Epigenetic processes interpret the genomic sequences in a cell-type and extracellular environment-dictated mode. Epigenetic information is encoded through covalent modifications of histones and DNA, nucleosome position and substitution by histone variants. Epigenetics is one of the most intensively studied fields of biology today and represents a new paradigm for the pathophysiology, diagnosis and treatment of diseases. To advance epigenetic studies, we have introduced a novel chromatin immunoprecipitation platform, Matrix ChIP, that utilizes surface-immobilized antibodies in a 96-well plate, where the entire procedure from chromatin precipitation to PCR-ready DNA purification is done on the same plate without sample transfers. This high- throughput method allows for parallel profiling of an order of magnitude greater number of chromatin and transcription events than was previously possible. Matrix ChIP is more sensitive than traditional approach to detect DNA-protein interactions of less abundant proteins such as chromatin modifiers. ChIP assay consists of three separate steps: (1) sample harvesting/chromatin fragmentation, (2) immunoprecipitation and (3) DNA purification and analysis (qPCR/sequencing). Although in the Matrix ChIP, steps (2) and (3) are greatly simplified allowing one to run hundreds of assays at a time, sample harvesting/chromatin fragmentation is done in test tubes with low efficiency and high labor intensity. This project proposes an innovative multidisciplinary approach (Dr. Matula, ultrasound/engineering, Dr. Denisenko, molecular biology, and Dr. Bomsztyk, chromatin biology/biotechnology/instrumentation) to develop a platform, PIXUL-ChIP, that will integrate highly efficient sample harvesting and chromatin shearing with immunoprecipitation in microplates for high-throughput epigenetic analysis. The following aims are proposed.
Aim#1. To build a pixelated ultrasound (PIXUL) processor for high-throughput fast chromatin shearing.
Aim#2. To combine sample harvesting and chromatin shearing with immunoprecipitation into an integrated microplate platform for epigenetic analysis, PIXUL-ChIP.
Aim#3. To test PIXUL-ChIP as a platform for studies of epigenetic changes at selected gene loci associated with drug-mediated human embryonic stem cell (hESC) renewal and differentiation. Epigenetics is a fast growing field, but nonetheless, there are significant unmet needs that require further technological advances to facilitate exploration of epigenetic processes and exploit this knowledge in translational research and clinical applications. A highly efficient technology, PIXUL-ChIP will provide powerful means to simultaneously study multiple chromatin modifications and modifiers at gene loci in a wide range of systems, and as such will be a valuable platform for studying epigenetic processes, discovering epigenetic biomarkers, and testing novel drugs and their combinations.
(provided by the applicant): The chromatin (ChIP) immunoprecipitation assay is a powerful technique to study epigenetic events but some of steps are time consuming and are done in test tubes. We propose to develop an integrated PIXUL ChIP platform for analysis of chromatin changes where all the steps from cell culture or tissues to DNA purification are done in microplates. PIXUL ChIP platform will increase the assay sensitivity, throughput and reproducibility, and will be user-friendly.
|Haynes, Premi; Bomsztyk, Karol; Miller, Daniel G (2018) 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. Epigenetics Chromatin 11:47|
|Denisenko, Oleg; Mar, Daniel; Trawczynski, Matthew et al. (2018) Chromatin changes trigger laminin genes dysregulation in aging kidneys. Aging (Albany NY) 10:1133-1145|
|Bomsztyk, Karol; Denisenko, Oleg; Wang, Yuliang (2018) DNA methylation yields epigenetic clues into the diabetic nephropathy of Pima Indians. Kidney Int 93:1272-1275|
|Kotha, Surya S; Hayes, Brian J; Phong, Kiet T et al. (2018) Engineering a multicellular vascular niche to model hematopoietic cell trafficking. Stem Cell Res Ther 9:77|
|Denisenko, Oleg; Lucas, Emma S; Sun, Congshan et al. (2016) Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation. Biochim Biophys Acta 1859:906-13|
|Gharib, Sina A; Mar, Daniel; Bomsztyk, Karol et al. (2016) SYSTEM-WIDE MAPPING OF ACTIVATED CIRCUITRY IN EXPERIMENTAL SYSTEMIC INFLAMMATORY RESPONSE SYNDROME. Shock 45:148-56|
|Mikula, M; Skrzypczak, M; Goryca, K et al. (2016) Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy. Nucleic Acids Res 44:10150-10164|
|Bomsztyk, Karol; Mar, Daniel; An, Dowon et al. (2015) Experimental acute lung injury induces multi-organ epigenetic modifications in key angiogenic genes implicated in sepsis-associated endothelial dysfunction. Crit Care 19:225|
|Mar, Daniel; Gharib, Sina A; Zager, Richard A et al. (2015) Heterogeneity of epigenetic changes at ischemia/reperfusion- and endotoxin-induced acute kidney injury genes. Kidney Int 88:734-44|
|Mikula, Michal; Rubel, Tymon; Karczmarski, Jakub et al. (2015) Beads-free protein immunoprecipitation for a mass spectrometry-based interactome and posttranslational modifications analysis. Proteome Sci 13:23|
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