| The regulatory factor X (RFX) that belongs to transcription factors (TFs) gene family with a highly conserved76-amino acid long DNA binding domain with a wing-helix structure by which it can bind to a scope of DNA sequences was first discovered in mammals more than20years ago as a regulatory factor. RFX binds to X-box motif that is a conserved cis-regulatory element with typically14-mer DNA sequences. X-box was primarily identified in the promoter regions of major histocompatibility complex (MHC) class II genes. RFX1was the prototype of the RFX family, then it was found that RFX1also known as enhancer factor C (EF-C; also called EP) had the function to be a transactivator of the hepatitis B virus enhancer. RFX1was widespread in mammals, and expressed in various tissues. It played a variety of key regulation control in activating or suppression genes expression in many biological processes. In the mouse genome, it is located on chromosome8C3. In the current research, it was very clear that RFX1regulated in the occurrence and development of cilia in mammals cells. However there are few reports about RFX1whether direct or indirect regulation in genes activity, or coordinate with other factors in some biological processes. In this context, based on present research, RFX1was preliminarily investigated by computational prediction and experimental validation to show that RFX1will have characters in ICR mouse genome.(1) By using the bioinformatics methods, compared with factor CTCF-, p53-binding sites, analysis RFX1binding sites, its targeted genes classification by different functional categories of biologicalprocess and chromosome distribution in mouse genome. The binding sites of RFX1, CTCF and p53were scanned with the matrixes of RFX1and p53in finding Transcription factor binding sites with computer programming. Then, acquired Refseq genes of these putative sites were analyzed their annotations after blasting on line. Genome-wide distribution of identified binding sites was determined using reference RefSeq genes downloaded from UCSC genome browser. A5-kb long region of the transcription start site (TSS) is described as the promoter of the gene. To characterize how RFX1-binding sites are located in the mouse genome, their localization is compared with the complete well-annotated mouse genes. There are majority of clusters of approximately9173possible binding sites in mouse genome. Twenty nine percent of these sequences are located far from target genes,21%in the nearest genes,16%in the introns,20%in both sides of enhancer, and few in the exons, UTR and promoter. In addition, the biological processes and chromosomal localization of RFX1target genes are analyzed. There was no notable enrichment within promoter regions.The local distribution of RFX1binding sites on chromosomes is very interesting. Previous studies have not revealed clusters of RFX1location on chromosomes. In this work, a comprehensive analysis has shown that RFX1binding sites spread across each chromosome. Most of them resided in chromosomes1,2,3,4,5,7,9and11. During comparison with CTCF and p53binding sites under the same circumstances, the binding sites of RFX1overlapped with CTCF predominantly clustered on chromosome11, secondly on chromosome4and7, when the distance of sites between RFX1and CTCF were within2000bp. There was no conspicuous distribution on the remaining chromosomes. Additionally, there were just approximate results in the case of RFX1bindig sites overlapped with p53, and no site was found on chromosome10.(2) It was illustrated that the expression of RFX1had temporal and spatial characteristics and the particular protein structure. Q-PCR analysis revealed that: RFX1gene mRNA expressed highly in brain, compared with other organs and tissues during the same period in mice. And RFX1gene mRNA also expressed highly in E17.5brain, compared between embryonic and neonatal mouse. And the expression of RFX1reduced at postpartum day, specifically at postpartum day2. The expression of RFX1protein in E17.5brain was the same as mRNA.(3) Analysis of RFX1protein domain: RFX1protein contains several domains, including domain A as an activation domain (residues275-302); DBD as a DNA binding domain (423-498); domain B (588-623) and C (671-710) which play important role in dimerization and are thus called the extended dimerization domain; as well as the domain D(727-893) as dimerization domain. RFX1DBD proved to be nuclear factor, with winged-helix conformation belongs to subfamily of HTH (helix-turn-helix) transcription factors. The DBD is composed of three structures, including of three α-helices (H), three β-strands (S) and three connecting loops (L), and their order is H1-S1-H2-L1-H3-L2-S2-W1-S3. S2and S3form the third loop, namely W1of the winged-helix motif. H1, S1, H2, H3, L2and S3form the hydrophobic core of the DBD. Fifteen out of nineteen acids in the winged-helix motif are either identical or conserved among RFX proteins. And motif consensus of RFX1in mouse was analyzed and verified by experiment at the same time.In this context, based on present research, RFX1was preliminarily investigated by computational prediction and experimental validation to show that RFX1had79binding sites in the promoters of323. In order to verify the feasibility of the method, the regulatory motifs in1kb promoter regions of323RBPs genes was only analyzed. Which expressed in E13.5mouse brain,65genes showed conserved binding sites, and10of65genes have2motifs,3have3motifs, and dispersal both sides of TSS.(4) Chromatin immunoprecipitation (ChIP) is a powerful technique for studying protein–DNA interactions within the cell, which was widely used in the past decade to map the location of post-translationally modified histones, transcription factors, chromatin modifiers and other nonhistone DNA-associated proteins, either in restricted genomic regions or at the genome-wide level. In this context, the genes that were regulated by RFX1were precipitated by RFX1-antibody (ChIP grade) and purified by DNA purification kit, then tested by PCR and Q-PCR, would be send to sequence. The data would be used to analyze and test factors interacted with RFX1and genes regulated by RFX1in the future. |
