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 orthologue of the human Breast Cancer Resistance Protein (BCRP) gene. BCRP is an ATP-binding cassette (ABC) transporter, formally designated ABCG2^2,3. As an apical plasma membrane protein, BCRP/Bcrp1 functions to efflux a wide variety of natural and xenobiotic substrates^3,4. In humans and mice, BCRP/Bcrp1 is highly expressed in pharmacologically relevant organs such as liver (bile canaliculi), intestine, and kidney, as well as organs with blood-tissue barriers such as placenta, brain and testis^2,5-12. Expression of BCRP/Bcrp1 in hematopoietic and other stem cells, including cancer stem cells, may confer resistance of these cells to xenobiotics and cancer chemotherapeutic drugs³.

In early work to understand the regulation of BCRP expression in normal and neoplastic cells, 5' rapid amplification of cDNA ends (5'-RACE) analysis of BCRP mRNA was performed to determine its exact transcriptional start site¹³. Not only were multiple transcriptional start sites found; also encountered were three alternative forms of the first exon, which in BCRP is untranslated. These alternative first exons — designated E1a, E1b, E1c — were expressed differently in a variety of human tissues. Two additional first exon variants were discovered in a BLAST search of the human EST database using the second exon of BCRP¹³. Four matches revealed a first exon >70 kb upstream from exon 2 which were designated E1u; four other matches revealed BCRP mRNA that lacked a first exon entirely, which were designated E1-¹³. The presence of alternative leader exons is considered to be a manifestation of alternative promoter usage¹⁴.

In mice, four alternative first exons of Bcrp1 are described that may correspond to alternative promoters that govern Bcrp1 transcription in different mouse tissues; these exons/promoters are designated E1U, E1A, E1B and E1C, and are located approximately 70, 58, 15, and 5 kb upstream from Bcrp1 exon 2^5,15. The E1A mRNA isoform was found to be predominant in murine hematopoietic stem cells, heart, lung, spleen, and brain, whereas the E1B isoform was expressed in mouse intestine, fetal liver cells, and erythroid precursor cells in bone marrow^5,15. The promoter upstream from E1B was shown to be the major alternative promoter governing Bcrp1 transcription in mouse intestine, regulated at least in part, by phospho-cyclic-AMP response element binding protein (p-CREB) and a CREB response element unique to that alternative Bcrp1 promoter¹⁶. The E1C mRNA isoform is predominantly expressed in adult murine liver and kidney⁵. The E1U isoform is undetectable in most tissues tested except for murine testis, where it is the predominant isoform expressed⁵. Bcrp1 expressed in rat testes is found in both somatic (endothelial tight junctions, peritubular myoid cells, and Sertoli cells) and germ cells (in the seminiferous endothelium, where it may protect late-stage spermatids⁴). The region upstream from E1U contains a functional response element for steroidogenic factor-1 (SF-1)⁵. Bcrp1 mRNA and protein are markedly reduced in the testes of Sertoli cell-specific SF-1 knockout mice, suggesting that Bcrp1 expression in murine Sertoli cells is controlled by SF-1⁵.

The protocols presented detail methods to detect alternative first exons of Bcrp1 in-silico, and to establish luciferase-based reporter assays for putative promoters upstream from the alternative first exons identified.

