Nuclear Matrix Interactions within the Sperm Genome

Analysis of the haploid-expressed human PRM1 → PRM2 → TNP2 genic domain has revealed two regions of attachment to the sperm nuclear matrix. These sperm nuclear matrix attachment regions delimit the DNase I-sensitive domain of this haploid-expressed locus. The domain is intermediately associated with but not attached to the nuclear matrix. DNase I-sensitive genes within the mature sperm nucleus, such as protamine 1, protamine 2, transition protein 2, α-globin, and β-actin, display this intermediate affinity for the sperm nuclear matrix. This may denote their role in templating the male genome prior to fertilization, thus ensuring the formation of a viable male pronucleus during early embryonic development. Analysis of the haploid-expressed human PRM1 → PRM2 → TNP2 genic domain has revealed two regions of attachment to the sperm nuclear matrix. These sperm nuclear matrix attachment regions delimit the DNase I-sensitive domain of this haploid-expressed locus. The domain is intermediately associated with but not attached to the nuclear matrix. DNase I-sensitive genes within the mature sperm nucleus, such as protamine 1, protamine 2, transition protein 2, α-globin, and β-actin, display this intermediate affinity for the sperm nuclear matrix. This may denote their role in templating the male genome prior to fertilization, thus ensuring the formation of a viable male pronucleus during early embryonic development. INTRODUCTIONFor many years the nuclear matrix received little attention, as it was thought to act merely as a structural element(1.Baskin Y. Science. 1995; 268: 1564-1565Crossref PubMed Scopus (16) Google Scholar). It has now been suggested that the nuclear matrix may play a key role in genome organization and gene potentiation(2.Zlatanova J.S. van Holde K.E. Crit. Rev. Eukaryotic Gene Expression. 1992; 2: 211-224PubMed Google Scholar). As in the somatic nucleus, chromatin within the male gamete is organized into discrete loops, bound at the base by regions of attachment to the nuclear matrix(3.Ward W.S. Coffey D.S. Biochem. Biophys. Res. Commun. 1990; 173: 20-25Crossref PubMed Scopus (49) Google Scholar). These loops differ from their somatic counterparts with respect to the packaging of their DNA (4.Ward W.S. Biol. Reprod. 1993; 48: 1193-1201Crossref PubMed Scopus (111) Google Scholar) and their average size. Loops within the sperm nucleus are ∼27 kb 1The abbreviations used are: kbkilobase(s)SMARsperm nuclear matrix attachment regionMARsomatic nuclear matrix attachment regionPCRpolymerase chain reactionbpbase pair(s). in size (5.Barone J.G. De Lara J. Cummings K.B. Ward W.S. J. Androl. 1994; 15: 139-145PubMed Google Scholar) compared with ∼60 kb in all other types of cells studied to date(6.Vogelstein B. Pardoll D.M. Coffey D.S. Cell. 1980; 22: 79-85Abstract Full Text PDF PubMed Scopus (468) Google Scholar). We have termed these sperm nuclear matrix attachment regions (SMARs) (7.Kramer J.A. Krawetz S.A. Mamm. Genome. 1995; 6: 677-679Crossref PubMed Scopus (13) Google Scholar). The somatic nuclear matrix has come under intense study, as actively transcribed genes have been shown to be associated with the nuclear matrix(8.Ciejek E. Tsai M.-J. O'Malley B.W. Nature. 1983; 306: 607-609Crossref PubMed Scopus (297) Google Scholar). Somatic nuclear matrix attachment regions (MARs) have been identified in or near introns(9.Jarman A.P. Higgs D.R. EMBO J. 1988; 7: 3337-3344Crossref PubMed Scopus (165) Google Scholar), enhancers(10.Gasser S.M. Laemmli U.K. Cell. 1986; 46: 521-530Abstract Full Text PDF PubMed Scopus (433) Google Scholar), origins of replication(11.Kalandadze A.G. Bushara S.A. Vassetzky Y.S. Razin S.V. Biochem. Biophys. Res. Commun. 1990; 168: 9-15Crossref PubMed Scopus (63) Google Scholar), and sites of transcription initiation(12.Dijkwel P.A. Hamlin J.L. Mol. Cell. Biol. 1988; 8: 5398-5409Crossref PubMed Scopus (162) Google Scholar), as well as other regulatory elements(9.Jarman A.P. Higgs D.R. EMBO J. 1988; 7: 3337-3344Crossref PubMed Scopus (165) Google Scholar). MARs have also been identified at the ends of the DNase I-sensitive domain in numerous loci (13.Levy-Wilson B. Fortier C. J. Biol. Chem. 1989; 264: 21196-21204Abstract Full Text PDF PubMed Google Scholar, 14.Loc P.