The H3K4 methyltransferase SETD1A is required for proliferation of non-small cell lung cancer cells by promoting S-phase progression.

Epigenetic dysregulation has been strongly implicated in carcinogenesis and is one of the mechanisms that contribute to the development of lung cancer. Using genome-wide CRISPR/Cas9 library screening, we showed SET domain-containing protein 1A (SETD1A) is an essential epigenetic modifier of the proliferation of NSCLC H1299 cells. Depletion of SETD1A strikingly inhibited the proliferation of NSCLC cells. IHC staining and bioinformatics showed that SETD1A is upregulated in lung cancer. Kaplan-Meier survival analysis indicated that high expression of SETD1A is associated with poor prognosis of patients with NSCLC. We revealed that loss of SETD1A inhibits DNA replication and induces replication stress accompanied by impaired fork progression. In addition, transcription of CDC7 and TOP1, which are involved in replication origin activation and fork progression, respectively, was significantly reduced by knockdown of SETD1A. Taken together, these findings demonstrated SETD1A is a critical epigenetic modifier of NSCLC cell proliferation by promoting the transcription of a subset of DNA replication-associated genes.

Methylation of UHRF1 by SET7 is essential for DNA double-strand break repair.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is a key epigenetic regulator of DNA methylation maintenance and heterochromatin formation. The roles of UHRF1 in DNA damage repair also have been emphasized in recent years. However, the regulatory mechanism of UHRF1 remains elusive. In this study, we showed that UHRF1 is methylated by SET7 and demethylation is catalyzed by LSD1. In addition, methylation of UHRF1 is induced in response to DNA damage and its phosphorylation in S phase is a prerequisite for interaction with SET7. Furthermore, UHRF1 methylation catalyzes the conjugation of polyubiquitin chains to PCNA and promotes homologous recombination for DNA repair. SET7-mediated UHRF1 methylation is also shown to be essential for cell viability against DNA damage. Our data revealed the regulatory mechanism underlying the UHRF1 methylation status by SET7 and LSD1 in double-strand break repair pathway.

KDM2B is a histone H3K79 demethylase and induces transcriptional repression via sirtuin-1-mediated chromatin silencing.

The methylation of histone H3 lysine 79 (H3K79) is an active chromatin marker and is prominent in actively transcribed regions of the genome; however, demethylase of H3K79 remains unknown despite intensive research. Here, we show that KDM2B, also known as FBXL10 and a member of the Jumonji C family of proteins known for its histone H3K36 demethylase activity, is a di- and trimethyl H3K79 demethylase. We demonstrate that KDM2B induces transcriptional repression of HOXA7 and MEIS1 via occupancy of promoters and demethylation of H3K79. Furthermore, genome-wide analysis suggests that H3K79 methylation levels increase when KDM2B is depleted, which indicates that KDM2B functions as an H3K79 demethylase in vivo. Finally, stable KDM2B-knockdown cell lines exhibit displacement of NAD+-dependent deacetylase sirtuin-1 (SIRT1) from chromatin, with concomitant increases in H3K79 methylation and H4K16 acetylation. Our findings identify KDM2B as an H3K79 demethylase and link its function to transcriptional repression via SIRT1-mediated chromatin silencing.-Kang, J.-Y., Kim, J.-Y., Kim, K.-B., Park, J. W., Cho, H., Hahm, J. Y., Chae, Y.-C., Kim, D., Kook, H., Rhee, S., Ha, N.-C., Seo, S.-B. KDM2B is a histone H3K79 demethylase and induces transcriptional repression via sirtuin-1-mediated chromatin silencing.

INHAT subunit SET/TAF-Iß regulates PRC1-independent H2AK119 mono-ubiquitination via E3 ligase MIB1 in colon cancer.

SET/TAF-Iß, a subunit of the inhibitor of acetyltransferases (INHAT) complex, exhibits transcriptional repression activity by inhibiting histone acetylation. We find that SET/TAF-Iß regulates mono-ubiquitination of histone H2A at lysine 119 (H2AK119ub), which is involved in polycomb-mediated transcriptional repression, in HCT116 cells. In this report, we demonstrate that SET/TAF-Iß acts as an E2 ubiquitin-conjugating enzyme for PRC1-independent H2AK119ub. Furthermore, we identify that MIB1 is the E3 ligase partner for SET/TAF-Iß using LC-MS/MS and in vitro ubiquitination assays. Transcriptome analysis reveals that SET/TAF-Iß and MIB1 regulate the expression of genes related to DNA replication and cell cycle progression in HCT116 cells, and knockdown of either protein reduces proliferation of HCT116 cells by impeding cell cycle progression. Together, our study reveals a novel PRC1-independent epigenetic regulatory mechanism for H2AK119ub by SET/TAF-Iß and MIB1 in colon cancer.

