Nuclear Matrix Interactions at the Human Protamine Domain
2004; Elsevier BV; Volume: 279; Issue: 50 Linguagem: Inglês
10.1074/jbc.m409415200
ISSN1083-351X
AutoresRui Pires Martins, G. Charles Ostermeier, Stephen A. Krawetz,
Tópico(s)CRISPR and Genetic Engineering
ResumoThe compact eukaryotic genome must be selectively opened to grant trans-factor access to cis-regulatory elements to overcome the primary barrier to gene transcription. The mechanism that governs the selective opening of chromatin domains (i.e. potentiation) remains poorly understood. In the absence of a well defined locus control region, the nuclear matrix is considered the primary candidate regulating the opening of the multigenic PRM1 → PRM2 → TNP2 human protamine domain. To directly examine its role, four lines of transgenic mice with different configurations of flanking nuclear matrix attachment regions (MARs) encompassing the protamine domain were created. We show that upon removal of the MARs, the locus becomes subject to position effects. The 3′ MAR alone may be sufficient to protect against silencing. In concert, the MARs bounding this domain likely synergize to regulate the expression of the various members of this gene cluster. Interestingly, the MARs may convey a selective reproductive advantage, such that constructs bearing both 5′ and 3′ MARs are passed to their offspring with greater frequency. Thus, the MARs bounding the PRM1 → PRM2 → TNP2 protamine domain have many and varied functions. The compact eukaryotic genome must be selectively opened to grant trans-factor access to cis-regulatory elements to overcome the primary barrier to gene transcription. The mechanism that governs the selective opening of chromatin domains (i.e. potentiation) remains poorly understood. In the absence of a well defined locus control region, the nuclear matrix is considered the primary candidate regulating the opening of the multigenic PRM1 → PRM2 → TNP2 human protamine domain. To directly examine its role, four lines of transgenic mice with different configurations of flanking nuclear matrix attachment regions (MARs) encompassing the protamine domain were created. We show that upon removal of the MARs, the locus becomes subject to position effects. The 3′ MAR alone may be sufficient to protect against silencing. In concert, the MARs bounding this domain likely synergize to regulate the expression of the various members of this gene cluster. Interestingly, the MARs may convey a selective reproductive advantage, such that constructs bearing both 5′ and 3′ MARs are passed to their offspring with greater frequency. Thus, the MARs bounding the PRM1 → PRM2 → TNP2 protamine domain have many and varied functions. The final stages of mammalian spermatogenesis are marked by a considerable morphological change, reflective of genomic restructuring mediated by the replacement of the majority of histones with protamines (PRM). 1The abbreviations used are: PRM, protamine; HS, DNase I-hypersensitive site; LCR, locus control region; MAR, nuclear matrix attachment region. These small, basic, arginine-rich proteins compact the genome into the sperm nucleus. This is accomplished in many varied ways throughout the phylogenetic kingdom (reviewed in Ref. 1Oliva R. Dixon G.H. Prog. Nucleic Acids Res. Mol. Biol. 1991; 40: 25-94Crossref PubMed Scopus (362) Google Scholar). For example, mammals, birds, and reptiles utilize a PRM or PRM1-like nuclear protein to repackage their sperm genomes. Compaction can be augmented through the formation of disulfide bonds between adjacent protamines and with the use of a second protamine PRM2 (2Balhorn R. Corzett M. Mazrimas J.A. Arch. Biochem. Biophys. 