An 8q24 gene desert variant associated with prostate
cancer risk confers differential in vivo activity
to a MYC enhancer
Nora F. Wasserman, Ivy Aneas, and Marcelo A. Nobrega1
Department of Human Genetics, University of Chicago, Chicago, Illinois 60637, USA
Genome-wide association studies (GWAS) routinely identify risk variants in noncoding DNA, as exemplified by reports of
multiple single nucleotide polymorphisms (SNPs) associated with prostate cancer in five independent regions in a gene
desert on 8q24. Two of these regions also have been associated with breast and colorectal cancer. These findings implicate
functional variation within long-range cis-regulatory elements in disease etiology. We used an in vivo bacterial artificial
chromosome (BAC) enhancer-trapping strategy in mice to scan a half-megabase of the 8q24 gene desert encompassing the
prostate cancer-associated regions for long-range cis-regulatory elements. These BAC assays identified both prostate and
mammary gland enhancer activities within the region. We demonstrate that the 8q24 cancer-associated variant rs6983267
show that the cancer risk allele increases prostate enhancer activity invivo relative to the non-risk allele. This allele-specific
enhancer activity is detectable during early prostate development and throughout prostate maturation, raising the pos-
efficient strategy to build experimentally on GWAS findings with an in vivo method for rapidly scanning large regions of
noncoding DNA for functional cis-regulatory sequences harboring variation implicated in complex diseases.
[Supplemental material is available online at http:/ /www.genome.org.]
Genome-wide association studies (GWAS) routinely implicate
variation within gene deserts and other types of noncoding DNA
in the etiology of disease (Houlston et al. 2008; Silverberg et al.
2009; Yang et al. 2009; Liu et al. 2010). A recent meta-analysis of
;1200 disease-associated single nucleotide polymorphisms (SNPs)
found that in 40% of cases, known exonic sequences were absent
from the associated linkage disequilibrium (LD) blocks (Visel et al.
2009). While the presence of nonannotated transcripts or non-
coding RNAs may explain some of the noncoding disease associ-
ations, these observations also have been interpreted as evidence
alter the activity of long-range cis-regulatory elements controlling
gene expression. Enhancers are one such type of long-range ele-
ment, functioning over up to megabase-long genomic distances to
regulate the temporal and tissue-specific expression patterns of
their target gene(s) (Nobrega et al. 2003). A large number of genes
be controlled by an array of enhancers, with each individual cis-
regulatory element driving a subset of its gene’s entire expression
profile (Carroll 2008). This modular nature of enhancer activity
makes them ideal candidates for involvement in complex diseases,
as functional variants in an individual cis-element would result in
changes to gene expression only in specific organs/tissue types.
Despite the plethora of GWAS signals implicating noncoding
regions in complex disease risk, strategies to experimentally follow
up on such findings are lacking. This deficiency stems principally
from the difficulty in identifying functional noncoding sequences
that map remotely from their target genes. Programs such as
ENCODE have been addressing this deficiency by developing and
applying technologies to identify these elusive types of long-range
regulatory elements (The ENCODE Project Consortium 2007).
While these technologies have been invaluable in the identifica-
tionofputativefunctionalnoncoding sequences, theyrelyheavily
on cell culture and other in vitro and in silico methodology to
identify and experimentally validate enhancers and other ele-
ments. Thus, although these techniques are ideal for functionally
following up on noncoding GWAS results when the relevant cell
type of interest is obvious and accessible, problems can arise if the
putative element under investigation imparts its transcriptional
regulatory effects in a cell type of unpredicted origin or one that is
not amenable to routine culture. Necessary, but lagging, is the de-
velopment of simpler in vivo strategies that can concurrentlyquery
the spatial and temporal properties of functional cis-regulatory se-
quences within large segments of noncoding DNA. Our goal in this
study is to describe one such strategy for following up on GWAS
results, and to test its ability to uncover noncoding risk variants in
loci associated with complex diseases.
