scholarly journals ­­­Proteomic analysis of H3K36me3 and PSIP1/p75 (LEDGF) complexes reveal their wider role in DNA repair

2017 ◽  
Vol 2 ◽  
pp. 83
Author(s):  
Madapura M. Pradeepa ◽  
Gillian C.A. Taylor ◽  
Alex von Kriegsheim
Keyword(s):  
Dna Repair ◽  
Proteomic Analysis ◽  
Embryonic Stem ◽  
Adaptor Proteins ◽  
Transcriptional Elongation ◽  
Pwwp Domain ◽  
Stable Isotope Labelling ◽  
Interacting Protein ◽  
Dna Repair Proteins

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses a  H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). Furthermore, we performed stable isotope labelling with amino acids in cell culture (SILAC) for a longer isoform of PSIP1 (p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts (MEFS). Results: Proteomic analysis of H3K36me3 chromatin show association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP1/p75. We validated the association of PSIP1/p75 with gamma H2A.X, an early marker of DNA damage and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP1/p75 in promoting HDR in mammals, our data supports the wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting several DNA repair proteins to transcribed gene bodies.

scholarly journals ­­­Proteomic analysis of H3K36me3 and PSIP1/p75 (LEDGF) complexes reveal their wider role in DNA repair

2017 ◽  
Vol 2 ◽  
pp. 83
Author(s):  
Madapura M. Pradeepa ◽  
Gillian C.A. Taylor ◽  
Alex von Kriegsheim
Keyword(s):  
Dna Repair ◽  
Proteomic Analysis ◽  
Embryonic Stem ◽  
Adaptor Proteins ◽  
Transcriptional Elongation ◽  
Pwwp Domain ◽  
Stable Isotope Labelling ◽  
Interacting Protein ◽  
Dna Repair Proteins

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses a  H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). Furthermore, we performed stable isotope labelling with amino acids in cell culture (SILAC) for a longer isoform of PSIP1 (p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts (MEFS). Results: Proteomic analysis of H3K36me3 chromatin show association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP1/p75. We validated the association of PSIP1/p75 with gamma H2A.X, an early marker of DNA damage and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP1/p75 in promoting HDR in mammals, our data supports the wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting several DNA repair proteins to transcribed gene bodies.

scholarly journals H3K36me3 and PSIP1/LEDGF associate with several DNA repair proteins, suggesting their role in efficient DNA repair at actively transcribing loci

2021 ◽  
Vol 2 ◽  
pp. 83
Author(s):  
Jayakumar Sundarraj ◽  
Gillian C.A. Taylor ◽  
Alex von Kriegsheim ◽  
Madapura M Pradeepa
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Embryonic Stem ◽  
Adaptor Proteins ◽  
Transcriptional Elongation ◽  
Pwwp Domain ◽  
Stable Isotope Labelling ◽  
Homology Directed Repair ◽  
Dna Repair Proteins

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses an H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology-directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry (qMS) to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). We also performed stable isotope labelling with amino acids in cell culture (SILAC) followed by qMS for a longer isoform of PSIP1 (PSIP/p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts ( MEFs). Furthermore, immunoprecipitation followed by western blotting was performed to validate the qMS data. DNA damage in PSIP1 knockout MEFs was assayed by a comet assay. Results: Proteomic analysis shows the association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP /p75. We further validated the association of PSIP/p75 with PARP1, hnRNPU and gamma H2A.X  and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP/p75 in promoting homology-directed repair (HDR), our data support a wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting proteins involved in DNA damage response pathways to the actively transcribed loci.

scholarly journals H3K36me3 and PSIP1/LEDGF associate with several DNA repair proteins, suggesting their role in efficient DNA repair at actively transcribing loci