1992; 296: 384-393Crossref PubMed Scopus (38) Google Scholar). Repackaging of the genome occurs in a stepwise manner with some somatic histones first being replaced by germ cell-specific histone variants (reviewed in Ref. 3Braun R.E. Nat. Genet. 2001; 28: 10-12Crossref PubMed Google Scholar). Through a series of DNA strand-breaks (4McPherson S. Longo F.J. Eur. J. Histochem. 1993; 37: 109-128PubMed Google Scholar), supercoiling is lost (5Ward W.S. Partin A.W. Coffey D.S. Chromosoma. 1989; 98: 153-159Crossref PubMed Scopus (97) Google Scholar), and some of the histones are displaced by the transition nuclear proteins (TNP1 and TNP2). Finally, the majority of the histones, along with the TNPs, are exchanged for protamines. The human protamine PRM1 → PRM2 → TNP2 gene cluster maps to chromosome 16p13.13 (6Nelson J.E. Krawetz S.A. DNA Seq. 1995; 5: 163-168Crossref PubMed Scopus (12) Google Scholar). Protamine 1 (PRM1), protamine 2 (PRM2), and transition protein 2 (TNP2) are expressed solely in the testes during a defined stage of spermiogenesis. The specific temporal and spatial pattern of expression of the various members of the PRM1 → PRM2 → TNP2 gene cluster and the ability to isolate purified populations of spermatogenic cells at each stage of differentiation render this system well suited to dissecting the regulatory mechanisms that underlie facultatively expressed genes. This suite of genes resides in a single DNase I-sensitive domain (7Choudhary S.K. Wykes S.M. Kramer J.A. Mohamed A.N. Koppitch F. Nelson J.E. Krawetz S.A. J. Biol. Chem. 1995; 270: 8755-8762Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) that forms by the late pachytene spermatocyte stage and is maintained in this state in the mature spermatozoa (8Kramer J.A. McCarrey J.R. Djakiew D. Krawetz S.A. Mol. Reprod. Dev. 2000; 56: 254-258Crossref PubMed Google Scholar, 9Kramer J.A. McCarrey J.R. Djakiew D. Krawetz S.A. Development. 1998; 125: 4749-4755Crossref PubMed Google Scholar). In part, this is believed to reflect the incomplete replacement of the histones with protamines in this region that may be required to initiate repackaging of the male genome to a somatic-like structure upon fertilization (10Wykes S.M. Krawetz S.A. J. Biol. Chem. 2003; 278: 29471-29477Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The domain is flanked by two haploid-specific nuclear matrix attachment regions (MARs) (11Kramer J.A. Krawetz S.A. J. Biol. Chem. 1996; 271: 11619-11622Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) spanning a distance similar to that of the short sperm chromatin loops (5Ward W.S. Partin A.W. Coffey D.S. Chromosoma. 1989; 98: 153-159Crossref PubMed Scopus (97) Google Scholar). The association of these select regions of the genome with the sperm nuclear matrix appears vital to the formation of the male pronucleus (12Ward W.S. Kimura Y. Yanagimachi R. Biol. Reprod. 1999; 60: 702-706Crossref PubMed Scopus (95) Google Scholar). The members of the PRM1 → PRM2 → TNP2 gene cluster are regulated at both the levels of transcription and translation. Both temporal and tissue transcriptional specificity are modulated by the association of various testes-specific factors with their respective promoters (13Zambrowicz B.P. Harendza C.J. Zimmermann J.W. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5071-5075Crossref PubMed Scopus (92) Google Scholar, 14Zambrowicz B.P. Palmiter R.D. Biol. Reprod. 1994; 50: 65-72Crossref PubMed Scopus (34) Google Scholar). Although each member is transcribed at the round spermatid stage, translation is not initiated until the spermatid begins to elongate (reviewed in Ref. 3Braun R.E. Nat. Genet. 