BACKGROUND
a dramatic increase in brain size is one of the hallmarks of human evolution, but despite the significance of this trait, the causal molecular mechanism underlying this expansion is unclear  <cit> .
until recently, addressing this question with genetic tools has been difficult because the dramatically enlarged brain is a human-specific trait.
genetic studies of a rare brain developmental disorder, human autosomal primary microcephaly syndrome , have uncovered a set of genes that regulate brain development.
to date, seven genes have been identified as being responsible for this syndrome: mcph <dig>  also known as brit <dig>   <cit> , wdr <dig>   <cit> , cdk5rap <dig>   <cit> , cep <dig>   <cit> , aspm   <cit> , and cenpj   <cit>  and stil   <cit> .
previous evolutionary analyses of these microcephaly genes showed that four of them, aspm, cdk5rap <dig>  cenpj and mcph <dig>  evolved rapidly under darwinian positive selection during the evolution of human and non-human primates  <cit> .
aspm also experienced positive selection across anthropoids  <cit> , while cdk5rap <dig> and cenpj showed accelerated rates of non-synonymous substitutions over the course of primate evolution  <cit> .
the signal of positive selection on mcph <dig> was observed in the common ancestor of great apes and humans as well as in the human lineage  <cit> , although another study on mcph <dig> only detected positive selection in the anthropoids as a whole and no particular acceleration in the human lineage  <cit> .
this rapid evolution suggests these genes may have had a key role in the evolutionary enlargement of the brain, although the link of cenpj and mcph <dig> to the evolution of gross brain size was not confirmed in the association analysis of absolute neonatal brain size among primates  <cit> .
among the four rapidly evolving microcephaly genes, only aspm has been experimentally studied to detect the evolutionary consequence of protein sequence changes; mice carrying a truncated aspm protein were shown to have reductions of both brain and testis size, while the transgenic mouse carrying human aspm could rescue this phenotype, but did not cause any additional enlargement of the brain  <cit> .
mcph <dig> was the first gene identified as being responsible for autosomal recessive primary microcephaly, characterized by significantly reduced brain volume, mental retardation and premature chromosome condensation  syndrome  <cit> .
the mcph <dig> gene encodes a  <dig> -bp-long coding sequence  with  <dig> exons, spanning about  <dig> kb at 8p <dig>  the mcph <dig> protein contains three brca1-carboxyl terminal  domains, including one n terminal brct domain and a tandem pair at the c terminus.
numerous studies have implied that the brct domains of mcph <dig> function as the key component for protein-protein interaction; this seems likely as the interaction of the mcph <dig> tandem brct domains with proteins like e2f <dig> and r-h2ax is required for the activation of cell cycle checkpoint, dna repair and apoptosis  <cit> .
several studies have likewise suggested that mcph <dig> may also function as a tumor suppressor  <cit> .
evolutionary studies of mcph <dig> have demonstrated a rapid change in protein sequence associated with the brain enlargement during primate evolution and human origin.
interestingly, during two key taxonomic transitions in primates, that is, between lesser apes and great apes, and between great apes and humans, absolute brain volume was greatly enlarged, and mcph <dig> might be involved in this process  <cit> .
additionally, mcph <dig> is also highly polymorphic in human populations and still carries the molecular signature of on-going positive selection  <cit> .
human population studies have reported a sex-specific association between a mcph <dig> sequence variant and brain volume  <cit> .
these results suggest that the protein sequence changes, especially the human-specific changes of mcph <dig> may have caused the functional changes that explain the genetic basis for the evolution of brain size in primates.
previously, the mcph <dig> protein has been shown to play an essential role during cell cycle and cell apoptosis and it can physically interact with e2f <dig> to form a complex and bind the promoters of the target genes for regulating their transcriptional activities  <cit> .
beyond this, mcph <dig> alone can also function as a transcriptional regulator, and we previously demonstrated mcph <dig> could function as a transcriptional repressor  <cit> .
together, these regulatory mechanisms allow the experimental testing of the functional changes of mcph <dig> during primate evolution.
to detect if the protein sequence divergence of mcph <dig> among primates may confer any functional alterations, we selected eight known down-stream genes regulated by e2f <dig> and mcph1: p <dig> <cit> , p <dig> <cit> , p <dig> <cit> , p <dig> <cit> , p14arf <cit> , caspase <dig> <cit> , cycline <dig> <cit>  and htert <cit> .
these genes are involved in cell proliferation and apoptosis, critical processes regulating brain development .
we tested the activating effects  and the repressing effects  of mcph <dig> on these genes’ promoter when introducing mutations at the sites containing human- and great-ape-specific amino acid changes.
our results demonstrated that most of the human-specific amino acid substitutions could influence the regulatory effects of mcph <dig> on the down-stream genes, and a similar effect was also seen for one of the four great-ape-specific changes, suggesting that the species and lineage specific mutations of mcph <dig> are indeed functional and potentially contributed to brain enlargement over the course of primate evolution.
