletion by restoring BRCA1 expression and G2-M checkpoint activity. Conversely, BARD1 protects BRCA1 from HERC2-mediated ubiquitination. Collectively, our findings identify a function for HERC2 in regulating BRCA1 stability in opposition to BARD1. The HERC2 expression in breast epithelial cells and breast carcinomas suggests that this mechanism may play a role in breast carcinogenesis. Cancer Res; 70(15); 6384–92. ©2010 AACR.

Supplementary Figure 4 from Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

Supplementary Figure 1 from BRCA1-Associated Protein 1 Interferes with BRCA1/BARD1 RING Heterodimer Activity

Supplementary Figure 3 from HERC2 Is an E3 Ligase That Targets BRCA1 for Degradation

Supplementary Figure 2 from HERC2 Is an E3 Ligase That Targets BRCA1 for Degradation

Supplementary Table 2 from Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

The breast and ovarian tumor suppressor BRCA1 catalyzes untraditional polyubiquitin chains that could be a signal for processes other than proteolysis. However, despite intense investigations, the mechanisms regulated by the enzyme activity remain only partially understood. Here, we report that BRCA1-BARD1 mediates polyubiquitination of RPB8, a common subunit of RNA polymerases, in response to DNA damage. A proteomics screen identified RPB8 as a protein modified after epirubicin treatment in BRCA1-dependent manner. RPB8 interacted with BRCA1-BARD1 and was polyubiquitinated by BRCA1-BARD1 in vivo and in vitro. BRCA1-BARD1 did not destabilize RPB8 in vivo but rather caused an increase in the amount of soluble RPB8. Importantly, RPB8 was polyubiquitinated immediately after UV irradiation in a manner sensitive to BRCA1 knockdown by RNA interference. Substitution of five lysine residues of RPB8 with arginine residues abolished its ability to be ubiquitinated while preserving its polymerase activity. HeLa cell lines stably expressing this ubiquitin-resistant form of RPB8 exhibited UV hypersensitivity accompanied by up-regulated caspase activity. Our findings suggest that ubiquitination of a common subunit of RNA polymerases is a mechanism underlying BRCA1-dependent cell survival after DNA damage. [Cancer Res 2007;67(3):951–8]

DNA replication, recombination, and repair are highly interconnected processes the disruption of which must be coordinated in cancer. HERC2, a large HECT protein required for homologous recombination repair, is an E3 ubiquitin ligase that targets breast cancer suppressor BRCA1 for degradation. Here, we show that HERC2 is a component of the DNA replication fork complex that plays a critical role in DNA elongation and origin firing. In the presence of BRCA1, endogenous HERC2 interacts with Claspin, a protein essential for G2–M checkpoint activation and replication fork stability. Claspin depletion slowed S-phase progression and additional HERC2 depletion reduced the effect of Claspin depletion. In addition, HERC2 interacts with replication fork complex proteins. Depletion of HERC2 alleviated the slow replication fork progression in Claspin-deficient cells, suppressed enhanced origin firing, and led to a decrease in MCM2 phosphorylation. In a HERC2-dependent manner, treatment of cells with replication inhibitor aphidicolin enhanced MCM2 phosphorylation. Taken together, our results suggest that HERC2 regulates DNA replication progression and origin firing by facilitating MCM2 phosphorylation. These findings establish HERC2 as a critical function in DNA repair, checkpoint activation, and DNA replication. Cancer Res; 71(17); 5621–5. ©2011 AACR.

Supplementary Figures S1-S13. Interactions of exogenously overexpressed proteins in vivo (S1); Purified recombinant proteins used in the experiments shown in Figure 2 (S2); SPR analyses of the HP1α, β, or HP1γ− chromoshadow domain binding to BARD1-BRCT (S3); SPR analyses of the BRCA1 fragments binding to HP1α, β, or γ (S4); Inhibition of HP1γ only mildly decreases the stable retention of BRCA1/BARD1 at sites of DNA damage (S5); Inhibition of HP1γ increases the interaction of BARD1 with HP1α (S6); BARD1 is dispensable for recruitment of HP1γ at DSB sites (S7); ATM-dependent interaction of BARD1 with HP1γ/H3K9me2 (S8); Ectopic accumulation of RIF1 and inhibition of RAD51 retention at DSB sites by depletion of all three isoforms of HP1 or BARD1 mutant that does not bind HP1 (S9); Dissolution kinetics of γH2AX after DSB damage in G2 cells (S10); Cell cycle progression of cells treated with UNC0638 (S11); An H3K9-specific HKMT inhibitor UNC0638 disrupts BRCA1/BARD1 retention at sites of DNA damage (S12); A model for the role of BRCA1/BARD1/HP1 complex in the DSB repair pathways (S13).

