Study On The Expression Map Of Mouse Transcriptional Factor Mdfic And Its Interaction With Rhox5

Mdfic(MyoD family inhibitor domain containing) gene was a newly discovered gene in recent years.It coded theⅠ-mfa domain,but its function research still was blank.In our prior research,we found Rhox5 could interact with Mdfic in yeast,which gave some clues to their function study.Mouse reproductive homeobox on the X chromosome(Rhox) is a novel homeobox gene cluster.Rhox5,also called Peru,belongs to theβsubcluster of Rhox.Despite of its important role during embryogenesis and reproductive development,more and more studies implied a critical role for Rhox5 on the determination and differentiation during myogenesis.The interaction between Mdfic and Rhox5 protein implied that they may form into complex for transcriptional control and then regulate the determination and differentiation during myogenesis.In our research,we focus not only on the function research of this novel transcriptional factor Mdfic,but also on its functional interaction with Rhox5 protein.Mapping the expression level of this novel transcriptional factor Mdfic,predicting its functions by bioinformatics methods will provide a solid basis for further study on the function role of Mdfic.In addition, advanced research about Rhox5/Mdfic functional interaction in vivo,the critical structure for its interaction and its putative interaction model may help us to elucidate the possible mechanism of this complex net of myogenesis.The main results are as follows:1.Rhox5 could interact with Mdfic directly.In our previous study,we obtained a novel interaction partner of Rhox5:No.1-99. yeast two-hybrid assay and GST-pull down assay suggested that Rhox5 could interact with No.1-99 in yeast and in vitro.NCBI blasting indicated that the sequence of No.1-99 contained a whole open reading frame that absolutely matched with No.1-99(100%) Then the full-lenth ofMdfic gene was cloned by PCR from the 7-day mouse embryo library and subcloned into BD(pGBKT7) and AD(pGADT7) vector separately,yeast two-hybrid assay and GST-pull down assay suggested that Rhox5 could also interact with Mdfic in yeast and in vitro.Furthermore,Coimmunoprecipation experiments confirmed that Mdfic could bind to Rhox5 protein directly in vivo.2.The expression map of Mdfic and its bioinformatical analysisIn order to map the expression level of this novel transcriptional factor Mdfic,total RNA was isolated from 10 mouse tissues including heart,liver,spleen,lung,kidney, testiculus,epididymis,small intestine,the 7-8days embryo,skeletal muscle and 3 cells including NIH3T3 cells,the un-induced and induced C2C 12 cells separately.RT-PCR and real-time PCR results indicated that the highest expression level can be detected in mouse liver,spleen,kidney and testiculus,lower expression can be detected in mouse heart,lung, embryo and muscle,while the lowest expression can be detected in epididymis and small intestine.In addition,the expression level of Mdfic in un-induced C2C 12 cells was a little higher than the expression level in induced C2C12 cells,however,compared to this two kinds of cells,the expression level of Mdfic in muscle was lowest.In order to predict the function of Mdfic protein,we analyzed its genomic structure, homology and its basic property by bioinformatics methods.The results indicated that:①Mdfic gene is located at the positive strand of chromosome 6(15670661-15752165 bp),and has a full length of 81454 bp composed of 5 exons and four introns.The junction sequences between introns and extrons of Mdfic gene is consistent with the "gt-ag" role.②The synthesis of Mdfic mRNA may be controlled at the translational level by two different codons,an AUG and three upstream non-AUG translational initiators(located at No 37-39 bp,70-72 bp and 142-144 bp respectively) which could extend the protein N-terminal by 82, 106 or 117 aa,allowing the production of four protein isoforms,Mdfic p26,p34,p37 and p38 respectively.③The result of the posttranslational modification sites and the presumable secondary structure of this four Mdfic protein isoforms showed that the 247aa this four Mdfic protein isoforms shared displayed the same posttranslational modification sites and the same secondary structure.④All of this four Mdfic protein isoforms was predicted to be located in nucleus prominently(the validity is 52.2%).⑤Compared to Mdfic p26 protein, the extended amino acids in the protein N-terminal of Mdfic p34,p37 and p38 respectively have little effect on the posttranslational modification sites,the presumable secondary structure and the subcellular localization.⑥The result of multiple sequence alignment indicated that Mdfic p26 protein had a similarity over 81%and 59%with HIC p32 and XIC protein.Their translation is initiated at an AUG codon and they have the similar size,247,246 and 246 amino acids in length.Most importantly,the similarity of their C terminal amounted to over 95%.Meantime,the result of multiple sequence alignment indicated Mfic p26 prtoein had a high similarity with mouseⅠ-mfa protein and humanⅠ-mfa protein,especially for their C terminal.Therefore,Mdfic gene may be a mouse orthologue of HIC.Both of them belong to theⅠ-mfa domain family.Ⅰ-mfa domain may be critical for Mdfic protein. 3.The critical structure for Rhox5/Mdfic interaction.In order to determine the minimal domain of Mdfic for Rhox5/Mdfic interaction,Mdfic A truncated fragment(from 72 aa to 247aa),including theⅠ-mfa domain,was abtained. Two-ways yeast two-hybrid assay and GST-pull down assay indicated that Mdfic A truncated fragment displayed a little higher affinity with Rhox5 than the Mdfic protein,which suggested that the minimal domain of Mdfic for Rhox5/Mdfic interaction may be in the Mdfic A truncated fragment and the amino acids from 1 to 71 of Mdfic may affect the Rhox5/Mdfic interaction.Furthermore,Mdfic A truncated fragment was divided into two part: Mdfic B,without the conservativeⅠ-mfa domain,included the amino acids from 72 to 191; Mdfic C,with the conservativeⅠ-mfa domain,included the amino acids from 192 to 247. Surprisingly,the Mdfic C truncated fragment WithⅠ-mfa domain loosed the ability of interacting with Rhox5,while the Mdfic B truncated fragment withoutⅠ-mfa domain could also bind to Rhox5.In addition,Two-ways yeast two-hybrid assay and GST-pull down assay suggested Rhox5 N truncated mutants(without homeobox domain) couldn't interact with Mdfic. However,the Rhox5 N truncated mutants(without homeobox domain) keep the ability of binding to Mdfic protein.This indicated the homeodomain of Rhox5 protein was critical for its interaction with Mdfic.In this study,we detected the expression level of Mdfic in the 10 mouse tissues including heart,liver,spleen,lung,kidney,testiculus,epididymis,small intestine,the 7-gdays embryo, skeletal muscle and 3 cells including NIH3T3 cells,the un-induced and induced C2C12 cells separately using RT-PCR and real-time PCR methods and proposed that the expression level of Mdfic in un-induced C2C 12 cells was a little higher than the expression level in induced C2C 12 cells,however,compared to this two kinds of cells,the expression level of Mdfic in muscle was lowest for the first time.These results suggested that Mdfic may act as a suppressor during the myogenesis.The conservative I-tufa domain may be critical for the function of Mdfic protein according to the bioinformatics analysis.In addition,we first characterized the interaction Rhox5 and Mdfic in vitro and in vivo and difined the critical structure for its interaction.Most importantly,we postulate:theⅠ-mfa domain of Mdfic may bind with other kinds of transcriptional factors and regulate its activity,while the interaction between Rhox5 and Mdfic may regulate the event controlled by Mdfic and other transcriptional factors.They may form a complex regulatory net to regulate the determination and differentiation during myogenesis.