2001; 28: 10-12Crossref PubMed Google Scholar). This elaborate orchestration of synthesis can only be accomplished once the chromatin domain has formed an open structure (8Kramer J.A. McCarrey J.R. Djakiew D. Krawetz S.A. Mol. Reprod. Dev. 2000; 56: 254-258Crossref PubMed Google Scholar, 9Kramer J.A. McCarrey J.R. Djakiew D. Krawetz S.A. Development. 1998; 125: 4749-4755Crossref PubMed Google Scholar). The packaging of chromatin into higher ordered structures in the eukaryotic genome is mediated by DNA-DNA, DNA-histone, protein-protein, and other interactions. These higher ordered structured segments tend to be silent but can, in some cases, permit basal transcriptional activity (15Georgel P.T. Fletcher T.M. Hager G.L. Hansen J.C. Genes Dev. 2003; 17: 1617-1629Crossref PubMed Scopus (28) Google Scholar). The transition from a higher ordered conformation to one that is relaxed and amenable to high levels of transcription is termed potentiation (16Scholer H.R. Gruss P. EMBO J. 1985; 4: 3005-3013Crossref PubMed Scopus (43) Google Scholar). This was first observed by increased nuclease sensitivity (17Wu C. Bingham P.M. Livak K.J. Holmgren R. Elgin S.C. Weintraub H. Rose S.M. Garrard W.T. Cell. 1979; 16: 797-806Abstract Full Text PDF PubMed Scopus (284) Google Scholar, 18Varshavsky A.J. Sundin O. Bohn M. Scott W.A. Wigmore D.J. Wu C. Bingham P.M. Livak K.J. Holmgren R. Elgin S.C. Weintraub H. Rose S.M. Garrard W.T. Cell. 1979; 16: 453-466Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 19Weintraub H. Groudine M. Science. 1976; 193: 848-856Crossref PubMed Scopus (1203) Google Scholar, 20Scott W.A. Wigmore D.J. Wu C. Bingham P.M. Livak K.J. Holmgren R. Elgin S.C. Weintraub H. Rose S.M. Garrard W.T. Cell. 1978; 15: 1511-1518Abstract Full Text PDF PubMed Scopus (135) Google Scholar) that led to the development of the physical concept of a chromatin domain (19Weintraub H. Groudine M. Science. 1976; 193: 848-856Crossref PubMed Scopus (1203) Google Scholar, 21Keene M.A. Corces V. Lowenhaupt K. Elgin S.C.R. Eur. J. Cell Biol. 1980; 22: 95Google Scholar, 22Lawson G.M. Knoll B.J. March C.J. Woo S.L.C. Tsai M.J. Omalley B.W. J. Biol. Chem. 1982; 257: 1501-1507Abstract Full Text PDF PubMed Google Scholar), whereby changes in local chromatin structure must occur before transcription can begin (23Weintraub H. Rose S.M. Garrard W.T. Cell. 1985; 42: 705-711Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 24Freeman L.A. Garrard W.T. Crit. Rev. Eukaryotic Gene Expression. 1992; 2: 165-209PubMed Google Scholar, 25Rose S.M. Garrard W.T. J. Biol. Chem. 1984; 259: 8534-8544Abstract Full Text PDF PubMed Google Scholar, 26Gross D.S. Garrard W.T. Trends Biol. Sci. 1987; 12: 293-297Abstract Full Text PDF Scopus (84) Google Scholar). Housekeeping genes are partitioned into chromosomal segments that bear a constitutively open structure, whereas tissue-specific genes tend to exist in a facultatively open conformation. For example, the α-globin cluster (27Vickers M.A. Vyas P. Harris P.C. Simmons D.L. Higgs D.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3437-3441Crossref PubMed Scopus (59) Google Scholar) exists in a constitutively potentiated conformation, since it also contains the 3-methyladenine DNA glycosylase gene, a housekeeping gene that is active in DNA damage repair. However, the α-globin genes are not expressed in all cell types. They are regulated through an elaborate series of histone modifications and the specific binding of several transcription factor complexes (28Anguita E. Hughes J. Heyworth C. Blobel G.A. Wood W.G. Higgs D.R. EMBO J. 2004; 23: 2841-2852Crossref PubMed Scopus (179) Google Scholar). In contrast, the genes of the PRM1 → PRM2 → TNP2 domain exist in a transcriptionally silent, compacted chromatin conformation in most cell types. It is facultatively potentiated in the pachytene spermatocyte just prior to their expression in the round spermatid (9Kramer J.A. McCarrey J.R. Djakiew D. Krawetz S.A. Development. 