| An important aim of current molecular biological research is to understand how gene expression is regulated in the context of chromatin.Current opinion on gene regulation have developed to a new level that the gene's position in the genome and in the whole nucleus has important affection on its expression.Exploring the regulation mechanisms on the gene's localization level,including both the localization on genome and localization in nucleus,is important in understanding the conception of "regulatory network".In addition,the gene's localization on genome also has impact on its position in the whole nucleus.Bioinformatic analysis of the whole genome sequence contributes significantly to the understanding of the gene's position on the genome.But the strategies in studying the gene's position in the nucleus are very less.A new method of 3C(chromatin conformation capture) have opened a window on the structural organization of an active endogenous locus in vivo.It has been proposed the long range regulatory elements could interact directly with its target sequences by "looping out" the interval region.Furthermore,the 3C assay has also been applied in confirming the suspected interehromosomal chromatin associations.However,the 3C assay can only be applied to confirming the suspected chromatin associations.In fact,many of chromatin associations couldn't be predicted with current knowledge.To search the unknown chromatin associations,we described a new strategy (ACT-QACC) by incorporating a modified 3C assay with a quantitative assay to capture all the possible association partners of one given chromatin fragment.Based on 3C template DNA,we introduce another restrictive enzyme and obtain the relative short hybridized fragments.The associate chromatin fragments were obtained by inverse PCR. To calculate the frequency of associated chromatin partners,a 19/20 bp MmeI restrictive fragment was obtained and ligated into concatemers for sequencing.The appearance frequency of sequenced tags represents the ligation frequency of this tag to the leader fragment.By comparing the appearance frequencies of different tags,the association frequency,which represents the close degree of the tags to the leader fragment,could be evaluated.This strategy was applied to the murineα-globin cluster with the main enhancer element HS26 as the leader fragment.Totally 300 tags were obtained and 218 tags were mapped to current mouse genome sequence.Analysis of these tags disclosed that the HS26 associated chromatin mainly come from theα-globin gene cluster.In an overview,we have confirmed our previous study onα-globin cluster with normal 3C assay.For instance, the promoters ofαl andα2 globin genes have been sequenced for 28 times.HS21 and HS8,the other two important regulatory elements near HS26 have been sequenced for 16 times and 3 times respectively.There is no any other known HSs upstream of HS26 have been detected.Therefore,we proved that HS26,together with other HSs,regulate only theα-globin genes in 14.Sdpc fetal liver and no other unknown genes have incorporated.Also, the frequency of associated chromatin partners is well consistent with our previous reported results.In addition,the "transcriptional factory" formed byα-globin gene cluster and its upstream housekeeping genes have been proved with our new strategy.Both the MPG gene promoter and the Mare gene promoter has been detected more than 1 time. Interestingly,the loop structure of HS26 located NcoI fragment could also be envisaged with the sequencing results.Another important result is that we have identified one previous unknown regulatory element 475A8 located 500kb upstream ofα-globin gene cluster,which could associate with HS26 with a significant high frequency.Bioinformatic analysis of this fragment suggested that it may contain a lot of GATA-1 and at least one NF- E2 binding sites. Further 3C assay disclosed that this regulatory element could associate with bothα1 andα2 promoter with a high frequency in both murine fetal liver and undifferentiated MEL cells.This result suggested that the 475A8 fragment is a highly conserved regulatory element that plays an important role in regulating the expression ofα-globin genes in adult erythroid cells.To identify the function of this regulatory element,we further performed transfection assay with reporter system in both differentiated and undifferentiated MEL cells.The results showed that the element could repress theα1 globin gene promoter significantly in undifferentiated MEL cells,but has no repression function in differentiated MEL cells.Therefore,we proposed a model to depict the "switching on" ofα-globin genes in late stage erythroid cells in mouse.At the early stage of differentiation,theα-globin genes were repressed by a novel silencer through the "looping" manner.After the terminal differentiation,the repression function of the silencer loses,which may owe to the disappearance of necessary repressive proteins,though the silencer is still associated withα-globin gene promoter.We also choose ApoAⅤgene promoter as the leader fragment to study the spatial organization of this ApoAⅤlocus.ApoAⅤis one member of ApoAⅤ-AⅠ-AⅣ-CⅢgene cluster.However,ApoAⅤis not regulated by the ApoCⅢenhancer in BAC mediated transgenic study.We obtained more than 200 tags with the ACT-QACC assay with the ApoAⅤgene promoter as the leader fragment.Interestingly,the captured associated chromatin are mainly from other chromosomes.No any chromatin fragments from ApoAⅤ-AⅠ-AⅣ-CⅢgene cluster have been observed.The associated chromatin from other chromosomes distributed evenly.No specific chromatin fragment with marked high frequency was observed.Therefore,we hypothesized that the ApoAⅤgene loops away from ApoAⅤ-AⅠ-AⅣ-CⅢgene cluster and participate in a transcriptional factory that contribute to its basal level expression.Comparing analysis ofα-globin gene cluster and ApoAⅤ-AⅠ-AⅣ-CⅢgene cluster suggested that different genes could participate in distinct transcriptional factory.As our previous report,theα-globin genes are important members of a transcriptional factory comprised ofα-globin genes and its upstream housekeeping genes.While ApoAⅤgene move to a transcriptional factory comprised of members from other chromosomes. Concentration of active transcribed gene in the subdomain may contribute significantly to ApoAⅤgene expression.
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