A striking example of GWAS implicating noncoding variants
in the etiology of complex diseases can be seen on chromosome
8q24, wherenumerousstudieshave reportedassociations between
multiple types of cancer—including prostate, colorectal, breast,
and urinary bladder—and variants concentrated within 620 kb of
a 1.2-Mb gene desert (Amundadottir et al. 2006; Easton et al. 2007;
Gudmundsson et al. 2007; Haiman et al. 2007; Tomlinson et al.
2007; Zanke et al. 2007; Ghoussaini et al. 2008; Kiemeney et al.
2008; Al Olama et al. 2009). Evidence for prostate cancer associa-
tion within the region is particularly strong, with five distinct LD
blocks spanning a 440-kb interval on 8q24 harboring risk variants
(Fig. 1A, all shaded regions; Ghoussaini et al. 2008; Al Olama et al.
2009). One of these prostate cancer-associated variants, rs6983267,
is independently associated with colorectal cancer (Fig. 1A, green;
Tomlinson et al. 2007), and a second prostate cancer-associated LD
Article published online before print. Article and publication date are at
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20:1191–1197 ? 2010 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/10; www.genome.org
block harbors a distinct SNP (rs13281615) that shows association
with breast cancer (Fig. 1A, pink; Easton et al. 2007). Although no
well-annotated genes lie within this interval, the independent
associated variants (or linked functional elements within the as-
sociated regions) may all be regulating the expression patterns of
a single gene involved in cancer tumorigenesis and/or progression
in various tissue types. The proto-oncogene MYC lies immediately
downstream of this gene desert, raising the possibility that the
associated regions of risk may harbor long-range cis-regulatory el-
ements involved in the tissue-specific transcriptional regulation of
MYC expression; under this hypothesis, each distinct association
interval mightharbora functional noncoding elementinvolvedin
regulating MYC expression in the corresponding tissue type for
each implicated cancer. A summary of the 8q24 gene desert and its
numerous cancer loci is shown in Figure 1. Here, we have chosen
to specifically focus on the multiple independent associations
between this 8q24 gene desert and prostate cancer.
Encoding a well-known transcription factor essential to the
regulation of cell proliferation and growth, MYC is up-regulated at
both the mRNA and protein levels in aggressive prostate cancers
(DeMarzo et al. 2003). In addition, copy-number analyses in pros-
MYC as the most common recurrent region of chromosomal gain
(Lapointe et al. 2007). These findings show that prostate cancers
employ multiple mechanisms for achieving MYC overexpression,
through transcriptional up-regulation or through amplification of
gene copy number. We hypothesized that variation within MYC’s
long-range cis-regulatory elements could disrupt the quantitative,
temporal, or spatial expression patterns of MYC in the prostate,
possibly underlying the GWAS signals identified in the 8q24 gene
desert. In this study, we describe how an in vivo bacterial artificial
chromosome (BAC) enhancer-trapping strategy efficiently scan-
ned the 8q24 gene desert for cis-regulatory sequences, and report
on the identification of both prostate and mammary gland en-
prostate enhancer interval, showing that it harbors the prostate
cancer risk SNP rs6983267, and demonstrate that the two resul-
tant allelic variants display functionally polymorphic prostate
enhancer properties in vivo.
Surveying the regulatory landscape of the 8q24 gene desert
To initially examine the 8q24 gene desert for regulatory elements,
we surveyed the region using a broad-scale BAC scan approach
shown to be associated with prostate cancer (all shaded regions; blue denotes a prostate-only association), with one locus independently associated with
breast cancer (pink) and a second associated with colorectal cancer (green). (B) Breast cancer–associated region, (CR) colorectal cancer–associated region,
(P) prostate cancer–associated region. (Blue circle) MYC, (red asterisk) SNP rs6983267. (Below) The three human lacZ-tagged BACs encompassing the
reference). (B) The male genitourinary apparatus in P8 mice, shown as a cartoon (left) and in wild-type, nontransgenic mice (right). (Dashed line, right)
staining in the SV and DD. (C) Representative P8 prostates from transgenic mice containing BAC RP11-124F15 or CTD-2533C10 showing prostate and
urogenital apparatus enhancer activity. (Dashed lines) Outlines of prostates. (D) The mammary gland in midgestational pregnant females, shown as
a cartoon (left) and in wild-type, nontransgenic mice (right). The enlargement (left) illustrates a lymph node, ducts, and alveoli and in a mammary fat pad.