RESULTS
identification of lineage-specific mcph <dig> amino acid substitutions
in order to identify lineage-specific amino acid substitutions, mcph <dig> orthologs of representative primate and other mammalian species were obtained from the ncbi, embl and uniprot databases.
we used a total of  <dig> species including  <dig> primates  and  <dig> other representative mammalian species  .
using muscle and clustal w, we aligned the mcph <dig> protein sequences .
during primate evolution, there are two key taxonomic transitions accompanied by brain enlargements .
we identified nine sites containing substitutions considered as human-specific sites that occur in the human lineage but are relatively conserved in the other species .
we further selected four sites containing substitutions considered as great-ape-specific since they occurred in the ancestor of hominidae .
the physicochemical properties of the  <dig> lineage-specific amino acid substitutions are shown in additional file 3: table s <dig> and the schematic map of the mcph <dig> protein domains labeled with the lineage-specific substitutions are shown in figure  <dig>  all these lineage-specific sites were selected to generate mutant mcph <dig> proteins for the reporter gene assays in order to test their functional effects.
test of protein-protein interaction between mcph <dig> and e2f1
previous studies suggested human mcph <dig>  could interact with e2f1in vitro and in vivo <cit> .
to test if the non-human primate mcph <dig> can also interact with e2f <dig>  we cloned the rhesus macaque mcph <dig> .
the results of the co-immunoprecipitation assay showed that mmpch <dig> can also directly interact with e2f <dig>  and no difference was observed for the intensity of the interaction with e2f <dig> between hmcph <dig> and mmcph <dig> .
given the established mcph1-e2f <dig> interaction in human cell lines  <cit> , this protein-protein interaction mechanism seems likely to have been conserved during primate evolution.
we also conducted a cellular co-localization assay for both mmcph <dig> and hmcph <dig>  and the results indicated that both were co-localized with e2f <dig> , consistent with the results of the co-immunoprecipitation assay.
divergent effects of hmcph <dig> and mmcph <dig> on transcriptional regulation
mcph <dig> interacts with e2f <dig> to enhance its transactivation activity by forming a complex, which binds to the promoters of the e2f <dig> target genes, including p <dig>  caspase and rad <dig>  among others  <cit> .
we, therefore, tested the enhancing effect of mcph <dig> using the e2f4b reporter vector commonly used as the positive control in testing the transactivation activity of e2f <dig> <cit> .
the expression of the ha-tagged mcph <dig> and e2f <dig> were verified by western blot using anti-ha monoclonal antibodies .
our results showed that hmcph <dig> could significantly enhance the transactivation activity of e2f <dig>  but no significant effect was observed for mmcph <dig> and gmcph <dig>  .
in addition, the difference between e2f <dig> + hmcph <dig> and e2f <dig> + mmcph <dig> is significant .
the same trend was also seen when comparing e2f <dig> + hmcph <dig> and e2f <dig> + gmcph <dig>  though not statistically significant .
interestingly, the difference between e2f <dig> + gmcph <dig> and e2f <dig> + mmcph <dig> is significant , suggesting continuum of functional divergence of mcph <dig> during primate evolution with a major shift in function between old world monkeys and apes and a probable further shift during human evolution .
to check the conservation of the e2f <dig> binding sites , we aligned the promoter sequences of the target genes among representative primate species.
we found that most of the e2f <dig> binding sites are highly conserved from humans to marmosets , ruling out the possibility of binding bias due to promoter sequence divergence.
the e2f <dig> protein sequences are also highly conserved among primate species , ruling out the possibility that the different effect of mcph <dig> is caused by mutations in e2f <dig>  collectively, these results suggest a functional divergence of mcph <dig> between humans and nonhuman primates.
we further tested a set of e2f <dig> target genes .