-V. Strätling W.H. EMBO J. 1988; 7: 655-664Crossref PubMed Scopus (226) Google Scholar) and shown to facilitate position-independent gene activity(15.Stief A. Winter D.M. Strätling W.H. Sippel A.E. Nature. 1989; 341: 343-345Crossref PubMed Scopus (446) Google Scholar). The function of the sperm nuclear matrix is comparatively unknown.An ∼40-kb region of human chromosome 16p13.13 has recently been sequenced in its entirety and shown to contain the genes for the sperm-specific protamine 1, protamine 2, and transition protein 2 proteins(16.Nelson J.E. Krawetz S.A. J. Biol. Chem. 1994; 269: 31067-31073Abstract Full Text PDF PubMed Google Scholar). DNase I sensitivity analysis has delineated the boundaries of the domain in the mature spermatozoan, and transgenic analysis has shown that this region of the genome contains all the elements necessary for the appropriate spatial and temporal expression of the genes of this cluster in a position-independent, copy number-dependent manner(17.Choudhary 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 (62) Google Scholar). To characterize structural elements that mediate this response, we have identified regions of genomic interaction with the sperm nuclear matrix. Further, we demonstrate that a specific subset of both haploid-specific and constitutively expressed genes are associated with the mature sperm nuclear matrix. These genes assume an altered structural conformation as evidenced by their increased sensitivity to DNase I. Thus, the mature sperm genome is organized in a specific non-random manner. This could provide the means to template the male genome for ordered protamine replacement immediately subsequent to fertilization.MATERIALS AND METHODSPhysical characterization of each of the candidate MARs employed nuclei prepared from frozen sperm essentially as described(19.Ward W.S. Partin A.W. Coffey D.S. Chromosoma. 1989; 98: 153-159Crossref PubMed Scopus (96) Google Scholar). Nuclei were resuspended in 50 mM HEPES, pH 7.5, buffer containing 10 mM NaCl, 5 mM MgOAc, and 25% glycerol, at ∼1 × 107/ml, and then used immediately or stored flash frozen at −80°C. DNA halos were prepared from fresh or frozen sperm nuclei as described(5.Barone J.G. De Lara J. Cummings K.B. Ward W.S. J. Androl. 1994; 15: 139-145PubMed Google Scholar). In brief, sperm nuclei were mixed with an equal volume of 2 M NaCl buffered with 25 mM Tris, pH 7.4, and then pelleted at 4°C for 30 min at 1,600 × g. The pellet was resuspended in 200 μl of 25 mM Tris, pH 7.4, buffer containing 2 M NaCl and then adjusted to contain 10 mM dithiothreitol. The nuclei were then incubated on ice for 30 min. The resulting halos were centrifuged at 4°C for 30 min at 1,600 × g and then resuspended in 50 mM Tris-HCl, pH 7.5, buffer containing 100 mM NaCl and 10 mM MgCl2. Aliquots were stained with propidium iodide and then visualized by fluorescent illumination using a Leitz DIAPLAN microscope. The remaining halo DNA was subsequently digested with BstXI, EcoRI, HindIII, or StyI for 4 h at 37°C. Successful restriction enzyme digestion was assayed by the inability to amplify across known sites. Following digestion, an equal aliquot of 4 M NaCl was added, and the samples were incubated for an additional 10 min at 37°C. The loop and matrix fractions were then separated by centrifugation for 30 min at 9,000 × g at 4°C. The fractions thus separated were subsequently purified using Prep-A-Gene matrix (Bio-Rad) and then resuspended in deionized water. PCR amplification was performed on both the loop and matrix-associated fractions utilizing primer pairs directed to the PRM1 → PRM2 → TNP2 locus, many of which have been described previously. 