PTEN decreases NR2F1 expression to inhibit ciliogenesis during EGFRL858R-induced lung cancer progression.

Lung cancer is the major cause of death worldwide. Activation of oncogenes or inhibition of tumor suppressors causes cancer formation. Previous studies have indicated that PTEN, as a tumor suppressor, inhibits cancer formation. In this study, we studied the role of PTEN in EGFRL858R-induced lung cancer in vivo. Interestingly, loss of PTEN increased bronchial cell hyperplasia but decreased alveolar cell hyperplasia in EGFRL858R*PTEN-/--induced lung cancer. Systematic analysis of gene expression by RNA-seq showed that several genes related to ciliogenesis were upregulated in EGFRL858R*PTEN-/--induced lung cancer and subsequently showed that bronchial ciliated cells were hyperplastic. Several critical ciliogenesis-related genes, such as Mucin5A, DNAI2, and DNAI3, were found to be regulated by NR2F1. Next, NR2F1 was found to be inhibited by overexpression of PTEN, indicating that PTEN negatively regulates NR2F1, thereby inhibiting the expression of ciliogenesis-related genes and leading to the inhibition of bronchial cell hyperplasia during EGFRL858R-induced lung cancer progression. In addition, we also found that PTEN decreased AKT phosphorylation in A549, KRAS mutant, and H1299 cells but increased AKT phosphorylation in PC9, EGFRL858R, and H1299L858R cells, suggesting that PTEN may function as a tumor suppressor and an oncogene in lung cancers with KRAS mutation and EGFR mutation, respectively. PTEN acts as a double-edged sword that differentially regulates EGFRL858R-induced lung cancer progression in different genomic backgrounds. Understanding the PTEN in lung cancer with different genetic backgrounds will be beneficial for therapy in the future.

USP24-i-101 targeting of USP24 activates autophagy to inhibit drug resistance acquired during cancer therapy.

Drug resistance in cancer therapy is the major reason for poor prognosis. Addressing this clinically unmet issue is important and urgent. In this study, we found that targeting USP24 by the specific USP24 inhibitors, USP24-i and its analogues, dramatically activated autophagy in the interphase and mitotic periods of lung cancer cells by inhibiting E2F4 and TRAF6, respectively. USP24 functional knockout, USP24C1695A, or targeting USP24 by USP24-i-101 inhibited drug resistance and activated autophagy in gefitinib-induced drug-resistant mice with doxycycline-induced EGFRL858R lung cancer, but this effect was abolished after inhibition of autophagy, indicating that targeting USP24-mediated induction of autophagy is required for inhibition of drug resistance. Genomic instability and PD-L1 levels were increased in drug resistant lung cancer cells and were inhibited by USP24-i-101 treatment or knockdown of USP24. In addition, inhibition of autophagy by bafilomycin-A1 significantly abolished the effect of USP24-i-101 on maintaining genomic integrity, decreasing PD-L1 and inhibiting drug resistance acquired in chemotherapy or targeted therapy. In summary, an increase in the expression of USP24 in cancer cells is beneficial for the induction of drug resistance and targeting USP24 by USP24-i-101 optimized from USP24-i inhibits drug resistance acquired during cancer therapy by increasing PD-L1 protein degradation and genomic stability in an autophagy induction-dependent manner.

NCI677397 targeting USP24-mediated induction of lipid peroxidation induces ferroptosis in drug-resistant cancer cells.

Cancer represents a profound challenge to healthcare systems and individuals worldwide. The development of multiple drug resistance is a major problem in cancer therapy and can result in progression of the disease. In our previous studies, we developed small-molecule inhibitors targeting ubiquitin-specific peptidase 24 (USP24) to combat drug-resistant lung cancer. Recently, we found that the USP24 inhibitor NCI677397 induced ferroptosis, a type of programmed cell death, in drug-resistant cancer cells by increasing lipid reactive oxygen species (ROS) levels. In the present study, we investigated the molecular mechanisms and found that the targeting of USP24 by NCI677397 increased gene expression of most lipogenesis-related genes, such as acyl-CoA synthetase long-chain family member 4 (ACSL4), and activated autophagy. In addition, the activity of several antioxidant enzymes, such as glutathione peroxidase 4 (GPX4) and dihydrofolate reductase (DHFR), was inhibited by NCI677397 treatment via an increase in protein degradation, thereby inducing lipid ROS production and lipid peroxidation. In summary, we demonstrated that NCI677397 induced a marked increase in lipid ROS levels, subsequently causing lipid peroxidation and leading to the ferroptotic death of drug-resistant cancer cells. Our study provides new insights into the clinical use of USP24 inhibitors as ferroptosis inducers (FINs) to block drug resistance during chemotherapy.