the regulatory network of these genes is shown in additional file 1: figure s <dig>  we found that the enhancing effect of hmcph <dig> was significantly stronger than mmcph <dig> for cycline <dig> and p <dig>  and a similar trend was also observed for p14arf, though not significantly .
no significant enhancing effects were detected for the other four genes, p <dig>  p <dig>  p <dig> and caspase <dig> .
consistent with the result from the e2f4b assay, these data also suggest a functional divergence between hmcph <dig> and mmcph <dig> in enhancing the transactivation activity of e2f <dig>
to further test the functional divergence between hmcph <dig> and mmcph <dig>  we performed another assay to detect the regulatory effect of mcph <dig> alone on eight down-stream genes .
among the eight genes tested, seven showed a repressing effect for both hmcph <dig> and mmcph <dig>  while only one, caspase <dig>  showed an activating effect.
when comparing the effects between hmcph <dig> and mmcph <dig>  three genes  showed significant differences  of repressing effect , the same three which showed enhancing differences between hmcph <dig> and mmcph <dig> in the e2f1-mcph <dig> transactivation assay .
for cycline <dig> and p14arf, hmcph <dig> showed a stronger repressing effect than mmcph <dig>  while the opposite effect was observed for p <dig>  we did not detect significant between-species differences for the other four genes with repressing effect  though the trend was the same .
caspase  <dig> was the only gene with an activating effect, and we also observed a significant difference between hmcph <dig> and mmcph <dig>  .
collectively, the data from the two reporter gene assays suggest a clear functional divergence of mcph <dig> between humans and rhesus macaques.
detection of regulatory changes of mcph <dig> for the human-specific sites
in order to test the functional effect of the human specific amino acids changes, we first constructed two mutant hmcph1s by introducing a backward mutation of changing the human-specific amino acid to an ancestral amino acid  and introducing a random mutation  at site- <dig> .
with the use of the e2f4b transactivation reporter assay, we measured the enhancing effect of the two mutant hmcph1s.
as shown in figure 4a, both mutant hmcph1s showed a significantly decreased enhancing effect as compared with the wild-type hmcph <dig>  similar to the difference between hmcph <dig> and mmcph <dig>  suggesting that the human-specific amino acid change at site- <dig> can influence the enhancing effect of mcph <dig>
we next generated a series of mutant hmcph1s carrying backward mutations at the other eight human-specific sites , and tested their enhancing effects.
five of the eight mutant hmcph1s  showed significantly decreased enhancing effects compared with the wild-type hmcph <dig>   and the three non-significant sites  showed the same trend.
among the nine human specific sites tested, the majority  showed a significantly decreased enhancing effect when mutated to the ancestral amino acids, suggesting most of the human-specific mcph <dig> amino acid changes are in fact functional.
we also tested the repressing effect on p14arf for the human-specific sites.
as shown in figure 4d, e, we detected a significant decrease of the p14arf repressing effect for five sites , and the same trend was observed for the other four, though it was not significant.
among the five significant sites, three  overlapped with the six sites showing decreased enhancing effect.
taken together, we detected regulatory changes for most  human-specific sites, suggesting hmcph <dig> has acquired functional modifications during the origin of humans.
detection of regulatory changes of mcph <dig> for the great-ape-specific sites
to detect the functional consequences of mcph1’s four great-ape-specific mutations , we conducted analyses similar to that used for the human-specific sites.
the results indicated that for the enhancing assay, although all four great-ape-specific sites showed a decreased level as compared with the wild-type of hmcph <dig>  none were statistically significant .
in contrast, in the repressing assay, one great-ape-specific site  showed a significant decrease of the repressing effect , suggesting that this great-ape-specific mutation may have also caused functional modifications of mcph <dig> during the evolution of the ancestor of hominidae.
discussion
mcph <dig> has experienced strong darwinian positive selection during primate evolution, but there are no data showing functional divergence.
here we demonstrated evidence of functional divergence of mcph <dig> between humans and nonhuman primates.
most of the human-specific amino acid changes could alter the regulatory effects of mcph <dig> on the transcription of the down-stream genes, and a similar effect was observed for one of the four great-ape-specific amino acid changes.
accordingly, our data support the hypothesis that selection on mcph <dig> has resulted in functional divergence at the protein level, which potentially contributes to changes in the development and evolution of brain size.
