A Preliminary Study On The Relationship Between Specific Mutations In Conserved Noncoding Elements Of The Cetacean PITX1 Gene And Hindlimb Degeneration

Cetaceans,originally from terrestrial artiodactyls,experienced their land-to-sea transition 53 million years ago.In order to adapt to a fully aquatic habitat,their morphology has undergone significant changes,including streamlined body,loss of hind limbs and transformation of forelimbs into flippers.Previous studies have shown that well-developed hind limb and forelimb buds are observed at an earlier stage,while the hind limb buds have regressed by around seven weeks.However,the specific mechanism of the degeneration is still unclear.PITX1,as a gene specifically expressed in hind limbs,mutation,ectopic expression and changes of its enhancers can significantly affect limb morphogenesis.Based on this,we selected PITX1,a mammalian hindlimb specific gene,as a candidate gene in our study.Through comparative genomics,bioinformatics and in vitro functional verification,the following scientific questions are explored: 1)whether coding region of the gene evolved under functional constraints or positive selection? 2)whether there are some cetaceans-specific mutations in the conserved noncoding elements(CNEs)of PITX1 and thus lead to activity change? Based on the above analysis,the study reveals the relationship between PITX1 gene and changes in its regulatory region,and the molecular mechanism of hind limb degeneration in cetaceans will be discussed.In this research,the orthologous PITX1 coding sequences of 56 mammalian species and 16 cetaceans were obtained by downloading from databases(NCBI and Ortho Ma M)and local BLAST program.Selective pressure analysis using the branch model showed that strong purifying selection and functional constraint have dominantly shaped PITX1 during the evolution in cetaceans.To investigate whether cetaceans-specific mutation had occurred to the PITX1 non-coding region,35 genomes were aligned to the homo sapiens and then screened out conserved non-coding elements(CNEs).CNEs covering a larger region than the pre-exist enhancers important for PITX1 expression such as PELB,PDE,RA4,PEN,etc,were selected as our genomic window of interest.Therefore,192 CNEs were generated within 670 kb of sequence upstream of the PITX1 and 330 kb of sequence downstream of the PITX1.Further analysis revealed 87 of 192 CNEs containing cetaceans-specific mutations,including 9 CNEs with specific deletions,1 CNE with specific insertions and 80 CNEs with site-mutations,three of them containing more than one type of mutations.To explore the function of these elements,we combined the Chip-seq and ATAC-seq data of the limb E11.5 of mouse and further revealed 32 CNEs related to limb development,including three CNEs overlapping with PITX1 enhancer,namely CNE19 overlapping with PELB enhancer and CNE127 and CNE128 overlapping with PEN enhancer.Therefore,we speculated that these CNEs containing cetaceans-specific mutations in putative regulatory landscape of PITX1 may affect the expression of limb associated genes,and thus affecting the degeneration of hindlimbs in cetaceans.To investigate the regulatory mechanism of these 32 CNEs in cetaceans,Hi-C data of bottlenose dolphins were combined in our analysis.The data showed that 4genes were annotated within the Topologically associating domains(TAD)containing PITX1 locus.Most importantly,only PITX1 gene and 19 cetaceans-specific CNEs were found to be associated with limb development in this TAD,thus it is further speculated that these 19 cetaceans-specific CNEs play an important role in regulating PITX1.To further verify the activity of these CNEs within the TAD and the function of mutations in these CNEs,we selected two CNEs,namely CNE54(containing cetaceans-specific site-mutations and deletions)and CNE127(containing cetaceans-specific site-mutations)for in vitro experiments.Dual fluorescence report assay showed that CNE54 had enhancer activity.In addition,PEN,the previous identified cis-regulatory elements of PITX1,showed significant enhancer activity in mouse hindlimb,while mouse homologous subregion of this sequence,CNE127,did not show enhancer activity in this study.However,the activity of bottlenose dolphin CNE127 was significantly higher than the control and homologous region in mouse,indicating that CNE127 serve as an enhancer in cetaceans.We further introduced the naturally-occurring mutation G45 A in cetaceans CNE127 into the homologous sequence of mouse.Dual fluorescence report assay showed that the activity of CNE127 was significantly increased,suggesting that the activity difference in CNE127 between cetaceans and mouse was mainly caused by the G45 A in CNE127 of cetaceans.Previous studies have shown that PITX1 negatively regulates the SHH gene,and the arrest of hind limb in spotted dolphin(S.attenuata)is associated with the cessation of SHH expression at an earlier stage.Therefore,CNEs with cetaceans-specific mutations may inhibit the expression of SHH gene by increasing the expression of PITX1,and ultimately contribute to the degeneration of hind limbs in cetaceans.Together,this study focused on the analysis of the coding and non-coding region of hindlimb specific PITX1 and found that coding region of PITX1 showed strongly functional constraints.While 87 CNEs containing cetaceans-specific mutations were screened out in our analysis.After combining mouse hindlimb Chip-seq and ATAC-seq data at E11.5,we further selected 32 CNEs associating with limb development.Comparing with Hi-C data of bottlenose dolphin with these CNEs,1limb associated gene-PITX1 and 19 CNEs regulating limb development were discovered in TAD containing PITX1 locus,indicting these CNEs may function as cis-regulatory elements in regulating PITX1.Two CNEs containing cetaceans-specific mutations,CNE54 and CNE127,were further selected for luciferase activity experiments,and it was found that both CNEs had significant regulatory activities.When the G45 A mutation in cetaceans CNE127 was introduced into the mouse sequence,the activity was significantly higher than that of the mouse homologous sequence.Therefore,G45 A mutations in CNE127 may upregulate the expression of PITX1 expression in cetaceans.Previous study revealed that PITX1 negatively regulate SHH expression in mouse.Therefore,we speculate that upregulated PITX1 express may contribute to hindlimb loss in cetaceans by downregulating the expression of SHH at an earlier stage.This study preliminary reveals the molecular mechanism of degeneration of hindlimb in cetaceans from the perspective of cis-regulatory region.