1998; 125: 4749-4755Crossref PubMed Google Scholar). Potentiation is necessary but not sufficient for transcription; it provides an essential first step toward expression, granting access to cis-regulatory elements. The mechanism of partitioning the genome into inactive and potentially active segments remains poorly understood. Several classes of regulatory regions including boundary elements, locus control regions (LCRs), and MARs are known to influence transcription through the modification of chromatin structure. For example, tissue and temporal specificity of the β-globin gene cluster is attributed to the LCR, characterized by five upstream DNase I-hypersensitive sites (HSs). These HSs interact with other elements upstream of each of the genes of this cluster and can be assembled into an active chromatin hub (29Patrinos G.P. De Krom M. De Boer E. Langeveld A. Imam A.M. Strouboulis J. De Laat W. Grosveld F.G. Genes Dev. 2004; 18: 1495-1509Crossref PubMed Scopus (150) Google Scholar, 30Palstra R.J. Tolhuis B. Splinter E. Nijmeijer R. Grosveld F. de Laat W. Nat. Genet. 2003; 35: 190-194Crossref PubMed Scopus (438) Google Scholar, 31de Laat W. Grosveld F. Chromosome Res. 2003; 11: 447-459Crossref PubMed Scopus (298) Google Scholar). The formation of the hub correlates with changes in the patterns of nuclear matrix association (32Ostermeier G.C. Liu Z. Martins R.P. Bharadwaj R.R. Ellis J. Draghici S. Krawetz S.A. Nucleic Acids Res. 2003; 31: 3257-3266Crossref PubMed Scopus (38) Google Scholar). This has led to the proposal that nuclear matrix facilitates and/or mediates the formation of the active chromatin hub. The nuclear matrix is a proteinaceous network that has been implicated in a myriad of processes while serving as a structural organizer within the cell nucleus. For example, by direct DNA-nuclear matrix interactions, MARs organize chromatin into loop domains (33Bode J. Kohwi Y. Dickinson L. Joh T. Klehr D. Mielke C. Kohwi-Shigematsu T. Science. 1992; 255: 195-197Crossref PubMed Scopus (383) Google Scholar) and by nuclear matrix association maintain chromosomal territories (34Ma H. Siegel A.J. Berezney R. J. Cell Biol. 1999; 146: 531-542Crossref PubMed Scopus (146) Google Scholar, 35Dundr M. Misteli T. Biochem. J. 2001; 356: 297-310Crossref PubMed Scopus (330) Google Scholar, 36Jackson D.A. J. Cell. Biochem. Suppl. 2000; 79: 69-77Crossref Google Scholar, 37Wolffe A.P. Urnov F.D. Guschin D. Biochem. Soc. Trans. 2000; 28: 379-386Crossref PubMed Google Scholar). In addition to its structural role, evidence from a number of loci suggests that the nuclear matrix may assume a more dynamic role modulating the activity of a host of genes and domains (32Ostermeier G.C. Liu Z. Martins R.P. Bharadwaj R.R. Ellis J. Draghici S. Krawetz S.A. Nucleic Acids Res. 2003; 31: 3257-3266Crossref PubMed Scopus (38) Google Scholar, 38Samuel S.K. Minish T.M. Davie J.R. J. Cell. Biochem. 1997; 66: 9-15Crossref PubMed Scopus (33) Google Scholar, 39Samuel S.K. Minish T.M. Davie J.R. Cancer Res. 1997; 57: 147-151PubMed Google Scholar, 40Spencer V.A. Davie J.R. J. Biol. Chem. 2001; 276: 34810-34815Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 41Sun J.M. Chen H.Y. Davie J.R. J. Biol. Chem. 2001; 276: 49435-49442Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 42Alvarez J.D. Yasui D.H. Niida H. Joh T. Loh D.Y. Kohwi-Shigematsu T. Genes Dev. 2000; 14: 521-535PubMed Google Scholar, 43Yasui D. Miyano M. Cai S. Varga-Weisz P. Kohwi-Shigematsu T. Nature. 