(LN) Lymph node, (MG) mammary gland. (E) Representative mammary fat pad from a day 14.5 pregnant female harboring BAC RP11-124F15.
1192 Genome Research
Wasserman et al.
(Spitz et al. 2003). This strategy allows for the rapid and effective
examination of large genomic regions for cis-regulatory elements,
and can be readily applied to any locus of interest. We identified
three overlapping human BACs encompassing the prostate cancer
risk regions (Fig. 1A), which together span 480 kb of noncoding
DNA. Each BAC carried the prostate cancer-associated risk haplo-
type and was tagged through a Tn7 transposon-mediated random
minimal promoter (Spitz et al. 2003). The transposon-mediated
insertion was performed using simple, commercially available kits
(see Methods) and occurs in vitro; the protocol yields rapid results
and can be easily scaled up for the simultaneous tagging of nu-
The lacZ cassette integration converts the BACs into en-
hancer-trapping systems, whereby any long-range enhancer(s)
contained within each ;180-kb BAC can act upon the reporter
gene to drive tissue- and temporal-specific beta-galactosidase ex-
pression. Any enhancers present within a given BAC are then si-
multaneously interrogated using a reporter assay system, allowing
for the concurrent examination of large genomic regions for
functional noncoding elements. The design of overlapping BACs
aids in the efficiency of the system to narrow the critical region of
to uniquely contained sequences; conversely, identical expression
patterns present in overlapping BACs suggest that the functional
element driving beta-galactosidase expression must be contained
in the shared genomic region. Modified BACs were analyzed by
of the Tn7b-lacZ reporter cassette. To mitigate any possible effects
of unknown insulator or silencer elements within the BAC se-
quence, we selected clones with at least two Tn7b-lacZ integration
events. Each BAC was then injected into fertilized mouse oocytes
to generate transgenic mice in accordance with IACUC regulatory
standards. For each BAC, a minimum of two independent trans-
genic founders were obtained and studied; this is necessary to
overcome potential position-dependent expression effects result-
ing from random integration of the transgene (BAC).
We assayed lacZ expression at multiple points in prostate or-
ganogenesis and maturation; postnatal days 0 and 8 (P0 and P8)
during prostate development, and P21, when prostate maturation
stage, prostates were dissected and stained for beta-galactosidase
expression using X-gal (Fig. 1B,C; Kothary et al. 1989).
These in vivo BAC transgenic reporter assays identified pros-
tate enhancer activity contained within the 8q24 gene desert (Fig.
1C). While we did not observe beta-galactosidase prostate expres-
sion in BAC CTD-2506D10 transgenic mice (12 independent
transgenics), animals harboring BACs CTD-2533C10 and RP11-
124F15 displayed beta-galactosidase prostate expression at days P0
(data not shown), P8 (Fig. 1C), and P21 (data not shown). As il-
lustratedin Figure1C, thebeta-galactosidaseexpressiondomainof
both BAC RP11-124F15 and BAC CTD-2533C10 extends to other
components of the urogenital system, including the coagulating
glands, urethra, and the lining of the urinary bladder. While the
seminal vesicles and ductus deferens also exhibit X-gal staining,
we and others observed this expression pattern in both wild-type
(Fig. 1B) and transgenic animals, reflecting the presence of en-
dogenous beta-galactosidase in these structures (Wang et al. 2002;
Krajnc-Franken et al. 2004). As 80% of the prostatic ducts are
that the enhancer(s) contained within these two BACs are active
both during and after prostate organogenesis and maturation.