Tp53 haploinsufficiency is involved in hotspot mutations and cytoskeletal remodeling in gefitinib-induced drug-resistant EGFRL858R-lung cancer mice.

Tumor heterogeneity is the major factor for inducing drug resistance. p53 is the major defender to maintain genomic stability, which is a high proportion mutated in most of the cancer types. In this study, we established in vivo animal models of gefitinib-induced drug-resistant lung cancer containing EGFRL858R and EGFRL858R*Tp53+/- mice to explore the molecular mechanisms of drug resistance by studying the genomic integrity and global gene expression. The cellular morphology of the lung tumors between gefitinib-induced drug-resistant mice and drug-sensitive mice were very different. In addition, in drug-resistant mice, the expression of many cytoskeleton-related genes were changed, accompanied by decreased amounts of actin filaments and increased amounts of microtubule, indicating that significant cytoskeletal remodeling is induced in gefitinib-induced drug-resistant EGFRL858R and EGFRL858R*Tp53+/- lung cancer mice. The gene expression profiles and involved pathways were different in gefitinib-sensitive, gefitinib-resistant and Tp53+/--mice. Increases in drug resistance and nuclear size (N/C ratio) were found in EGFRL858R*Tp53+/- drug-resistant mice. Mutational hotspot regions for drug resistance via Tp53+/+- and Tp53+/--mediated pathways are located on chromosome 1 and chromosome 11, respectively, and are related to prognosis of lung cancer cohorts. This study not only builds up a gefitinib-induced drug-resistant EGFRL858R lung cancer animal model, but also provides a novel mutation profile in a Tp53+/+- or Tp53+/--mediated manner and induced cytoskeleton remodeling during drug resistance, which could contribute to the prevention of drug resistance during cancer therapy.

Estradiol-mediated inhibition of DNMT1 decreases p53 expression to induce M2-macrophage polarization in lung cancer progression.

Previous studies indicate that estrogen positively regulates lung cancer progression. Understanding the reasons will be beneficial for treating women with lung cancer in the future. In this study, we found that tumor formation was more significant in female EGFRL858R mice than in male mice. P53 expression levels were downregulated in the estradiol (E2)-treated lung cancer cells, female mice with EGFRL858R-induced lung cancer mice, and premenopausal women with lung cancer. E2 increased DNA methyltransferase 1 (DNMT1) expression to enhance methylation in the TP53 promoter, which led to the downregulation of p53. Overexpression of GFP-p53 decreased DNMT1 expression in lung cancer cells. TP53 knockout in mice with EGFRL858R-induced lung cancer not only changed gene expression in cancer cells but also increased the polarization of M2 macrophages by increasing C-C motif chemokine ligand 5 (CCL5) expression and decreasing growth differentiation factor 15 (GDF15) expression. The TP53 mutation rate was increased in females with late-stage but not early-stage lung cancer compared to males with lung cancer. In conclusion, E2-induced DNMT1 and p53 expression were negatively regulated each other in females with lung cancer, which not only affected cancer cells but also modulated the tumor-associated microenvironment, ultimately leading to a poor prognosis.

Estradiol-mediated inhibition of Sp1 decreases miR-3194-5p expression to enhance CD44 expression during lung cancer progression.

