ATM-dependent phosphorylation of heterogeneous nuclear ribonucleoprotein K promotes p53 transcriptional activation in response to DNA damage

Double-strand breaks (DSBs) of chromosomal DNA trigger the cellular response that activates the pathways for DNA repair and cell-cycle checkpoints, and sometimes the pathways leading to cell death if the damage is too severe to be tolerated. Evidence indicates that, upon generation of DNA DSBs, many nuclear proteins that are involved in DNA repair and checkpoints are recruited to chromatin around the DNA lesions. In the present study we used a proteomics approach to identify DNA-damage-induced chromatin-binding proteins in a systematic way. Two-dimensional gel analysis for protein extracts of chromatin from DNA-damage-induced and control HeLa cells identified four proteins as the candidates for DNA-damage-induced chromatin-binding proteins. MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS analysis identified these proteins to be NPM (nucleophosmin), hnRNP (heterogeneous nuclear ribonucleoprotein) C1, hnRNP C2 and 37-kDa laminin-receptor precursor, and the identity of these proteins was further confirmed by immunoblot analysis with specific antibodies. We then demonstrated with chromatin-binding assays that NPM and hnRNP C1/C2, the abundant nuclear proteins with pleiotropic functions, indeed bind to chromatin in a DNA-damage-dependent manner, implicating these proteins in DNA repair and/or damage response. Immunofluorescence experiments showed that NPM, normally present in the nucleoli, is mobilized into the nucleoplasm after DNA damage, and that neither NPM nor hnRNP C1/C2 is actively recruited to the sites of DNA breaks. These results suggest that NPM and hnRNP C1/C2 may function at the levels of the global context of chromatin, rather than by specifically targeting the broken DNA.

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Heterogeneous nuclear ribonucleoprotein E1 binds polycytosine DNA and monitors genome integrity

Life Science Alliance ◽

10.26508/lsa.202000995 ◽

2021 ◽

Vol 4 (9) ◽

pp. e202000995

Author(s):

Bidyut K Mohanty ◽

Joseph AQ Karam ◽

Breege V Howley ◽

Annamarie C Dalton ◽

Simon Grelet ◽

...

Keyword(s):

Dna Damage ◽

Binding Sites ◽

Protein Translation ◽

Genome Integrity ◽

Nuclear Antigen ◽

Dna Integrity ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Hydroxyurea Treatment ◽

Nuclear Ribonucleoprotein ◽

Hnrnp E1

Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) is a tumor suppressor protein that binds site- and structure-specifically to RNA sequences to regulate mRNA stability, facilitate alternative splicing, and suppress protein translation on several metastasis-associated mRNAs. Here, we show that hnRNP E1 binds polycytosine-rich DNA tracts present throughout the genome, including those at promoters of several oncogenes and telomeres and monitors genome integrity. It binds DNA in a site- and structure-specific manner. hnRNP E1-knockdown cells displayed increased DNA damage signals including γ-H2AX at its binding sites and also showed increased mutations. UV and hydroxyurea treatment of hnRNP E1-knockdown cells exacerbated the basal DNA damage signals with increased cell cycle arrest, activation of checkpoint proteins, and monoubiquitination of proliferating cell nuclear antigen despite no changes in deubiquitinating enzymes. DNA damage caused by genotoxin treatment localized to hnRNP E1 binding sites. Our work suggests that hnRNP E1 facilitates functions of DNA integrity proteins at polycytosine tracts and monitors DNA integrity at these sites.

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Arginine methylation of hnRNPK negatively modulates apoptosis upon DNA damage through local regulation of phosphorylation

Nucleic Acids Research ◽

10.1093/nar/gku705 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9908-9924 ◽

Cited By ~ 23

Author(s):

Jen-Hao Yang ◽

Yi-Ying Chiou ◽

Shu-Ling Fu ◽

I-Yun Shih ◽

Tsai-Hsuan Weng ◽

...

Keyword(s):

Dna Damage ◽

Rna Processing ◽

Tumor Development ◽

Arginine Methylation ◽

Wild Type ◽

Post Translational Modifications ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Damage Response ◽

Nuclear Ribonucleoprotein

Abstract Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA/DNA-binding protein involved in chromatin remodeling, RNA processing and the DNA damage response. In addition, increased hnRNPK expression has been associated with tumor development and progression. A variety of post-translational modifications of hnRNPK have been identified and shown to regulate hnRNPK function, including phosphorylation, ubiquitination, sumoylation and methylation. However, the functional significance of hnRNPK arginine methylation remains unclear. In the present study, we demonstrated that the methylation of two essential arginines, Arg296 and Arg299, on hnRNPK inhibited a nearby Ser302 phosphorylation that was mediated through the pro-apoptotic kinase PKCδ. Notably, the engineered U2OS cells carrying an Arg296/Arg299 methylation-defective hnRNPK mutant exhibited increased apoptosis upon DNA damage. While such elevated apoptosis can be diminished through addition with wild-type hnRNPK, we further demonstrated that this increased apoptosis occurred through both intrinsic and extrinsic pathways and was p53 independent, at least in part. Here, we provide the first evidence that the arginine methylation of hnRNPK negatively regulates cell apoptosis through PKCδ-mediated signaling during DNA damage, which is essential for the anti-apoptotic role of hnRNPK in apoptosis and the evasion of apoptosis in cancer cells.