Because some of the prostate cancer-associated regions also
chose to additionally assay the mammary glands, colon, and rec-
tum of those animals transgenic for BACs containing the relevant
regions (BAC RP11-124F15 for breast cancer, and both BACs RP11-
124F15 and CTD-2533C10 for colorectal cancer). Mammary
glands were examined at embryonic day 14.5 (E14.5), when the
virgin females with mature branched glands, and in prelactating
females 14 d after conception, when the mammary gland un-
dergoes extensive hyperplasia and tissue remodeling (Hens and
Wysolmerski 2005; Oakes et al. 2006; Sternlicht 2006).
We observed in vivo mammary gland enhancer activity in
mice transgenic for BAC RP11-124F15 (Fig. 1E), which harbors
associated intervals for not only prostate but also breast and co-
lorectal cancer. Transgenic animals displayed beta-galactosidase
expression in the epithelial compartment—ducts and alveoli
(Hennighausen and Robinson 2005)—of the mammary glands of
midgestational pregnant and 11-wk-old virgin females (Fig. 1E;
data not shown). No enhancer activity was seen in E14.5 embryos.
Of note, Jia et al. (2009) recently identified a noncoding element
within this region capable of in vitro enhancer activity in breast
cancer cell lines; this element should be viewed as a strong can-
didate for the mammary gland activity we see in vivo.
Characterizing the prostate enhancer
We next aimed to refine the location of the prostate enhancer(s)
within the BACs driving prostateexpression.Because of the highly
similar reporter expression patterns obtained from BACs RP11-
124F15 and CTD-2533C10, including prostate, coagulating gland,
and urethral/bladder lining, we hypothesized that our BAC trans-
genic assayswereidentifyinga singleprostateenhancer within the
59-kb shared genomic segment of these two BACs. Interestingly,
one of the most strongly associated prostate cancer risk SNPs,
disrupts an evolutionarily conserved sequence (Fig. 1A).
To directly test the rs6983267-containing evolutionarily
conserved element for regulatory potential in vivo, we cloned a
5-kb DNA fragment containing each allele of this SNP in a lacZ
reporter cassette using Invitrogen’s Gateway cloning system
(Kothary et al. 1989). Transgenic mice harboring either the risk or
the non-risk variant of rs6983267 were generated and analyzed.
We determined that the conserved sequence containing the
led to consistent, stronger beta-galactosidase expression in pros-
tates and coagulating glands than the non-risk allele, rs6983267-T,
in P0, P8, and P21 transgenic mice (Figs. 2A,B, 3B,C). The expres-
sion pattern driven by the rs6983267-G risk allele in three in-
dependent mousetransgeniclinesclosely resembled that observed
in BACs RP11-124F15 and CTD-2533C10—both of which also
harbor the risk allele. In contrast, the rs6983267-T non-risk allele
led to weakened prostate and coagulating gland expression in
three independent transgenic lines (Fig. 2B). For each allelic vari-
ant evaluated, those transgenic founders exhibiting enhancer ac-
tivity showed highly concordant beta-galactosidase expression in
the prostate, with a clear qualitative difference between the risk
and non-risk variants.
To test whether this spatial reporter expression pattern of the
rs6983267-containing enhancer correlates with endogenous MYC
expression in prostate and other components of the urogenital
Allelic-specific enhancer has prostate cancer SNP
system, we performed whole mount in situ hybridizations using
a full-length Myc probe in mouse prostates at P8 (Wilkinson and
Nieto 1993). We observed Myc expression in the male genitouri-
nary apparatus, including the prostate, in a pattern closely mim-
icking the reporter expression of the rs6983267-G enhancer and
BACs CTD-2533C10 and RP11-124F15, both of which harbor the
G risk allele as well (Fig. 2C).
This same prostate enhancer that we have characterized also
has been shown to act as an allelic-specific long-range MYC en-
hancer in colorectal cancer cells (Jia et al. 2009; Pomerantz et al.
