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Jianbo Diao - One of the best experts on this subject based on the ideXlab platform.

  • dna damage regulates uhrf1 Stability via the scfβ trcp e3 ligase
    Molecular and Cellular Biology, 2013
    Co-Authors: Hao Chen, Hiroyuki Inuzuka, Jianbo Diao
    Abstract:

    UHRF1 (ubiquitin-like, with PHD and RING finger domains 1) is a critical epigenetic player involved in the maintenance of DNA methylation patterns during DNA replication. DysRegulation of the UHRF1 level is implicated in cancer onset, metastasis, and tumor recurrence. Previous studies demonstrated that UHRF1 can be stabilized through USP7-mediated deubiquitylation, but the mechanism through which UHRF1 is ubiquitylated is still unknown. Here we show that proteasomal degradation of UHRF1 is mediated by the SCFβ-TrCP E3 ligase. Through bioinformatic and mutagenesis studies, we identified a functional DSG degron in the UHRF1 N terminus that is necessary for UHRF1 Stability Regulation. We further show that UHRF1 physically interacts with β-TrCP1 in a manner dependent on phosphorylation of serine 108 (S108UHRF1) within the DSG degron. Furthermore, we demonstrate that S108UHRF1 phosphorylation is catalyzed by casein kinase 1 delta (CK1δ) and is important for the recognition of UHRF1 by SCFβ-TrCP. Importantly, we demonstrate that UHRF1 degradation is accelerated in response to DNA damage, coincident with enhanced S108UHRF1 phosphorylation. Taken together, our data identify SCFβ-TrCP as a bona fide UHRF1 E3 ligase important for regulating UHRF1 steady-state levels both under normal conditions and in response to DNA damage.

  • m phase phosphorylation of the epigenetic regulator uhrf1 regulates its physical association with the deubiquitylase usp7 and Stability
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Hao Chen, Xue Guo, Zhentian Wang, Mathew E Sowa, Lijuan Zheng, Pingyao Zeng, Rui Guo, Jianbo Diao, Fei Lan, Wade J Harper
    Abstract:

    UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) plays an important role in DNA CpG methylation, heterochromatin function and gene expression. Overexpression of UHRF1 has been suggested to contribute to tumorigenesis. However, Regulation of UHRF1 is largely unknown. Here we show that the deubiquitylase USP7 interacts with UHRF1. Using interaction-defective and catalytic mutants of USP7 for complementation experiments, we demonstrate that both physical interaction and catalytic activity of USP7 are necessary for UHRF1 ubiquitylation and Stability Regulation. Mass spectrometry analysis identified phosphorylation of serine (S) 652 within the USP7-interacting domain of UHRF1, which was further confirmed by a UHRF1 S652 phosphor (S652ph)-specific antibody. Importantly, the S652ph antibody identifies phosphorylated UHRF1 in mitotic cells and consistently S652 can be phosphorylated by the M phase-specific kinase CDK1-cyclin B in vitro. UHRF1 S652 phosphorylation significantly reduces UHRF1 interaction with USP7 in vitro and in vivo, which is correlated with a decreased UHRF1 Stability in the M phase of the cell cycle. In contrast, UHRF1 carrying the S652A mutation, which renders UHRF1 resistant to phosphorylation at S652, is more stable. Importantly, cells carrying the S652A mutant grow more slowly suggesting that maintaining an appropriate level of UHRF1 is important for cell proliferation Regulation. Taken together, our findings uncovered a cell cycle-specific signaling event that relieves UHRF1 from its interaction with USP7, thus exposing UHRF1 to proteasome-mediated degradation. These findings identify a molecular mechanism by which cellular UHRF1 level is regulated, which may impact cell proliferation.

Hao Chen - One of the best experts on this subject based on the ideXlab platform.

