A Novel CpG-free Vertebrate Insulator Silences the Testis-specific SP-10 Gene in Somatic Tissues

Regulation of cell type-specific gene transcription is central to cellular differentiation and development. During spermatogenesis, a number of testis-specific genes are expressed in a precise spatiotemporal order. How these genes remain silent in the somatic tissues is not well understood. Our previous studies using the round spermatid-specific mouse SP-10 gene, which codes for an acrosomal protein, revealed that its proximal promoter acts as an insulator and prevents expression in the somatic tissues. Here we report that the insulator tethers the SP-10 gene to the nuclear matrix in somatic tissues, sequestering the core promoter in the process, thus preventing transcription. In round spermatids where the SP-10 gene is expressed, this tethering is released. TAR DNA-binding protein of 43 kDa (TDP-43), previously shown to interact with the SP-10 insulator, was found to be in the 2 m NaCl-insoluble nuclear matrix fraction. TDP-43 prevented enhancer-promoter interactions when artificially recruited between the two by Gal4 strategy. Knockdown of TDP-43 using small interfering RNA released the enhancer-blocking effect of the SP-10 insulator in a stable cell culture model. Mutation of TDP-43 binding sites abolished this effect. Finally, a 50-bp subfragment of the SP-10 insulator, which includes TDP-43 binding sites, functioned as a minimal insulator in transgenic mice and silenced an otherwise ectopically expressed transgene in somatic tissues. The SP-10 insulator lacks CpG dinucleotides or CTCF binding sites. Thus, the present study characterized a novel vertebrate insulator in a physiological context and showed for the first time how a testis-specific gene is silenced in the somatic tissues by an insulator. Regulation of cell type-specific gene transcription is central to cellular differentiation and development. During spermatogenesis, a number of testis-specific genes are expressed in a precise spatiotemporal order. How these genes remain silent in the somatic tissues is not well understood. Our previous studies using the round spermatid-specific mouse SP-10 gene, which codes for an acrosomal protein, revealed that its proximal promoter acts as an insulator and prevents expression in the somatic tissues. Here we report that the insulator tethers the SP-10 gene to the nuclear matrix in somatic tissues, sequestering the core promoter in the process, thus preventing transcription. In round spermatids where the SP-10 gene is expressed, this tethering is released. TAR DNA-binding protein of 43 kDa (TDP-43), previously shown to interact with the SP-10 insulator, was found to be in the 2 m NaCl-insoluble nuclear matrix fraction. TDP-43 prevented enhancer-promoter interactions when artificially recruited between the two by Gal4 strategy. Knockdown of TDP-43 using small interfering RNA released the enhancer-blocking effect of the SP-10 insulator in a stable cell culture model. Mutation of TDP-43 binding sites abolished this effect. Finally, a 50-bp subfragment of the SP-10 insulator, which includes TDP-43 binding sites, functioned as a minimal insulator in transgenic mice and silenced an otherwise ectopically expressed transgene in somatic tissues. The SP-10 insulator lacks CpG dinucleotides or CTCF binding sites. Thus, the present study characterized a novel vertebrate insulator in a physiological context and showed for the first time how a testis-specific gene is silenced in the somatic tissues by an insulator. Regulation of transcription of a tissue-specific gene is central to cellular differentiation and development. Mechanisms must be in place not only to activate the gene in the correct cell type but also to keep the gene silenced in all other tissues. We study this problem using the mouse testis-specific SP-10 gene as a model. The SP-10 gene codes for an acrosomal protein conserved in mammals and is exclusively expressed in round spermatids (1Reddi P.P. Flickinger C.J. Herr J.C. Biol. Reprod. 