2009a; Tuupanen et al. 2009; Wright et al. 2010). Although we did
not observe colorectal enhancer activity in our initial BAC screen
of the region, we again assayed transgenic animals harboring ei-
ther the risk or non-risk rs6983267-containing enhancer element
for in vivo enhancer activity in the colorectal area at three de-
velopmental time points. We observed no beta-galactosidase
expression in E14.5 intestines for either construct tested, and co-
lorectal X-gal staining at P8 and P21 was indistinguishable be-
tween wild-type mice and transgenic animals harboring either
enhancer variant (Supplemental material). Strong endogenous
beta-galactosidase expression is observed
in intestines of both wild-type and trans-
genic animals starting at E15.5, limiting
our ability to identify in vivo colorectal
enhancers in late embryogenesis and
postnatally. These findings highlight the
difficulty in assaying postnatal in vivo
intestinal enhancers using lacZ reporter
Investigations into the embryonic
activity of the rs6983267-containing
element demonstrated that while this
enhancer has several spatial domains of
expression, its allele-specific activity is
restricted to the prostate and coagulat-
ing glands. Both the rs6983267-G and
rs6983267-T enhancer elements drove
expression in several spatial domains of
E11.5 and E14.5 embryos, with no ap-
parent allelic-specific enhancer activity
(Fig. 3A). Transgenics harboring either
haplotype variant showed similar X-gal
staining in the limbs and tail at E11.5, consistent with previously
reported patterns (data not shown; Tuupanen et al. 2009). We also
observed enhancer activity in the developing urinary bladder,
genital tubercle, and limbs in the E14.5 embryos. This pattern,
which precedes prostate development, is also indistinguishable
between the allelic variants of this enhancer (Fig. 3A).
Taken together, our data posit that the rs6983267-containing
enhancer is part of MYC’s regulatory landscape, and that the var-
iant within this enhancer may increase the risk of prostate cancer
through its role in allelic-specific control of MYC expression in the
The BAC enhancer-trapping strategy that we employed allowed us
noncoding DNA for cis-regulatory elements. We effectively
screened a half-megabase genomic interval in vivo using only
three constructs, identifying the existence of mammary gland and
prostate enhancers in the interval associated with each respective
plasmids driven by either the G (risk) allele (A) or T (non-risk) allele (B) are shown at P8. (Dashed lines) Outlines of prostates; (CG) coagulating glands. The
prostate cancer risk allele leads to consistently stronger beta-galactosidase expression in prostates and coagulating glands than the non-risk allele in vivo.
(C) MYC in situ hybridization at P8 correlates with the reporter expression pattern driven by the rs6983267-containing enhancer.
SNP rs6983267 mediates allelic-specific enhancer activity in mouse prostates. Three independent transgenic founders harboring reporter
Representative G (risk, top) and T (non-risk, bottom) transgenics are shown at a series of developmental
time points. (A) E14.5 transgenic embryos exhibit beta-galactosidase expression in the genital tubercle
and limbs, with no apparent allele-specific enhancer activity. (GT) Genital tubercle. (B,C) Allele-specific
coagulating gland beta-galactosidase expression qualitatively stronger in the risk allele (top) line than
the non-risk variant (bottom). (CG) Coagulating gland, (P) prostate.
The rs6983267-containing enhancer demonstrates distinct temporal regulatory abilities.
Wasserman et al.