absolute brain size has increased in parallel across primate evolution.
along the two branches we focused on in this analysis, absolute brain volume increased from  <dig> to  <dig> ml to  <dig> to  <dig> ml during the transition between lesser apes and great apes  <cit> , and from  <dig> to  <dig> ml to  <dig>  to  <dig>  ml during the transition between great apes and humans  <cit> .
previous studies indicated that there were accelerated amino acid substitutions during both the origin of hominidae’s ancestor and of our own species, paralleling the two brain enlargements  <cit> , suggesting that the amino acid substitutions of mcph <dig> were probably adaptive and may have contributed to the brain expansion during primate evolution.
in addition, the gradient change of mcph1’s transcription regulation from macaque to gibbon, and to humans  seems to imply a continuum of functional divergence rather than a number of discrete shifts, which calls for further functional tests in extensive primate lineages.
interestingly, all the human- and great-ape-specific mutations are located in the non-brct domains .
since the three brct domains of mcph <dig> are critical for protein-protein interaction, the amino acid changes during primate evolution seems not to have caused drastic functional alteration, but rather a modification of the existing function.
inferring the exact functional alterations of the human-specific and great-ape-specific mutations is difficult.
previous studies have shown that the middle domain  of mcph <dig>  where the four human-specific mutations are located, is the binding domain by condensin ii for homologous recombination repair  <cit> , an important mechanism for cell cycle checkpoints and genome integrity.
concurrently, all four human-specific sites located in this middle domain showed altered regulatory effects when mutated into ancestral amino acids, suggesting that the human-specific mutations may have changed the binding property with condensin ii.
additionally, all four human-specific mutations caused changes in physicochemical properties of amino acids .
we also found that for the regulatory changes of the down-stream genes, almost half  of the tested genes  had significant differences between humans and rhesus macaques, either in the enhancing assay with mcph1-e2f <dig> or in the repressing assay with mcph <dig> alone , indicating a functional divergence between humans and non-human primates.
the protein p <dig> is involved in both cell cycle regulation and induction of apoptosis  <cit> .
e2f <dig> is an important regulator of p <dig>  especially during brain development  <cit> .
cycline <dig>  meanwhile, is involved in cell cycle and is a key target gene of e2f <dig> <cit>  and has been shown to take part in the determination of the number of neurons during mouse corticogenesis by regulating the g <dig> mode of cell division  <cit> .
p14arf is an alternate reading frame product of cdkn2a involved in cell cycle regulation that is also involved in self-renewal of neural stem cells and neural development  <cit> .
additionally, human population studies have reported that the mcph <dig> sequence variants were associated with brain volume in a sex-specific manner  <cit> .
recently, it was also reported that mcph <dig> might have contributed to the evolution of sexual dimorphism in brain mass across anthropoid primates  <cit> .
in fact, two of the down-stream genes regulated by mcph <dig>  p <dig> and cycline <dig>  were reported to be associated with sex dimorphism during germ line development  <cit> , suggesting that the regulation of mcph <dig> on brain development may differ between males and females.
taken together, the strengthened transactivation effect of human mcph <dig> on these down-stream genes may contribute to the greatly enlarged neuro-progenitor pool in the human brain during neurogenesis, which is in line with recent studies that suggest mcph1’s functional role in neuro-progenitor cells through the chk1-cdc25-cdk <dig> pathway  <cit> .
conversely, when mcph <dig> acts alone as a transcription repressor, there were also differences between humans and rhesus macaques on the repressing effect of the down-stream genes , implying that a homeostasis of gene expression regulation by mcph <dig> is required during neurogenesis.
although we observed functional divergence of mcph <dig> due to its protein sequence changes during primate evolution and human origin, it should be stressed that we did not establish a direct link between the adaptive changes of mcph <dig> and the ever-increasing brain size in primates.
as shown in the mcph <dig> knock-out mice analysis, the truncated mcph <dig> not only caused a reduction in brain size, but also resulted in a reduction of testis size  <cit> , suggesting that mcph <dig> may also play a role during testis development.
accordingly, we cannot rule out the possibility that the adaptive evolution of mcph <dig> in primates may be caused by selection on other phenotypes, though the current evidences mostly favor enlargement of the brain.
initially proposed by king and wilson  <cit> , the importance of cis-regulatory changes in human evolution has recently been tested and confirmed  <cit> .
however, our functional data of mcph <dig> suggests that protein sequence changes may also have significant phenotypic effects.
hence, the evolution of an important trait like brain function may require genetic alterations at multiple regulatory levels.