Negative Regulation of Erythroid Differentiation via the CBX8-TRIM28 Axis.

Although the mechanism of chronic myeloid leukemia (CML) initiation through BCR/ABL oncogene has been well characterized, CML cell differentiation into erythroid lineage cells remains poorly understood. Using CRISPR-Cas9 screening, we identify Chromobox 8 (CBX8) as a negative regulator of K562 cell differentiation into erythrocytes. CBX8 is degraded via proteasomal pathway during K562 cell differentiation, which activates the expression of erythroid differentiation-related genes that are repressed by CBX8 in the complex of PRC1. During the differentiation process, the serine/threonine-protein kinase PIM1 phosphorylates serine 196 on CBX8, which contributes to CBX8 reduction. When CD235A expression levels are analyzed, the result reveals that the knockdown of PIM1 inhibits K562 cell differentiation. We also identify TRIM28 as another interaction partner of CBX8 by proteomic analysis. Intriguingly, TRIM28 maintains protein stability of CBX8 and TRIM28 loss significantly induces proteasomal degradation of CBX8, resulting in an acceleration of erythroid differentiation. Here, we demonstrate the involvement of the CBX8-TRIM28 axis during CML cell differentiation, suggesting that CBX8 and TRIM28 are promising novel targets for CML research.

SRF is a nonhistone methylation target of KDM2B and SET7 in the regulation of skeletal muscle differentiation.

The demethylation of histone lysine residues, one of the most important modifications in transcriptional regulation, is associated with various physiological states. KDM2B is a demethylase of histones H3K4, H3K36, and H3K79 and is associated with the repression of transcription. Here, we present a novel mechanism by which KDM2B demethylates serum response factor (SRF) K165 to negatively regulate muscle differentiation, which is counteracted by the histone methyltransferase SET7. We show that KDM2B inhibited skeletal muscle differentiation by inhibiting the transcription of SRF-dependent genes. Both KDM2B and SET7 regulated the balance of SRF K165 methylation. SRF K165 methylation was required for the transcriptional activation of SRF and for the promoter occupancy of SRF-dependent genes. SET7 inhibitors blocked muscle cell differentiation. Taken together, these data indicate that SRF is a nonhistone target of KDM2B and that the methylation balance of SRF as maintained by KDM2B and SET7 plays an important role in muscle cell differentiation.

Histone H3K79 demethylation by KDM2B facilitates proper DNA replication through PCNA dissociation from chromatin.

OBJECTIVES: The level of histone H3 lysine 79 methylation is regulated by the cell cycle and involved in cell proliferation. KDM2B is an H3K79 demethylase. Proliferating cell nuclear antigen (PCNA) is a component of the DNA replication machinery. This study aimed at elucidating a molecular link between H3K79me recognition of PCNA and cell cycle control. MATERIALS AND METHODS: We generated KDM2B-depleted 293T cells and histone H3-K79R mutant-expressing 293T cells. Western blots were primarily utilized to examine the H3K79me level and its effect on subsequent PCNA dissociation from chromatin. We applied IP, peptide pull-down, isothermal titration calorimetry (ITC) and ChIP experiments to show the PCNA binding towards methylated H3K79 and DNA replication origins. Flow cytometry, MTT, iPOND and DNA fibre assays were used to assess the necessity of KDM2B for DNA replication and cell proliferation. RESULTS: We revealed that KDM2B-mediated H3K79 demethylation regulated cell cycle progression. We found that PCNA bound chromatin in an H3K79me-dependent manner during S phase. KDM2B was responsible for the timely dissociation of PCNA from chromatin, allowing to efficient DNA replication. Depletion of KDM2B aberrantly enriched chromatin with PCNA and caused slow dissociation of residual PCNA, leading to a negative effect on cell proliferation. CONCLUSIONS: We suggested a novel interaction between PCNA and H3K79me. Thus, our findings provide a new mechanism of KDM2B in regulation of DNA replication and cell proliferation.

Methylated-UHRF1 and PARP1 interaction is critical for homologous recombination.