cancer type. We believe that this methodology provides a signifi-
cant advance to current genomic techniques for following up on
GWAS results in noncoding regions, as it can be easily adapted to
examine loci in vivo on a megabase scale. As demonstrated by our
results, this strategy can be used to concurrently identify spatially
be useful in refining the critical regions for enhancer mapping,
while still permitting the use of a whole-systems, in vivo animal
These relatively straightforward BAC transgenic reporter as-
says also provide a way to more closely approximate the genomic
context of relevant enhancers. By testing ;200 kb of sequence
simultaneously, enhancers are assayed in a context much closer to
their true genomic environment, one where they are subjected to
(largely unknown) modifications by neighboring repressors, in-
sulators, chromatin changes, and/or various other interactions
with nearby cis sequences. In traditional plasmid-based reporter
assays, this important genomic context is lost. We conducted our
clone selection strategy so as to minimize the potential negative
effects of such insulators or repressors; tagged BACs containing at
each end of the BAC sequence—were selected for experimental
use. We hypothesized that this would diminish false-negative re-
sults caused by repressive elements in a single-copy integration
clone. When compared with BACs tagged with just a single Tn7b-
lacZ cassette, we observed more reproducible results in mice
transgenicfor BACs harboringtwo Tn7b-lacZ integrations(M.A.N,
Because we observed the same urogenital system spatial pat-
tern of expression in both of the overlapping BACs tested, we de-
duced that the enhancer was within the small interval shared be-
tween those BACs. However, it is possible that other prostate
enhancers also exist within the BACs we tested. To formally ex-
clude this possibility, other approaches could have been used, in-
cluding the analysis of additional enhancer-trapping BACs with
complementary overlapping patterns. Alternatively, BAC recom-
bineering could have been employed to specifically delete our
known enhancer from the BACs assayed. Both approaches are
logical follow-ups to the in vivo BAC transgenic reporter assays,
in their genomic environments.
Recent studies have reported on the colorectal and prostate
enhancer activities of the rs6983267-containing sequence we de-
scribe here (Jia et al. 2009; Pomerantz et al. 2009a; Tuupanen et al.
2009; Sotelo et al. 2010; Wright et al. 2010). Using a combination
as possessing attributes of an enhancer, including specific chro-
matin modifications and binding of transcription factors. Several
groups have demonstrated that in colorectal cancer cell lines,
TCF7l2 (TCF4) binds preferentially to the risk allele (rs6983267-G)
of this enhancer (Pomerantz et al. 2009a; Tuupanen et al. 2009;
Wright et al. 2010). Reports regarding the enhancer properties of
this sequence in prostate cancer cell lines have been mixed, how-
ever. Whentestedin LNCaPandPC3 prostatecancercelllines, this
sequence displayed enhancer properties only in the former, pos-
sibly due to the PC-3 line’s lack of androgen receptor expression
(Jia et al. 2009). In a second study, this rs6983267-containing en-
hancer was unable to drive luciferase expression above promoter-
only levels in LNCaP or PC-3 cells, unless cells were cotransfected
with Tcf4 and beta-catenin expression vectors (Sotelo et al. 2010).
Under those conditions, the rs6983267-containing element dem-
the non-risk rs6983267-T variant driving stronger expression than
the risk rs6983267-G allele.
Our in vivo results—showing the cancer risk allele demon-
strating stronger enhancer potential than the non-risk allele—
et al. 2009a; Tuupanen et al. 2009; Wright et al. 2010), and are con-
cordant with MYC’s known role as a proto-oncogene. Our whole-
animal experimental strategy obviated the experimental variation
added by cell lines to clearly show that this element is a functional
prostate enhancer in vivo, while also adding the ability to in-
vestigate enhancer activity throughout organogenesis. We believe
that this broad spatial and temporal characterization of regulatory
potential is ideally afforded by in vivo experimentation, and pro-
pose this as the standard in the follow-up to GWAS risk variants
implicated in human disease.
The rs6983267-containing element physically interacts with
MYC’s promoter in both colorectal cancer and prostate cancer cell
MYC expression in these two tissue types (Pomerantz et al. 2009a;
Sotelo et al. 2010; Wright et al. 2010). Despite these compelling
findings and the fact that altered MYC expression has been impli-
cated repeatedly in the pathogenesis of prostate cancers (Williams
et al. 2005), no association has been seen between rs6983267 ge-
notype and MYC mRNA levels in normal prostate cells or prostate
correlation implies that steady-state MYC mRNA levels in adult
prostate tissue may not be the correct biological entity underlying
risk. Our findings demonstrate that the rs6983267-containing
enhancer exhibits differential in vivo activity throughout prostate
organogenesis, and raise the possibility that this variant asserts
its influence on prostate cancer risk long before tumorigenesis
occurs. With widely varying risk allele frequencies in different
Americans (HapMap, merged Phase 1, 2, and 3 frequencies)—this
SNP may also have an effect on the population prevalence of both
prostate cancer and colorectal cancer (Jemal et al. 2009).
