| BackgroundPolycystic ovary syndrome(PCOS)is one of the most common heterogeneous endocrine disorders,and accounts for most of the infertility cases of female origin.PCOS is primarily characterized by polycystic ovaries on ultrasound,oligo-ovulation or anovulation,and biochemical or clinical hyperandrogenism.Among them,hyperandrogenism is the most common characteristic of PCOS,and has been used as a criterion of the diagnosis for this disease.Hyperandrogenism itself may contribute to the development of PCOS,and paradoxically,the hyperinsulinemia,which is the accompanied disease of PCOS,is likely to aggravate androgen biosynthesis through amplifying LH signaling on theca cells.It is safe to say that a vicious cycle between PCOS and hyperandrogenism could have been established at certain stage of PCOS developmentThe excessive androgen is biosynthesis by ovaries and adrenal glands,and especially,theca cells in ovarian follicles.Until now,the etiology and pathogenesis of PCOS are not fully understood.To further demonstrate the molecular mechanism of hyperandrogenism in PCOS,we performed cDNA microarray to compare ovarian transcriptomes between PCOS patients(n=3)and normal female(n=3).We found that SET(SE translocation,SET)was one of the upregulated genes in PCOS and its increase was verified with quantitative real-time PCR and Western blot.Immunological Histological Chemistry verified that SET was expressed in oocytes and theca cells.Through inhibiting the phosphatase activity of PP2A(protein phosphatase 2A),SET upregulated the transcriptional expression of steroidogenic enzymes cytochrome P450 17 alpha hydroxylase/lyase(P450c17)and 3 beta-hydroxysteroid dehydrogenase 2(HSD3B2),and exerts a possitive action on P450c17 lyase activity.In addtion,SET can bind to the CYP17(the gene encoding P450c17)promoter and initiate the P450cl7 transcription,which may also lead to the increased androgen biosynthesis.Together,SET overexpression upregulates the androgen biosynthesis and may participate in the development of hyperangrogenism in PCOS.However,the upstream regulational mechanism(s)of SET gene is not well understood.Whether SET is the primary cause or a result of PCOS is an issue of debate.Identification of the binding proteins and cis-elements controlling SET expression will help us to better understand the SET regulation as a possible mechanism underlying the pathogenesis of aforementioned diseases and providing new therapeutic methods for hyperandrogenism in PCOS.SET(SE translocation,SET),also known as Template Activating Factor-1β(TAF-1β),was first identified in 1992,as a component of SET-CAN(also termed nup214)fusion protein generated by chromosomal translocation in a patient SE with acute undifferentiated leukemia.It is involved in gene transcription,histone modification and cell proliferation.As a multifunctional oncoprotein,SET is overexpressed in tumor cells and may contribute to the malignant transformation.In the brain neurons of patients with Alzheimer’s disease(AD),SET overexpression lead to the formation of intracellular neurofibrillary tangles(NFTs).Together,SET is widely expressed and participates in various pathological or physiological process.Through the use of alternative promoters,SET gene produces four mRNA variants with distinct transcription start site(TSS).Among the four transcription variants,transcript 1(SETa/TAFa)and transcript 2(SETβ/TAFβ)represent two major transcripts.Studies have shown that transcript 2 is expressed more widely than transcript 1(SETα/TAFα).In porcine testes and xenopus oocytes,transcript 2 is the major variant participating in the gonad development.The early stage hemopoietic lineages,including Jurkat and PEER,only express transcript 2.Besides,the expression levels of transcript 2 tend to be more stable than those of transcript 1.Several studies have focused on the transcriptional regulation of transcript 1,and found NF-κB and Spl to be the major factors upregulating transcript 1 promoter activity.However,the molecular mechanism(s)of SETβ transcriptional regulation is not well understood.The current study is designed to characterize the transcription regulation of SET transcript 2.Objectives1.Identify the core promoter of human SET gene and demonstrate the transcriptional regulatory mechanism of huamn SET geneClone and analysis the promoter of human SET gene transcript 2.Identify the core promoter region regulating SETβ expression.Screen out crucial transcription factors controlling SET promoter activity.Verify the direct binding between transcription factors ZFX and the cis-acting element,and define the binding sequence on SET core promoter.2.Using clinical specimen to verify the ZFX-SET axisObserve the location of ZFX and SET in human ovary.Compare the expression of ZFX and SET in the endometrial stromal cells(ESCs)among normal ESCs,ectopic ESCs and eutopic ESCs.Illustrate the effect of ZFX on ESCs proliferation.MethodsPart One:Identify the core promoter of SET gene1.We use the Promoter 2.0(http://www.cbs.dtu.dk/services/Promoter/)software and the promoter marker,including H3K4Me3 and Pol-Ⅱ,to elucidate the promoter sequence of human SET gene.2.The predict promoter region was PCR-amplified using human DNA from HeLa cells and inserted into the polycloning site preceding the luciferase coding sequences in pGL3-Basic plasmid.The promoter activity of the whole SET promoter was identified using Dual-luciferase reporter assays.3.A series of 5’deletion promoter plasmids were used to identify the proximal minimal promoter of SET.Seven sequentially truncated promoter plasmids were transfected in HeLa and HEBK293 cells.The promoter activity was detected with Dual-luciferase reporter assays.4.The core promoter region was analyzed by Genomatix(http://www.genomatix.com/cgi-bin//eldorado/main)database