A recent study suggested that methylation of ubiquitin-like with PHD and RING finger domain 1 (UHRF1) is regulated by SET7 and lysine-specific histone demethylase 1A (LSD1) and is essential for homologous recombination (HR). The study demonstrated that SET7-mediated methylation of UHRF1 promotes polyubiquitination of proliferating cell nuclear antigen (PCNA), inducing HR. However, studies on mediators that interact with and recruit UHRF1 to damaged lesions are needed to elucidate the mechanism of UHRF1 methylationinduced HR. Here, we identified that poly [ADP-ribose] polymerase 1 (PARP1) interacts with damage-induced methylated UHRF1 specifically and mediates UHRF1 to induce HR progression. Furthermore, cooperation of UHRF1-PARP1 is essential for cell viability, suggesting the importance of the interaction of UHRF1-PARP1 for damage tolerance in response to damage. Our data revealed that PARP1 mediates the HR mechanism, which is regulated by UHRF1 methylation. The data also indicated the significant role of PARP1 as a mediator of UHRF1 methylation-correlated HR pathway. [BMB Reports 2020; 53(2): 112-117].

Proteosomal degradation of NSD2 by BRCA1 promotes leukemia cell differentiation.

The human myelogenous leukemic cell line, K562 undergoes erythroid differentiation by exposure to hemin. Here, we uncovered NSD2 as an innate erythroid differentiation-related factor through a genome-wide CRISPR library screen and explored the regulatory role of NSD2 during myeloid leukemia cell differentiation. We found that NSD2 stability was disrupted by poly-ubiquitination in differentiated K562 cells. Proteomic analysis revealed an interaction between NSD2 and an E3 ubiquitin ligase, BRCA1, which ubiquitylates NSD on K292. Depletion of BRCA1 stabilized NSD2 protein and suppressed K562 cell differentiation. Furthermore, BRCA1 protein level was decreased in bone marrow tumor, while NSD2 level was elevated. Surprisingly, among BRCA1 mutation(s) discovered in lymphoma patients, BRCA1 K1183R prevented its translocation into the nucleus, failed to reduce NSD2 protein levels in hemin-treated K562 cells and eventually disrupted cell differentiation. Our results indicate the regulation of NSD2 stability by BRCA1-mediated ubiquitination as a potential therapeutic target process in multiple myeloma.

Acetylation of UHRF1 Regulates Hemi-methylated DNA Binding and Maintenance of Genome-wide DNA Methylation.

UHRF1 is a key regulator in DNA methylation maintenance. It binds histone H3K9me2/3 and hemi-methylated DNA and recruits DNMT1 to DNA replication forks during S phase. However, the regulatory mechanism of hemi-methylated DNA binding activity of UHRF1 remains unknown. In this study, we reveal that acetylation of UHRF1 is regulated by PCAF and HDAC1. We show that UHRF1 acetylation at K490 attenuates its binding affinity to hemi-methylated DNA. We analyze genome-wide DNA methylation and gene-expression patterns using stable cell lines and discover that cells where the endogenous UHRF1 is replaced with an acetyl-mimetic (UHRF1 K490Q) mutant show deficiencies in inherited DNA methylation and show different gene-expression patterns in genes related to cell survival. These results reveal that precise regulation of UHRF1 acetylation is required to maintain DNA methylation during cell division and control cell survival.

Inhibition of FoxO1 acetylation by INHAT subunit SET/TAF-Iß induces p21 transcription.

Post-translational modification of forkhead family transcription factor, FoxO1, is an important regulatory mode for its diverse activities. FoxO1 is acetylated by HAT coactivators and its transcriptional activity is decreased via reduced DNA binding affinity. Here, we report that SET/TAF-Iß inhibited p300-mediated FoxO1 acetylation in an INHAT domain-dependent manner. SET/TAF-Iß interacted with FoxO1 and activated transcription of FoxO1 target gene, p21. Moreover, SET/TAF-Iß inhibited acetylation of FoxO1 and increased p21 transcription induced by oxidative stress. Our results suggest that SET/TAF-Iß inhibits FoxO1 acetylation and activates its transcriptional activity toward p21.

H3K9 histone methyltransferase G9a-mediated transcriptional activation of p21.

We report that H3K9 HMTase G9a activates transcription of the cell cycle regulatory gene, p21, in p53-null H1299 cells. Positive regulation of p21 by G9a is independent of its HMTase activity. We demonstrate that G9a upregulates p21 via interaction with PCAF, and provide evidence that the activating complex is recruited to the p21 promoter upon DNA damage-inducing agent etoposide treatment. Our study suggests that G9a decreases proliferation and cell viability by increasing the level of p21-mediated apoptosis. Our results suggest that G9a functions as a coactivator for p21 transcription, and directs cells to undergo apoptosis.