CONCLUSIONS
we demonstrated the existence of functional alterations caused by the lineage-specific mutations of mcph <dig> during the evolution of primates, especially during the origin of humans.
the functional changes of mcph <dig> are likely executed by regulating several key down-stream genes.

 a dramatic increase in brain size is one of the hallmarks of human evolution, but despite the significance of this trait, the causal molecular mechanism underlying this expansion is unclear. until recently, addressing this question with genetic tools has been difficult because the dramatically enlarged brain is a human-specific trait. genetic studies of a rare brain developmental disorder, human autosomal primary microcephaly syndrome, have uncovered a set of genes that regulate brain development. to date, seven genes have been identified as being responsible for this syndrome: mcph also known as brit, wdr, cdk5rap, cep, aspm, and cenpj and stil. previous evolutionary analyses of these microcephaly genes showed that four of them, aspm, cdk5rap cenpj and mcph evolved rapidly under darwinian positive selection during the evolution of human and non-human primates. aspm also experienced positive selection across anthropoids, while cdk5rap and cenpj showed accelerated rates of non-synonymous substitutions over the course of primate evolution. the signal of positive selection on mcph was observed in the common ancestor of great apes and humans as well as in the human lineage, although another study on mcph only detected positive selection in the anthropoids as a whole and no particular acceleration in the human lineage. this rapid evolution suggests these genes may have had a key role in the evolutionary enlargement of the brain, although the link of cenpj and mcph to the evolution of gross brain size was not confirmed in the association analysis of absolute neonatal brain size among primates. among the four rapidly evolving microcephaly genes, only aspm has been experimentally studied to detect the evolutionary consequence of protein sequence changes; mice carrying a truncated aspm protein were shown to have reductions of both brain and testis size, while the transgenic mouse carrying human aspm could rescue this phenotype, but did not cause any additional enlargement of the brain. mcph was the first gene identified as being responsible for autosomal recessive primary microcephaly, characterized by significantly reduced brain volume, mental retardation and premature chromosome condensation syndrome. the mcph gene encodes a -bp-long coding sequence with exons, spanning about kb at 8p the mcph protein contains three brca1-carboxyl terminal domains, including one n terminal brct domain and a tandem pair at the c terminus. numerous studies have implied that the brct domains of mcph function as the key component for protein-protein interaction; this seems likely as the interaction of the mcph tandem brct domains with proteins like e2f and r-h2ax is required for the activation of cell cycle checkpoint, dna repair and apoptosis. several studies have likewise suggested that mcph may also function as a tumor suppressor. evolutionary studies of mcph have demonstrated a rapid change in protein sequence associated with the brain enlargement during primate evolution and human origin. interestingly, during two key taxonomic transitions in primates, that is, between lesser apes and great apes, and between great apes and humans, absolute brain volume was greatly enlarged, and mcph might be involved in this process. additionally, mcph is also highly polymorphic in human populations and still carries the molecular signature of on-going positive selection. human population studies have reported a sex-specific association between a mcph sequence variant and brain volume. these results suggest that the protein sequence changes, especially the human-specific changes of mcph may have caused the functional changes that explain the genetic basis for the evolution of brain size in primates. previously, the mcph protein has been shown to play an essential role during cell cycle and cell apoptosis and it can physically interact with e2f to form a complex and bind the promoters of the target genes for regulating their transcriptional activities. beyond this, mcph alone can also function as a transcriptional regulator, and we previously demonstrated mcph could function as a transcriptional repressor. together, these regulatory mechanisms allow the experimental testing of the functional changes of mcph during primate evolution. to detect if the protein sequence divergence of mcph among primates may confer any functional alterations, we selected eight known down-stream genes regulated by e2f and mcph1: p, p, p, p, p14arf, caspase, cycline and htert. these genes are involved in cell proliferation and apoptosis, critical processes regulating brain development. we tested the activating effects and the repressing effects of mcph on these genes’ promoter when introducing mutations at the sites containing human- and great-ape-specific amino acid changes. our results demonstrated that most of the human-specific amino acid substitutions could influence the regulatory effects of mcph on the down-stream genes, and a similar effect was also seen for one of the four great-ape-specific changes, suggesting that the species and lineage specific mutations of mcph are indeed functional and potentially contributed to brain enlargement over the course of primate evolution.  