2002; 419: 641-645Crossref PubMed Scopus (394) Google Scholar, 44de Belle I. Cai S. Kohwi-Shigematsu T. J. Cell Biol. 1998; 141: 335-348Crossref PubMed Scopus (127) Google Scholar). For example, the initial observation that many MARs contain topoisomerase II binding sites immediately suggested that association with the nuclear matrix could provide a means to relieve torsional stress in active chromatin domains (24Freeman L.A. Garrard W.T. Crit. Rev. Eukaryotic Gene Expression. 1992; 2: 165-209PubMed Google Scholar, 45Cockerill P.N. Garrard W.T. Cell. 1986; 44: 273-282Abstract Full Text PDF PubMed Scopus (743) Google Scholar). Other activities including the nuclear matrix acting as a primary transcriptional organizer have recently become well delineated. For example, the SATB1 network binds the MARs of a host of T-cell-specifying genes aiding in the recruitment of chromatin-modifying complexes and transcription-promoting elements (43Yasui D. Miyano M. Cai S. Varga-Weisz P. Kohwi-Shigematsu T. Nature. 2002; 419: 641-645Crossref PubMed Scopus (394) Google Scholar, 46Kieffer L.J. Greally J.M. Landres I. Nag S. Nakajima Y. Kohwi-Shigematsu T. Kavathas P.B. J. Immunol. 2002; 168: 3915-3922Crossref PubMed Scopus (26) Google Scholar). Without this interaction, the genes are misregulated, and T-cell differentiation and spermatogenesis fail (42Alvarez J.D. Yasui D.H. Niida H. Joh T. Loh D.Y. Kohwi-Shigematsu T. Genes Dev. 2000; 14: 521-535PubMed Google Scholar, 47Cai S. Kohwi-Shigematsu T. Methods. 1999; 19: 394-402Crossref PubMed Scopus (19) Google Scholar, 48Cai S.T. Han H.J. Kohwi-Shigematsu T. Nat. Genet. 2003; 34: 42-51Crossref PubMed Scopus (345) Google Scholar). In this report, we have used transgenic analysis to evaluate the function of nuclear matrix association in the expression of the human protamine domain. This is the first direct assessment of the role of the nuclear matrix for any haploid expressed gene. We present evidence that the PRM1 → PRM2 → TNP2 domain becomes subject to position effects without the complement of haploid-specific MARs. In the absence of the 5′ MAR, and with only the 3′ MAR about the protamine domain, transcription is reduced but not ablated. This suggests that the 3′ MAR may provide a dominant protective effect against silencing in the male haploid genome, whereas a regulatory synergism between upstream and downstream elements bounded by the MARs ensures the appropriate regulation of this suite of genes. Constructs and Transgenic Animals—Four constructs bearing different arrangements of MARs about the human PRM1 → PRM2 → TNP2 protamine domain (GenBank™ U15422.1) were generated by restriction endonuclease digestion of cosmid hp3.1 (49Nelson J.E. Krawetz S.A. J. Biol. Chem. 1994; 269: 31067-31073Abstract Full Text PDF PubMed Google Scholar). As shown in Fig. 1, this cosmid contains an ∼40-kb fragment of human chromosome 16p13.13 (6Nelson J.E. Krawetz S.A. DNA Seq. 1995; 5: 163-168Crossref PubMed Scopus (12) Google Scholar). Purified DNA was microinjected into fertilized eggs obtained by mating (C57BL/6 × SJL)F1 or C57BL/6 female mice with (C57BL/6 × SJL)F1 male mice essentially as described for single or low copy transgene insertion (50Brinster R.L. Chen H.Y. Trumbauer M.E. Yagle M.K. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4438-4442Crossref PubMed Scopus (778) Google Scholar). Transgenes were maintained hemizygous on a C57BL/6 background (Jackson Laboratories, Bar Harbor, ME). Genotype and copy number verification was performed by real time PCR, as described (51Martins R.P. Krawetz S.A. Anal. Biochem. 