Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Summary

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

Abstract

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique — usually untranslated — first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

Introduction

More than half of human genes are regulated by alternative promoters¹. Each alternative promoter can contain regulatory elements that may be different from those in other alternative promoters. The promoter(s) utilized in one tissue may differ from those used in another tissue. For example, it is possible that activation of a given signaling pathway may trigger the alternative promoter for a gene utilized in one tissue, yet have no effect on or repress a separate alternative promoter for the same gene that is utilized in another tissue.

Expression of the Bcrp1 gene is governed by alternative promoters. Bcrp1 is the murine ortholo....

Protocol

1. In Silico Prediction of Alternative First Exons of Bcrp1 Using BLAST Analysis of Mouse EST Database

Note: This protocol describes how to search the mouse expressed sequence tag (EST) database for ESTs with sequence similarity to exon 2 of Bcrp1 (which contains the translational start site) and then how to align the matching EST sequences to genomic sequences to ascertain their location in the mouse Bcrp1 gene relative to the 5' end of Bcrp1 exon 2.

1. Obtain the sequence for mouse Bcrp1 exon 2 by inputting the mRNA reference sequence ID (NM_011920.3) into the search window of the Ensembl Genome Browser¹⁷,....

Results

Identification of Bcrp1 alternative promoter utilization in mouse testis by analysis of leader exons

When the EST database at NCBI was probed (April 2015) using the steps outlined in Protocol 1, the ESTs found that were contiguous with the 5' end of exon 2 in Bcrp1 mRNA are shown in Table 1, along with their position in genomic DNA relative to .......

Discussion

The majority of the methods and representative results presented are described in previous work entitled "Bcrp1 transcription in mouse testis is controlled by a promoter upstream of a novel first exon (E1U) regulated by steroidogenic factor-1," which was published in 2013⁵. In addition to the representative results depicted here, the previous paper provided estimates of alternative first exon utilization using 5'-RACE PCR and RT-PCR methodology. Furthermore, in-silico identificati.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
COMMERCIAL BIOLOGIC MATERIALS AND KITS:
First Choice RLM-RACE kit	Ambion Inc., Austin, TX, currently available through Life Technologies	AM1700	5'-RACE PCR kit
TOPO TA cloning kit	Life Technologies	K450001	This kit contains the TOP10 chemically competent E. coli bacteria.
T4 DNA ligase quick ligation kit	New England Biolabs	M2200
Faststart high-fidelity Taq- DNA polymerase	Sigma-Aldrich/Roche	3553400001/RMB-4738284001	Contains a high-fidelity Taq polymerase enzyme blend
XtremeGENE HP DNA transfection reagent	Roche	6366236001
Dual-luciferase reporter assay kit	Promega	E1910	Includes the pGL3-basic empty reporter vector (firefly luciferase), pRLTK renilla luciferase expressing vector, and other control vectors.
QIAprep	Qiagen	27104	For plasmid miniprep - isolation and purification of plasmid DNA from bacterial colonies
pCR2.1 vector	part of the TOPO TA cloning kit
pGL3-basic luciferase containing vector	Promega	E1751
pRL-TK Renilla luciferase expressing vector	Promega	E2241
Bacterial artificial chromosome	BACPAC Resources Center, Children’s Hospital Oakland Research Institute, Oakland, CA	RP23-285A12 and RP24-314E24	http://bacpac.chori.org/clones.htm
REAGENTS/CHEMICALS/MEDIA/SUPPLIES
TRIzol	Life Technologies	15596026
Wizard® SV Gel and PCR Clean-Up System	Promega	A9281	Useful for purifying PCR products following digestion with restriction endonucleases
CRYOVIALS	Denville Scientific	V9012
SOFTWARE:
Ensembl software	Ensembl project	http://Ensembl.org	The Ensembl project produces genome databases for vertebrates and other eukaryotic species, and makes this information freely available online.
Blast software	NCBI	http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD= Web&PAGE_TYPE=BlastHome
Primer 3 software	Simgene	http://simgene.com/Primer3	Useful for designing primers for 5'-RACE PCR
NCBI Nucleotide database	NCBI	http://www.ncbi.nlm.nih.gov/nucleotide/
CELL LINES:
TM4 murine Sertoli cells	ATCC, Manassas, Virginia	CRL-1715™
INSTRUMENTS:
Luminometer	Turner Biosystems	TD-20/20	This is a relatively inexpensive, manually operated luminometer. Automated systems are also available from the same manufacturer that utilize 96 well plates.
NanoDrop spectrophotometers	Thermo Scientific, Inc.	NanoDrop 2000	Spectrophotometer can use very small quantities of sample
DU 800 UV/VIS spectrophotometer and Nucleic Acid Analysis II software	BeckmanCoulter Inc, Fullerton, CA	DU 800