1999; 61: 1256-1266Crossref PubMed Scopus (53) Google Scholar). In this report, we address the mechanism of transcriptional silencing of the SP-10 gene in the somatic tissues. Our previous work in transgenic mice showed that the SP-10 proximal promoter performs dual functions; it activates testis-specific transcription and also acts as an insulator preventing the possibility of transcription in the somatic tissues (2Reddi P.P. Shore A.N. Shapiro J.A. Anderson A. Stoler M.H. Acharya K.K. Dev. Biol. 2003; 262: 173-182Crossref PubMed Scopus (22) Google Scholar). Insulators are DNA sequences located at gene boundaries (3Kellum R. Schedl P. Cell. 1991; 64: 941-950Abstract Full Text PDF PubMed Scopus (495) Google Scholar). Their action prevents ectopic expression of genes they flank. Insulators are operationally defined as enhancer blockers and barrier elements. Enhancer-blocking insulators prevent a foreign enhancer from inappropriately encroaching into the promoter of the neighboring gene. The barrier insulators prevent the spread of heterochromatin and thus prevent untimely shut down of gene expression. Insulators may possess only one or both of the above properties (4West A.G. Gaszner M. Felsenfeld G. Genes Dev. 2002; 16: 271-288Crossref PubMed Scopus (515) Google Scholar). The chicken β-globin HS4 insulator (cHS4), 3The abbreviations used are:cHS4chicken β-globin HS4 insulatorDBDDNA binding domainCMVcytomegalovirusTKthymidine kinasesiRNAsmall interfering RNATDP-43TAR DNA-binding protein of 43 kDa. a prototypic vertebrate insulator, possesses both of the above activities (5Chung J.H. Whitney M. Felsenfeld G. Cell. 1993; 74: 505-514Abstract Full Text PDF PubMed Scopus (763) Google Scholar, 6Pikaart M.J. Recillas-Targa F. Felsenfeld G. Genes Dev. 1998; 12: 2852-2862Crossref PubMed Scopus (344) Google Scholar). Binding sites for the transcription factors CTCF and USF1 have been shown to be responsible for the enhancer-blocking and barrier functions of the cHS4 insulator, respectively (7Bell A.C. West A.G. Felsenfeld G. Cell. 1999; 98: 387-396Abstract Full Text Full Text PDF PubMed Scopus (866) Google Scholar, 8West A.G. Huang S. Gaszner M. Litt M.D. Felsenfeld G. Mol. Cell. 16: Full Text Full Text PDF PubMed Scopus Google Scholar). The located between the and genes and the the are two other well characterized vertebrate insulators with enhancer-blocking The the of expression of and or not it is by the protein, CTCF A.C. Felsenfeld G. PubMed Scopus Google Scholar, C.J. PubMed Scopus Google Scholar). the acts as an enhancer when by CTCF and prevents gene thus the 2002; PubMed Scopus Google Scholar). the and the insulators CpG the of which by number of other vertebrate insulators also CpG dinucleotides and binding sites for the insulators at the RNA genes of A. M. Mol. Biol. PubMed Scopus Google the chicken insulator of the β-globin gene A.C. Recillas-Targa F. West A.G. M. M. Felsenfeld G. PubMed Scopus Google the at the (5Chung J.H. Whitney M. Felsenfeld G. Cell. 1993; 74: 505-514Abstract Full Text PDF PubMed Scopus (763) Google the β-globin insulator G. PubMed Google the insulator of the PubMed Scopus Google and the insulator located between the and genes PubMed Scopus Google Scholar). chicken β-globin HS4 insulator DNA binding small interfering RNA TAR DNA-binding protein of 43 kDa In the SP-10 insulator, which functioned as an enhancer in transgenic not CpG dinucleotides or CTCF binding sites (2Reddi P.P. Shore A.N. Shapiro J.A. Anderson A. Stoler M.H. Acharya K.K. Dev. Biol. 2003; 262: 173-182Crossref PubMed Scopus (22) Google Scholar). We that the SP-10 insulator to TDP-43 DNA-binding protein of 43 and that of TDP-43 binding sites enhancer-blocking K.K. Shore A.N. Stoler M. P.P. Dev. Biol. PubMed Scopus Google Scholar). we that the study of the SP-10 insulator novel of insulator we have the mechanism of SP-10 insulator in the the of TDP-43 for SP-10 insulator a 50-bp subfragment as a minimal insulator of silencing a transgene in the somatic tissues. and number and in with and in with stable cell from In from mouse and round spermatids as Cell. Full Text PDF PubMed Scopus Google Scholar, Felsenfeld G. S. A. PubMed Scopus Google Scholar). the with for at by for at with and in and at The DNA with these was DNA was also in was using matrix as well as DNA as The SP-10 was using an at as shown in of of and of in a The for by of for for for with a at