2Primer sequences, PCR conditions, and the PRM1 → PRM2 → TNP2 domain sequence will be made available at the internet address “http://compbio.med.wayne.edu/”. PCR was maintained within the linear range of amplification. DNA halos were prepared from HeLa cells essentially as described(21.Gerdes M.G. Carter K.C. Moen P.T. Bently-Lawrence J. J. Cell Biol. 1994; 126: 289-304Crossref PubMed Scopus (146) Google Scholar), digested to completion with HindIII, and then treated as described above for sperm halos.RESULTS AND DISCUSSIONTo begin to elucidate the elements necessary to potentiate this domain, candidate regions of sperm nuclear matrix association within the PRM1 → PRM2 → TNP2 biological locus were identified utilizing a computational strategy. Characteristic MAR motifs were gathered from the literature (7.Kramer J.A. Krawetz S.A. Mamm. Genome. 1995; 6: 677-679Crossref PubMed Scopus (13) Google Scholar, 22.Boulikas T. J. Cell. Biochem. 1993; 52: 14-22Crossref PubMed Scopus (170) Google Scholar) and then expressed as unique sequence patterns as described(18.Kramer, J. A., Singh, G. B., Krawetz, S. A. (1996) Genomics, in pressGoogle Scholar). In this manner, the ∼40-kb sequence containing the PRM1 → PRM2 → TNP2 biological locus was queried for the presence of various sequence patterns indicative of MARs. Motifs were then weighted according to their expected frequency in a random sequence of the same base composition as that of the sequence queried. A weighted sum was subsequently applied to each region along the locus using a sliding window of 1000 bp with a 100-bp step size. The results are presented graphically in Fig. 1. Regions above a likelihood of 50% were considered as candidates to have strong nuclear matrix binding potential. This computer analysis predicted two SMARs centered at nucleotide positions 8,175 and 34,100 (Fig. 1). These potential SMARs were similar to those previously identified in this locus (7.Kramer J.A. Krawetz S.A. Mamm. Genome. 1995; 6: 677-679Crossref PubMed Scopus (13) Google Scholar) and were used to guide their physical identification.DNA “halos” were prepared by extracting sperm nuclei with a high ionic strength reducing buffer(5.Barone J.G. De Lara J. Cummings K.B. Ward W.S. J. Androl. 1994; 15: 139-145PubMed Google Scholar). This displaced the histones and protamines from the chromatin, while leaving the DNA attached at discrete points to the intact nuclear matrix. The resulting halo structures were then stained with propidium iodide and visualized by fluorescence microscopy as shown in Fig. 2. The intact nuclei stained in a uniform manner, consistent with tightly packaged sperm chromatin, while the halo structures showed a more dispersed pattern of staining. Regions of the sperm chromatin that remained associated with the nuclear matrix possessed a brightly staining center, while the unassociated loop DNA stained dimly. This was manifested as a broad fibrous “halo” of fluorescence surrounding the brightly stained nuclear matrix.Figure 2:Fluorescence microscopy of human sperm nuclei and DNA halos. Panel a, sperm nuclei; panel b, the corresponding DNA halo. Non-matrix-associated chromatin loops out from the proteinaceous matrix upon the depletion of the protamines and histones. The non-matrix-associated DNA appears as a halo around the more brightly stained nuclear scaffold.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To separate the nuclear matrix-bound and unbound DNA, halos were digested with various restriction endonucleases, and then the nuclear matrix-bound DNA was pelleted. fractions were purified and then to PCR amplification using unique of to discrete regions of the haploid-expressed PRM1 → PRM2 → TNP2 locus and the somatic expressed locus (Fig. The of each showed of The at of the non-matrix-associated loop DNA, to the of the DNA with the nuclear In intermediately matrix-associated DNA into both the and pellet Regions that the DNase I-sensitive domain to the non-matrix-associated loop as the DNase locus. Regions within the domain were intermediately associated with the nuclear matrix. This intermediate affinity for the sperm nuclear matrix is similar to that for the human C. Y. T. J. 1990; PubMed Scopus Google Scholar). The region surrounding and the gene nuclear matrix association and is by points of with the nuclear matrix. Regions near the ends of the PRM1 → PRM2 → TNP2 DNase I-sensitive domain were bound to the sperm nuclear matrix. These to be in a similar to the MARs of the (13.Levy-Wilson B. Fortier C. J. Biol. Chem. 1989; 264: 21196-21204Abstract Full Text PDF PubMed Google Scholar) and human P.