We have described how a noncoding SNP strongly associated
with disease can in fact alter the in vivo activity of its encom-
passing cis-regulatory element, suggesting a possible impact on
cancer risk before tumorigenesis actually occurs. Although further
studies are warranted, our in vivo temporal data hint at an un-
derlying molecular explanation for this nongenic SNP’s contribu-
tion to prostate cancer risk. These findings emphasize the notion
that thorough investigations into the regulatory impact of poly-
morphisms are an indispensable component to the functional
follow-up of GWAS scans, andstress the importance of conducting
these experiments using in vivo systems.
Transposon-mediated BAC modification
BACs CTD-2506D10, RP11-124F15, and CTD-2533C10 were
2003). BAC DNA was extracted by using the Nucleobond AX Kit
(Macherey-Nagel). Twenty nanograms of Tn7b-lacZ vector was
mixed with 20–40 ng of BAC DNA, GPS buffer, and TnsABC trans-
37°C. Start solution was added and the reaction was extended for
1 h. After heat inactivation for 10 min at 75°C and a 1-h dialysis,
electrocompetent DH10B cells were transformed with 2 mL of the
transposition reaction. Cells were plated on LB agar containing
Allelic-specific enhancer has prostate cancer SNP
20 mg/mL kanamycin and 20 mg/mL chloramphenicol. Positive
colonies were first identified by polymerase chain reaction (PCR)
using beta-globin and lacZ primers (Tn7b-lacZ beta-globin F: AGCA
TCTATTGCTTACATTTGC; Tn7b-lacZ lacZ R: ATAGGTTACGTTGG
TGTAGATGG). Modified BAC clones were then digested with NotI
and separated by pulsed-field gel electrophoresis overnight on a 1%
agarose gel to determine the number of copies and the position(s)
of the integrated Tn7b-lacZ cassette. Clones with two copies of the
cassette were chosen for further analysis to minimize the possible
influence of silencer or insulator elements with the BACs.
lacZ plasmid generation
The 5 kb of sequence surrounding the rs6983267-containing
conserved element was PCR amplified from human genomic DNA
heterozygous for the rs6983267 SNP (rs6983267 F: TCTTGACCTG
ATTGCTGAAAAAT; rs6983267 R: TCTGGGGGTGAGTTAAATGA
TAA). The fragment was then purified using the QIAquick PCR
entry vector (Invitrogen). Colonies were analyzed by restriction
enzyme analysis for successful fragment insertion, and positive
clones were sequenced to determine the allelic status of SNP
rs6983269 (rs6983267-seq F: TAGACACCAAGAGGGAGGTATCA;
rs6983267-seq R: CCAGGTTAAAGGAAACTGAACTG). Clones con-
taining sequence harboring both the risk (G) and non-risk (T)
rs6983267 allele were transferred to a Gateway-HSP68-lacZ reporter
vector using the LR recombination reaction (Invitrogen) (Poulin
et al. 2005). All plasmids were again verified by restriction analysis
and direct sequencing prior to pronuclear mouse injections.
Production of transgenic mice
Tn7b-lacZ tagged BAC DNA was purified using the Nucleobond
BAC 100 Kit (Macherey-Nagel), rehydrated in injection buffer (10
mM Tris at pH 7.5; 0.1 mM EDTA), and diluted to a concentration
of 2 ng/mL. BAC DNA was injected in its circular form.
Plasmid DNA was purified using the Plasmid Maxi Kit (Qia-
gen), and 50 mg of each plasmid was digested with SalI to excise
the vector backbone. Following a gel purification step using the
QIAquick Gel Extraction Kit (Qiagen), the DNA to be injected was
further purified using a standard ethanol precipitation. The puri-
at pH 7.5; 0.1 mM EDTA), and its concentration was determined
fluorometrically and by agarose gel electrophoresis. The DNA was
diluted to a concentration of 2 ng/mL. Purified BAC and plasmid
DNA were then used for pronuclear injections of CD1 mouse em-
bryos in accordance with standard protocols approved by the
University of Chicago.
