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Grant Details

Grant Number: 5R33CA191135-03 Interpret this number
Primary Investigator: Bomsztyk, Karol
Organization: University Of Washington
Project Title: Next Generation Technology to Detect Epigenetic Alterations in Human Tissues
Fiscal Year: 2017


 DESCRIPTION (provided by applicant): Increasingly, there has been a realization of the importance of accurate assessment of chromatin modifications for both clinical care (for example for cancer detection, prognosis, determining treatment, designing new therapies and predicting response) as well as for understanding carcinogenesis. Currently available approach for identification of specific genome-associated proteins or DNA modifications, the chromatin Immunoprecipitation (ChIP) assay, is of limited clinical utility given that it requires time consuming/labor intensive pre-analytical sample preparation, provides low throughput, and is associated with epitope loss (raising concerns about data accuracy/reproducibility). To address some of these issues, we developed the Matrix ChIP, a microplate-based method which accelerates the analytical process, and increases assay's throughput, sensitivity and reproducibility. We now (Aims#1&2) propose to develop next generation microplate based platform, PIXUL-ChIP, which will integrate cancer sample preparation/ chromatin fragmentation with immunoprecipitation. PIXUL-ChIP will provide high throughput, simultaneous, and automated analysis of a wide range of chromatin marks which will allow us to dramatically expand our understanding of the role of chromatin alterations in health and disease. Current approaches to collect and prepare clinical specimens are not optimized for chromatin analysis. To assure the success of the PIXUL-ChIP, in Aim&3 we will develop and validate methods for collection, fixation, storage and ultrasound treatment of human normal and cancer (tissues/fluid) specimens. Specifically we propose: Aim#1. To build a pixelated ultrasound (PIXUL) processor for fast and high-throughput chromatin fragmentation. We will utilize our recently pioneered ultrasound technology to build a PIXUL microplate processor for cell culture and tissue chromatin fragmentation in 96 well plates. Aim#2. To combine sample preparation/chromatin with immunoprecipitation into an integrated microplate platform for high throughput epigenetic analysis, PIXUL-ChIP. The ability to shear chromatin in a microplate will streamline sample preparation, immunoprecipitation and ChIP DNA analysis. Aim#3. Using PIXUL technology we will develop and validate practical cost-effective approaches for sample collection from human cancer tissues and their preparation for analysis of epigenetic alterations. Sample preparation and storage conditions dramatically affect the epitope loss, thus determining how to best collect/process/store biospecimens is of great importance. To this end we will develop protocols for sample collection/ preparation/storage and analysis for snap-frozen fresh and for formalin fixed paraffin embedded (FFPE) tissues. The user-friendly, high sensitivity/throughput PIXUL-ChIP technology will greatly increase the utility of new and archived cancer biospecimens and will facilitate basic and translational research to exploit epigenetic information from the vastly unexplored human cancer biospecimes.


PIXUL-ChIP: integrated high-throughput sample preparation and analytical platform for epigenetic studies.
Authors: Bomsztyk K. , Mar D. , Wang Y. , Denisenko O. , Ware C. , Frazar C.D. , Blattler A. , Maxwell A.D. , MacConaghy B.E. , Matula T.J. .
Source: Nucleic acids research, 2019-07-09; 47(12), p. e69.
PMID: 30927002
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Distinct patterns of transcriptional and epigenetic alterations characterize acute and chronic kidney injury.
Authors: Sharifian R. , Okamura D.M. , Denisenko O. , Zager R.A. , Johnson A. , Gharib S.A. , Bomsztyk K. .
Source: Scientific reports, 2018-12-14; 8(1), p. 17870.
EPub date: 2018-12-14.
PMID: 30552397
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Sporadic DUX4 expression in FSHD myocytes is associated with incomplete repression by the PRC2 complex and gain of H3K9 acetylation on the contracted D4Z4 allele.
Authors: Haynes P. , Bomsztyk K. , Miller D.G. .
Source: Epigenetics & chromatin, 2018-08-20; 11(1), p. 47.
EPub date: 2018-08-20.
PMID: 30122154
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DNA methylation yields epigenetic clues into the diabetic nephropathy of Pima Indians.
Authors: Bomsztyk K. , Denisenko O. , Wang Y. .
Source: Kidney international, 2018 Jun; 93(6), p. 1272-1275.
PMID: 29792271
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Chromatin changes trigger laminin genes dysregulation in aging kidneys.
Authors: Denisenko O. , Mar D. , Trawczynski M. , Bomsztyk K. .
Source: Aging, 2018-05-29; 10(5), p. 1133-1145.
PMID: 29846172
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Engineering a multicellular vascular niche to model hematopoietic cell trafficking.
Authors: Kotha S.S. , Hayes B.J. , Phong K.T. , Redd M.A. , Bomsztyk K. , Ramakrishnan A. , Torok-Storb B. , Zheng Y. .
Source: Stem cell research & therapy, 2018-03-23; 9(1), p. 77.
EPub date: 2018-03-23.
PMID: 29566751
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Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy.
Authors: Mikula M. , Skrzypczak M. , Goryca K. , Paczkowska K. , Ledwon J.K. , Statkiewicz M. , Kulecka M. , Grzelak M. , Dabrowska M. , Kuklinska U. , et al. .
Source: Nucleic acids research, 2016-12-01; 44(21), p. 10150-10164.
EPub date: 2016-09-01.
PMID: 27587583
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Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation.
Authors: Denisenko O. , Lucas E.S. , Sun C. , Watkins A.J. , Mar D. , Bomsztyk K. , Fleming T.P. .
Source: Biochimica et biophysica acta, 2016 Jul; 1859(7), p. 906-13.
EPub date: 2016-04-07.
PMID: 27060415
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Authors: Gharib S.A. , Mar D. , Bomsztyk K. , Denisenko O. , Dhanireddy S. , Liles W.C. , Altemeier W.A. .
Source: Shock (Augusta, Ga.), 2016 Feb; 45(2), p. 148-56.
PMID: 26536201
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Quadruplex formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8•(GGCCCC)8 repeat: effect of CpG methylation.
Authors: Zamiri B. , Mirceta M. , Bomsztyk K. , Macgregor R.B. , Pearson C.E. .
Source: Nucleic acids research, 2015-11-16; 43(20), p. 10055-64.
EPub date: 2015-10-01.
PMID: 26432832
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Heterogeneity of epigenetic changes at ischemia/reperfusion- and endotoxin-induced acute kidney injury genes.
Authors: Mar D. , Gharib S.A. , Zager R.A. , Johnson A. , Denisenko O. , Bomsztyk K. .
Source: Kidney international, 2015 Oct; 88(4), p. 734-44.
EPub date: 2015-06-10.
PMID: 26061546
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Experimental acute lung injury induces multi-organ epigenetic modifications in key angiogenic genes implicated in sepsis-associated endothelial dysfunction.
Authors: Bomsztyk K. , Mar D. , An D. , Sharifian R. , Mikula M. , Gharib S.A. , Altemeier W.A. , Liles W.C. , Denisenko O. .
Source: Critical care (London, England), 2015-05-11; 19(1), p. 225.
EPub date: 2015-05-11.
PMID: 25959381
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Beads-free protein immunoprecipitation for a mass spectrometry-based interactome and posttranslational modifications analysis.
Authors: Mikula M. , Rubel T. , Karczmarski J. , Statkiewicz M. , Bomsztyk K. , Ostrowski J. .
Source: Proteome science, 2015; 13, p. 23.
EPub date: 2015-09-02.
PMID: 26336360
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