identification of lineage-specific mcph amino acid substitutions in order to identify lineage-specific amino acid substitutions, mcph orthologs of representative primate and other mammalian species were obtained from the ncbi, embl and uniprot databases. we used a total of species including primates and other representative mammalian species. using muscle and clustal w, we aligned the mcph protein sequences. during primate evolution, there are two key taxonomic transitions accompanied by brain enlargements. we identified nine sites containing substitutions considered as human-specific sites that occur in the human lineage but are relatively conserved in the other species. we further selected four sites containing substitutions considered as great-ape-specific since they occurred in the ancestor of hominidae. the physicochemical properties of the lineage-specific amino acid substitutions are shown in additional file 3: table s and the schematic map of the mcph protein domains labeled with the lineage-specific substitutions are shown in figure  all these lineage-specific sites were selected to generate mutant mcph proteins for the reporter gene assays in order to test their functional effects. test of protein-protein interaction between mcph and e2f1 previous studies suggested human mcph could interact with e2f1in vitro and in vivo. to test if the non-human primate mcph can also interact with e2f we cloned the rhesus macaque mcph. the results of the co-immunoprecipitation assay showed that mmpch can also directly interact with e2f and no difference was observed for the intensity of the interaction with e2f between hmcph and mmcph. given the established mcph1-e2f interaction in human cell lines, this protein-protein interaction mechanism seems likely to have been conserved during primate evolution. we also conducted a cellular co-localization assay for both mmcph and hmcph and the results indicated that both were co-localized with e2f, consistent with the results of the co-immunoprecipitation assay. divergent effects of hmcph and mmcph on transcriptional regulation mcph interacts with e2f to enhance its transactivation activity by forming a complex, which binds to the promoters of the e2f target genes, including p caspase and rad among others. we, therefore, tested the enhancing effect of mcph using the e2f4b reporter vector commonly used as the positive control in testing the transactivation activity of e2f. the expression of the ha-tagged mcph and e2f were verified by western blot using anti-ha monoclonal antibodies. our results showed that hmcph could significantly enhance the transactivation activity of e2f but no significant effect was observed for mmcph and gmcph. in addition, the difference between e2f + hmcph and e2f + mmcph is significant. the same trend was also seen when comparing e2f + hmcph and e2f + gmcph though not statistically significant. interestingly, the difference between e2f + gmcph and e2f + mmcph is significant, suggesting continuum of functional divergence of mcph during primate evolution with a major shift in function between old world monkeys and apes and a probable further shift during human evolution. to check the conservation of the e2f binding sites, we aligned the promoter sequences of the target genes among representative primate species. we found that most of the e2f binding sites are highly conserved from humans to marmosets, ruling out the possibility of binding bias due to promoter sequence divergence. the e2f protein sequences are also highly conserved among primate species, ruling out the possibility that the different effect of mcph is caused by mutations in e2f collectively, these results suggest a functional divergence of mcph between humans and nonhuman primates. we further tested a set of e2f target genes. the regulatory network of these genes is shown in additional file 1: figure s we found that the enhancing effect of hmcph was significantly stronger than mmcph for cycline and p and a similar trend was also observed for p14arf, though not significantly. no significant enhancing effects were detected for the other four genes, p p p and caspase. consistent with the result from the e2f4b assay, these data also suggest a functional divergence between hmcph and mmcph in enhancing the transactivation activity of e2f to further test the functional divergence between hmcph and mmcph we performed another assay to detect the regulatory effect of mcph alone on eight down-stream genes. among the eight genes tested, seven showed a repressing effect for both hmcph and mmcph while only one, caspase showed an activating effect. when comparing the effects between hmcph and mmcph three genes showed significant differences of repressing effect, the same three which showed enhancing differences between hmcph and mmcph in the e2f1-mcph transactivation assay. for cycline and p14arf, hmcph showed a stronger repressing effect than mmcph while the opposite effect was observed for p we did not detect significant between-species differences for the other four genes with repressing effect though the trend was the same. caspase was the only gene with an activating effect, and we also observed a significant difference between hmcph and mmcph. collectively, the data from the two reporter gene assays suggest a clear functional divergence of mcph between humans and rhesus macaques. detection of regulatory changes of mcph for the human-specific sites in order to test the functional effect of the human specific amino acids changes, we first constructed two mutant hmcph1s by introducing a backward mutation of changing the human-specific amino acid to an ancestral amino acid and introducing a random