2021 ◽  
Vol 2 ◽  
pp. 83
Author(s):  
Jayakumar Sundarraj ◽  
Gillian C.A. Taylor ◽  
Alex von Kriegsheim ◽  
Madapura M Pradeepa
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Embryonic Stem ◽  
Adaptor Proteins ◽  
Transcriptional Elongation ◽  
Pwwp Domain ◽  
Stable Isotope Labelling ◽  
Homology Directed Repair ◽  
Dna Repair Proteins

Background: Trimethylation at histone H3 at lysine 36 (H3K36me3) is associated with expressed gene bodies and recruit proteins implicated in transcription, splicing and DNA repair. PC4 and SF2 interacting protein (PSIP1/LEDGF) is a transcriptional coactivator, possesses an H3K36me3 reader PWWP domain. Alternatively spliced isoforms of PSIP1 binds to H3K36me3 and suggested to function as adaptor proteins to recruit transcriptional modulators, splicing factors and proteins that promote homology-directed repair (HDR), to H3K36me3 chromatin. Methods: We performed chromatin immunoprecipitation of H3K36me3 followed by quantitative mass spectrometry (qMS) to identify proteins associated with H3K36 trimethylated chromatin in mouse embryonic stem cells (mESCs). We also performed stable isotope labelling with amino acids in cell culture (SILAC) followed by qMS for a longer isoform of PSIP1 (PSIP/p75) and MOF/KAT8 in mESCs and mouse embryonic fibroblasts ( MEFs). Furthermore, immunoprecipitation followed by western blotting was performed to validate the qMS data. DNA damage in PSIP1 knockout MEFs was assayed by a comet assay. Results: Proteomic analysis shows the association of proteins involved in transcriptional elongation, RNA processing and DNA repair with H3K36me3 chromatin. Furthermore, we show DNA repair proteins like PARP1, gamma H2A.X, XRCC1, DNA ligase 3, SPT16, Topoisomerases and BAZ1B are predominant interacting partners of PSIP /p75. We further validated the association of PSIP/p75 with PARP1, hnRNPU and gamma H2A.X  and also demonstrated accumulation of damaged DNA in PSIP1 knockout MEFs. Conclusions: In contrast to the previously demonstrated role of H3K36me3 and PSIP/p75 in promoting homology-directed repair (HDR), our data support a wider role of H3K36me3 and PSIP1 in maintaining the genome integrity by recruiting proteins involved in DNA damage response pathways to the actively transcribed loci.

scholarly journals BRCT Domain-Containing Protein PTIP Is Essential for Progression through Mitosis

2003 ◽  
Vol 23 (5) ◽  
pp. 1666-1673 ◽  
Author(s):  
Eun Ah Cho ◽  
Marc J. Prindle ◽  
Gregory R. Dressler
Keyword(s):  
Cell Proliferation ◽  
Dna Repair ◽  
Cell Cycle Control ◽  
Embryonic Stem ◽  
Essential Element ◽  
Brct Domain ◽  
Transactivation Domain ◽  
Mouse Development ◽  
Nuclear Condensation ◽  
Interacting Protein

ABSTRACT The Pax transactivation domain-interacting protein (PTIP) is a large nuclear protein with multiple BRCT domains that was identified on the basis of its interaction with transcription factors of the Pax and Smad families. To address the function of PTIP during mouse development, we generated a constitutive null allele. Homozygous PTIP mutants are developmentally retarded, disorganized, and embryonic lethal by day 9.5 of embryonic development (E9.5). PTIP mutant cells appear to replicate DNA but show reduced levels of mitosis and widespread cell death by E8.5. DNA damage appears to precede nuclear condensation at E7.5, suggesting a defect in DNA repair. Neither embryonic fibroblast nor embryonic stem cells from PTIP mutants proliferate in culture, suggesting a fundamental defect in cell proliferation. Trophoblast cells from PTIP mutants are more sensitive to DNA-damaging agents. Condensation of chromatin and expression of phospho-histone H3 are also affected in PTIP mutants, and this may underlie the inability of PTIP mutants to progress through mitosis. Given the role of BRCT domain proteins in DNA repair and cell cycle control, we propose that PTIP is an essential element of the cell proliferation machinery, perhaps by functioning in the DNA repair pathways.

