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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.

TIP60 can act as a transcriptional activator or a repressor depending on the cellular context. However, little is known about the role of the chromodomain in the functional regulation of TIP60. In this study, we found that TIP60 interacted with H3K4me3 in response to TNF-α signaling. TIP60 bound to H3K4me3 at the promoters of the NF-κB target genes IL6 and IL8. Unlike the wild-type protein, a TIP60 chromodomain mutant did not localize to chromatin regions. Because TIP60 binds to histones with specific modifications and transcriptional regulators, we used a histone peptide assay to identify histone codes recognized by TIP60. TIP60 preferentially interacted with methylated or acetylated histone H3 and H4 peptides. Phosphorylation near a lysine residue significantly reduced the affinity of TIP60 for the modified histone peptides. Our findings suggest that TIP60 acts as a functional link between the histone code and transcriptional regulators.

Rhythmic gene expression is central to the circadian control of physiology in mammals. Transcriptional activation of Per and Cry genes by heterodimeric bHLH-PAS proteins is a key event in the feedback loop that drives rhythmicity; however, the mechanism is not clearly understood. Here we show the transcriptional coactivators and histone acetyltransferases, p300/CBP, PCAF, and ACTR associate with the bHLH-PAS proteins, CLOCK and NPAS2, to regulate positively clock gene expression. Furthermore, Cry2 mediated repression of NPAS2:BMAL1 is overcome by overexpression of p300 in transactivation assays. Accordingly, p300 exhibits a circadian time-dependent association with NPAS2 in the vasculature, which precedes peak expression of target genes. In addition, a rhythm in core histone H3 acetylation on the mPer1 promoter in vivo correlates with the cyclical expression of their mRNAs. Temporal coactivator recruitment and HAT-dependent chromatin remodeling on the promoter of clock controlled genes in the vasculature permits the mammalian clock to orchestrate circadian gene expression.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The oncogene protein DEK is an abundant and ubiquitous nuclear protein with implications in acute myelogenous leukemia, as translocation which results in the formation of a DEK-CAN fusion protein. In a previous study, we have identified that DEK negatively regulated peroxiredoxin 6 (Prdx 6) transcription synergistically with the p65 subunit of NF-κB. In this study, we further investigated DEK-mediated transcriptional regulation of Prdx 6 during leukemia cell differentiation. Using Chromatin Immunoprecipitation analysis and Prdx 6 reporter assays, we found that DEK operated as a negative regulator of Prdx 6 transcription during leukemia cell differentiation. DEK was highly expressed and recruited to Prdx 6 promoter along with p65 and repressed transcription after leukemia cell differentiation. [ABSTRACT FROM PUBLISHER]

Abstract: The proto-oncogene protein DEK has been implicated in the t(6;9) chromosomal translocation associated with a subtype of acute myelogenous leukemia (AML), which results in the formation of a DEK-CAN fusion protein. Histone acetylation is an important post-translational modification which is involved in transcriptional regulation. In this study, we report that the acidic domain containing protein DEK interacts with histones and exerts a potent inhibitory effect on both p300 and PCAF-mediated histone acetyltransferase activity and transcription. Using chromatin immunoprecipitation assays, we have demonstrated that the recruitment of DEK to the appropriate promoter induces the histone H3 and H4 hypoacetylation of chromatin. Collectively, our data illustrate the important regulatory role played by protein DEK in transcriptional regulation, and suggest that transcription-regulating acidic domain regions may play a role in leukemogenesis. [Copyright &y& Elsevier]

Summary: Lysine methylation within histones is crucial for transcriptional regulation and thus links chromatin states to biological outcomes. Although recent studies have extended lysine methylation to nonhistone proteins, underlying molecular mechanisms such as the upstream signaling cascade that induces lysine methylation and downstream target genes modulated by this modification have not been elucidated. Here, we show that Reptin, a chromatin-remodeling factor, is methylated at lysine 67 in hypoxic conditions by the methyltransferase G9a. Methylated Reptin binds to the promoters of a subset of hypoxia-responsive genes and negatively regulates transcription of these genes to modulate cellular responses to hypoxia. [Copyright &y& Elsevier]

Abstract: A family of PRDM proteins are similar to histone methyltransferases (HMTases) with SET domain in that they modulate different cellular processes, including transcriptional regulation, through chromatin modifying activities. By applying a bioinformatic approach, we searched for proteins containing the SET domain and identified a double zinc-finger domain containing PRDM8 with HMTase activity. In vitro HMTase assay and immunoblot analysis revealed that PRDM8 specifically methylates H3K9 of histones which indicates transcriptional repression activity of PRDM8. Direct recruitment of PRDM8 to the promoter mediated transcriptional repression and indicated no involvement of HDAC. Tissue blot analyses identified PRDM8 transcripts from brain and testis in adult mouse. Consistent with these observations, we demonstrate that PRDM8 repressed the expression of steroidogenic markers, p450c17c and LHR, which indicates its regulatory role in mouse testis development. [Copyright &y& Elsevier]

Skeletal muscle differentiation is well regulated by a series of transcription factors. We reported previously that enhancer of polycombi (Epcl), a chromatin protein, can modulate skeletal muscle differentiation, although the mechanisms of this action have yet to be defined. Here we report that Epcl recruits both serum response factor (SRF) and p300 to induce skeletal muscle differentiation. Epcl interacted physically with SRF. Transfection of Epcl to myoblast cells potentiated the SRF-induced expression of skeletal muscle-specific genes as well as multinucleation. Proximal CArG box in the skeletal α-actin promoter was responsible for the synergistic activation of the promoter-luciferase. Epcl knockdown caused a decrease in the acetylation of histones associated with serum response element (SRE) of the skeletal a-actin promoter. The EpcFSRF complex bound to the SRE, and the knockdown of Epcl resulted in a decrease in SRF binding to the skeletal n-actin promoter. Epcl recruited histone acetyltransferase activity, which was potentiated by cotransfection with p300 but abolished by si-p300. Epcl directly bound to p300 in myoblast cells. Epc1[sup+/-]mice showed distortion of skeletal α-actin, and the isolated myoblasts from the mice had impaired muscle differentiation. These results suggest that Epcl is required for skeletal muscle differentiation by recruiting both SRF and p300 to the SRE of muscle-specific gene promoters. [ABSTRACT FROM AUTHOR]