©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Haploid Expressed Gene Cluster Exists as a Single Chromatin Domain in Human Sperm (*)

Suresh K. Choudhary (1) (3)(§), Susan M. Wykes (1) (3)(§) (2), Jeffrey A. Kramer (1) (3) (2)(¶), Anwar N. Mohamed (4), Fred Koppitch (4), James E. Nelson (2)(**), , and Stephen A. Krawetz (1) (3) (2)(§§)

From the (1) Department of Obstetrics and Gynecology and Centers for (2) Molecular Medicine and Genetics and (3) Human Growth and Development, Wayne State University School of Medicine, and (4) Department of Pathology, Harper Hospital, Detroit, Michigan 48201

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mammalian spermiogenesis is marked by the initial disruption of the nuclear-histone-DNA complex by the transition proteins for ultimate replacement with protamines. The genes for three of these low molecular weight basic nuclear proteins exist as a single linear array of PRM1, PRM2, and TNP2 on human chromosome 16p13.2. To begin to address the mechanism governing their transcriptional potentiation, a region of 40 kilobases of the human genome encompassing these genes was introduced into the germ line of mice. Fluorescence in situ hybridization and Southern analysis showed that this segment of the human genome integrated into independent chromosomal sites while maintaining its fidelity. Transcript analysis demonstrated that the expression of the endogenous mouse protamine Prm1 and Prm2 genes as well as the mouse transition protein Tnp2 gene were expressed along with their human transgene counterparts. The pattern of expression of these transgenic human genes within this multigenic cluster faithfully represented that observed in vivo. In addition, all members of this transgenic gene cluster were expressed in proportions similar to those in human testis. Copy number-dependent and position-independent expression of the transgenic construct demonstrated that the corresponding biological locus was contained within this segment of the human genome. Furthermore, DNase I sensitivity established that in sperm the human PRM1 PRM2 TNP2 genic domain was contained as an 28.5-kilobase contiguous segment bounded by an array of nuclear matrix associated topoisomerase II consensus sites. This is the first description of a multigenic male gamete-specific domain as a fundamental gene regulatory unit. A model of haploid-specific gene determination is presented.


INTRODUCTION

Protamines are highly basic, sperm-specific nuclear proteins that are characterized by an arginine rich core segment ( cf. Refs. 1 and 2). In mammals there are two classes of protamine proteins, PRM1 and PRM2, and at least three distinct classes of transition proteins, TNP1, TNP2, and TNP4 (3, 4) . During the terminal phase of spermiogenesis, the nucleohistone array is replaced with a nucleoprotamine complex. A compact and essentially quiescent genome results. This process is mediated by the transition proteins ( cf. Ref. 5), which in humans leads to the final replacement of 85% of the nuclear histone complement with protamines (6) . The functionally related PRM1, PRM2, and TNP2 genes exist as an ordered linear array on human chromosome 16p13.2 (7, 8) . The complete primary structure of the 40,573-bp()region that encompasses these genes has been determined (9) . Extensive sequence analysis of this chromosomal segment has provided many insights into the biology and evolution of the various members of the cluster and this region of the human genome.

Phylogenetic footprinting (2, 10, 11) and transgenic strategies have been used to identify a series of sequences flanking the 5` region of the protamine genes that confer tissue specificity. An 859-bp sequence, 5` of the initiator codon of the mouse Prm2 gene (12) and a 465-bp sequence, 5` of the initiator codon of the mouse Prm1 gene (13, 14) can direct tissue-specific expression in a homologous system. Deletion analysis has shown that the tissue-specific element(s) resides within a 113-bp segment, 5` of the initiator codon of the Prm1 gene (15) encompassing nucleotides -77 to -37 (16) . Their significance remains to be assessed, since both testis-specific and ubiquitous protein factors bind to this segment. Although this directly addressed transcriptional regulation, the issue of how these linked genes are selected from the genome for expression remains.