  • dna damage regulates uhrf1 Stability via the scfβ trcp e3 ligase
    Molecular and Cellular Biology, 2013
    Co-Authors: Hao Chen, Hiroyuki Inuzuka, Jianbo Diao
    Abstract:

    UHRF1 (ubiquitin-like, with PHD and RING finger domains 1) is a critical epigenetic player involved in the maintenance of DNA methylation patterns during DNA replication. DysRegulation of the UHRF1 level is implicated in cancer onset, metastasis, and tumor recurrence. Previous studies demonstrated that UHRF1 can be stabilized through USP7-mediated deubiquitylation, but the mechanism through which UHRF1 is ubiquitylated is still unknown. Here we show that proteasomal degradation of UHRF1 is mediated by the SCFβ-TrCP E3 ligase. Through bioinformatic and mutagenesis studies, we identified a functional DSG degron in the UHRF1 N terminus that is necessary for UHRF1 Stability Regulation. We further show that UHRF1 physically interacts with β-TrCP1 in a manner dependent on phosphorylation of serine 108 (S108UHRF1) within the DSG degron. Furthermore, we demonstrate that S108UHRF1 phosphorylation is catalyzed by casein kinase 1 delta (CK1δ) and is important for the recognition of UHRF1 by SCFβ-TrCP. Importantly, we demonstrate that UHRF1 degradation is accelerated in response to DNA damage, coincident with enhanced S108UHRF1 phosphorylation. Taken together, our data identify SCFβ-TrCP as a bona fide UHRF1 E3 ligase important for regulating UHRF1 steady-state levels both under normal conditions and in response to DNA damage.

  • m phase phosphorylation of the epigenetic regulator uhrf1 regulates its physical association with the deubiquitylase usp7 and Stability
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Hao Chen, Xue Guo, Zhentian Wang, Mathew E Sowa, Lijuan Zheng, Pingyao Zeng, Rui Guo, Jianbo Diao, Fei Lan, Wade J Harper
    Abstract:

    UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) plays an important role in DNA CpG methylation, heterochromatin function and gene expression. Overexpression of UHRF1 has been suggested to contribute to tumorigenesis. However, Regulation of UHRF1 is largely unknown. Here we show that the deubiquitylase USP7 interacts with UHRF1. Using interaction-defective and catalytic mutants of USP7 for complementation experiments, we demonstrate that both physical interaction and catalytic activity of USP7 are necessary for UHRF1 ubiquitylation and Stability Regulation. Mass spectrometry analysis identified phosphorylation of serine (S) 652 within the USP7-interacting domain of UHRF1, which was further confirmed by a UHRF1 S652 phosphor (S652ph)-specific antibody. Importantly, the S652ph antibody identifies phosphorylated UHRF1 in mitotic cells and consistently S652 can be phosphorylated by the M phase-specific kinase CDK1-cyclin B in vitro. UHRF1 S652 phosphorylation significantly reduces UHRF1 interaction with USP7 in vitro and in vivo, which is correlated with a decreased UHRF1 Stability in the M phase of the cell cycle. In contrast, UHRF1 carrying the S652A mutation, which renders UHRF1 resistant to phosphorylation at S652, is more stable. Importantly, cells carrying the S652A mutant grow more slowly suggesting that maintaining an appropriate level of UHRF1 is important for cell proliferation Regulation. Taken together, our findings uncovered a cell cycle-specific signaling event that relieves UHRF1 from its interaction with USP7, thus exposing UHRF1 to proteasome-mediated degradation. These findings identify a molecular mechanism by which cellular UHRF1 level is regulated, which may impact cell proliferation.

Kevin G. Becker - One of the best experts on this subject based on the ideXlab platform.

  • control of gene expression during t cell activation alternate Regulation of mrna transcription and mrna Stability
    BMC Genomics, 2005
    Co-Authors: Chris Cheadle, Jin Shui Fan, Myriam Gorospe, Yoon S Chochung, Thomas Werner, Jill M Ray, Kevin G. Becker
    Abstract:

    Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. The effective correlation of observed changes in gene expression with shared transcription regulatory elements remains difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels. In order to investigate the relative contribution of gene transcription and changes in mRNA Stability Regulation to standard analyses of gene expression, we used two distinct microarray methods which individually measure nuclear gene transcription and changes in polyA mRNA gene expression. Gene expression profiles were obtained from both polyA mRNA (whole-cell) and nuclear run-on (newly transcribed) RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin. Comparative analysis revealed that Regulation of mRNA Stability may account for as much as 50% of all measurements of changes in polyA mRNA in this system, as inferred by the absence of any corresponding Regulation of nuclear gene transcription activity for these groups of genes. Genes which displayed dramatic elevations in both mRNA and nuclear run-on RNA were shown to be inhibited by Actinomycin D (ActD) pre-treatment of cells while large numbers of genes regulated only through altered mRNA turnover (both up and down) were ActD-resistant. Consistent patterns across the time course were observed for both transcribed and Stability-regulated genes. We propose that Regulation of mRNA Stability contributes significantly to the observed changes in gene expression in response to external stimuli, as measured by high throughput systems.

