Loss-of-Function Mutation in a Repressor Module of HumanSpecifically Activated Enhancer HACNS1
Kenta Sumiyama* and Naruya Saitou
Division of Population Genetics, National Institute of Genetics, Mishima, Japan*Corresponding author: E-mail: ksumiyam@lab.nig.ac.jp.
Associate editor: Yoko Satta

Abstract

Key words: HACNS1 cis-regulatory element, loss of function.
2001). The observation that all the 13 human-specific substitutions within the 81-bp region are G or C (11 are A or T
to G or C, and 2 are G to C) may be consistent with the ideathat BGC took place in this region (Duret and Galtier 2009).
It is possible that the 13 mutations were caused by BGC andresulted in a disruption of repressor function of the 81-bpregion (loss of function). It could eventually enhance theactivity of human HACNS1.
To see which process, gain or loss of function, is moreplausible, we conducted transgenic mice assay by using
two transgene constructs to characterize the 81-bp region
in HACNS1 that harbors 13 human-specific GC-biased substitutions. One, HACNS1-full, has an intact human HACNS1
sequence that is combined with heat shock protein 68 promoter (hsp68p) and lacZ reporter gene with SV40 polyA signal sequence. Another construct, HACNS1-D81bp, isbasically the same as HACNS1-full except a mutation that
replaced the 81-bp region that harbors 13 human-specificGC-biased substitutions with 5#-GAATTC-3# sequence. A
total of 367 and 426 fertilized eggs were microinjected with
the HACNS1-full construct and the HACNS1-D81bp construct, respectively, and they were transferred into oviductof recipient mice, and then, embryos were dissected outat E11.5. We recovered 72 and 49 E11.5 embryos for
HACNS1-full and HACNS1-D81bp constructs, respectively.
We then obtained eight lacZ-positive embryos for theHACNS1-full construct in which five showed strong to middle-level expression in limb, and three showed weak expression (fig. 1A). This result is consistent with the previously
reported pattern (Prabhakar et al. 2008), although the expressions may be slightly less intensive overall in our experiment. As for mice injected HACNS1-D81bp construct, weobtained five lacZ-positive embryos. Three of them showed© The Author 2011. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, pleasee-mail: journals.permissions@oup.com

Mol. Biol. Evol. 28(11):3005–3007. 2011 doi:10.1093/molbev/msr231

Advance Access publication September 22, 2011 3005

Letter

Genomic changes responsible for creating humanness after
divergence from the human–chimpanzee common ancestor
are of great interest to human evolutionary studies (e.g.,
Saitou 2005). Cis-regulatory mutations in noncoding genomic regions are thought to be responsible for generationof human-specific developmental programs (e.g., Carroll2003). Pollard, Salama, and king (2006) and Pollard, Salama,and Lambert (2006) found a series of short DNA sequencesthat are highly conserved in vertebrates but show accelerated
evolution only in human and named them ‘‘HAR.’’ Prabhakar
et al. (2006) and Bird et al. (2007) also conducted similar genome-wide studies. Prabhakar et al. (2008) found one suchsequence, HACNS1, 239-bp fragment located neighbor tothe GBX2 limb-expressing gene. HACNS1 includes 119-bp
HAR2 and was shown to act as a limb bud enhancer withenhanced limb enhancer activity specifically in human. Thischange was caused by 13 human-specific substitutions
within an 81-bp region and was interpreted that accumulation of these positively selected substitutions created multiple novel transcription factor (TF)–binding sites (gain offunction) and that the deposition of those facilitated thehuman-specific enhanced activity (Prabhakar et al. 2008).
However, loss of function in a repressor element within
HACNS1 can be another explanation for it. A GC-biased
gene conversion (BGC) may be an alternative explanationfor fixation of such mutations without experiencing adaptive evolution (Galtier and Duret 2007; Duret and Galtier2009; Galtier et al. 2009; Katzman et al. 2010). BGC is consequence of DNA double-strand break repair between homologous chromosomal regions, and the alleles from one
chromosome are converted to the other with a bias of A or
T to G or C (Strathern et al. 1995). Neutral or evendeleterious alleles could be fixed by BGC (Galtier et al.

