Phylogenetic And Evolutionary Analysis On Protein Families With Fz-CRD Domain And Two Families Of BHLH Transcription Factors

GPCR （G protein coupled receptors） is a receptor superfamily with7-transmemberdomains. Frizzleds （FZD） are a kind of GPCR proteins acting as Wnt receptors andessential for Wnt signaling pathways in embryonic development, such as cell proliferation,differentiation, migration and polarity. The N-terminal domain of FZD including anextracellular Cysteine-rich domain （CRD） which contains ten Cysteines to formdisulphide bonds, can interact with Wnt，activating and mediating the Wnt signalingpathway. The CRD of Frizzled （Frizzled-like CRD, Fz-CRD） exists not only in FZDs butalso in many other proteins. The transcription factor family of bHLH （Basichelix-loop-helix） is widely distributed in animals, plants and fungi, which is involved inneurogenesis, myogenesis, cell proliferation, tissue differentiation, and other essentialdevelopmental processes. The HES/HEY genes including HES, HEY, HESL and DECcontain both bHLH and Orange domain. The bHLH/PAS is another kind of bHLHproteins containing both bHLH and PAS domains.There are three parts in this thesis. First, we studied the evolution of the FZDs and otherproteins with the Fz-CRD domain. Second, the evolution of HES/HEY was stated. Third,we studied the evolution of bHLH/PAS.The first part is about the evolution of the FZDs and other proteins containing theFz-CRD domain. Our results showed that there were several protein families with Fz-CRDdomain, which include FZD, SFRP, RTK, MFRP, CPZ, CORIN, COL18A1and others.Our systematic analysis revealed that the Fz-CRD domain was found in Dictyosteliumdiscoideum and was fused with Frizzled-seven-transmembrane domain （7TM） to form theFrizzled receptor. By phylogenetic analysis, we found that the Frizzled proteinsexperienced the processes of duplication, diversification and intron loss during theevolution, and formed11members in Vertebrates. We also analyzed the SFRPs and RTKswith Fz-CRD （ROR and MuSK）, which were present in sponge and had a larger familysize in Vertebrates than in Invertebrates. MFRP, CPZ, CORIN and COL18A1emergedduring vertebrate evolution through domain fusion. Moreover, we found a glycosylationsite and other sites in Fz-CRD, which may be related with Wnt interaction and play vital roles mediated by Wnt signaling pathway. Based on our results, a model showing that tworounds of expansion in Fz-CRD proteins occurred in Metazoa and Vertebrate wasproposed.The second part is about the evolution of HES/HEY. HES/HEY genes belong to thebHLH transcription factor superfamily. HES/HEY proteins are direct targets of the Notchsignaling pathway and are essential in developmental decisions, such as the developmentsof nervous system, somitogenesis, blood vessel and heart. In this study, we identifiedgenes of the HES/HEY family in more than10species representing the main lineages andperformed evolutionary analysis to elucidate their origin and evolutionary process. Ourresults showed that the HES/HEY genes only existed in metazoans and may originatefrom the common ancestor of metazoans. Combining the bHLH and Orange domainsequences, we constructed the phylogenetic trees by different methods （Bayesian, ML, NJand ME） and classified the HES/HEY gene family into four groups. Our results indicatedthat this gene family had undergone three expansions, which were along with the originsof Eumetazoa, vertebrate, and teleost. Gene structure analysis revealed that the HES/HEYgenes were involved in exon and/or intron loss in different species lineages. We proposeda model to show the evolution of this gene family with processes of expansion,exon/intron loss, and motif loss. Moreover, we also studied the teleost-specificduplications in zebrafish and investigated the expression pattern of duplicated genes indifferent tissues by RT-PCR. This study will provide clues for the research of Notchfunction in development.The third part is about the evolution of bHLH/PAS. The bHLH/PAS proteins areinvolved in the environment transcriptional response and neural development. Despitetheir important functions, the origin and evolution of this bHLH/PAS gene family has yetto be elucidated. Firstly, we identified the bHLH/PAS in various representative species.We constructed the phylogenetic trees by different methods （Bayesian, ML, NJ and ME）and classified the bHLH/PAS gene family into several groups, which are supported withhigh bootstraps. The exon-intron structure shows that eight exons with a conservativeexon phase of the "1001-0210" are present in many genes of bHLH/PAS, and retained orloss to the form the mammals bHLH/PAS members in the evolutionary process. Thefunctional divergence based on full sequences indicated that most θ>0with p<0.05in comparisons, suggesting that a site-specific rate shift after gene duplication is a commonphenomenon in the evolution of bHLH/PAS. No positively selected site was detected inthe three domains （bHLH, PAS and PAS₃domains）, suggested that strong purifyingselection may play a major role in their evolution. Our study provides a new insight forunderstanding the evolution and origination of bHLH/PAS.