mutation at site-. with the use of the e2f4b transactivation reporter assay, we measured the enhancing effect of the two mutant hmcph1s. as shown in figure 4a, both mutant hmcph1s showed a significantly decreased enhancing effect as compared with the wild-type hmcph similar to the difference between hmcph and mmcph suggesting that the human-specific amino acid change at site- can influence the enhancing effect of mcph we next generated a series of mutant hmcph1s carrying backward mutations at the other eight human-specific sites, and tested their enhancing effects. five of the eight mutant hmcph1s showed significantly decreased enhancing effects compared with the wild-type hmcph and the three non-significant sites showed the same trend. among the nine human specific sites tested, the majority showed a significantly decreased enhancing effect when mutated to the ancestral amino acids, suggesting most of the human-specific mcph amino acid changes are in fact functional. we also tested the repressing effect on p14arf for the human-specific sites. as shown in figure 4d, e, we detected a significant decrease of the p14arf repressing effect for five sites, and the same trend was observed for the other four, though it was not significant. among the five significant sites, three overlapped with the six sites showing decreased enhancing effect. taken together, we detected regulatory changes for most human-specific sites, suggesting hmcph has acquired functional modifications during the origin of humans. detection of regulatory changes of mcph for the great-ape-specific sites to detect the functional consequences of mcph1’s four great-ape-specific mutations, we conducted analyses similar to that used for the human-specific sites. the results indicated that for the enhancing assay, although all four great-ape-specific sites showed a decreased level as compared with the wild-type of hmcph none were statistically significant. in contrast, in the repressing assay, one great-ape-specific site showed a significant decrease of the repressing effect, suggesting that this great-ape-specific mutation may have also caused functional modifications of mcph during the evolution of the ancestor of hominidae. discussion mcph has experienced strong darwinian positive selection during primate evolution, but there are no data showing functional divergence. here we demonstrated evidence of functional divergence of mcph between humans and nonhuman primates. most of the human-specific amino acid changes could alter the regulatory effects of mcph on the transcription of the down-stream genes, and a similar effect was observed for one of the four great-ape-specific amino acid changes. accordingly, our data support the hypothesis that selection on mcph has resulted in functional divergence at the protein level, which potentially contributes to changes in the development and evolution of brain size. absolute brain size has increased in parallel across primate evolution. along the two branches we focused on in this analysis, absolute brain volume increased from to ml to to ml during the transition between lesser apes and great apes, and from to ml to to ml during the transition between great apes and humans. previous studies indicated that there were accelerated amino acid substitutions during both the origin of hominidae’s ancestor and of our own species, paralleling the two brain enlargements, suggesting that the amino acid substitutions of mcph were probably adaptive and may have contributed to the brain expansion during primate evolution. in addition, the gradient change of mcph1’s transcription regulation from macaque to gibbon, and to humans seems to imply a continuum of functional divergence rather than a number of discrete shifts, which calls for further functional tests in extensive primate lineages. interestingly, all the human- and great-ape-specific mutations are located in the non-brct domains. since the three brct domains of mcph are critical for protein-protein interaction, the amino acid changes during primate evolution seems not to have caused drastic functional alteration, but rather a modification of the existing function. inferring the exact functional alterations of the human-specific and great-ape-specific mutations is difficult. previous studies have shown that the middle domain of mcph where the four human-specific mutations are located, is the binding domain by condensin ii for homologous recombination repair, an important mechanism for cell cycle checkpoints and genome integrity. concurrently, all four human-specific sites located in this middle domain showed altered regulatory effects when mutated into ancestral amino acids, suggesting that the human-specific mutations may have changed the binding property with condensin ii. additionally, all four human-specific mutations caused changes in physicochemical properties of amino acids. we also found that for the regulatory changes of the down-stream genes, almost half of the tested genes had significant differences between humans and rhesus macaques, either in the enhancing assay with mcph1-e2f or in the repressing assay with mcph alone, indicating a functional divergence between humans and non-human primates. the protein p is involved in both cell cycle regulation and induction of apoptosis. e2f is an important regulator of p especially during brain development. cycline meanwhile, is involved in cell cycle and is a key target gene of e2f and has been shown to take part in the determination of the number of neurons during mouse corticogenesis by regulating the g mode of cell