For the Tn7b-lacZ tagged BACs, multiple stable transgenic
lines were generated for each construct, and F1animals were ana-
lyzed for each line at multiple postnatal developmental time
points. BAC CTD-2506D10 DNA injections yielded 12 indepen-
dent lines (0/12 positive for prostate beta-galactosidase expres-
sion); injections of RP11-124F15 and CTD-2533C10 both resulted
in two independent beta-galactosidase-expressing lines.
For the rs6983267-containing enhancer plasmid, a total of
three beta-galactosidase-expressing independent transgenics was
obtained for rs6983267-G; three beta-galactosidase-expressing
independent transgenic animals/lines were also obtained for
rs6983267-T. For severalof these independent lines, the F0animals
themselves were analyzed at P8; this excluded any analysis of the
line at other time points. For the risk allele, rs6983267-G, we
obtained two F0animals positive for beta-galactosidase expression
in the prostate. The third independent rs6983267-G transgenic
one F0transgenic animal was obtained; the remaining two in-
dependent transgenics were maintained as stable lines.
Mouse in vivo transgenic reporter assay
Prostates and mammary glands were harvested from mice at P0, P8,
and P21 and dissected into cold 100 mM phosphate buffer (PBS)
(pH 7.3), followed by 30–45 min of incubation with 4% parafor-
maldehyde at 4°C. E14.5 embryos were incubated in 4% parafor-
maldehyde for 2 h. Tissues were then washed two times for 20 min
with wash buffer (2 mM MgCl2; 0.01% deoxycholate; 0.02% NP-40;
100 mM phosphate buffer at pH 7.3), and stained for 18 h at room
temperature with freshly made staining solution (0.8 mg/mL X-gal;
timesfor 20 min inPBS and post-fixed in 4%paraformaldehyde. For
each animal analyzed, tail samples were taken at the time of dis-
section and DNA was isolated through the addition of lysis buffer
(100 mM Tris-HCl at pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl,
and 1 mg/mL proteinase K) and incubation overnight at 55°C.
Genotyping was performed by PCR with primers within the re-
porter cassette/vector (using beta-globin and lacZ primers for the
Tn7b-lacZ tagged BACs, rs6983267-seq primers for the plasmids).
kept constant between structure- and aged-matched samples. Im-
ages displayed in the paper were generated using an image pro-
of extended depth of field images. Multiple pictures of each
structure were taken at varying depth of fields and then compu-
tationally integrated; the focus areas are blended to create a com-
posite high-resolution image with an extended depth of field. This
allowed for the production of images where all the multiple plains
of the urogenital apparatus appear well focused and defined.
In situ hybridization
In situ hybridization analysis on whole P8 prostates using digox-
igenin-labeled Myc antisense and sense riboprobes was performed
according to standard protocols (Wilkinson and Nieto 1993). The
probes were generated from a full-length mouse Myc cDNA clone
(IMAGE ID 3962047). Staining was performed for 48 h, and the
stained prostates were then transferred to 10% buffered formalin
phosphate prior to imaging.
We thank Franc xois Spitz for kindly providing us with the beta-
globin-Tn7 vector and Linda Degenstein for assistance in gener-
ating transgenic animals. We also thank James Noonan, Rick Kit-
tles, and Gail Prins for their consultation and support. The uro-
genital apparatus and mammary gland cartoons in Figure 1, B and
D, were kindly drawn by John Westlund. This work was partially
supported by grant HG004428 (M.A.N.). N.F.W. is supported by
a DoD Prostate Cancer Training Award (PC094251).
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Received January 15, 2010; accepted in revised form June 3, 2010.
Allelic-specific enhancer has prostate cancer SNP