and Jarspar(http://jaspar.binf.ku.dk/cgi-bin/iaspar)software.5.Co-transfection was performed with plasmids expressing predict transcription factors(TFs)and SET promoter plasmid.Dual-luciferase reporter assays were used to demonstrate the transcriptional regulation of these TFs on SET promoter activity.Part Two:ZFX transactivates SET expression1.RNA interference and overexpression experiments were used to demonstrate and verify the regulation of the predict transcription factors on SET promoter.2.Jarspar(http://jaspar.binf.ku.dk/cgi-bin/jaspar)database was used to predict the the cis-acting element on SET The direct binding between ZFX and the specific cis-acting element on SET promoter was verified by the mutational analysis.3.ChIP and the innovative Transient ChIP assays were carried out to confirm binding between ZFX and the specific cis-acting element on SET promoter.Part Three:ZFX-SET participates in the development of PCOS and may be involved in other SET-related diseases1.Collecting the ovaries from nomal female and PCOS patients,and examine the location and expression of SET and ZFX using immunohistochemistry(IHC).2.Compared the expression of SET and ZFX in endometrial stromal cells(ESCs)among normal ESCs,ectopic ESCs and eutopic ESCs with real-time RT-PCR.MTT assay was used to illustrate the effect of ZFX on ESCs proliferation.ResultsPart One:Identify the core promoter of SET geneThe SET isoforms are generated by alternative splicing.SET transcript 2(SETβ)has higher promoter activity than transcript 1.The full-length SETβ promoter plasmid(pGL3-855)generated strong luciferase activities in both HeLa and HEK 293cells.When the sequences were deleted from-855 bp to-157 bp,the truncated region still exhibited high promoter activity.Further deletion of the promoter led to a stepwise reduction of promoter activities.A significant reduction was noted to occur between pGL3-157 and pGL3+47.Thus,the core promoter of SETβ gene was located within the region-157/+47 bp.The 204 bp region harbors strong positivecis-active elements crucial for the promoter activity of the human SETβ gene.The JASPAR database and Genomatix software were used to predict transcription factor binding sites.Several highly scored binding sites were identified within the 204 bp(-157/+47)region,including those for E2F3,E2F4,Sp1,E2F1,ZFX and EGR1.Part Two:ZFX transactivates SET expressionCo-transfection was performed to determine whether these putative sites are functional.E2F3a and E2F3b overexpression significantly suppressed SET promoter activity(p<0.05),while E2F4 and ZFX overexpression significantly increased SET promoter activity,to 1.8-fold and 2.3-fold(p<0.05),respectively.E2F1,Sp1 and EGR1 overexpression had no significant effect(p>0.05).ZFX accounts for a large portion of the SET promoter activity and the transactive function is dose-dependent.Manipulation of ZFX levels by siRNA knockdown or overexpression confirmed the specificity and significance of the ZFX-mediated SET promoter activation.There are four ZFX-binding sites(-146/-133,-99/-86,-93/-80 and-69/-56 bp)located on core promoter region.Four plasmids with each containing a mutation on individual ZFX-binding site were constructed.Only mutation of the fourth binding site led to a 50%reduction of promoter activity,whereas mutation of the first,second and third binding sites had no significant effect on promoter activity.Co-transfection experiments with reporters containing mutant promoters and ZFX expression vector confirmed the significant role of the fourth site in ZFX regulation on SET promoter.ChIP and Transient ChIP results verified the binding of ZFX to its cognate sequences of Site4 in the SET promoter.Part Three:ZFX-SET participates in the development of PCOS and may be involved in other SET-related diseasesZFX and SET were co-located in oocytes and theca cells both in the ovaries of PCOS patients and normal female.We detected the expression levels of ZFX and SET in Endometriosis(EMs).The results showed that ectopic ESCs had higher ZFX and SET level than normal controls and eutopic ESCs.Besides,ZFX was positively related to the cell number of ESCs.Conclusion1.The core promoter of SETβ is located with the-157/+47 bp region relative to theTSS.Several highly scored binding sites were identified within this 204 bpregion,including those for E2F3,E2F4,Sp1,E2F1,ZFX and EGR1.Amongthem,E2F1,Spl and EGR1 had no significant effect on SET promoter,E2F3 significantly suppressed SET promoter activity,while E2F4 and ZFX significantly increased SET promoter activity.2.ZFX,the Zinc finger and X-linked transcription factor,was able to transactivate the SETβ promoter in the dose-dependent way through directly binds to its cognate sequences of Site4(-69/-56 bp,CAGGCCAATGGCGC)on the SET promoter.3.Using clinical specimen,we demonstrated that the ZFX and SET were co-located on the theca cells and oocytes in ovaries of PCOS patients and normal female.It provided the histological basis for the ZFX-SET axis involved in the development of hyperandrogenism in PCOS.Besides,we demonstrated that the expression levels of ZFX and SET were consistent with each other in ESCs of EMs patients.Thus,the ZFX-SET axis is likely to participate in various SET-related disease through regulating mutiple downstream physical or biological processes.More studies are required to define the in vivo significance of this mechanism.4.Together,we demonstrate the core promoter region of human SET transcript 2.It would facilitate the further investigation on SET gene regulation in PCOS and other SET-related benign and malignant diseases.The ZFX-SET axis improves the regulation web of SET,which is the central joint in the development of hyperandrogenism in PCOS,and may provide new insight into therapeutic method for SET-related diseases. |