division. p14arf is an alternate reading frame product of cdkn2a involved in cell cycle regulation that is also involved in self-renewal of neural stem cells and neural development. additionally, human population studies have reported that the mcph sequence variants were associated with brain volume in a sex-specific manner. recently, it was also reported that mcph might have contributed to the evolution of sexual dimorphism in brain mass across anthropoid primates. in fact, two of the down-stream genes regulated by mcph p and cycline were reported to be associated with sex dimorphism during germ line development, suggesting that the regulation of mcph on brain development may differ between males and females. taken together, the strengthened transactivation effect of human mcph on these down-stream genes may contribute to the greatly enlarged neuro-progenitor pool in the human brain during neurogenesis, which is in line with recent studies that suggest mcph1’s functional role in neuro-progenitor cells through the chk1-cdc25-cdk pathway. conversely, when mcph acts alone as a transcription repressor, there were also differences between humans and rhesus macaques on the repressing effect of the down-stream genes, implying that a homeostasis of gene expression regulation by mcph is required during neurogenesis. although we observed functional divergence of mcph due to its protein sequence changes during primate evolution and human origin, it should be stressed that we did not establish a direct link between the adaptive changes of mcph and the ever-increasing brain size in primates. as shown in the mcph knock-out mice analysis, the truncated mcph not only caused a reduction in brain size, but also resulted in a reduction of testis size, suggesting that mcph may also play a role during testis development. accordingly, we cannot rule out the possibility that the adaptive evolution of mcph in primates may be caused by selection on other phenotypes, though the current evidences mostly favor enlargement of the brain. initially proposed by king and wilson, the importance of cis-regulatory changes in human evolution has recently been tested and confirmed. however, our functional data of mcph suggests that protein sequence changes may also have significant phenotypic effects. hence, the evolution of an important trait like brain function may require genetic alterations at multiple regulatory levels.  we demonstrated the existence of functional alterations caused by the lineage-specific mutations of mcph during the evolution of primates, especially during the origin of humans. the functional changes of mcph are likely executed by regulating several key down-stream genes. 
to test this hypothesis, we used co-immunoprecipitation and reporter gene assays to examine the activating and repressing effects of mcph on a set of its down-stream genes and then compared the functional outcomes of a series of mutant mcph proteins that carry mutations at the human- and great-ape-specific sites. collectively, we propose that during primate evolution in general and human evolution in particular, the divergence of mcph protein sequences under darwinian positive selection led to functional modifications, providing a possible molecular mechanism of how mcph contributed to brain enlargement during primate evolution and human origin. the results demonstrate that the regulatory effects of human mcph and rhesus macaque mcph are different in three of eight down-stream genes tested, suggesting a functional divergence of mcph between human and non-human primates. further analyses of the mutant mcph proteins indicated that most of the human-specific mutations could change the regulatory effects on the down-stream genes. during this evolution, the divergence of mcph protein sequences among primates may have caused functional changes that contribute to brain enlargement. a similar result was also observed for one of the four great-ape-specific mutations. one of the key genes that regulate human brain size, mcph has evolved under strong darwinian positive selection during the evolution of primates.
 one of the key genes that regulate human brain size, mcph has evolved under strong darwinian positive selection during the evolution of primates. during this evolution, the divergence of mcph protein sequences among primates may have caused functional changes that contribute to brain enlargement.  to test this hypothesis, we used co-immunoprecipitation and reporter gene assays to examine the activating and repressing effects of mcph on a set of its down-stream genes and then compared the functional outcomes of a series of mutant mcph proteins that carry mutations at the human- and great-ape-specific sites. the results demonstrate that the regulatory effects of human mcph and rhesus macaque mcph are different in three of eight down-stream genes tested, suggesting a functional divergence of mcph between human and non-human primates. further analyses of the mutant mcph proteins indicated that most of the human-specific mutations could change the regulatory effects on the down-stream genes. a similar result was also observed for one of the four great-ape-specific mutations.  collectively, we propose that during primate evolution in general and human evolution in particular, the divergence of mcph protein sequences under darwinian positive selection led to functional modifications, providing a possible molecular mechanism of how mcph contributed to brain enlargement during primate evolution and human origin. mcph1e2f1brain sizeprimatesevolutionfunctional divergence 