for The was in a these by of of from matrix using of of spermatids was using as previously K.K. Shore A.N. Stoler M. P.P. Dev. Biol. PubMed Scopus Google Scholar, J.C. M. Biol. 74: PubMed Scopus Google Scholar). from mice to to round from mice used as a of round spermatids from mice and to and to the and with spermatids from used to nuclear from as Felsenfeld G. S. A. PubMed Scopus Google Scholar). In in for with a by using a and at for at in and m in and 2 m and at for in and m in m and and for with at a the at for and was with an of from to of the with of by at for The at and with of was used for The 2 m nuclear matrix was in in an and to TDP-43 in as K.K. Shore A.N. Stoler M. P.P. Dev. Biol. PubMed Scopus Google Scholar). number and number from to number was from using an from DNA TDP-43 was from mouse and in was used as a to or using Gal4 DNA binding or of TDP-43 in as in with the Gal4 of the in was by The SP-10 was the was a from of of The was as The was from and was into of in P.P. Shore A.N. Shapiro J.A. Anderson A. Stoler M.H. Acharya K.K. Dev. Biol. 2003; 262: 173-182Crossref PubMed Scopus (22) Google Scholar). Thus, the enhancer and mouse SP-10 core of the between the enhancer and the core using the to for a of all The for mouse SP-10 promoter are as in P.P. Shore A.N. Shapiro J.A. Anderson A. Stoler M.H. Acharya K.K. Dev. Biol. 2003; 262: 173-182Crossref PubMed Scopus (22) Google number The SP-10 core promoter was with and sites using the (1Reddi P.P. Flickinger C.J. Herr J.C. Biol. Reprod. 1999; 61: 1256-1266Crossref PubMed Scopus (53) Google as and into (2Reddi P.P. Shore A.N. Shapiro J.A. Anderson A. Stoler M.H. Acharya K.K. Dev. Biol. 2003; 262: 173-182Crossref PubMed Scopus (22) Google The insulator of the SP-10 from was with sites both and into the of The 50-bp SP-10 insulator of the SP-10 was with sites from and into the of the 50-bp insulator in both and 2 and in a to of the 50-bp insulator all in was to of The 50-bp insulator sites both was into which the insulator of the enhancer in the correct as well as to and with the core a to the to the transcriptional of the gene promoter was from with and sites and into (2Reddi P.P. Shore A.N. Shapiro J.A. Anderson A. Stoler M.H. Acharya K.K. Dev. Biol. 2003; 262: 173-182Crossref PubMed Scopus (22) Google Scholar). was used to in the 50-bp SP-10 insulator in both as and by in or using in and the of DNA well was The was at a with the to for activities by the to the with the in and with of and The was of stable cell using the as the In two at and and in and into used to these expression with and as the all was by not RNA or from was into by using as the for of mouse using transgene was as a by the of and using the Scholar). DNA was from mice using the and transgenic by using to the of the and used as and of a The transgenic by using the and the enhancer as for was used to the number of the transgene in all of the mouse that used for the of expression. of DNA was to with a and to from was using and the DNA was using the The was to an and in an number the was into a was the and was all the of The was to to of the of the of a at the of the core promoter in all used for transgenic in a above 2 of the of a at the of the to transgene in a at for to in a at for all of the The transgenic mouse which a that is at and the of that was as a to the number of the of the The of from was by that to the number of the The for of the was to the number of that mouse number was and using the enhancer from (2Reddi P.P. Shore A.N. Shapiro J.A. Anderson A. Stoler M.H. Acharya K.K. Dev. 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Full Text PDF PubMed Scopus Google Scholar, Felsenfeld G. S. A. PubMed Scopus Google Scholar). from mouse gene or round spermatids gene with with and with the nuclear as and the DNA was the SP-10 gene insulator is to the we a of at the to of the mouse SP-10 gene and the In where the SP-10 gene is not expressed, the to was found to in the nuclear matrix as with the sequences using to the also showed as the not was in the of round spermatids where the SP-10 gene is expressed that the proximal promoter of the SP-10 gene is the nuclear matrix in that not SP-10 and that it is released the thus SP-10 