Our understanding of how a multigenic locus assumes a potentiated conformation, i.e. readied for expression, or how this state is maintained for transcription is limited to somatic cells. The concept that transcriptionally competent chromatin assumed an altered nuclear structure was validated when it was shown that the -globin gene acquired a DNase I-sensitive conformation within cells in which it was expressed (17) . These studies were extended to the chicken ovalbumin, x, y locus, which was one of the first chromatin domains containing a multigenic loci to be defined (18) . The complete characterization of the -globin locus has extended the concept of the genic domain as the primary regulatory unit to mammalian systems (19, 20) . The subsequent identification of the LCR, i.e. locus control region (21) , provided a key element requisite to understanding the mechanism of gene potentiation.

In contrast to the well characterized in vivo and in vitro -globin locus, our understanding of haploid expressed genic domains and how these genes, i.e. PRM1 PRM2 TNP2, assume a potentiated conformation is lacking. Significant progress toward understanding the underlying mechanism governing male germ cell gene expression has been hampered by the lack of a permanent spermatogenic cell line and the short life span of primary cells in culture. A coculture system using virus-transformed 15-p1 cells has been developed (22) , although it has yet to be widely adopted. This has left cell purification by separation or transgenic analysis as the only tools that can be used to address these issues within the differentiating sperm cell.

To permit the dissection of the molecular potentiative mechanism that governs the expression of the male gamete a transgenic mouse model was constructed. A segment of human chromosome 16 harboring the PRM1 PRM2 TNP2 gene cluster (8, 9) was introduced into fertilized mouse eggs. This 40,255-bp fragment contained sufficient information to faithfully express all of the members of this multigene family in a copy number-dependent, position-independent manner in the heterologous system. A DNase I-sensitive domain of 28.5 kb encompassing the h PRM1h PRM2h TNP2 gene cluster is bounded by an array of topoisomerase II consensus sites. These results establish that like somatic cells, the genes transcribed by the male gamete are organized at specific loci as functional domains. The role of organizing the male nucleus in this manner as a means of achieving first order control during male gametogenesis is discussed in the proposed model of haploid-specific gene determination.


MATERIALS AND METHODS

Mouse Cot-1 DNA and the Bio-Nick labeling system were purchased from Life Technologies, Inc. CENTRI-SEP spin columns were from Princeton Separations, and the biotin-FITC detection kit and PBD solution were purchased from Oncor. Positively charged Quiabrane nylon membranes were purchased from Quiagen. Lyophilized bovine pancreatic DNase I (EC 3.1.21.1) was purchased from Amersham/USB. The mouse pTRI--actin probe was from Ambion. All other reagents were of ACS or molecular biology grade and purchased from sources described previously (23) .

Creation of the Human Protamine Transgenic Mouse Model

A 40,573-bp genomic DNA segment encompassing the human protamine PRM1 gene, PRM2 gene, and the transition protein TNP2 gene cluster from chromosome 16p13.2 was isolated and characterized (24) . The corresponding 40,255-bp genomic fragment containing this human gene cluster was isolated by electroelution of the NotI restriction-digested hP3.1 cosmid DNA, then purified by DEAE-ion exchange chromatography. The purified DNA was then microinjected into the pronuclei of 20 C57BL/6 SJL F2 hybrid recipient fertilized mouse eggs. Transgenic offspring were identified by slot blot analysis of mouse tail tip DNA using the human-specific TNP2 probe. Their identity was confirmed by gene fingerprint analysis.