The role of DNA repair proteins in the assessment of thyroid nodules

2014 ◽  
Author(s):  
Bahri Evren ◽  
Nese Karadag ◽  
Sami Yilmaz ◽  
Ayse Cikim Sertkaya
Keyword(s):  
Dna Repair ◽  
Thyroid Nodules ◽  
Dna Repair Proteins

scholarly journals Ape1 regulates hematopoietic differentiation of embryonic stem cells through its redox functional domain

Blood ◽  
2006 ◽  
Vol 109 (5) ◽  
pp. 1917-1922 ◽  
Author(s):  
Gang-Ming Zou ◽  
Mei-Hua Luo ◽  
April Reed ◽  
Mark R. Kelley ◽  
Mervin C. Yoder
Keyword(s):  
Gene Expression ◽  
Dna Repair ◽  
Redox Regulation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Redox Activity ◽  
Normal Embryo ◽  
Functional Domain ◽  
Regulation Of Transcription

Abstract Ape1 is a molecule with dual functions in DNA repair and redox regulation of transcription factors. In Ape1-deficient mice, embryos do not survive beyond embryonic day 9, indicating that this molecule is required for normal embryo development. Currently, direct evidence of the role of Ape1 in regulating hematopoiesis is lacking. We used the embryonic stem (ES) cell differentiation system and an siRNA approach to knockdown Ape1 gene expression to test the role of Ape1 in hematopoiesis. Hemangioblast development from ES cells was reduced 2- to 3-fold when Ape1 gene expression was knocked down by Ape1-specific siRNA, as was primitive and definitive hematopoiesis. Impaired hematopoiesis was not associated with increased apoptosis in siRNA-treated cells. To begin to explore the mechanism whereby Ape1 regulates hematopoiesis, we found that inhibition of the redox activity of Ape1 with E3330, a specific Ape1 redox inhibitor, but not Ape1 DNA repair activity, which was blocked using the small molecule methoxyamine, affected cytokine-mediated hemangioblast development in vitro. In summary, these data indicate Ape1 is required in normal embryonic hematopoiesis and that the redox function, but not the repair endonuclease activity, of Ape1 is critical in normal embryonic hematopoietic development.

scholarly journals Beyond the Double-Strand Breaks: The Role of DNA Repair Proteins in Cancer Stem-Cell Regulation

Cancers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 4818
Author(s):  
Jacqueline Nathansen ◽  
Felix Meyer ◽  
Luise Müller ◽  
Marc Schmitz ◽  
Kerstin Borgmann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Tumor Evolution ◽  
Regulation Of Transcription ◽  
Mesenchymal Transition ◽  
Damage Response ◽  
Dna Repair Proteins

Cancer stem cells (CSCs) are pluripotent and highly tumorigenic cells that can re-populate a tumor and cause relapses even after initially successful therapy. As with tissue stem cells, CSCs possess enhanced DNA repair mechanisms. An active DNA damage response alleviates the increased oxidative and replicative stress and leads to therapy resistance. On the other hand, mutations in DNA repair genes cause genomic instability, therefore driving tumor evolution and developing highly aggressive CSC phenotypes. However, the role of DNA repair proteins in CSCs extends beyond the level of DNA damage. In recent years, more and more studies have reported the unexpected role of DNA repair proteins in the regulation of transcription, CSC signaling pathways, intracellular levels of reactive oxygen species (ROS), and epithelial–mesenchymal transition (EMT). Moreover, DNA damage signaling plays an essential role in the immune response towards tumor cells. Due to its high importance for the CSC phenotype and treatment resistance, the DNA damage response is a promising target for individualized therapies. Furthermore, understanding the dependence of CSC on DNA repair pathways can be therapeutically exploited to induce synthetic lethality and sensitize CSCs to anti-cancer therapies. This review discusses the different roles of DNA repair proteins in CSC maintenance and their potential as therapeutic targets.

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