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, click on "GO." In the next screen, select a full-length sequence (contains 16 exons):
   1. In the next screen ("Transcript-based Display"), select "16 Exons." This will display a screen containing the sequence of all exons of Bcrp1. Exon 2 of Bcrp1 will read "5'-AAAGGC…TATCAA-3'." The results obtained will be similar to those shown in reference ¹⁸.
   2. Click on the "Download sequence" option. In the next screen, choose the FASTA format, and then click on "Preview." In the next screen, copy the Preview sequence of exon 2 into the clipboard.
2. Navigate to the BLAST homepage¹⁹ on the National Center for Biotechnology Information (NCBI) website²⁰.
   1. Select "Mouse" genome. Paste the exon 2 sequence from the clipboard into the query box.
   2. Select "ESTs" from the database dropdown, optimize for "highly similar sequences," and then choose "BLAST." Run time will take a few minutes. When the BLAST run is complete, the "Results" page will appear.
   3. Under the "Descriptions" subheading of the "Results" page, select "ALL," then "Download," then "FASTA (complete sequence)" in the dropdown, and then choose "Continue." A .txt file will appear; open and copy the entire file into the clipboard. The .txt file contains the sequences of all ESTs with high sequence similarity with the mouse Bcrp1 exon2 but not their position in relation to exon 2 in the Bcrp1 gene.
      Note: An analysis performed on April 15, 2015 identified 14 murine ESTs that aligned with Bcrp1 exon 2. These are listed in Table 1.
3. Identify the location of the EST sequence that is 5' to exon 2 in the Bcrp1 gene. The mouse Bcrp1 gene is located in the chromosome 6 contig NC_000072.6 (GI:372099104).
   1. On the BLAST homepage under the "Specialized Blast" subheading, select "Align two (or more) sequences using BLAST (bl2seq)."
   2. Paste the text file from the clipboard into the query box and enter 372099104 in the subject sequence box. Optimize for "highly similar sequences" under program selection, and run BLAST.
   3. Once the results window appears, view the alignments graphically by clicking on "Graphics" in the "Alignments" window. Use the right and left arrows and zoom to focus on Bcrp1/Abcg2 and the sequence alignments.
   4. Save the sequence alignments: select "ALL" in the "Descriptions" box, then under the "download" dropdown select "Hit table (CSV)," and then click on "continue." This file contains the sequence alignment of Bcrp1 exon 2 and the alignment of all the EST sequences with sequence similarity to Bcrp1 Exon2 relative to the numbering of the nucleotides in the mouse chromosome 6 contig. Each complete EST sequence might generate multiple alignments spanning regions 5' and 3' to exon 2 including the sequences overlapping with Bcrp1 exon 2.
   5. As the position of the 5' end of exon 2 will correspond to nucleotide 58,655,638 in the chromosome 6 contig, designate this as +1, and then calculate the position of the partial sequences of each EST 5' to 58,655,638. The results for the 14 ESTs are given in Table 1.
      Note: Be careful to analyze EST sequences that are 5' to +1 (i.e., have a negative nucleotide value) as potential first exons. For example, in two of the ESTs that aligned with Bcrp1 exon 2 shown in Table 1 (AI647825 and AI664571) the remaining sequence was 3' to exon 2.