Supplementary Figure 6 from HERC2 Is an E3 Ligase That Targets BRCA1 for Degradation

Supplementary Figure 4 from HERC2 Is an E3 Ligase That Targets BRCA1 for Degradation

Supplementary Table 1 from Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

Supplementary Figure 3 from Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

Supplementary Figure 2 from BRCA1 Ubiquitinates RPB8 in Response to DNA Damage

Supplementary Figure 3 from BRCA1 Ubiquitinates RPB8 in Response to DNA Damage

Supplementary Figure 8 from Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

Stable retention of BRCA1/BARD1 complexes at sites of DNA damage is required for the proper response to DNA double-strand breaks (DSB). Here, we demonstrate that the BRCT domain of BARD1 is crucial for its retention through interaction with HP1. In response to DNA damage, BARD1 interacts with Lys9-dimethylated histone H3 (H3K9me2) in an ATM-dependent but RNF168-independent manner. This interaction is mediated primarily by HP1γ. A conserved HP1-binding motif in the BARD1 BRCT domain directly interacted with the chromoshadow domain of HP1 in vitro. Mutations in this motif (or simultaneous depletion of all three HP1 isoforms) disrupted retention of BARD1, BRCA1, and CtIP at DSB sites and allowed ectopic accumulation of RIF1, an effector of nonhomologous end-joining, at damaged loci in S-phase. UNC0638, a small-molecule inhibitor of histone lysine methyltransferase (HKMT), abolished retention and cooperated with the PARP inhibitor olaparib to block cancer cell growth. Taken together, our findings show how BARD1 promotes retention of the BRCA1/BARD1 complex at damaged DNA sites and suggest the use of HKMT inhibitors to leverage the application of PARP inhibitors to treat breast cancer. Cancer Res; 75(7); 1311–21. ©2015 AACR.

Supplementary Figure 10 from Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

Supplementary Materials and Methods, Figure Legends 1-8 from HERC2 Is an E3 Ligase That Targets BRCA1 for Degradation

Supplementary Figure 5 from HERC2 Is an E3 Ligase That Targets BRCA1 for Degradation

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Supplementary Figure 5 from Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

Supplementary Figure 2 from Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

Supplementary Methods and Figure Legends 1-4 from BRCA1 Ubiquitinates RPB8 in Response to DNA Damage

Supplementary Figure 1 from HERC2 Is an E3 Ligase That Targets BRCA1 for Degradation

Supplementary Figure 9 from Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

Supplementary Figure 4 from BRCA1 Ubiquitinates RPB8 in Response to DNA Damage

Supplementary Figure 8 from HERC2 Is an E3 Ligase That Targets BRCA1 for Degradation

Supplementary Figure 7 from Recruitment of Phosphorylated NPM1 to Sites of DNA Damage through RNF8-Dependent Ubiquitin Conjugates

Legends for Supplementary Figures S1-S13.

Supplementary Figure 7 from HERC2 Is an E3 Ligase That Targets BRCA1 for Degradation

Supplemenatary Methods and References. Description of additional methods and procedures used in the study. Also includes Supplementary References.