Southern Analysis and Copy Number Determination

Transgene copy number was determined using concentration standardized mouse tail tip DNAs isolated essentially as described (25) . Mouse tail tip DNAs and cosmid hP3.1 DNA were digested to completion with EcoRI. Equal genomic equivalents of the various transgenic mouse genomic DNAs and cosmid DNA were electrophoretically resolved then transferred onto positively charged nylon membranes. The membranes were then prehybridized in the hybridization solution of 5 SSPE containing 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400, 50% formamide, 1% SDS, 0.1 mg/ml yeast tRNA, plus 1.25% polyethylene glycol 8000 for at least 1 h at 42 °C. The membranes were then hybridized overnight at 42 °C in fresh hybridization solution that contained 10cpm/ml of each individual [-P]dCTP-radiolabeled probe. The human PRM1 probe (3F1), PRM2 probes (7D9 plus 8B5), and the TNP2 probe (7F8) were purified from pTZ18R-subcloned fragments of cosmid hP3.1. They encompass nucleotide positions 14,637-15,802, 19,556-20,003, 19,635-20,164, and 26,736-27,307, respectively. Following hybridization, the membranes were washed for 30 min at room temperature in 2 SSPE with 0.1% SDS, then for 30 min at 65 °C in 0.1 SSPE with 0.1% SDS, prior to autoradiography for 7 days at -70 °C. Copy number was then determined utilizing cosmid hP3.1 as the Whole Band Analysis copy number calibration standard, as described (26) .

Fluorescence in Situ Hybridization

Primary cell cultures were established as explants from either mouse tail tissue or muscle tissue (27) . The cells were grown in -minimal essential medium, Earles' salts, supplemented with 10% fetal bovine serum, 2 m M glutamine, and 1% penicillin/streptomycinin in Corning T-25 flasks. Cells were then harvested and metaphase chromosomal spreads prepared (28) . To permit chromosomal assignment of the integrated human loci and karyotype analysis, the transgenic mouse chromosomes were QFQ-banded (29) prior to FISH analysis.

Approximately 1 µg of the purified 40,255-bp NotI-digested fragment from cosmid hP3.1 was labeled with biotin-14-dATP by nick translation using the Bio-Nick labeling system as recommended by the manufacturer. After incubation for 1 h at room temperature, the reaction was terminated. Unincorporated nucleotides were then separated from the labeled DNA using CENTRI-SEP spin columns. The eluted biotinylated DNA probe was then brought to a final volume of 100 µl with TE for storage at -20 °C.

The slides containing the various preparations of transgenic metaphase chromosomes were prewarmed in 2 SSPE at 60 °C for 10 min. After warming, the slides were denatured in a solution of 2 SSPE containing 70% formamide at 70 °C for 2 min. They were then dehydrated through a series of graded -20 °C ethanol washes of 70%, 80% then 95%, then air dried. After drying, 50 µl of the hybridization solution containing 100 ng of the heat-denatured biotinylated hP3.1 probe containing 2.5 ng of nonspecific sequence competitor mouse Cot-1 DNA was applied to each slide. The slides were coverslipped and sealed with rubber cement, then incubated at 37 °C overnight in a moisture retentive chamber. Subsequent to hybridization, the slides were washed in 2 SSPE at 72 °C for 5 min, followed by a 5-min wash in 1 PBD solution at room temperature. Hybridization products were then detected using biotin-FITC, and the chromosomes were counterstained with propidium iodide. Chromosomes were viewed with an Axioskop fluorescence microscope using a Zeiss wide-pass FITC filter and 50-watt mercury vapor bulb. Assignment of each integrant to its respective chromosome was determined as a function of coincident QFQ banding.

Transcript Analysis

Total RNA was isolated using the method of Chomczynski and Sacchi (30) , and then equal amounts of each sample were fractionated on a 1.2% agarose-formaldehyde gel (31) . RNAs thus resolved were then electroblotted onto positively charged nylon membranes.

The corresponding human sequences and the mouse -actin sequence were detected using a specific series of random primed probes. The h PRM1, h PRM2, and h TNP2 probes were as described above. The mouse actin probe was a 250-bp fragment of the pTRI--actin clone spanning codons 220-303. The membranes were prehybridized in the hybridization solution for at least 8 h at 42 °C. They were subsequently hybridized overnight at 42 °C in fresh hybridization solution that contained 10cpm/ml of each individual [-P]dCTP-radiolabeled probe corresponding to the various members of the gene cluster. Following hybridization the membranes were washed at room temperature in a solution of 2 SSPE containing 0.1% SDS for 30 min, then at 65 °C in 0.1 SSPE containing 0.1% SDS for 30 min. The membranes were then autoradiographed -70 °C for various times to visualize the human PRM1, PRM2, TNP2, and mouse actin mRNAs.

