Primate-specific evolution of an LDLR enhancer Page: 7 of 9
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Genome Biology 2006, Volume 7, Issue 8, Article R68 Wang et al. R68.7
11061099) was PCR amplified (see Additional data file 4 for
primers used).
Transient transfection reporter assay
Cells of human embryonic kidney cell line 293T (ATCC CRL-
11268) were grown at 37 C and 5% carbon dioxide CO2 in
Dulbecco's modified Eagle's medium (ATCC), supplemented
with io% fetal bovine serum (Hyclone, Logan, UT, USA), 1-
glutamine, and penicillin-streptomycin. Cells with a passage
number below 15 were used. The cells were grown in 12-well
plates (4 x 104 cells/well) and transfected using Fugene
(Roche Molecular Biochemicals, Indianapolis, IN, USA), in
accordance with the manufacturer's protocol. Briefly, 100 ng
of each assayed plasmid and 10 ng pCMV3 (BD Biosciences,
Franklin Lakes, NJ, USA) were mixed with 1.5 l Fugene and
added to each well. Following 42-48 hours of incubation, cells
were harvested and lysed. Activity of luciferase and p-galac-
tosidase was measured using the Luciferase Assay System
(Promega) and the Galacto-Light Plus (Applied Biosystems,
Foster City, CA, USA), respectively. Luciferase activity for
each sample was normalized to the p-galactosidase assay con-
trol. For co-transfection experiments, 100 ng of the lulcif-
erase reporter gene construct, 3 ng of SREBP-2 expression
vector, and 10 ng of pCMV3 were used. Transfections were
carried out in duplicate. All experiments are representative of
at least three independent transfections.
Tail vein plasmid DNA transfer assays
Tail vein injection was performed as described by Herweijer
and Wolff ['8], following the TransITIn Vivo Gene Delivery
System (Mirus Corporation, Madison, WI, USA) protocol. Six
to nine FVB male mice (Charles River Laboratory, Wilming-
ton, MA, USA) at age 7-8 weeks were used for each reporter
gene construct. Ten micrograms of each reporter construct,
along with 2 g of pCMV3 (BD Biosciences) to correct for
delivery efficiency, were injected into each mouse. The entire
content of the syringe was delivered in 3-5 s. Animals were
killed 24 hours later, livers extracted, measured to correct for
size, homogenized, and centrifuged for 15 min at 4 C, 14,000
rpm. Activities of luciferase and p-galactosidase were meas-
ured as described above. All P values are from the two-sample
Wilcoxon rank-sum (Mann-Whitney) test using STATA
(STATA Corporation, College Station, TX, USA). All experi-
mental results are representative of two independent plasmid
DNA transfer assays.
Chromatin immunoprecipitation
HepG2 cells (ATCC HB-8o65) were cultured in DMSF
(defined medium serum free) medium for 24 hours for induc-
tion of endogenous SREBPs [31]. Chromatin immunoprecip-
itaion assays were performed as described previously [32].
Cross-linked chromatin was immunoprecipitated with a spe-
cific SREBP-2 antibody (Santa Cruz sc-8151) [8] or IgG con-
trol.N
L..
C-
-
0
U101
9.2
1-
0~Control PS2 LDLR
enhancer promoterIfflL III
Figure 5
Anti-SREBP-2 antibody specifically enriches human PS2 DNA sequences in
HepG2 cells in a ChIP assay. DNA precipitates were measured by real-
time polymerase chain reaction using primers spanning the indicated
regions. Control region (control) corresponds to the first coding exon of
the neighboring gene SPBC24, which is approximately 7.5 kilobases away
from the PS2 sequence. The results are presented as fold increase in the
enrichment of precipitated DNA by anti-SREBP-2 (anti-sterol regulatory
element binding protein-2) antibody over nonspecific IgG. Graphical
representations of the mean standard error from three independent
experiments are shown.
Sources of sequence data
Draft sequences of baboon, colobus, dusky titi, marmoset,
owl monkey, lemur, and galago bacterial artificial chromo-
somes (BACs) were determined by sequencing ends of 3 kb
subclones to 8- to 10-fold coverage using BigDye terminators
(Applied Biosystems) and assembling reads into contigs with
the Phred-Phrap-Consed suite, as described previously [33].
All BAC sequences were submitted to GenBank with the fol-
lowing species and accession numbers: baboon (Papio
hamadryas), AC14o74; colobus (Colobus guereza),
ACis54a; marmoset (Callithrix jacchus), AC14.'80; dusky
titi (Callicebus moloch), AC1446.5; owl monkey (Aotus
hybrid), AC17nn3; squirrel monkey (Saimiri boliviensis
boliviensis), AC146467; lemur (lemur catta), ACii8o69;
mouse lemur (microcebus murinus), AC17%6.6; and galago
(otolemur garnetti), AC17% 6.). Human, chimpanzee, rhe-
sus, mouse, rat, and dog sequences were downloaded from
the UCSC Genome Bioinformatics website [34].
Analysis of sequence conservation
We aligned the human LDLR locus (chrl9:11,o55,219-
11,117,169; NCBI Build 35) to its orthologs in baboon, colobus,
dusky titi, marmoset, owl monkey, lemur, mouse, and dog
using MLAGAN [35]. Because incomplete primate sequenceGenome Biology 2006, 7:R68
http://genomebi ology.com/2006/7/8/ R68
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Wang, Qian-Fei; Prabhakar, Shyam; Wang, Qianben; Moses, Alan M.; Chanan, Sumita; Brown, Myles et al. Primate-specific evolution of an LDLR enhancer, article, December 1, 2005; Berkeley, California. (https://digital.library.unt.edu/ark:/67531/metadc885857/m1/7/: accessed April 23, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.