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The cis-regulatory element contributed to gaining humanness is of great interest in human evolutionary studies. A humanaccelerated region exceeding neutral evolutionary rates, termed HACNS1, was recently reported as a positively selectedsequence acquiring novel TF-binding sites responsible for human-specific gain of limb enhancer function. However,another possibility is loss of function in repressor element in HACNS1. Signature of the human substitutions in the 81-bp
region infers that a GC-biased gene conversion (BGC) might create these seemingly excessive substitutions. To evaluate the81-bp function, we performed transgenic mouse assay of the HACNS1 construct lacking the 81-bp region. The deleted
construct showed similar enhancer activity to the intact human HACNS1, suggesting that the function of the human 81bp region is not an activating enhancer but rather a disrupted repressor. This result infers that loss of function in theHACNS1 81-bp region, possibly via a BGC, played an important role in human-specific evolution.

Sumiyama and Saitou · doi:10.1093/molbev/msr231

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strong to middle-level expression in limb, whereas the othertwo showed middle to weak expression (fig. 1B). We did notobserve apparent difference in expression pattern nor trend
in expression intensity between those two constructs.
The purpose of this study is to see if removal of the 81-bp
region results in reduction of enhancer activity in limb or unchanged. The positive selection model predicts that accumulation of a series of de novo synthesis of TF-binding sites byadaptive substitutions made the 81-bp region to become an
active enhancer module. Therefore, removal of the 81-bp region should decrease HACNS1 enhancer activity significantly
to the level of that seen in other primates. Loss-of-function
model assumes that the 81-bp region originally functioned asa repressor element in nonhuman primates, and perhaps,a BGC event that occurred in the human lineage disrupted
the repressor function of the 81-bp region. The latter model
predicts that removal of the 81-bp region from HACNS1
should cause no effect on total enhancer activity.
Our result clearly favors the latter, loss-of-function
model (fig. 2). A synthetic human enhancer replaced with the81-bp region of the chimpanzee and macaque indeed showedreduced expression (Prabhakar et al. 2008), which is consistent
with our repressor loss-of-function model. BGC may thus bea major player shaping this human-specific ‘‘accelerated’’ region(HAR2 or HACNS1). Nucleotide substitutions that occurred
within accelerated region may not be positively selected due
to generation of multiple adaptive TF-binding sites. Therefore,prediction of positively selected de novo TF-binding sites based
onexcessiveaccumulationofpointmutationsassumingasimple
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substitution model (e.g., Prabhakar et al. 2008) is potentially erroneous.Weshouldpayattentioninchoosingmodelspredicting
genuine positively selected sequences.
The loss-of-function model for the HACNS1-81bp region
does not exclude the possibility that the entire HACNS1 isadaptive. We know little about its benefit at individual andpopulation level, or even we do not know if HACNS1 is

FIG. 2. The proposed model of HACNS1 evolution in primates. The 81-bp
element was originally a repressor element within HACNS1, and humanspecific 13 substitutions possibly caused by BGC disrupted repressor
function and HACNS1 expression expanded. The expression patterns forchimpanzee and macaque were based on Prabhakar et al. (2008).

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FIG. 1. Transgenic mice assay showed indistinguishable X-gal staining pattern between the two constructs. (A) E11.5 transgenic mice embryos with
X-gal staining using intact HACNS1 construct (HACNS1-full). All the eight lacZ-positive embryos are shown. (B) E11.5 transgenic mice embryoswith X-gal staining using the 81-bp region–deleted HACNS1 construct (HACNS1-D81bp). All the five lacZ-positive embryos are shown.

Loss of Function in HACNS1 Evolution · doi:10.1093/molbev/msr231

indeed an enhancer element of the human GBX2 gene.
More extensive functional analysis will be necessary to conclude whether HACNS1 contributed to increasing fitness inthe course of human evolution.

Methods

Acknowledgments
We thank Ms. Atuko Ide for technical assistance. We also
thank Dr. Aya Takahashi for comments on the manuscript.
This study was partially supported by Grants-in-Aids from
MEXT, Japan, to K.S. and N.S.

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Human HACNS1 was polymerase chain reaction (PCR) amplified by using two primers, HACNS1HSAupHindIII: 5#GCGAAGCTTACCCCAGCATACAGTAGCTTCA-3# and HA
CNS1HSAloHindIII: 5#-GCAAGCTTATCAAGTGTGGCAAAAATGAC-3#. The 81-bp deletion construct was made using anoligomer (5#-TCGTATAAAGCCCCGATTGAATTCGCCACACACAGGTCAG-3#) together with PCR primers listed above.
Those PCR products were digested with HindIII and cloned
into minimal general promoter-reporter system vector HSF51
containing hsp68 heat shock protein promoter and lacZ gene.
ScaI digested–linearized constructs were adjusted to 10 ng/lland injected into pronuclei of one-cell mouse embryos. Transgenesis and X-gal staining of transgenic embryos were performed according to Sumiyama and Ruddle (2003).

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