gene transcription. We this tethering by the of the cell to of the SP-10 proximal promoter was the nuclear matrix in culture as well not We used mouse as an of a of the in mouse be round spermatids of the of the first of to the round spermatids from the spermatids also showed of SP-10 promoter in the nuclear matrix not this is the first of properties of an insulator in two of gene expression. the SP-10 in the previously of TDP-43 DNA-binding protein of 43 using a of the SP-10 insulator to a expression K.K. Shore A.N. Stoler M. P.P. Dev. Biol. PubMed Scopus Google Scholar). TDP-43 was using the TAR and shown to be a transcriptional F. PubMed Google Scholar). and Biol. Full Text Full Text PDF PubMed Scopus (495) Google showed a for TDP-43 in and that it to We previously showed that TDP-43 to the SP-10 promoter two located the of these in of TDP-43 binding in and the and expression of a gene in transgenic mice K.K. Shore A.N. Stoler M. P.P. Dev. Biol. 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We the Gal4 DNA binding to the type TDP-43 or its the or the The Gal4 protein with the above into and the of the to the enhancer was activities from used as type TDP-43 of the enhancer effect was to the TDP-43 not a effect. of the the enhancer to a for this in In the to a in The showed enhancer the protein, a transcriptional gene showed that TDP-43 prevent the enhancer-promoter and a to as an insulator protein in between TDP-43 and SP-10 Knockdown of TDP-43 of an to TDP-43 is in SP-10 insulator we used the this first we a of stable cell in the of the gene with or the SP-10 insulator an enhancer and a The used to these and activities are shown in The enhancer SP-10 core transgene expressed a of The of the SP-10 insulator but not a of the enhancer Mutation of the TDP-43 binding sites at and the enhancer-blocking of the SP-10 insulator. to the of TDP-43 for the above enhancer-blocking we of TDP-43 in all of the above stable cell for we used a that down the TDP-43 protein, the effect stable cell from the as Knockdown of TDP-43 the gene in the the SP-10 insulator as with the the TDP-43 effect when TDP-43 binding sites in the SP-10 insulator these that TDP-43 a at in in the enhancer-blocking of the SP-10 insulator. 50-bp of the SP-10 as a we a the SP-10 insulator as a minimal insulator. Our previous work using of the insulator that the but not the as an enhancer (2Reddi P.P. Shore A.N. Shapiro J.A. Anderson A. Stoler M.H. Acharya K.K. Dev. Biol. 2003; 262: 173-182Crossref PubMed Scopus (22) Google Scholar). the by which includes TDP-43 sites, as a minimal insulator, we the between the enhancer M. F. G. Cell. Full Text PDF PubMed Scopus Google and the SP-10 core promoter and enhancer-blocking in The the SP-10 insulator was also used for the enhancer-blocking in into and The the enhancer and SP-10 core promoter expressed of of a of the between the enhancer and core promoter in a in that the 50-bp subfragment by was to enhancer DNA to enhancer not by the 50-bp minimal insulator was with that of the SP-10 insulator, that the for enhancer of the 50-bp showed an effect enhancer and In when in the the minimal insulator its to the enhancer Finally, for a of insulator the 50-bp SP-10 was of the insulator not with enhancer when of the a transcriptional be to the enhancer from this Enhancer-blocking showed that the 50-bp SP-10 not enhancer when of the enhancer in of the insulator of the 50-bp SP-10 the above showed that the 50-bp SP-10 functions as a minimal insulator. The 50-bp both and for the 50-bp insulator from a testis-specific gene, we the enhancer-blocking of the SP-10 insulator was to the context of the SP-10 core promoter we the SP-10 core promoter in and of with the core promoter enhancer-blocking activities in the context of core that the mechanism of enhancer by the SP-10 minimal insulator is not to the context of the SP-10 core promoter with the of a the of the 50-bp SP-10 insulator to enhancer in the context of the promoter as well and Thus, the above using the core promoter that the 50-bp minimal SP-10 insulator is both and for enhancer-blocking of the 50-bp SP-10 minimal insulator and the chicken β-globin cHS4 insulator (7Bell A.C. West A.G. Felsenfeld G. Cell. 1999; 