BACKGROUND: Sp1, an important transcription factor, is involved in the progression of various cancers. Our previous studies have indicated that Sp1 levels are increased in the early stage of lung cancer progression but decrease during the late stage, leading to poor prognosis. In addition, estrogen has been shown to be involved in lung cancer progression. According to previous studies, Sp1 can interact with the estrogen receptor (ER) to coregulate gene expression. The role of interaction between Sp1 and ER in lung cancer progression is still unknown and will be clarified in this study. METHODS: The clinical relevance between Sp1 levels and survival rates in young women with lung cancer was studied by immunohistochemistry. We validated the sex dependence of lung cancer progression in EGFRL858R-induced lung cancer mice. Wound healing assays, chamber assays and sphere formation assays in A549 cells, Taxol-induced drug-resistant A549 (A549-T24) and estradiol (E2)-treated A549 (E2-A549) cells were performed to investigate the roles of Taxol and E2 in lung cancer progression. Luciferase reporter assays, immunoblot and q-PCR were performed to evaluate the interaction between Sp1, microRNAs and CD44. Tail vein-injected xenograft experiments were performed to study lung metastasis. Samples obtained from lung cancer patients were used to study the mRNA level of CD44 by q-PCR and the protein levels of Sp1 and CD44 by immunoblot and immunohistochemistry. RESULTS: In this study, we found that Sp1 expression was decreased in premenopausal women with late-stage lung cancer, resulting in a poor prognosis. Tumor formation was more substantial in female EGFRL858R mice than in male mice and ovariectomized female mice, indicating that E2 might be involved in the poor prognosis of lung cancer. We herein report that Sp1 negatively regulates metastasis and cancer stemness in E2-A549 and A549-T24 cells. Furthermore, E2 increases the mRNA and protein levels of RING finger protein 4 (RNF4), which is the E3-ligase of Sp1, and thereby decreases Sp1 levels by promoting Sp1 degradation. Sp1 can be recruited to the promoter of miR-3194-5p, and positively regulate its expression. Furthermore, there was a strong inverse correlation between Sp1 and CD44 levels in clinical lung cancer specimens. Sp1 inhibited CD44 expression by increasing the expression of miR-3194-5p, miR-218-5p, miR-193-5p, miR-182-5p and miR-135-5p, ultimately resulting in lung cancer malignancy. CONCLUSION: Premenopausal women with lung cancer and decreased Sp1 levels have a poor prognosis. E2 increases RNF4 expression to repress Sp1 levels in premenopausal women with lung cancer, thus decreasing the expression of several miRNAs that can target CD44 and ultimately leading to cancer malignancy.

USP24 promotes drug resistance during cancer therapy.

Drug resistance has remained an important issue in the treatment and prevention of various diseases, including cancer. Herein, we found that USP24 not only repressed DNA-damage repair (DDR) activity by decreasing Rad51 expression to cause the tumor genomic instability and cancer stemness, but also increased the levels of the ATP-binding cassette (ABC) transporters P-gp, ABCG2, and ezrin to enhance the pumping out of Taxol from cancer cells, thus resulted in drug resistance during cancer therapy. A novel USP24 inhibitor, NCI677397, was screened for specific inhibiting the catalytic activity of USP24. This inhibitor was identified to suppress drug resistance via decreasing genomic instability, cancer stemness, and the pumping out of drugs from cancer cells. Understanding the role and molecular mechanisms of USP24 in drug resistance will be beneficial for the future development of a novel USP24 inhibitor. Our studies provide a new insight of USP24 inhibitor for clinically implication of blocking drug resistance during chemotherapy.

USP24 stabilizes bromodomain containing proteins to promote lung cancer malignancy.

Bromodomain (BRD)-containing proteins are important for chromatin remodeling to regulate gene expression. In this study, we found that the deubiquitinase USP24 interacted with BRD through its C-terminus increased the levels of most BRD-containing proteins through increasing their protein stability by the removal of ubiquitin from Lys391/Lys400 of the BRD. In addition, we found that USP24 and BRG1 could regulate each other through regulating the protein stability and the transcriptional activity, respectively, of the other, suggesting that the levels of USP24 and BRG1 are regulated to form a positive feedback loop in cancer progression. Loss of the interaction motif of USP24 eliminated the ability of USP24 to stabilize BRD-containing proteins and abolished the effect of USP24 on cancer progression, including its inhibition of cancer cell proliferation and promotion of cancer cell migration, suggesting that the interaction between USP24 and the BRD is important for USP24-mediated effects on cancer progression. The targeting of BRD-containing proteins has been developed as a strategy for cancer therapy. Based on our study, targeting USP24 to inhibit the levels of BRD-containing proteins may inhibit cancer progression.

Sp1 in Astrocyte Is Important for Neurite Outgrowth and Synaptogenesis.

In this study, we found that Sp1 was highly expressed in astrocytes, implying that Sp1 might be important for the function of astrocytes. Sp1/GFAP-Cre-ERT2 conditional knockout mice were constructed to study the role of Sp1 in astrocytes. Knockout of Sp1 in astrocytes altered astrocytic morphology and decreased GFAP expression in the cortex and hippocampus but did not affect cell viability. Loss of Sp1 in astrocytes decreased the number of neurons in the cortex and hippocampus. Conditioned medium from primary astrocytes with Sp1 knockout disrupted neuronal dendritic outgrowth and synapse formation, resulting in abnormal learning, memory, and motor behavior. Sp1 knockout in astrocytes altered gene expression, including decreasing the expression of Toll-like receptor 2 and Cfb and increasing the expression of C1q and C4Bp, thereby affecting neurite outgrowth and synapse formation, resulting in disordered neuron function. Studying these gene regulations might be beneficial to understanding neuronal development and brain injury prevention.