2004; 329: 337-339Crossref PubMed Scopus (2) Google Scholar). RNA Isolation and Nuclease Protection—Total RNA was isolated from whole organs by homogenization in 4 m guanidine thiocyanate, buffered with 100 mm Tris-HCl, pH 6.5, containing 2% β-mercaptoethanol, 17 mm sarcosyl, using a PRO Scientific 200 homogenizer (PRO Scientific Inc., Oxford, CT). Homogenates were treated with 0.1 volumes of 3.0 m sodium acetate, pH 5.2, and then extracted with 1 volume of pH ∼4.3, saturated phenol/chloroform/isoamyl alcohol (50:49:1). The aqueous phase was removed, and then nucleic acids were precipitated following the addition of 1 volume of isopropyl alcohol. The nucleic acid pellet was then suspended in resuspension solution that contained 6 m guanidine-HCl, buffered with 100 mm Tris-HCl, pH 7.0, containing 20 mm EDTA, 10 mm dithiothreitol. RNA was selectively precipitated overnight at 4 °C following the addition of 0.18 m sodium acetate and 2 m LiBr. The RNA was pelleted by centrifugation and then suspended in the guanidine-HCl resuspension solution, and purity was verified by spectrophotometry. This was repeated until the A260/A280 ratio of at least 1.6 was obtained. When this was achieved, RNA was precipitated a final time at –20 °C for at least 2 h following the addition of 0.18 m sodium acetate and 0.75 volumes of ethanol. Following precipitation, the RNA was recovered by centrifugation and then suspended in RNase-free water. RNA integrity was judged by the relative ratio of the 28 and 18 S rRNAs as analyzed by 2% formaldehyde, 1% agarose gel electrophoresis. RNAs with a ratio of 28 to 18 S of at least 1.8 was deemed acceptable. Antisense RNA probe templates for human PRM1, PRM2, and TNP2 as well as mouse Prm1, Prm2, and Tnp2 were generated by ligation-mediated PCR using Lig'n'scribe (Ambion, Austin, TX) as shown in Fig. 1. A eukaryotic 18 S rRNA probe template was purchased from Ambion. Probes were prepared by PCR using Hot Star Taq (Qiagen, La Jolla, CA). A 15-min hot start at 95 °C was followed by 35 cycles of denaturing at 95 °C for 30 s, a 30-s annealing step at TA1, as described in Table I, and then elongation at 72 °C for 30 s. A final 10-min extension step at 72 °C for 10 min terminated the reaction.Table ILigation-mediated PCR strategy to produce antisense probes for the ribonuclease protection assay Open table in a new tab The primary PCR product was then ligated to a T7 promoter adapter (Ambion). Following ligation, the product was subjected to a second round of PCR as above, at an annealing temperature of TA2 (Table I) using the Ambion primer along with the nested primer. Antisense probes, labeled with α-[32P]dCTP, were then generated by in vitro transcription using the MAXIscript protocol (Ambion) and then purified by polyacrylamide gel electrophoresis. RNase protection assays were performed with 10 μg of total sample RNA, using the RPAIII kit (Ambion), essentially as recommended by the manufacturer. RNA was hybridized to antisense probes at 48 °C for 16 h. The hybridization mixture containing the hybridized products was then digested with a mixture of RNase A and T1 at a ratio of 1 unit of A/3 units of T1 for 2 h at 37 °C. Protected fragments were then denatured and resolved using a 5% polyacrylamide, 8 m urea denaturing gel electrophoresis. The protected and resolved products were then visualized by phosphorimaging using a Typhoon 9210 (Amersham Biosciences). Using the probes described, a protected fragment for the human TNP2 message was not detected in any assay, even when 50 μg of total transgenic RNA or 6 μg of normal human testes poly(A)+-enriched RNA obtained from normal males (7Choudhary S.K. Wykes S.M. Kramer J.A. Mohamed A.N. Koppitch F. Nelson J.E. Krawetz S.A. J. Biol. Chem. 1995; 270: 8755-8762Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) were used (data not shown). Image analysis was then carried out using the Quantity One (Bio-Rad) software suite. Statistical Analyses—A χ2 test was applied to binomially distributed data (52Conover W.J. Practical Nonparametric Statistics. 