| 其他摘要 | Human evolution is characterized by a dramatic increase in brain size, but the underling molecular mechanism that caused this expansion is unknown. It had been difficult to address this question using genetic tools because the dramatically enlarged brain is one of the human-specific traits. Fortunately, the dissections of a rare brain developmental disorder, i.e. the human primary microcephaly (MCPH) syndrome have discovered a set of genes regulating brain development. MCPH1 is the first identified microcephalin gene. Evolutionary studies of MCPH1 have suggested a rapid evolution of the protein coding sequences in primates, which is associated with the brain enlargement during primate evolution and human origin, especially in the lineage leading to humans. It has been reportedthat MCPH1 is highly polymorphic in human populations with a signature of on-going positive selection. However, sequence comprison analysis of gene coding regions could not give us functional hints. Hence, in this study, we aim to conduct a systematic dissection of the function of MCPH1, including not only the in vitro assays of revealing the role of MCPH1 for down-stream gene regulation, but also the construction of transgenic animal models (transgenic mouse and rhesus macaque models). The have performed the following four studies of MCPH1 and the major findings are: Firstly, we identified MCPH1 as a transcriptional repressor. We examined the role of MCPH1 in regulating the hTERT promoter in vitro. Co-transfection of the hTERT promoter with MCPH1 in Hela cells could inhibit the hTERT promoter activity. The EMSA assay demonstrated that MCPH1 could bind to the proximal hTERT promoter. Over expression of MCPH1 could repress telomerase activity, and the repression was abolished by knocking down the MCPH1 expression using siRNA in U2OS cells. We propose that MCPH1 functions as a transcriptional repressor of hTERT in vitro. Secondly, we cloned the human MCPH1 promoter and we identified a novel E2F1 binding motif located in the proximal promoter region of MCPH1. The experiments using electrophoretic mobility shift and promoter assays (EMSA) showed that E2F1 could stimulate MCPH1 transcription by direct binding to the E2F1 motif. Over-expression of E2F1 led to the up-regulation of MCPH1 transcription, and knocking down the endogenous E2F1 resulted in the inhibition of the MCPH1 promoter activity. Surprisingly, sequence comparison of vertebrate species suggested that the identified E2F1 binding motif is primate specific, consistent with the previous observation of rapid evolution of MCPH1 protein sequence in primates. We propose that during primate evolution, MCPH1 has acquired a novel E2F1 binding motif in its promoter which may act as a parallel mechanism acting together with the rapid protein sequence changes in primates, and eventually contributed to brain enlargement during primate evolution and human origin. Thirdly, we have constructed the human MCPH1 transgenic mouse and the macaque MCPH1 transgenic mouse. These mice are generally healthy, and they are different from wild type mice on brain weight and testis weight. Phenotype analysis indicates that the human MCPH1 transgenic mouse is not different from the macaque MCPH1 transgenic mouse in view of brain weight. The primary differences are reflected by the regulatory effect of MCPH1 on the expression of its downstream target genes. Finally, We have constructed the human MCPH1 transgenic monkey to test wether human MCPH1 could affect macaque’s brain size. Our preliminary functional experiments indicate that we successfully constructed the human MCPH1 transgenic macaque. The MRI data suggests that the total brain volume of the transgenic macaque is larger than that of the wild-type macaque, so are the white matter volume and the grey matter volume. In summary, we have shown that through interaction with E2F1, MCPH1 can regulate a series of genes involved in cell cycle control, cell proliferation and differentiation, and apoptosis, and eventually contribute to brain development in primates. The preliminary data from our transgenic animal studies suggest that over expression of MCPH1 can influence the development of brain and testis, and there might be functional divergence of MCPH1 between humans and nonhuman primates. |
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