Correction: A novel derivative of betulinic acid, SYK023, suppresses lung cancer growth and malignancy.

[This corrects the article DOI: 10.18632/oncotarget.3701.].

The role of ubiquitin-specific peptidases in cancer progression.

Protein ubiquitination is an important mechanism for regulating the activity and levels of proteins under physiological conditions. Loss of regulation by protein ubiquitination leads to various diseases, such as cancer. Two types of enzymes, namely, E1/E2/E3 ligases and deubiquitinases, are responsible for controlling protein ubiquitination. The ubiquitin-specific peptidases (USPs) are the main members of the deubiquitinase family. Many studies have addressed the roles of USPs in various diseases. An increasing number of studies have indicated that USPs are critical for cancer progression, and some USPs have been used as targets to develop inhibitors for cancer prevention. Herein we collect and organize most of the recent studies on the roles of USPs in cancer progression and discuss the development of USP inhibitors for cancer therapy in the future.

USP24 induces IL-6 in tumor-associated microenvironment by stabilizing p300 and ß-TrCP and promotes cancer malignancy.

We have previously demonstrated that USP24 is involved in cancer progression. Here, we found that USP24 expression is upregulated in M2 macrophages and lung cancer cells. Conditioned medium from USP24-knockdown M2 macrophages decreases the migratory and chemotactic activity of lung cancer cells and the angiogenic properties of human microvascular endothelial cell 1 (HMEC-1). IL-6 expression is significantly decreased in USP24-knockdown M2 macrophages and lung cancer cells, and IL-6-replenished conditioned medium restores the migratory, chemotactic and angiogenetic properties of the cells. USP24 stabilizes p300 and ß-TrCP to increase the levels of histone-3 acetylation and NF-κB, and decreases the levels of DNMT1 and IκB, thereby increasing IL-6 transcription in M2 macrophages and lung cancer cells, results in cancer malignancy finally. IL-6 has previously been a target for cancer drug development. Here, we provide direct evidence to support that USP24 promotes IL-6 expression, which might be beneficial for cancer therapy.

Stress stimuli induce cancer-stemness gene expression via Sp1 activation leading to therapeutic resistance in glioblastoma.

It has been suggested that stress stimuli from the microenvironment maintain a subset of tumor cells with stem-like properties, including drug resistance. Here, we investigate whether Sp1, a stress-responsive factor, regulates stemness gene expression and if its inhibition sensitizes cancer cells to chemotherapy. Hydrogen peroxide- and serum deprivation-induced stresses were performed in glioblastoma (GBM) cells and patient-derived cells, and the effect of the Sp1 inhibitor mithramycin A (MA) on these stress-induced stem cells and temozolomide (TMZ)-resistant cells was evaluated. Sp1 and stemness genes were not commonly overexpressed in clinical GBM samples. However, their expression was highly induced by stress stimuli. Using MA, we demonstrated Sp1 as a critical stemness-related transcriptional factor protecting GBM cells against stress- and TMZ-induced death. Thus, Sp1 inhibition may prevent recurrence of malignant cells persisting after primary therapy.

Nm23-H1-stabilized hnRNPA2/B1 promotes internal ribosomal entry site (IRES)-mediated translation of Sp1 in the lung cancer progression.

Our recent studies have indicated that specificity protein-1 (Sp1) accumulates substantially in the early stage of lung cancer but is partially decreased in the late stages, which is an important factor in the progression of the cancer. In this study, we found that Nm23-H1 and hnRNPA2/B1 could be recruited to the 5'UTR of Sp1 mRNA. In investigating the clinical relevance of Nm23-H1/Sp1 levels, we found a positive correlation between lung cancer patients with poor prognosis and low levels of Sp1 and Nm23-H1, suggesting an association between Nm23-H1/Sp1 levels and survival rate. Knockdown of Nm23-H1 inhibits lung cancer growth but increases lung cancer cell malignancy, which could be rescued by overexpression of Sp1, indicating that Nm23-H1-induced Sp1 expression is critical for lung cancer progression. We also found that Nm23-H1 increases the protein stability of hnRNPA2/B1and is thereby co-recruited to the 5'UTR of Sp1 mRNA to regulate cap-independent translational activity. Since the Sp1 level is tightly regulated during lung cancer progression, understanding the molecular mechanisms underlying the regulation by Nm23-H1/hnRNPA2B1 of Sp1 expression in the various stages of lung cancer will be beneficial for lung cancer therapy in the future.