The aldehyde degrading function of the ALDH2 enzyme is impaired by Glu504Lys polymorphisms (rs671, termed A allele), which causes alcohol flushing in east Asians, and elevates the risk of esophageal cancer among habitual drinkers. Recent studies suggested that the ALDH2 variant may lead to higher levels of DNA damage caused by endogenously generated aldehydes. This can be a threat to genome stability and/or cell viability in a synthetic manner in DNA repair-defective settings such as Fanconi anemia (FA). FA is an inherited bone marrow failure syndrome caused by defects in any one of so far identified 22 FANC genes including hereditary breast and ovarian cancer (HBOC) genes BRCA1 and BRCA2. We have previously reported that the progression of FA phenotypes is accelerated with the ALDH2 rs671 genotype. Individuals with HBOC are heterozygously mutated in either BRCA1 or BRCA2, and the cancer-initiating cells in these patients usually undergo loss of the wild-type BRCA1/2 allele, leading to homologous recombination defects. Therefore, we hypothesized that the ALDH2 genotypes may impact breast cancer development in BRCA1/2 mutant carriers. We genotyped ALDH2 in 103 HBOC patients recruited from multiple cancer centers in Japan. However, we were not able to detect any significant differences in clinical stages, histopathological classification, or age at clinical diagnosis across the ALDH2 genotypes. Unlike the effects in hematopoietic cells of FA, our current data suggest that there is no impact of the loss of ALDH2 function in cancer initiation and development in breast epithelium of HBOC patients.

Background Sacituzumab govitecan is an antibody–drug conjugate that delivers SN-38, an active metabolite of irinotecan, to the target molecule, trophoblast cell-surface antigen 2 (Trop-2). It is a promising drug for triple-negative breast cancer and is anticipated to be effective for luminal breast cancer. The efficacy of the agent relies on the expression of Trop-2 rather than its intracellular function. However, conditions that alter the Trop-2 expression have not been well investigated. Methods We tested a range of clinically related treatments for their effect on Trop-2 expression in cultured breast cancer cell lines. Results The expression level of Trop-2 differed among cell lines, independent of their subtypes, and was highly variable on treatment with kinase inhibitors, tamoxifen, irradiation, and chemotherapeutic agents including irinotecan. While inhibitors of AKT, RSK, and p38 MAPK suppressed the Trop-2 expression, tamoxifen treatment significantly increased Trop-2 expression in luminal cancer cell lines. Notably, luminal cancer cells with acquired resistance to tamoxifen also exhibited higher levels of Trop-2. We identified transcription factor EB (TFEB) as a possible mechanism underlying tamoxifen-induced elevation of Trop-2 expression. Tamoxifen triggers dephosphorylation of TFEB, an active form of TFEB, and the effect of tamoxifen on Trop-2 was prevented by depletion of TFEB. A luciferase reporter assay showed that Trop-2 induction by TFEB was dependent on a tandem E-box motif within the Trop-2 promoter region. Conclusions Overall, these results suggest that the effectiveness of sacituzumab govitecan could be altered by concomitant treatment and that tamoxifen could be a favorable agent for combined therapy.

Ubiquitin‐dependent protein degradation has been implicated in the control of various cellular processes such as cell cycle control, transcriptional regulation, DNA damage repair, and apoptosis, many of which are involved in the initiation, progression, metastasis, and drug resistance of cancers. E3 ubiquitin ligases are known to be the second most prevalent cancer‐related functional gene family next to protein kinases. Of these, FBXO22, an F‐box receptor subunit of SCF E3 ligase, has recently been proposed to play a critical role in multiple aspects related to cancer development and therapy response. Firstly, FBXO22 is a key regulator of senescence induction through ubiquitylation of p53 for degradation. FBXO22 also acts as a molecular switch for the antagonistic and agonistic actions of selective estrogen receptor modulators (SERM) and determines the sensitivity of breast cancer to SERM by ubiquitylating KDM4B complexed with unliganded or SERMs‐bound estrogen receptor (ER). Furthermore, FBXO22 binds to Bach1, a pro‐metastatic transcription factor, suppressing Bach1‐driven metastasis of lung adenocarcinoma, and loss of FBXO22 facilitates metastasis. These findings, as well as other reports, unveiled strikingly important roles of FBXO22 in cancer development and therapeutic strategy. In this review, we summarize recent findings of how FBXO22 regulates major cancer suppression pathways., Fbxo22 plays strikingly important roles in cancer development and therapeutic strategy through the regulation of p53, KDM4B, and Bach1.