The development of innovative high throughput genome-wide methodologies and technologies such as RNA-Seq and ChIP-Seq have made it possible to identify and analyze all coding and non-coding RNAs as well as map epigenetic marks and transcription factor binding sites. However, RNA-Seq is typically performed using total RNA isolated from cells or tissues and such analysis will generate signatures of steady-state levels of RNA but will not inform on whether particular gene expression alterations are due to altered transcription and/or altered RNA stability. Recently, a number of different approaches have been developed to estimate nascent transcription of genomic sequences such as GRO-Seq, NET-Seq and by metabolic labeling and isolated of nascent RNA. We will in this R01 application further develop and validate two approaches that we believe add important new capabilities to existing techniques for the comprehensive and high throughput exploration of gene expression signatures in human cells. BruChase-Seq is based on bromouridine pulse-chase labeling coupled to deep sequencing directly measuring the kinetics of synthesis and degradation of all primary and mature mRNAs and non-coding RNAs as well as determines splicing kinetics of all intron sequences. BrUV-Seq introduces random transcription-blocking lesions by UV light prior to BrU-labeling, identifying transcription start sites (TSS), putative enhancer elements and by stabilizing transcripts that might be labile if completely transcribed allowing their more sensitive detection. Each of these advances has the potential to greatly expand current genomic annotations by assigning additional functional assessments to known genes, showing how the parts of genes act independently, and identifying cryptic intergenic elements.

Public Health Relevance

We have initiated the development of two new approaches, named BruChase-Seq and BrUV-Seq, for the comprehensively analysis of gene expression signatures. In this R01 proposal, we will attempt to validate these approaches as well as determining their predictive power of determining RNA stability and identifying specific transcription regulatory elements.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG006786-03
Application #
8628859
Study Section
Special Emphasis Panel (ZHG1-HGR-M (J2))
Program Officer
Pazin, Michael J
Project Start
2012-05-01
Project End
2015-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
3
Fiscal Year
2014
Total Cost
$382,295
Indirect Cost
$129,293
Name
University of Michigan Ann Arbor
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Venkata Narayanan, Ishwarya; Paulsen, Michelle T; Bedi, Karan et al. (2017) Transcriptional and post-transcriptional regulation of the ionizing radiation response by ATM and p53. Sci Rep 7:43598
Zhang, Honglian; Gan, Haiyun; Wang, Zhiquan et al. (2017) RPA Interacts with HIRA and Regulates H3.3 Deposition at Gene Regulatory Elements in Mammalian Cells. Mol Cell 65:272-284
Kirkconnell, Killeen S; Paulsen, Michelle T; Magnuson, Brian et al. (2016) Capturing the dynamic nascent transcriptome during acute cellular responses: The serum response. Biol Open 5:837-47
Magnuson, Brian; Bedi, Karan; Ljungman, Mats (2016) Genome stability versus transcript diversity. DNA Repair (Amst) 44:81-86
Xin, Wei; Emadi, Sharareh; Williams, Stephanie et al. (2015) Toxic Oligomeric Alpha-Synuclein Variants Present in Human Parkinson's Disease Brains Are Differentially Generated in Mammalian Cell Models. Biomolecules 5:1634-51
Magnuson, Brian; Veloso, Artur; Kirkconnell, Killeen S et al. (2015) Identifying transcription start sites and active enhancer elements using BruUV-seq. Sci Rep 5:17978
Andrade-Lima, Leonardo C; Veloso, Artur; Paulsen, Michelle T et al. (2015) DNA repair and recovery of RNA synthesis following exposure to ultraviolet light are delayed in long genes. Nucleic Acids Res 43:2744-56
Kocab, A J; Veloso, A; Paulsen, M T et al. (2015) Effects of physiological and synthetic IAP antagonism on c-IAP-dependent signaling. Oncogene 34:5472-81
Muenyi, Clarisse S; Ljungman, Mats; States, J Christopher (2015) Arsenic Disruption of DNA Damage Responses-Potential Role in Carcinogenesis and Chemotherapy. Biomolecules 5:2184-93
Lefkofsky, Hailey B; Veloso, Artur; Ljungman, Mats (2015) Transcriptional and post-transcriptional regulation of nucleotide excision repair genes in human cells. Mutat Res 776:9-15

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