2. Evaluation of Bcrp1 Alternative Promoter Function

1. Design of reporter constructs for Bcrp1 E1U, E1A, E1B and E1C promoters
   1. Using the sequence of E1U obtained from searching the EST database, designate the first 5' nucleotide of E1U as +1.
   2. Obtain a bacterial artificial chromosome (BAC) clone of mouse chromosome 6 that contains the Bcrp1/Abcg2 gene sequence and the sequence at least 100 kb upstream of the Bcrp1/Abcg2 gene (see List of Materials and Equipment).
      1. Identify a suitable reporter vector such as the luciferase reporter plasmid pGL3-Basic.
      2. Determine the multiple cloning sites in the reporter vector. KpnI, SacI, Mlu1, NheI, SmaI, XhoI, BglII and HindIII are the restriction endonuclease sites in the multiple cloning site of the pGL3-Basic vector, in the 5' to 3' orientation.
         Note: The sequence of the digestion sites is available from product catalogs of companies that produce the restriction enzymes.
      3. Identify all digestion sites in the E1U exon and the 2 kb region 5' of E1U using software programs such as DNA5. Identify at least two digestion sites in the pGL3-Basic multiple cloning site that are not found in E1U or the 2 kb upstream region.
         Note: Be sure to choose a multiple cloning site restriction site for the forward primer that is upstream of the one chosen for the reverse primer to ensure that the insert will be in the proper orientation.
   3. Prepare forward and reverse primers for PCR that contain sequences for the selected pGL3-basic multiple cloning site restriction endonuclease sites using commercial gene/primer synthesis services or primer selection software (see List of Materials and Equipment). Select a forward primer located ~2 kb upstream from the E1U sequence and a reverse primer within the E1U sequence. The primers used in the authors' previous work incorporated a SacI site in the forward primer, and an NheI site in the reverse primer, and amplified the genomic region from approximately −1,906 to +64 with the first 5' nucleotide of E1U specified as +1 (see step 2.1.1 above), and are provided in reference ¹⁶.
      1. PCR amplify the E1U genomic region using these primers, 0.01 to 1 µg of the BAC, and PCR master mix containing high-fidelity Taq polymerases (see List of Materials and Equipment) with denaturing at 95 °C for 30 sec, then 35 to 40 cycles of PCR, with extension at 72 °C (2 min), and annealing and melting temperatures based on the melting characteristics of the primers used.
         Note: The use of high-fidelity Taq polymerases is essential for sequencing promoter regions that have high GC content. When using large genomic DNA fragments such as a BAC as the template, the secondary structure of the genomic DNA may make it difficult for PCR primers to bind. If this problem arises, better PCR results may be obtained if the long genomic DNA template is sheared gently by sonication before the PCR reaction.
      2. Verify the length of the amplified PCR product by comparing against a 1 kb DNA ladder using 0.8% TAE agarose gel electrophoresis.
   4. Digest the PCR product obtained and the pGL3-Basic vector, using the restriction endonucleases specific for the restriction digestion sites introduced into the forward and reverse primers as per instructions supplied with the restriction digestion enzymes.
      1. Purify the digestion reactions using commercial SV Gel and PCR Clean-Up Systems kit (see List of Materials and Equipment), following the kit protocol²¹.
         1. Verify digestion and linearization of the vector by comparing it against the uncut vector using agarose gel electrophoresis, then cut and purify the digested vector DNA band. Measure the concentration of the purified PCR product and the purified vector using UV spectrometry (see List of Materials and Equipment).
   5. Prepare the E1U reporter construct by ligating the purified, restriction enzyme digested PCR product and pGL3-Basic firefly luciferase reporter vector (contained in the reporter assay kit; see List of Materials and Equipment) at insert : vector ratios of 11, 21 and 31, using the "T4 DNA ligase quick ligation kit" as per kit instructions²², thereby producing the E1U reporter construct, named pGL3-E1U.
      1. Use the pGL3-E1U plasmid to transform bacteria to clonally expand pGL3-E1U following instructions in the TA cloning kit (see List of Materials and Equipment).
      2. Confirm the orientation and fidelity of the insert by sequence analysis.
   6. Prepare reporter constructs for E1A, E1B and E1C using similar methodology as for E1U. Confirm the fidelity of the inserts by sequencing as in section 2.1.5.2.
      Note: The promoter inserts for E1A, E1B and E1C should be approximately −1,875/+10, −1,847/+60, and −1,904/+83 relative to the first 5' nucleotide (designated as +1) of E1A, E1B, or E1C, respectively.
2. Reporter assay methods
   Note: General strategy of reporter assays: the putative promoter (5' upstream region) of a gene is inserted into the multiple cloning site of an empty "reporter" vector that contains a "reporter gene" (e.g., firefly luciferase) downstream from the multiple cloning site. The vector is then transfected into eukaryotic cells that express the gene of interest. The trans-acting factors that promote expression of the gene of interest in these cells should activate the promoter in the transfected reporter vector, causing expression of firefly luciferase, which can be easily quantified with a luminescence assay.
   1. Select the appropriate cell line to use for the reporter assay.
      Note: To evaluate the activity of the E1U alternative promoter reporter construct, it is necessary to transfect that construct into cells that express Bcrp1 under control of the E1U promoter. The TM4 murine Sertoli cell line (see List of Materials and Equipment) expresses Bcrp1 protein and Bcrp1 mRNA containing E1U, as well as E1A, E1B, and E1C⁵. As a negative control, consider using a cell line that does not express Bcrp1 protein or mRNA.
   2. Culture the TM4 cells in 24-well plates at an initial density of 200,000 cells/well in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium with 1.2 g/L sodium bicarbonate, 15 mM HEPES supplemented with 5% horse serum, 2.5% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml), at 37 °C, 5% CO₂, as described previously⁵.
   3. Six hours after placing the cells in culture, transfect the cells with 0.2 µg of empty pGL3-basic vector or with 0.2 µg of pGL3 vector containing the −1,906/+64 E1U or −1,875/+10 E1A or −1,847/+60 E1B or −1,904/+83 E1C Bcrp1 deletion construct along with 4 ng of pRL-TK (a Renilla luciferase-expressing vector) as internal control, using a commercial DNA transfection kit and the manufacturer's protocol²³ (see List of Materials and Equipment).
   4. 30 hr following transfection, remove the growth media from the cultured transfected cells. Wash the cells once with 200 µl of PBS, then remove the wash solution.
      1. Measure firefly and Renilla luciferase activity using a commercial kit according to manufacturer's protocols²⁴.
   5. Express the activity for each tube as the ratio of the firefly luciferase activity divided by the internal (Renilla luciferase) control; overall results are usually expressed as the activity of each reporter construct relative to that of cells transfected with the empty pGL3 basic vector, which is given a value of 1.

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