Studies Of NF-E2 In The Regulation Mechanisms Of β-globin Gene Expression

Eukaryotic gene expression is a complex process that relies on the collective action of genetic and epigenetic regulations.It can be viewed within a conceptual framework in which regulatory mechanisms are integrated at three hierarchical levels,i.e.the sequence level,the chromatin level and the nuclear level.At the sequence level,gene expresson is controlled by the interplay between DNA and DNA or DNAs and proteins.DNA mainly refers to the structural genes and cis-regulatory elements.Protens include RNA polymeraseⅡ,trans-acting factors and other co-regulatory proteins.Distal regulatory elements are the most important regulatory elements existing in gene cluster and widely dispersed in genome of higher eukaryotes.They occur so frequently and play critical roles in the process of cellular diffierentication and organic,development.They can exert their effects from several hundred bps to several hundred kbs far away from the promoters of their target genes.β-globin gene cluster is an exellent model for the study of distal enhance actions.The murineβ-globin locus contains fourβ-like globin genes(εy,βh1,β-maj,andβ-min) and an upstream LCR consisting of six DNaseⅠhypersensitive sites(HSs).How such distal LCR work is one of the basic and pivotal problems in the regulation of globin gene expression.Three models have been proposed to explain the action of LCR.They are:Looping modcl,Linking model and Tracking model. Recently,a notable advance has been achieved in the studies of distal enhance action. By using two novel techniques,RNA-TRAP(Tagging and Recovery of Associated Proteins) and 3C(Chromatin Conformation Capture),researchers demonstrated for thc first time that LCR spatial locates close to the active globin gene promoter.It is the first time that people get direct evidence supporting looping model.On this base, Grosvel et al put forward novel concept,the active chromatin hub(ACH),to explain the action of distal enhance action in nuclei.The ACH is local nucleic compartment formed by distal enhance elements and promoters which concentrates the transcription factors and isolates the gene from surrounding enviroment.The question is what factors involved in the looping and ACH formation ?GATA-1 and EKLF are two important factors which are involved in globin gene activation.EKLF mainly binds to conserved CACCC sequences,and regulates adult globin gene expression.GATA-1 distributed widely on the wholeβ-globin gene cluster,including LCR and gene promoter.In EKLF knocked-out mouse,the structure of ACH dispeared.While in G1E cell in which GATA-1 is absent, proximity between the LCR and theβ-major promoter reduced,indicating that GATA-1 and EKLF are two factors which are involved in the looping or ACH formation.NF-E2 is another important factor which played important roles in erythroid differentiation and globin gene expression.NF-E2 composed of a tissue-specific subunit,NF-E2 p45 and a smaller subunit NF-E2 p18 that is widely expressed. During erythroid differentiation,p18 formed heterodimer with p45 to activate globin genes expression.NF-E2 binds two'tandom AP-1/NF-E2 sites in HS2 which form the core of its enhancer activity.During globin gene activation,both hypersensitive sites of LCR and the promoters ofβ-major andβ-minor globin genes were occupied by NF-E2.In addition,the histone modification pattern and the transfer of RNA PolⅡfrom LCR toβ-major promoter have a close relationship with the transcription factor NF-E2.So it is interesting and meaningful to elucidate the mechanism about how NF-E2 involved in globin gene activation.In this reaserch,we tried to study the mechanism about how NF-E2 regulating globin gene expression.We first knocked down NF-E2p18 subunit by retrovirus vector-directed RNAi in MEL DS19 cell line in order to establish NF-E2 activity reduced cell line.Then we examined the function of NF-E2 in NF-E2p18 silenced cell pools.At last,we explored the'mechanism of NF-E2 in LCR-directed looping formation and discussed the relationship between NF-E2 and how RNA PolⅡinitiating globin gene transcription.The results showed that:In NF-E2p18 knocked-down cell line,the occupancy of NF-E2 p18 and p45 on both HSs of LCR and globin gene promoter declined, accompanied by low expression level of a andβglobin genes.In additon,the binding activity of RNA PolⅡat both HSs of LCR and globin gene promoters also reduced,indicating that the binding of RNA PolⅡatβglobin gene promoter relied on NF-E2 binding.To assay the effect of NF-E2 on LCR proximity with each active globin gene,a primer within a BglⅡfragment containing HS2 was used in pair-wise combination with a primer within a BglⅡfragment in the vicinity of eachβ-globin gene,such as:β—maj,β—min,Ey andβh1.The results showed that the relative proximity of eachβ-like globin gene with(the LCR) HS2 decreased in a distance-dependent manner in DS19 cell line before differentiation.After DMSO induction,the ligation frequency between HS2 andβ—maj,β—min increased, HS2-β-major ligation product increased 2.2-fold.A moderate increase in proximity was observed between HS2 and 13-minor,which likely reflects the LCR-dependent activity of this gene.Proximity of the LCR with Ey andβh1 embryonic globin genes was not significantly increased,consistent with low-level transcription of these genes in DS19sip18 cells.However,when NF-E2p18 knocked down,HS2-βmajor ligation product decreased 0.43 fold in accordance with low NF-E2 binding frequency and low expression of globin genes.The results of 3C analysis indicate that NF-E2 is necessary to the transition of globin locus into a looped conformation as determined by 3C analysis. Combined the research results on transcription factor NF-E2 in other labs and the research work about this factor in our lab,we think that the mechanism of NF-E2 involvement in activiatingβ-globin gene expression includes three steps:1. Before erythroid terminal differentiation,LCR was occupied by NF-E2p18 and BachI,globin gene expression was inhibited.During the process of globin gene activation,p18 heterodimerized with p45 forming NF-E2 complex.2.NF-E2 occupied NF-E2/AP-1 binding site on LCR,recruited other protein activators,RNA PolⅡetc,forming LCR preinitiation complex.At the same time,NF-E2 was also recruited toβ-maj promoter through protein-protein interaction,forming another protein complex.3.The binding of protein complex on LCR and promoter lead to chromatin structure alteration,forming loop structure,further interaction of these complexes promoted the transfer of RNA PolⅡfrom LCR toβ-globin gene promoter,leading to transcription initiation.