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Spotting new DNA damage-responsive chromatin-binding proteins

Biochemical Journal ◽

10.1042/bj20050503 ◽

2005 ◽

Vol 388 (1) ◽

Cited By ~ 2

Author(s):

Benjamin N. WARDLEWORTH ◽

Jessica A. DOWNS

Keyword(s):

Dna Damage ◽

Transcriptional Activation ◽

Cellular Response ◽

Cycle Arrest ◽

Biochemical Journal ◽

Proteomics Approach ◽

Repair Mechanisms ◽

Comprehensive Picture ◽

Nuclear Ribonucleoprotein ◽

Multicellular Organisms

In response to DNA damage, cells initiate multiple repair mechanisms that all contribute to the survival of both the cell and the organism. These responses are numerous and variable, and can include cell cycle arrest, transcriptional activation of DNA repair genes and relocalization of repair proteins to sites of DNA damage. If all else fails, in multicellular organisms the initiation of apoptosis is also a potential cellular response to DNA damage. Despite a wealth of information about these events, it is clear that we do not yet have a comprehensive picture of the cellular responses to DNA damage. In this issue of the Biochemical Journal, a proteomics approach was used by Lee et al. to identify proteins that bind to chromatin in a DNA damage-inducible manner. The proteins identified, nucleophosmin, hnRNP C1 (heterogeneous nuclear ribonucleoprotein C1) and hnRNP C2, were proteins that would not necessarily have been predicted to behave this way. These studies have the potential to be extended and contribute to our knowledge of the cellular response to DNA damage.

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Loss of heterogeneous nuclear ribonucleoprotein L (HNRNP L) leads to mitochondrial dysfunction, DNA damage response and caspase-dependent cell death in hematopoietic stem cells

Experimental Hematology ◽

10.1016/j.exphem.2016.06.154 ◽

2016 ◽

Vol 44 (9) ◽

pp. S78-S79 ◽

Cited By ~ 1

Author(s):

Anne Helness ◽

Marie-Claude Gaudreau ◽

Damien Grapton ◽

Charles Vadnais ◽

Jennifer Fraszczak ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Cell Death ◽

Mitochondrial Dysfunction ◽

Dna Damage Response ◽

Hematopoietic Stem ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Damage Response ◽

Dependent Cell ◽

Nuclear Ribonucleoprotein

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Heterogeneous nuclear ribonucleoprotein (hnRNP) L promotes DNA damage-induced cell apoptosis by enhancing the translation of p53

Oncotarget ◽

10.18632/oncotarget.17003 ◽

2017 ◽

Vol 8 (31) ◽

pp. 51108-51122 ◽

Cited By ~ 8

Author(s):

Ji-Young Seo ◽

Do-Yeon Kim ◽

Seong-Hoon Kim ◽

Hyo-Jin Kim ◽

Hye Guk Ryu ◽

...

Keyword(s):

Dna Damage ◽

Cell Apoptosis ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Nuclear Ribonucleoprotein

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Heterogeneous Nuclear Ribonucleoprotein A2 Is a Common Transcriptional Coactivator in the Nuclear Transcription Response to Mitochondrial Respiratory Stress

Molecular Biology of the Cell ◽

10.1091/mbc.e09-04-0296 ◽

2009 ◽

Vol 20 (18) ◽

pp. 4107-4119 ◽

Cited By ~ 37

Author(s):

Manti Guha ◽

Hua Pan ◽

Ji-Kang Fang ◽

Narayan G. Avadhani

Keyword(s):

Stress Response ◽

Transcriptional Activation ◽

C2c12 Cells ◽

Transcriptional Analysis ◽

Common Mechanism ◽

Nuclear Factor ◽

Mitochondrial Stress ◽

Heterogeneous Nuclear Ribonucleoprotein ◽

Nuclear Ribonucleoprotein ◽

Mitochondrial Respiratory

Mitochondrial dysfunction and altered transmembrane potential initiate a mitochondrial respiratory stress response, also known as mitochondrial retrograde response, in a wide spectrum of cells. The mitochondrial stress response activates calcineurin, which regulates transcription factors, including a new nuclear factor-κB (NF-κB) pathway, different from the canonical and noncanonical pathways. In this study using a combination of small interfering RNA-mediated mRNA knock down, transcriptional analysis, and chromatin immunoprecipitation, we report a common mechanism for the regulation of previously established stress response genes Cathepsin L, RyR1, and Glut4. Stress-regulated transcription involves the cooperative interplay between NF-κB (cRel: p50), C/EBPδ, cAMP response element-binding protein, and nuclear factor of activated T cells. We show that the functional synergy of these factors requires the stress-activated heterogeneous nuclear ribonucleoprotein (hnRNP) A2 as a coactivator. HnRNP A2 associates with the enhanceosome, mostly through protein–protein interactions with DNA-bound factors. Silencing of hnRNP A2 as well as other DNA binding signature factors prevents stress-induced transcriptional activation and reverses the invasiveness of mitochondrial DNA-depleted C2C12 cells. Induction of mitochondrial stress signaling by electron transfer chain inhibitors also involved hnRNPA2 activation. We describe a common mechanism of mitochondrial respiratory stress-induced activation of nuclear target genes that involves hnRNP A2 as a transcription coactivator.

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