2nd Ed. John Wiley & Sons, Inc., New York1980: 143-212Google Scholar). This included assessing whether the transgene was passed with a similar frequency among transgenic lines. A Mann-Whitney test (53Conover W.J. Practical Nonparametric Statistics. John Wiley & Sons, Inc., New York1980: 213-341Google Scholar) was employed to assess whether the levels of expression of each member of the endogenous and transgenic PRM1 → PRM2 → TNP2 protamine locus were similar between transgenic lines. MARs Convey a Selective Advantage for Transgene Passage—To assess how the MARs of the PRM1 → PRM2 → TNP2 domain impact expression, a series of transgenic constructs containing one, both, or no-MARs were created. The objective was to produce a series of single copy founders for each transgenic line. This is the most sensitive assay known to reveal position effects in higher ordered eukaryotic systems. The four transgenic constructs, no-MAR, 5′ MAR only, 3′ MAR only, and 5′ + 3′ MARs that encompass the PRM1 → PRM2 → TNP2 region of the human genome are summarized in Fig. 1. The transgenic lines were maintained in a hemizygous state on a C57BL/6 (Jackson Laboratories, Bar Harbor, ME) background. All of the different lines were fertile and showed no obvious abnormal phenotype. Of the 270 mice born, 31 were transgenic, and 14 achieved germ line transmission. As shown in Table II, single copy transgenic animals were obtained from the 3′ MAR only and 5′ + 3′ MAR lines, whereas low, two-copy transgenic animals were created in all other lines. Surprisingly, each construct yielded transgenic animals with varied efficiency (p < 0.05). A greater number of transgenic animals than expected were produced when the no-MAR construct (27%; 8 of 30) and the 5′ MAR only construct (36%; 5 of 14) were injected. In comparison, fewer transgenic animals than expected were produced when the 5′ + 3′ MAR construct was injected (10%; 11 of 105). Moreover, once created, each construct was passed from the founder to the progeny with different efficiencies (p < 0.05). Only the 5′ + 3′ MAR construct founders passed their transgene at the expected Mendelian frequency (56%; 29 of 52). All others passed the transgene at 50% of the expected efficiency. This suggests that constructs bounded by MARs impart a selective advantage onto the integrated locus, since they exhibit a greater stability when integrated into the genome. This is further evidenced by the long term (at least 6-year) stability and expression of similar PRM1 → PRM2 → TNP2 transgenic constructs that bear both MARs (7Choudhary S.K. Wykes S.M. Kramer J.A. Mohamed A.N. Koppitch F. Nelson J.E. Krawetz S.A. J. Biol. Chem. 1995; 270: 8755-8762Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 54Wykes S.M. Krawetz S.A. Mol. Biotechnol. 2003; 25: 131-138Crossref PubMed Scopus (7) Google Scholar).Table IITransgenic passage Open table in a new tab We and others have routinely observed that MAR-containing constructs are directly targeted to nuclear structures (55Bode J. Goetze S. Heng H. Krawetz S.A. Benham C. Chromosome Res. 2003; 11: 435-445Crossref PubMed Scopus (88) Google Scholar, 56Heng H.H.Q. Krawetz S.A. Lu W. Bremer S. Liu G. Yee C.J. Cytogenet. Cell Genet. 