ABSTRACT
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.
Subject(s)
DNA Replication , F-Box Proteins/metabolism , Histones/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Cell Cycle , Cell Proliferation , Chromatin , Demethylation , HEK293 Cells , Humans , S PhaseABSTRACT
Autophagy, a self-degradative physiological process, is critical for homeostasis maintenance and energy source balancing in response to various stresses, including nutrient deprivation. It is a highly conserved catabolic process in eukaryotes and is indispensable for cell survival as it involves degradation of unessential or excessive components and their subsequent recycling as building blocks for the synthesis of necessary molecules. Although the dysregulation of autophagy has been reported to broadly contribute to various diseases, including cancers and neurodegenerative diseases, the molecular mechanisms underlying the epigenetic regulation of autophagy are poorly elucidated. Here, we report that the level of lysine demethylase 3B (KDM3B) increases in nutrient-deprived HCT116 cells, a colorectal carcinoma cell line, resulting in transcriptional activation of the autophagy-inducing genes. KDM3B was found to enhance the transcription by demethylating H3K9me2 on the promoter of these genes. Furthermore, we observed that the depletion of KDM3B inhibited the autophagic flux in HCT116 cells. Collectively, these data suggested the critical role of KDM3B in the regulation of autophagy-related genes via H3K9me2 demethylation and induction of autophagy in nutrient-starved HCT116 cells.
Subject(s)
Autophagy/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Transcriptional Activation/genetics , Amino Acids/deficiency , Autophagy/drug effects , Epigenesis, Genetic/drug effects , HCT116 Cells , HEK293 Cells , Histones/metabolism , Humans , Promoter Regions, Genetic , Proteasome Endopeptidase Complex/metabolism , Protein Processing, Post-Translational/drug effects , Proteolysis/drug effects , Sirolimus/pharmacology , Transcriptional Activation/drug effects , Valosin Containing Protein/metabolismABSTRACT
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].
Subject(s)
CCAAT-Enhancer-Binding Proteins/metabolism , DNA Damage/genetics , Homologous Recombination/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , Ubiquitin-Protein Ligases/metabolism , CCAAT-Enhancer-Binding Proteins/chemistry , CCAAT-Enhancer-Binding Proteins/genetics , Cell Survival/genetics , DNA Damage/drug effects , DNA Methylation/drug effects , G2 Phase Cell Cycle Checkpoints/drug effects , G2 Phase Cell Cycle Checkpoints/genetics , HCT116 Cells , HEK293 Cells , Humans , Hydrogen Peroxide/pharmacology , Poly (ADP-Ribose) Polymerase-1/genetics , Protein Binding , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/geneticsABSTRACT
Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Owing to the chemotherapy associated side effects and toxicity, it is necessary to find a new mechanism, which can identify new potential therapeutic targets at the molecular level. Here, we identified new target genes that are induced during the TPA-induced HL-60 cell differentiation by ChIP-seq and microarray data analysis. Using q-PCR and ChIP assay, we confirmed that the target genes including USP3, USP35, TCF4, and SGK1 are upregulated during TPA-mediated HL-60 cell differentiation. Levels of USP3, one of the deubiquitinating enzymes (DUBs), increased by TPA treatment, resulting in the reduction of H2AK119ub levels. In addition, we revealed that depletion of USP3 inhibits TPA-mediated leukemia cell differentiation q-PCR and FACS analysis. Taken together, our data indicate that USP3 promotes TPA-mediated leukemia cell differentiation via regulating H2AK119ub levels.
ABSTRACT
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ABSTRACT
The human myeloid leukemia cell line HL-60 differentiate into monocytes following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the mechanism underlying the differentiation of these cells in response to TPA has not been fully elucidated. In this study, we performed ChIP-seq profiling of RNA Pol II, HDAC2, Acetyl H3 (AcH3), and H3K27me3 and analyzed differential chromatin state changes during TPA-induced differentiation of HL-60 cells. We focused on atypically active genes, which showed enhanced H3 acetylation despite increased HDAC2 recruitment. We found that HDAC2 positively regulates the expression of these genes in a histone deacetylase activity-independent manner. HDAC2 interacted with and recruited paired box 5 (PAX5) to the promoters of the target genes and regulated HL-60 cell differentiation by PAX5-mediated gene activation. Taken together, these data elucidated the specific-chromatin status during HL-60 cell differentiation following TPA exposure and suggested that HDAC2 can activate transcription of certain genes through interactions with PAX5 in a deacetylase activity-independent pathway.