Wade J Harper - One of the best experts on this subject based on the ideXlab platform.

  • m phase phosphorylation of the epigenetic regulator uhrf1 regulates its physical association with the deubiquitylase usp7 and Stability
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Hao Chen, Xue Guo, Zhentian Wang, Mathew E Sowa, Lijuan Zheng, Pingyao Zeng, Rui Guo, Jianbo Diao, Fei Lan, Wade J Harper
    Abstract:

    UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) plays an important role in DNA CpG methylation, heterochromatin function and gene expression. Overexpression of UHRF1 has been suggested to contribute to tumorigenesis. However, Regulation of UHRF1 is largely unknown. Here we show that the deubiquitylase USP7 interacts with UHRF1. Using interaction-defective and catalytic mutants of USP7 for complementation experiments, we demonstrate that both physical interaction and catalytic activity of USP7 are necessary for UHRF1 ubiquitylation and Stability Regulation. Mass spectrometry analysis identified phosphorylation of serine (S) 652 within the USP7-interacting domain of UHRF1, which was further confirmed by a UHRF1 S652 phosphor (S652ph)-specific antibody. Importantly, the S652ph antibody identifies phosphorylated UHRF1 in mitotic cells and consistently S652 can be phosphorylated by the M phase-specific kinase CDK1-cyclin B in vitro. UHRF1 S652 phosphorylation significantly reduces UHRF1 interaction with USP7 in vitro and in vivo, which is correlated with a decreased UHRF1 Stability in the M phase of the cell cycle. In contrast, UHRF1 carrying the S652A mutation, which renders UHRF1 resistant to phosphorylation at S652, is more stable. Importantly, cells carrying the S652A mutant grow more slowly suggesting that maintaining an appropriate level of UHRF1 is important for cell proliferation Regulation. Taken together, our findings uncovered a cell cycle-specific signaling event that relieves UHRF1 from its interaction with USP7, thus exposing UHRF1 to proteasome-mediated degradation. These findings identify a molecular mechanism by which cellular UHRF1 level is regulated, which may impact cell proliferation.

Chris Cheadle - One of the best experts on this subject based on the ideXlab platform.

  • control of gene expression during t cell activation alternate Regulation of mrna transcription and mrna Stability
    BMC Genomics, 2005
    Co-Authors: Chris Cheadle, Jin Shui Fan, Myriam Gorospe, Yoon S Chochung, Thomas Werner, Jill M Ray, Kevin G. Becker
    Abstract:

    Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. The effective correlation of observed changes in gene expression with shared transcription regulatory elements remains difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels. In order to investigate the relative contribution of gene transcription and changes in mRNA Stability Regulation to standard analyses of gene expression, we used two distinct microarray methods which individually measure nuclear gene transcription and changes in polyA mRNA gene expression. Gene expression profiles were obtained from both polyA mRNA (whole-cell) and nuclear run-on (newly transcribed) RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin. Comparative analysis revealed that Regulation of mRNA Stability may account for as much as 50% of all measurements of changes in polyA mRNA in this system, as inferred by the absence of any corresponding Regulation of nuclear gene transcription activity for these groups of genes. Genes which displayed dramatic elevations in both mRNA and nuclear run-on RNA were shown to be inhibited by Actinomycin D (ActD) pre-treatment of cells while large numbers of genes regulated only through altered mRNA turnover (both up and down) were ActD-resistant. Consistent patterns across the time course were observed for both transcribed and Stability-regulated genes. We propose that Regulation of mRNA Stability contributes significantly to the observed changes in gene expression in response to external stimuli, as measured by high throughput systems.