-V. Strätling W.H. EMBO J. 1988; 7: 655-664Crossref PubMed Scopus (226) Google Scholar) The region of attachment to the sperm nuclear matrix was by positions while the corresponding region was by positions The strong attachment to the nuclear matrix of these regions at the ends of the PRM1 → PRM2 → TNP2 DNase I-sensitive domain the presence of a these regions not and matrix-associated of the PRM1 → PRM2 → TNP2 The DNase I-sensitive that the human PRM1 → PRM2 → TNP2 domain is shown from 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 (62) Google The and TNP2 genes are as along the corresponding sequence of human chromosome 1995; Google Scholar). DNA halos were digested with various restriction endonucleases, separated into their loop and nuclear matrix-bound and PCR primer pairs that specific regions of the PRM1 → PRM2 → TNP2 domain are shown the as pairs delimit the corresponding within the loop and nuclear matrix A PCR primer directed to the locus was used as a non-matrix-associated This same region of the locus contains a somatic as shown in HeLa matrix-bound restriction in which of the with the matrix-bound are identified as matrix-associated are by for those that within both Non-matrix-associated are by for those that from to of the matrix Large restriction that contain the SMARs showed to the matrix of attachment to the nuclear matrix are as association for the could not be for the locus, as is a StyI the Large Image Figure ViewerDownload Hi-res image Download of intermediate nuclear matrix association are to in the organization of sperm It has been shown that of the chromatin in human sperm protamine replacement Science. PubMed Scopus Google Scholar). The intermediate association that is (Fig. may affinity of the sperm nuclear matrix for chromatin as compared with This is consistent with DNase of the PRM1 → PRM2 → TNP2 domain in human 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 (62) Google Scholar), increased of this of the genome to the DNase I sensitivity may be with the of interaction of each gene with the sperm nuclear matrix. To this mature loop and matrix-bound were to PCR analysis using primer directed numerous well loci the human As shown in Fig. PCR analysis of the regions containing the and TNP2 genes showed that these genes were associated with the nuclear matrix. amplification of regions of the and which are also DNase I-sensitive in mature A. Kramer and S. A. Krawetz, also showed an association with the sperm nuclear matrix. In the and and all of which are DNase in mature showed association with the sperm nuclear matrix. In somatic DNase I sensitivity has been shown to with the of genes for not to be transcription in mature the intermediate association of the DNase I-sensitive regions with the sperm nuclear matrix may a means by which the genome is for for protamine are necessary for the formation of a viable male I sensitivity and nuclear matrix association are in the male halos were separated into their loop and nuclear matrix-bound fractions and then is by the of to the loop or matrix by the The DNase and genes showed association with the sperm nuclear matrix The DNase I-sensitive PRM1 PRM2 and TNP2 gene as well as the and genes were associated with the nuclear matrix and showed an interaction with the HeLa nuclear matrix not Large Image Figure ViewerDownload Hi-res image Download is that MARs and SMARs sequence and the locus has been shown to contain a MAR that of the of somatic 1988; 7: PubMed Scopus Google Scholar). It that region of the genome to the somatic nuclear matrix. As shown for HeLa nuclei in Fig. this is with the human locus. the organization within the somatic nucleus, this region not with the mature sperm nuclear matrix. This is in with that for the sperm-specific PRM1 → PRM2 → TNP2 with the presented above and that of be more of association with the nuclear matrix. It is to assume that are at of nuclear matrix regulatory somatic and associated regulatory MARs an to be bound by the nuclear matrix as be identified by an in Cell. 1986; Full Text PDF PubMed Scopus Google Scholar). These MARs are not at the ends of the DNase I-sensitive have been to regions containing enhancers(10.Gasser S.M. Laemmli U.K. Cell. 1986; 46: 521-530Abstract Full Text PDF PubMed Scopus (433) Google Scholar), origins of replication(11.Kalandadze A.G. Bushara S.A. Vassetzky Y.S. Razin S.V. Biochem. Biophys. Res. Commun. 