98: 387-396Abstract Full Text Full Text PDF PubMed Scopus (866) Google showed the for as other well characterized vertebrate but the of the 50-bp SP-10 insulator not to the CTCF The insulator is and two CpG the 50-bp SP-10 insulator is of CpG In the 50-bp SP-10 insulator is of are for in a above enhancer-blocking using and in is that the of may be in for enhancer-blocking in and to the of the 50-bp insulator in preventing ectopic expression of the testis-specific SP-10 gene, we transgenic The of first transgenic mice in which a transgene was the of enhancer and the SP-10 core promoter was to a a foreign enhancer to the SP-10 core promoter in expression of the transgene was in these mice of the of the enhancer G. R. P. Mol. Biol. PubMed Scopus Google Scholar). of transgenic mice the 50-bp SP-10 insulator one or between the enhancer and the SP-10 core promoter 2 and the insulator prevent ectopic expression by the enhancer in a physiological The of the transgene and its number in mouse by as mice the enhancer of the SP-10 core promoter expressed the gene in a number of tissues, and that the SP-10 core promoter was in to by an located foreign expression of the transgenic somatic tissues, the of expression was in and of two and in and expressed of in somatic tissues with expression in and is in with previous using the enhancer in with a promoter G. R. P. Mol. Biol. PubMed Scopus Google Scholar, M. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. Dev. Scopus Google Scholar, R. PubMed Scopus Google Scholar). of the mouse expressed of in was between the number of the transgene and the of expression in or somatic tissues, that the of of the transgene may have to the in gene expression. for which an of the expressed with the of the transgene the in that the enhancer ectopic expression of SP-10 core in to the 50-bp SP-10 insulator the to prevent ectopic expression of the The 50-bp SP-10 in but in of to mice the of the transgenic mice the 50-bp SP-10 minimal insulator between the enhancer and the SP-10 core promoter expressed in somatic tissues one of the 50-bp insulator and two of the insulator of the mouse expressed in the somatic tissues above The of somatic expression in these mice that the 50-bp insulator enhancer in somatic tissues is that a 50-bp insulator was to a enhancer and prevent ectopic expression of in the somatic tissues mouse expressed in the of the 50-bp insulator, that the SP-10 insulator not as an enhancer in in showed that the 50-bp insulator was as an enhancer when in between the enhancer and the showed that the SP-10 insulator functions by tethering the gene to the nuclear this the of insulator one the of the DNA to be for In to is a for enhancer-blocking in we transgenic mice in which the SP-10 50-bp insulator was in the between the enhancer and the transgene The mice showed expression in somatic tissues, and The of expression in somatic tissues, not as with in mice the insulator only a in insulator that in the context of the 50-bp SP-10 insulator in is with the of action of the SP-10 insulator The present study is at how an insulator testis-specific transcription of the mouse SP-10 gene. We that an insulator located in the proximal promoter of the SP-10 gene tethers the gene to the nuclear matrix in tethering the SP-10 core promoter and prevents the possibility of transcription in somatic tissues. In round where the SP-10 gene is expressed, the gene is to the We that tethering to the nuclear matrix by the insulator testis-specific transcription of the SP-10 gene. Our for the of TDP-43 in SP-10 insulator Finally, we that a 50-bp subfragment of the SP-10 insulator functions as a minimal insulator in a physiological The SP-10 insulator lacks CpG dinucleotides and CTCF binding sites and the possibility to DNA of insulator in to the nuclear matrix as a mechanism of insulator been first by Mol. Cell. Full Text Full Text PDF PubMed Scopus Google showed that the insulator is to the nuclear with insulator of and that by the DNA to the nuclear and into in a that not between these elements. Felsenfeld G. