Primate-specific evolution of an LDLR enhancer Page: 3 of 9
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Genome Biology 2006, Volume 7, Issue 8, Article R68 Wang et al. R68.3
PS2 enhancer functional divergence correlates with sequence constraint
Sequence analysis Functional test
Species compared Conservation P value Species assayed Relative enhancer strength
PSI PS2 PSI PS2
Human/5 primates 4.8 x 10-s 10-s Human 0.9 5.1
Human/lemur -I 0.76 Lemur ND 2.6
Human/mouse -I -0.99 Mouse ND 1.5
Human/dog 0.28 0.45 Dog ND 2.6
Conservation P values are calculated using Gumby [ 12] under the null hypothesis of evolution at the neutral (background) rate. Low P values indicate
that the null model of neutrality should be rejected, with the lowest P values identify the most significantly conserved sequences. The sequences
analyzed for human-mammal conservation or enhancer activity correspond to the Gumby predicted conserved sequence and approximately 200
base pairs of flanking sequence on either side (see Materials and methods). Enhancer strength is shown as fold increase over promoter alone in
luciferase assays in 293T cells. ND, not done.
data not shown). The promoter region was the only noncod-
ing region consistently identified as being conserved in the
three pair-wise comparisons. In contrast, multiple sequence
comparisons between human and a set of five anthropoid pri-
mate species, chosen on the basis of their evolutionary rela-
tionship using the 'phylogenetic shadowing' strategy [i5],
identified two human noncoding DNA elements, named PS
(primate specific) l and 2, which were found to be highly sig-
nificantly conserved (P approximately 10-5) in primates (Fig-
ure ib). However, they were undetected in comparisons
involving human and each of the distant species (Figure 1 and
Additional data file 1).
To confirm independently the lack of significant conservation
of the PSi and PS2 elements between human and distant
mammals, we also analyzed human-mouse alignment using a
sliding-window percentage identity conservation criterion.
We found that the human-mouse percentage identities across
PSi and PS2 were below 5o% (Figure 1 and data not shown).
This is close to the background percentage identity in aligned
intergenic DNA and is well below the threshold of 70% iden-
tity that is normally applied to the detection of conserved
functional sequences [i6]. We further verified that the phast-
Cons program  detects no conserved sequences overlap-
ping PSi and PS2 (data not shown). Although the phastCons
predictions, obtained from the UCSC Genome Browser, are in
general based on alignment of 17 mammalian and nonmam-
malian species, conservation scores in the LDLR locus reflect
only mammalian conservation because more distant genomes
exhibit very limited nonexonic alignment in this locus.
To assess quantitatively the conservation level of PSi and PS2
between human and distant mammals, we identified the
orthologous aligned counterparts of the human PSi and PS2
elements in lemur, mouse, and dog. Gumby analysis of con-
servation scores indicated that each of these nonanthropoid
primate sequences exhibited a level of similarity to the human
sequence consistent with unconstrained evolution at the neu-
tral rate (conservation P value; Table i). Together, these anal-
yses strongly suggest a lack of significant sequence constraint
between the anthropoid primate and mammalian PSi and
The human LDLR PS2 element exhibits significantly
greater enhancer activity than its mammalian
To explore the potential regulatory function of these two pri-
mate-specific conserved elements, we examined their ability
to drive reporter gene expression in both a transient transfec-
tion assay in human 293T cells and in an in vivo mouse liver
gene transfer assay . Each human element plus approxi-
mately 200 base pairs (bp) of flanking sequence on either side
was cloned upstream of the human LDLR promoter  fused
to a luciferase reporter gene. Human element PS2, but not
PSi, consistently increased luciferase expression approxi-
mately fivefold relative to the human promoter alone in both
the in vitro and in vivo assays (Figure 2). The human element
PS2 also increased luciferase expression when cloned
upstream of the generic SV4o promoter, albeit to a lesser
extent (twofold; Additional data file 3). Enhancer activity of
this element was further confirmed by the finding that
genomic region corresponding to PS2, but not PSi, is a DNa-
seI hypersensitive site in human liver cells (Additional data
file 2 and data not shown).
To explore the regulatory function, if any, of mammalian
sequences orthologous to human PS2, we cloned the PS2-
aligned sequences from lemur, mouse, and dog into the luci-
ferase reporter vector described above and compared their
activities with that of the human sequence. Despite the lack of
statistically significant sequence constraint between the
human enhancer and its lemur, mouse, and dog orthologs,
the latter three sequences exhibited enhancer activity both in
vitro and in vivo (Figure 2). The human regulatory element,
Genome 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/m1/3/: accessed April 19, 2019), University of North Texas Libraries, Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.