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. 2013 May 22:11:62.
doi: 10.1186/1741-7007-11-62.

Functional divergence of the brain-size regulating gene MCPH1 during primate evolution and the origin of humans

Lei Shi  1 , Ming Li, Qiang Lin, Xuebin Qi, Bing Su
Affiliations

Functional divergence of the brain-size regulating gene MCPH1 during primate evolution and the origin of humans

Lei Shi et al. BMC Biol. .

Abstract

Background: One of the key genes that regulate human brain size, MCPH1 has evolved under strong Darwinian positive selection during the evolution of primates. During this evolution, the divergence of MCPH1 protein sequences among primates may have caused functional changes that contribute to brain enlargement.

Results: To test this hypothesis, we used co-immunoprecipitation and reporter gene assays to examine the activating and repressing effects of MCPH1 on a set of its down-stream genes and then compared the functional outcomes of a series of mutant MCPH1 proteins that carry mutations at the human- and great-ape-specific sites. The results demonstrate that the regulatory effects of human MCPH1 and rhesus macaque MCPH1 are different in three of eight down-stream genes tested (p73, cyclinE1 and p14ARF), suggesting a functional divergence of MCPH1 between human and non-human primates. Further analyses of the mutant MCPH1 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.

Conclusions: Collectively, we propose that during primate evolution in general and human evolution in particular, the divergence of MCPH1 protein sequences under Darwinian positive selection led to functional modifications, providing a possible molecular mechanism of how MCPH1 contributed to brain enlargement during primate evolution and human origin.

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Figures

Figure 1
Figure 1
Schematic map of MCPH1 protein domains labeled with lineage-specific amino acid substitutions. Sites containing human specific substitutions are marked with shadows, and the two dramatic brain size enlargement events that coincided with the molecular signatures of Darwinian positive selection during primate evolution are indicated.
Figure 2
Figure 2
Human and macaque MCPH1 can directly interact with E2F1. (A) Interaction of the transfected E2F1 and MCPH1 in HEK293T cells. (B) Co-localization of MCPH1-GFP and RFP-E2F1 in HEK293T cells. White arrows indicate co-staining areas. (C) Expression of the HA-tagged human and macaque MCPH1s were verified by Western blot using anti-HA monoclonal antibody (top panel), and the HA-tagged E2F1 was also verified by Western blot (middle panel). The bottom panel is the actin control. (D) Quantification of E2F1 and MCPH1 transcriptional activity using the E2F4B constructs. All histograms represent the mean ± SD of at least nine data points, including both biological and technical replicates (* P <0.05,** P <0.01, *** P <0.001, n.s, not significant).
Figure 3
Figure 3
Comparison of MCPH1 enhancing and repressing effects between humans and macaques. (A) The enhancing effect difference between hMCPH1 and mMCPH1 on the CyclinE1 promoter. (B) The enhancing effect difference between hMCPH1 and mMCPH1 on the p73 promoter. (C) The enhancing effect difference between hMCPH1 and mMCPH1 on the p14ARF promoter. For enhancing effect assay, 0.2 ug target gene promoter construct was co-transfected with 0.2 ug HA tagged E2F1 per/well or 0.2 ug HA tagged E2F1 and 0.2 ug HA tagged hMCPH1 per/well or 0.2 ug HA tagged E2F1 and 0.2 ug HA tagged mMCPH1 per well in 24-well plates in HEK923T cells, Renilla was used as the internal control. The HA-tagged empty vector was used as the negative control. (D) The repressing effect difference between hMCPH1 and mMCPH1 on the CyclinE1 promoter. (E) The repressing effect difference between hMCPH1 and mMCPH1 on the p73 promoter. (F) The repressing effect difference between hMCPH1 and mMCPH1 on the p14ARF promoter. For repression assay, 0.2 ug target gene promoter construct was co-transfected with 0.2 ug HA tagged hMCPH1 per/well or 0.2 ug HA tagged mMCPH1 per/well in 24-well plates in HEK923T cells, Renilla was used as the internal control. The HA-tagged empty vector was used as the negative control. All histograms represent the mean ± SD of at least nine data points including both biological and technical replicates (* P <0.05,** P <0.01, *** P <0.001, n.s, not significant).
Figure 4
Figure 4
Results of assays testing transcriptional regulations of mutant MCPH1s containing mutations at human-specific sites. (A-C) The results of the E2F4B assay testing the changes of enhancing effect. (D-E) The results of the p14ARF assay testing the repressing effect. All histograms represent the mean ± SD of at least nine data points including both biological and technical replicates (* P <0.05,** P <0.01, *** P <0.001, n.s, not significant).
Figure 5
Figure 5
Results of assays testing transcriptional regulations of mutant MCPH1s containing mutations at human-specific sites. (A) Results of the E2F4B assay testing the changes of enhancing effect. (B) Results of the p14ARF assay testing the repressing effect. All histograms represent the mean ± SD of at least nine data points, including both biological and technical replicates (* P <0.05,** P <0.01, *** P <0.001, n.s, not significant).
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