2001; 93: 155-161Crossref PubMed Scopus (46) Google Scholar). They remain stably associated with the nuclear matrix that is intimately involved with both replication and transcription. These unique properties of the nuclear matrix probably facilitate and reflect the observed long term stable integration of MAR-containing constructs. MARs Act as Boundary Elements to the PRM1 → PRM2 → TNP2 Locus—The spatial pattern of expression of the various members of the PRM1 → PRM2 → TNP2 gene cluster was assessed by ribonuclease protection. As shown in Fig. 2, in all cases, appropriate tissue-specific expression was recapitulated. Irrespective of transgene copy number, the PRM1 and PRM2 genes were exclusively expressed in testes but not in brain, heart, kidney, liver, and lung. The expression of the endogenous Prm1, Prm2, Tnp2, or 18 S rRNA genes was not altered even when their transgenic orthologs were expressed. A striking difference in the level of expression of the transgene was noted when founders within the no-MAR construct family were compared. As shown in Fig. 3C, transgenic expression could not be detected in founder F0155, although the transgene was present in the germ line and passed to subsequent generations (Table II). In contrast, founder F0147 (lane B) displayed an opposite effect. In the latter, both the PRM1 and PRM2 transcripts were detected at a significantly elevated level (p < 0.05) when compared with animals from the line bearing both MARs (Table III). This lack of expression and appropriate regulation was not observed in any of the other constructs. Without an end region MAR, the PRM1 → PRM2 → TNP2 locus is subject to the chromosomal context at the site of insertion.Table IIIMARs act as boundary elementsNo-MAR5′ + 3′ MARMedianF0147 (25th, 75th quartile)F0155Median(25th, 75th quartile)%%%%PRM1:Prm120.13(12.36, 22.19)ND4.8aExpression was significantly lower, comparing the no-MAR constructs with 5′ + 3′ MAR constructs, as determined by the Mann-Whitney test, p < 0.05.(3.36, 6.13)PRM2:Prm258.52(56.78, 61.78)ND33.51aExpression was significantly lower, comparing the no-MAR constructs with 5′ + 3′ MAR constructs, as determined by the Mann-Whitney test, p < 0.05.(26.54, 50.69)a Expression was significantly lower, comparing the no-MAR constructs with 5′ + 3′ MAR constructs, as determined by the Mann-Whitney test, p < 0.05. Open table in a new tab 3′ MAR Modifies the Expression of the Locus—To examine whether a single flanking MAR exhibited a dominant effect, homogenous MAR-containing lines were created that bore neither the 5′ MAR or the 3′ MAR (Fig. 1). The expression of the various members of the protamine domain was then assessed by ribonuclease protection. As shown in Table IV, the single copy 3′ MAR only line showed a significant down-regulation of PRM1 and PRM2 genes (p < 0.05) when compared with the single copy 5′ + 3′ MAR line. This is in accord with the view that the 3′ MAR tempered expression of the locus. In comparison, the tandem arrayed two-copy 5′ MAR only line showed no significant change in transgenic expression (Table V) when compared with the single-copy 5′ + 3′ MAR line. This suggested that the 5′ MAR from the second copy of the tandemly arrayed two-copy 5′ MAR line functionally substituted for the deleted 3′ MAR, essentially recapitulating the native domain. Accordingly, the PRM1 → PRM2 → TNP2 domain is functionally bound by upstream and downstream MARs.Table IV3′ MAR conveys suppression on the locus5′ + 3′ MAR3′ MAR onlyMedian(25th, 75th quartile)Median(25th, 75th quartile)%%%%PRM1:Prm122.65(18.30, 45.25)2.96aExpression was significantly lower, comparing the 5′ + 3′ MAR constructs with the 3′ MAR only constructs, as determined by the Mann-Whitney test, p < 0.05.(2.64, 3.68)PRM2:Prm265.02(54.37, 76.33)19.19aExpression was significantly lower, comparing the 5′ + 3′ MAR constructs with the 3′ MAR only constructs, as determined by the Mann-Whitney test, p < 0.05.(17.08, 24.56)a Expression was significantly lower, comparing the 5′ + 3′ MAR constructs with the 3′ MAR only constructs, as determined by the Mann-Whitney test, p < 0.05. Open table in a new tab Table VTwo
Referência(s)