Subject(s)
Cell Differentiation/drug effects , Cell Differentiation/genetics , Histone Deacetylase 2/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Transcriptional Activation/drug effects , Chromatin/drug effects , Chromatin/metabolism , HL-60 Cells , Humans , PAX5 Transcription Factor/metabolismABSTRACT
Recent studies have reported the ectopic expression of olfactory receptors (ORs) in non-olfactory tissues, however, their physiological roles were not well elucidated. ORs are expressed in and function in different types of cancers. Here, we identified that the H3K9me2 levels of several OR promoters decreased during differentiation in the HL-60, human myeloid leukaemia cell line, by all-trans-retinoic acid (ATRA). We found that the differential OR promoters H3K9me2 levels were regulated by G9a and LSD1, resulting in the decrease of ORs transcription during HL-60 differentiation. G9a and LSD1 could regulate the expression of ORs in several non-olfactory cells via the methylation and demethylation of H3K9me2. In addition, we demonstrated that knockdown of OR significantly reduced cell proliferation. Therefore, the epigenetic regulation of ORs transcription is critical for carcinogenesis.
Subject(s)
Gene Expression Regulation, Leukemic , Histocompatibility Antigens/metabolism , Histone Demethylases/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Leukemia/genetics , Leukemia/metabolism , Receptors, Odorant/genetics , Transcription, Genetic , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line, Tumor , Cell Proliferation , DNA Methylation , Gene Knockdown Techniques , HL-60 Cells , Histones/metabolism , Humans , Leukemia/pathology , Tretinoin/pharmacologyABSTRACT
Data derived from genomic and transcriptomic analyses have revealed that long noncoding RNAs (lncRNAs) have important roles in the transcriptional regulation of various genes. Recent studies have identified the mechanism underlying this function. To date, a variety of noncoding transcripts have been reported to function in conjunction with epigenetic regulator proteins. In this study, we investigated the function of linc00598, which is transcribed by a genomic sequence on chromosome 13, downstream of FoxO1 and upstream of COG6. Microarray analysis showed that linc00598 regulates the transcription of specific target genes, including those for cell cycle regulators. We discovered that linc00598 regulates CCND2 transcription through modulation of the transcriptional regulatory effect of FoxO1 on the CCND2 promoter. Moreover, we observed that knockdown of linc00598 induced G0/G1 cell cycle arrest and inhibited proliferation. These data indicate that linc00598 plays an important role in cell cycle regulation and proliferation through its ability to regulate the transcription of CCND2.
Subject(s)
Cyclin D2/biosynthesis , G1 Phase Cell Cycle Checkpoints/physiology , RNA, Long Noncoding/metabolism , Response Elements/physiology , Transcription, Genetic/physiology , Cyclin D2/genetics , Forkhead Box Protein O1/genetics , Forkhead Box Protein O1/metabolism , HEK293 Cells , HL-60 Cells , HeLa Cells , Hep G2 Cells , Humans , K562 Cells , MCF-7 Cells , RNA, Long Noncoding/genetics , Resting Phase, Cell Cycle/physiology , THP-1 CellsABSTRACT
Histone H3K9 methyltransferase (HMTase) G9a-mediated transcriptional repression is a major epigenetic silencing mechanism. UHRF1 (ubiquitin-like with PHD and ring finger domains 1) binds to hemimethylated DNA and plays an essential role in the maintenance of DNA methylation. Here, we provide evidence that UHRF1 is transcriptionally downregulated by H3K9 HMTase G9a. We found that increased expression of G9a along with transcription factor YY1 specifically represses UHRF1 transcription during TPA-mediated leukemia cell differentiation. Using ChIP analysis, we found that UHRF1 was among the transcriptionally silenced genes during leukemia cell differentiation. Using a DNA methylation profiling array, we discovered that the UHRF1 promoter was hypomethylated in samples from leukemia patients, further supporting its overexpression and oncogenic activity. Finally, we showed that G9a regulates UHRF1-mediated H3K23 ubiquitination and proper DNA replication maintenance. Therefore, we propose that H3K9 HMTase G9a is a specific epigenetic regulator of UHRF1.
Subject(s)
CCAAT-Enhancer-Binding Proteins/genetics , Cell Differentiation , DNA Modification Methylases/metabolism , Gene Expression Regulation , Leukemia/pathology , Transcription, Genetic , Cell Line , Chromatin Immunoprecipitation , Flow Cytometry , Humans , Leukemia/genetics , Ubiquitin-Protein LigasesABSTRACT
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.
