Isolation of putative FOXO1 genomic elements using an improved in vitro technique to isolate genomic regulatory sequences

Alpa Sidhu; Patrick J Miller; Andrew D Hollenbach

doi:10.1016/j.gene.2010.03.008

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Isolation of putative FOXO1 genomic elements using an improved in vitro technique to isolate genomic regulatory sequences

Journal article

Peer reviewed

Isolation of putative FOXO1 genomic elements using an improved in vitro technique to isolate genomic regulatory sequences

Alpa Sidhu, Patrick J Miller and Andrew D Hollenbach

Gene, Vol.458(1-2), pp.45-53

06/15/2010

DOI: 10.1016/j.gene.2010.03.008

PMCID: PMC3126678

PMID: 20338229

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https://www.ncbi.nlm.nih.gov/pmc/articles/3126678View

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Abstract

The regulation of gene expression drives many biological processes and alterations in normal regulation are integral in the development of the diseased state. Therefore, the ability to screen genomic DNA for direct targets of DNA binding proteins (DNA-BP) would provide valuable information about the mechanisms underlying these processes. At present chromatin immunoprecipitation (ChIP) and its variants are the primary methods for identifying regulatory elements. However, some DNA-BPs, such as the winged-helix transcription factor FOXO1, are difficult to ChIP thereby detracting from the use of this technique as a nonbiased screen to isolate regulatory sequences. In this report we use an improved in vitro method to Pull Out Regulatory Elements (PORE), which uses purified protein with a stable genomic library to isolate regulatory elements directly bound by a DNA-BP, to identify putative FOXO1 genomic regulatory sequences. We first validate this technique using two known DNA-BP (FOXO1 and Pax3) by demonstrating their ability to bind and amplify identified promoter elements when present in a genomic DNA context or when present in the context of our stable genomic library. Subsequent use of this technique with FOXO1 isolated regulatory elements associated with several genes known to be regulated in a FOXO1-dependent manner and multiple genes whose biological functions are consistent with the known biological functions of FOXO1 proving that the in vitro PORE is a valuable and easy to use alternative to ChIP for the isolation of genomic regulatory elements.

Paired Box Transcription Factors - genetics

PAX3 Transcription Factor

Transcriptional Activation

Genomic Library

DNA-Binding Proteins - genetics

Regulatory Sequences, Nucleic Acid - genetics

DNA-Binding Proteins - metabolism

Animals

Forkhead Transcription Factors - metabolism

Female

Forkhead Box Protein O1

Mice

Genome

Paired Box Transcription Factors - metabolism

Details

Title: Subtitle: Isolation of putative FOXO1 genomic elements using an improved in vitro technique to isolate genomic regulatory sequences
Creators: Alpa Sidhu - Department of Genetics, Louisiana State University Health Sciences Center, 533 Bolivar Street, CSRB 6th floor, New Orleans, LA 70112, USA
Patrick J Miller
Andrew D Hollenbach
Resource Type: Journal article
Publication Details: Gene, Vol.458(1-2), pp.45-53
DOI: 10.1016/j.gene.2010.03.008
PMID: 20338229
PMCID: PMC3126678
NLM abbreviation: Gene
ISSN: 0378-1119
eISSN: 1879-0038
Grant note: P20 RR020152-04 / NCRR NIH HHS R01 CA138656 / NCI NIH HHS R01 CA138656-02 / NCI NIH HHS P20 RR020152 / NCRR NIH HHS
Language: English
Date published: 06/15/2010
Academic Unit: Stead Family Department of Pediatrics; Medical Genetics and Genomics
Record Identifier: 9984093495302771

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