Specificity protein 1-modulated superoxide dismutase 2 enhances temozolomide resistance in glioblastoma, which is independent of O6-methylguanine-DNA methyltransferase.

Acquisition of temozolomide (TMZ) resistance is a major factor leading to the failure of glioblastoma (GBM) treatment. The exact mechanism by which GBM evades TMZ toxicity is not always related to the expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), and so remains unclear. In this study, TMZ-resistant variants derived from MGMT-negative GBM clinical samples and cell lines were studied, revealing there to be increased specificity protein 1 (Sp1) expression associated with reduced reactive oxygen species (ROS) accumulation following TMZ treatment. Analysis of gene expression databases along with cell studies identified the ROS scavenger superoxide dismutase 2 (SOD2) as being disease-related. SOD2 expression was also increased, and it was found to be co-expressed with Sp1 in TMZ-resistant cells. Investigation of the SOD2 promoter revealed Sp1 as a critical transcriptional activator that enhances SOD2 gene expression. Co-treatment with an Sp1 inhibitor restored the inhibitory effects of TMZ, and decreased SOD2 levels in TMZ-resistant cells. This treatment strategy restored susceptibility to TMZ in xenograft animals, leading to prolonged survival in an orthotopic model. Thus, our results suggest that Sp1 modulates ROS scavengers as a novel mechanism to increase cancer malignancy and resistance to chemotherapy. Inhibition of this pathway may represent a potential therapeutic target for restoring treatment susceptibility in GBM.

Chronic treatment with cisplatin induces chemoresistance through the TIP60-mediated Fanconi anemia and homologous recombination repair pathways.

The Fanconi anemia pathway in coordination with homologous recombination is essential to repair interstrand crosslinks (ICLs) caused by cisplatin. TIP60 belongs to the MYST family of acetyltransferases and is involved in DNA repair and regulation of gene transcription. Although the physical interaction between the TIP60 and FANCD2 proteins has been identified that is critical for ICL repair, it is still elusive whether TIP60 regulates the expression of FA and HR genes. In this study, we found that the chemoresistant nasopharyngeal carcinoma cells, derived from chronic treatment of cisplatin, show elevated expression of TIP60. Furthermore, TIP60 binds to the promoters of FANCD2 and BRCA1 by using the chromatin immunoprecipitation experiments and promote the expression of FANCD2 and BRCA1. Importantly, the depletion of TIP60 significantly reduces sister chromatid exchange, a measurement of HR efficiency. The similar results were also shown in the FNACD2-, and BRCA1-deficient cells. Additionally, these TIP60-deficient cells encounter more frequent stalled forks, as well as more DNA double-strand breaks resulting from the collapse of stalled forks. Taken together, our results suggest that TIP60 promotes the expression of FA and HR genes that are important for ICL repair and the chemoresistant phenotype under chronic treatment with cisplatin.

Positive feedback regulation between IL10 and EGFR promotes lung cancer formation.

The role of IL10 in the tumorigenesis of various cancer types is still controversial. Here, we found that increased IL10 levels are correlated with a poor prognosis in lung cancer patients. Moreover, IL10 levels were significantly increased in the lungs and serum of EGFRL858R- and Kras4bG12D-induced lung cancer mice, indicating that IL10 might facilitate lung cancer tumorigenesis. IL10 knockout in EGFRL858R and Kras4bG12D mice inhibited the development of lung tumors and decreased the levels of infiltrating M2 macrophages and tumor-promoting Treg lymphocytes. We also showed that EGF increases IL10 expression by enhancing IL10 mRNA stability, and IL10 subsequently activates JAK1/STAT3, Src, PI3K/Akt, and Erk signaling pathways. Interestingly, the IL10-induced recruitment of phosphorylated Src was critical for inducing EGFR through the activation of the JAK1/STAT3 pathway, suggesting that Src and JAK1 positively regulate each other to enhance STAT3 activity. Doxycycline-induced EGFRL858R mice treated with gefitinib and anti-IL10 antibodies exhibited poor tumor formation. In conclusion, IL10 and EGFR regulate each other through positive feedback, which leads to lung cancer formation.