1990; 168: 9-15Crossref PubMed Scopus (63) Google Scholar), and other regulatory elements A.P. Higgs D.R. EMBO J. 1988; 7: 3337-3344Crossref PubMed Scopus (165) Google Scholar) and may also regions is J.S. van Holde K.E. Crit. Rev. Eukaryotic Gene Expression. 1992; 2: 211-224PubMed Google Scholar). MARs contain specific by nuclear matrix In the nuclear matrix protein which to specific within the has recently been shown to be the transcription B. van J. S. J.L. S. A. 1995; PubMed Scopus (162) Google Scholar). of these MARs not act as or of the regulatory to the matrix-associated region and the nuclear matrix may all of the elements necessary for In of the regulatory sequence motifs and the of within the nuclear S. S. A. 1988; PubMed Scopus Google Scholar), it is that sequence for the MAR will be 2 somatic MARs are to the ends of DNase I-sensitive and act as elements in somatic may loci and in types of The MARs of the locus that delimit the DNase I-sensitive domain have been shown to mediate position-independent A. Winter D.M. Strätling W.H. Sippel A.E. Nature. 1989; 341: 343-345Crossref PubMed Scopus (446) Google Scholar). It has been suggested that region MARs may transcription by across the that J.S. van Holde K.E. Crit. Rev. Eukaryotic Gene Expression. 1992; 2: 211-224PubMed Google Scholar). A sequence for this of MAR as more are identified and many loci region MARs. The MAR may be of this regions of matrix association described above for the haploid-specific PRM1 → PRM2 → TNP2 domain are of nuclear matrix attachment This is the of a haploid-specific the 2 SMARs to act as the ends of chromatin to the sperm nuclear matrix. The of the computational that those of the 2 somatic MARs. MARs and SMARs are not 2 somatic MARs have been shown to be MARs of genes have been shown to binding of or S.M. Laemmli U.K. Cell. 1986; 46: 521-530Abstract Full Text PDF PubMed Scopus (433) Google Scholar). Further, MARs from the locus the of of the 1988; 7: PubMed Scopus Google Scholar), while MARs of the genes have been shown to be the S. Res. 1986; PubMed Scopus Google Scholar). These the SMARs and the and 2 MARs are in Figure Figure that contain a somatic MAR 1988; 7: PubMed Scopus Google Scholar) not with the sperm nuclear and SMARs of the haploid-expressed PRM1 → PRM2 → TNP2 domain not to the HeLa nuclear matrix (Fig. As with the 2 of a sequence for SMARs will upon the and of intermediate association with the sperm nuclear matrix of those genes that DNase I sensitivity in mature be considered to a 4 nuclear matrix This intermediate affinity for the sperm nuclear matrix may be similar to that for the somatic expressed in this be identified using an in and it appears that it is not on the presence of a It is not known this of association a structural specific to sperm the function of MARs has been the biological role for nuclear matrix attachment and nuclear matrix association within the male genome to be The region the 2 region MARs to act as It is not from as has been shown for 2 MARs A. Winter D.M. Strätling W.H. Sippel A.E. Nature. 1989; 341: 343-345Crossref PubMed Scopus (446) Google Scholar). of transgenic containing SMARs from the PRM1 → PRM2 → TNP2 locus have been shown to copy of 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 (62) Google Scholar). such expression also be by a locus as by the S. F. EMBO J. 1990; 7: Scopus Google Scholar). the human PRM1 → PRM2 → TNP2 locus contains an locus region the SMARs as a means of locus haploid-specific SMARs and the intermediately associated regions described above two of at of nuclear matrix-associated of the and of various nuclear matrix-associated regions will both and the of the of gene and genome INTRODUCTIONFor many years the nuclear matrix received little attention, as it was thought to act merely as a structural element(1.Baskin Y. Science. 1995; 268: 1564-1565Crossref PubMed Scopus (16) Google Scholar). It has now been suggested that the nuclear matrix may play a key role in genome organization and gene potentiation(2.Zlatanova J.S. van Holde K.E. Crit. Rev. Eukaryotic Gene Expression. 