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google showed that vertebrate insulators also the mechanism for enhancer showed that the chicken β-globin insulator is to a by of with CTCF and its and that the mechanism of insulator is conserved and Our present study using the mouse SP-10 insulator this insulators may in and to the nuclear or a to be a conserved mechanism of insulator the of action of it is that be of of The SP-10 insulator is its must be in the be the of the that the SP-10 insulator from the nuclear matrix in the of CpG dinucleotides DNA as a TDP-43 in SP-10 insulator TDP-43 been shown to be a transcriptional and a F. PubMed Google Scholar, Biol. Full Text Full Text PDF PubMed Scopus (495) Google Scholar). may be an for is not for transcription factors to have in gene the present study that TDP-43 is of the nuclear matrix where a insulator protein be to be present Our previous study showed that of TDP-43 binding sites the SP-10 insulator in expression K.K. Shore A.N. Stoler M. P.P. Dev. Biol. PubMed Scopus Google Scholar). with of TDP-43 in the of transgene in a stable cell this that which is present in must or in the to its and SP-10 gene expression. In the present study that TDP-43 the be as an enhancer Our previous study showed that round spermatids only the of TDP-43 with other cell and in K.K. Shore A.N. Stoler M. P.P. Dev. Biol. PubMed Scopus Google Scholar). In studies that and TDP-43 in M. A.C. M. PubMed Scopus Google Scholar). Our studies the of TDP-43 in must be that of TDP-43 in culture not in expression of SP-10 as by not Our for this is that in to of spermatid-specific be for the of the SP-10 gene. The of this study is that it characterized a 50-bp subfragment of the SP-10 insulator and showed its enhancer-blocking properties in a physiological this is the first in a mouse that a small 50-bp vertebrate insulator functions as a enhancer The that the 50-bp minimal insulator an otherwise expressed transgene in somatic tissues the transcriptional used by testis-specific In of its to as an enhancer in the context of the core promoter we a for the 50-bp SP-10 insulator, which is conserved between mice and in gene and for gene to in The enhancer-blocking of the SP-10 insulator in the somatic tissues of transgenic mice the as to the SP-10 gene from the of other located In mouse the testis-specific SP-10 gene is by the expressed gene R. PubMed Scopus Google one and a gene which codes for protein M.H. PubMed Scopus Google the other Thus, of genes with tissue-specific expression (1Reddi P.P. Flickinger C.J. Herr J.C. Biol. Reprod. 1999; 61: 1256-1266Crossref PubMed Scopus (53) Google Scholar, R. PubMed Scopus Google Scholar, M.H. PubMed Scopus Google the of of preventing enhancer-promoter it is that the SP-10 insulator characterized in the present study may a in the SP-10 gene from the of the of neighboring The for mouse and SP-10 genes and are and the proximal of mouse and SP-10 genes (1Reddi P.P. Flickinger C.J. Herr J.C. Biol. Reprod. 1999; 61: 1256-1266Crossref PubMed Scopus (53) Google the 50-bp insulator of the the of SP-10 insulator The SP-10 insulator, from other vertebrate insulators in of its in the gene. insulators are located at gene boundaries of the promoter and enhancer The HS4 chicken insulator is located of the gene and the (5Chung J.H. Whitney M. Felsenfeld G. Cell. 1993; 74: 505-514Abstract Full Text PDF PubMed Scopus (763) Google Scholar). the the the insulator is located from the of the and genes C.J. 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A. 1993; PubMed Scopus Google Scholar, M. Dev. Biol. PubMed Scopus Google Scholar, S. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google the mechanism by which testis-specific is understood. studies a between the of the promoter and gene expression for testis-specific genes Biol. Reprod. PubMed Scopus Google Scholar). study CTCF and in somatic of a testis-specific gene M. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Our study how an insulator located in a proximal promoter testis-specific gene transcription. proximal is to gene M. S. A. PubMed Scopus Google Scholar, S. S. A. PubMed Scopus Google Scholar, P. PubMed Scopus Google Scholar). The of testis-specific in and the of insulators to gene expression in somatic tissues the that insulators may be in testis-specific gene been We the for and of the for and