The sigma-1 receptor-zinc finger protein 179 pathway protects against hydrogen peroxide-induced cell injury.

The accumulation of reactive oxygen species (ROS) have implicated the pathogenesis of several human diseases including neurodegenerative disorders, stroke, and traumatic brain injury, hence protecting neurons against ROS is very important. In this study, we focused on sigma-1 receptor (Sig-1R), a chaperone at endoplasmic reticulum, and investigated its protective functions. Using hydrogen peroxide (H2O2)-induced ROS accumulation model, we verified that apoptosis-signaling pathways were elicited by H2O2 treatment. However, the Sig-1R agonists, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), reduced the activation of apoptotic pathways significantly. By performing protein-protein interaction assays and shRNA knockdown of Sig-1R, we identified the brain Zinc finger protein 179 (Znf179) as a downstream target of Sig-1R regulation. The neuroprotective effect of Znf179 overexpression was similar to that of DHEAS treatment, and likely mediated by affecting the levels of antioxidant enzymes. We also quantified the levels of peroxiredoxin 3 (Prx3) and superoxide dismutase 2 (SOD2) in the hippocampi of wild-type and Znf179 knockout mice, and found both enzymes to be reduced in the knockout versus the wild-type mice. In summary, these results reveal that Znf179 plays a novel role in neuroprotection, and Sig-1R agonists may be therapeutic candidates to prevent ROS-induced damage in neurodegenerative and neurotraumatic diseases.

Sp1-mediated ectopic expression of T-cell lymphoma invasion and metastasis 2 in hepatocellular carcinoma.

T-cell lymphoma invasion and metastasis 2 (TIAM2) is a neuron-specific protein that has been found ectopically expressed in hepatocellular carcinoma (HCC). Results from clinical specimens and cellular and animal models have shown that the short form of TIAM2 (TIAM2S) functions as an oncogene in the tumorigenesis of liver cancer. However, the regulation of TIAM2S ectopic expression in HCC cells remains largely unknown. This study aimed to identify the mechanism underlying the ectopic expression of TIAM2S in liver cancer cells. In this report, we provide evidence illustrating that Sp1 binds directly to the GC box located in the TIAM2S core promoter. We further demonstrated that overexpression of Sp1 in HepaRG cells promotes endogenous TIAM2S mRNA and protein expressions, and knockdown of Sp1 in 2 HCC cell lines, HepG2 and PLC/PRF/5, led to a substantial reduction in TIAM2S mRNA and protein in these cells. Of 60 paired HCC samples, 70% showed a significant increase (from 1.1- to 3.6-fold) in Sp1 protein expression in the tumor cells. The elevated Sp1 expression was highly correlated with both TIAM2S mRNA and protein expressions in these samples. Together, these results illustrate that Sp1 positively controls TIAM2S transcription and that Sp1-mediated transcriptional activation is essential for TIAM2S ectopic expression in liver cancer cells.

Combined Interactions of Plant Homeodomain and Chromodomain Regulate NuA4 Activity at DNA Double-Strand Breaks.

DNA double-strand breaks (DSBs) represent one of the most threatening lesions to the integrity of genomes. In yeast Saccharomyces cerevisiae, NuA4, a histone acetylation complex, is recruited to DSBs, wherein it acetylates histones H2A and H4, presumably relaxing the chromatin and allowing access to repair proteins. Two subunits of NuA4, Yng2 and Eaf3, can interact in vitro with methylated H3K4 and H3K36 via their plant homeodomain (PHD) and chromodomain. However, the roles of the two domains and how they interact in a combinatorial fashion are still poorly characterized. In this study, we generated mutations in the PHD and chromodomain that disrupt their interaction with methylated H3K4 and H3K36. We demonstrate that the combined mutations in both the PHD and chromodomain impair the NuA4 recruitment, reduce H4K12 acetylation at the DSB site, and confer sensitivity to bleomycin that induces DSBs. In addition, the double mutant cells are defective in DSB repair as judged by Southern blot and exhibit prolonged activation of phospho-S129 of H2A. Cells harboring the H3K4R, H3K4R, K36R, or set1Δ set2Δ mutant that disrupts H3K4 and H3K36 methylation also show very similar phenotypes to the PHD and chromodomain double mutant. Our results suggest that multivalent interactions between the PHD, chromodomain, and methylated H3K4 and H3K36 act in a combinatorial manner to recruit NuA4 and regulate the NuA4 activity at the DSB site.