1992; 2: 211-224PubMed Google Scholar). As in the somatic nucleus, chromatin within the male gamete is organized into discrete loops, bound at the base by regions of attachment to the nuclear matrix(3.Ward W.S. Coffey D.S. Biochem. Biophys. Res. Commun. 1990; 173: 20-25Crossref PubMed Scopus (49) Google Scholar). These loops differ from their somatic counterparts with respect to the packaging of their DNA (4.Ward W.S. Biol. Reprod. 1993; 48: 1193-1201Crossref PubMed Scopus (111) Google Scholar) and their average size. Loops within the sperm nucleus are ∼27 kb 1The abbreviations used are: kbkilobase(s)SMARsperm nuclear matrix attachment regionMARsomatic nuclear matrix attachment regionPCRpolymerase chain reactionbpbase pair(s). in size (5.Barone J.G. De Lara J. Cummings K.B. Ward W.S. J. Androl. 1994; 15: 139-145PubMed Google Scholar) compared with ∼60 kb in all other types of cells studied to date(6.Vogelstein B. Pardoll D.M. Coffey D.S. Cell. 1980; 22: 79-85Abstract Full Text PDF PubMed Scopus (468) Google Scholar). We have termed these sperm nuclear matrix attachment regions (SMARs) (7.Kramer J.A. Krawetz S.A. Mamm. Genome. 1995; 6: 677-679Crossref PubMed Scopus (13) Google Scholar). The somatic nuclear matrix has come under intense study, as actively transcribed genes have been shown to be associated with the nuclear matrix(8.Ciejek E. Tsai M.-J. O'Malley B.W. Nature. 1983; 306: 607-609Crossref PubMed Scopus (297) Google Scholar). Somatic nuclear matrix attachment regions (MARs) have been identified in or near introns(9.Jarman A.P. Higgs D.R. EMBO J. 1988; 7: 3337-3344Crossref PubMed Scopus (165) Google Scholar), enhancers(10.Gasser S.M. Laemmli U.K. Cell. 1986; 46: 521-530Abstract Full Text PDF PubMed Scopus (433) Google Scholar), origins of replication(11.Kalandadze A.G. Bushara S.A. Vassetzky Y.S. Razin S.V. Biochem. Biophys. Res. Commun. 1990; 168: 9-15Crossref PubMed Scopus (63) Google Scholar), and sites of transcription initiation(12.Dijkwel P.A. Hamlin J.L. Mol. Cell. Biol. 1988; 8: 5398-5409Crossref PubMed Scopus (162) Google Scholar), as well as other regulatory elements(9.Jarman A.P. Higgs D.R. EMBO J. 1988; 7: 3337-3344Crossref PubMed Scopus (165) Google Scholar). MARs have also been identified at the ends of the DNase I-sensitive domain in numerous loci (13.Levy-Wilson B. Fortier C. J. Biol. Chem. 1989; 264: 21196-21204Abstract Full Text PDF PubMed Google Scholar, 14.Loc P.-V. Strätling W.H. EMBO J. 1988; 7: 655-664Crossref PubMed Scopus (226) Google Scholar) and shown to facilitate position-independent gene activity(15.Stief A. Winter D.M. Strätling W.H. Sippel A.E. Nature. 1989; 341: 343-345Crossref PubMed Scopus (446) Google Scholar). The function of the sperm nuclear matrix is comparatively unknown.An ∼40-kb region of human chromosome 16p13.13 has recently been sequenced in its entirety and shown to contain the genes for the sperm-specific protamine 1, protamine 2, and transition protein 2 proteins(16.Nelson J.E. Krawetz S.A. J. Biol. Chem. 1994; 269: 31067-31073Abstract Full Text PDF PubMed Google Scholar). DNase I sensitivity analysis has delineated the boundaries of the domain in the mature spermatozoan, and transgenic analysis has shown that this region of the genome contains all the elements necessary for the appropriate spatial and temporal expression of the genes of this cluster in a position-independent, copy number-dependent manner(17.Choudhary 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 (62) Google Scholar). To characterize structural elements that mediate this response, we have identified regions of genomic interaction with the sperm nuclear matrix. Further, we demonstrate that a specific subset of both haploid-specific and constitutively expressed genes are associated with the mature sperm nuclear matrix. These genes assume an altered structural conformation as evidenced by their increased sensitivity to DNase I. Thus, the mature sperm genome is organized in a specific non-random manner. This could provide the means to template the male genome for ordered protamine replacement immediately subsequent to