The corresponding mouse Prm1, Prm2, and Tnp2 mRNAs were detected using a series of -P 5`-end labeled mouse-specific oligonucleotide probes. The sequences of the mouse-specific oligonucleotide probes were as follows: Prm1, CGGCGACGGCAGCATCTT; Prm2, CTGGGGAGGCTTAGTGATG and Tnp2, CTGATGCCCTCTCCTGGTGTGT. The corresponding membranes were prehybridized for 1 h at 37 °C in the oligonucleotide hybridization solution comprising 6 SSC containing 50 m M sodium phosphate, pH 6.5, buffer, 5 Denhardt's solution, 0.1% SDS, 2 m M EDTA, plus 0.1 mg/ml yeast tRNA. The membranes were then hybridized overnight at 37 °C in fresh oligonucleotide hybridization solution that contained a single labeled oligonucleotide probe. Following hybridization the membranes were washed at room temperature in a solution of 2 SSPE containing 0.1% SDS for 30 min, then at 58 °C in 0.1 SSPE containing 0.1% SDS for 45 min. The membranes were then autoradiographed at -70 °C for 14 days to visualize the mouse mRNAs.

Autoradiographic images that were within the linear response range of the film were quantitated using the Millipore 60S version 3.0 Whole Band Image Analysis system. Comparative values were calculated as a function of exposure time. RNA sample loading was normalized as a function of the intensity of the hybridization signal of the mouse -actin mRNA and/or the internal ethidium bromide-stained rRNAs. Deviation among samples averaged 16.7%, when compared to the expected values.

DNase I Sensitivity of the Human PRM1PRM2TNP2 Locus

Approximately 20 10permeabilized human sperm were treated with 250 units of DNase I at 37 °C and equal individual aliquots removed at 0, 5, 15, 30, and 60 min. The reactions were terminated with the addition of a 50 m M Tris-HCl, pH 8.5, buffered solution containing 50 m M NaCl, 25 m M EDTA plus 0.5% SDS. Proteinase K was then added to the DNase I-treated samples to a concentration of 200 µg/ml, and the resulting mixture incubated at 50 °C for 2 h. The DNA was subsequently purified by organic extraction then precipitated with ethanol. Purified DNAs were then denatured in 10 m M EDTA plus 0.4 N NaOH at 95 °C for 10 min prior to slot blotting onto positively charged nylon membranes. The membranes were hybridized at 42 °C for 18 h to eight different [-P]dCTP-labeled probes from unique sequence fragments of human genomic DNA essentially as above. Six of these fragments, denoted as A-F, spanned the region of human chromosome 16p13.2 that encompassed the PRM1 PRM2 TNP2 locus. Fragment A encompassed the region from 6,678-7,488; B, 15,035-15,304; C, 19,927-20,327; D, 22,516-23,088; E, 27,940-28,360; and F, 35,296-35,894. The 1,126-bp human, -actin probe was synthesized using Clontech PCR primer set 5402 and the 268-bp -globin probe using Perkin-Elmer PCR primer set PCO4/GH20. Both probes recognize sequences on different chromosomes and were used as controls. Subsequent to hybridization, the membranes were washed at 50 °C for 15 min in 0.1 SSPE that contained 0.1% SDS. The membranes were then autoradiographed at -70 °C overnight. Autoradiographic images were quantitated using the Millipore 60S slot blot image analysis system. The intensity of hybridization of each DNase I-treated sample data point was expressed relative to that of the zero time point. Mean percent hybridization as a function of time was then plotted. The relative DNase I sensitivity, i.e. t, of each fragment was defined as the time required to produce a 50% reduction in the total amount of hybridization signal detected.


RESULTS

Creation of Transgenic Mouse Model of the Human PRM1PRM2TNP2 Locus

A 40,255-bp NotI fragment of cloned human genomic DNA from cosmid hP3.1 was microinjected into the pronucleus of fertilized C57BL/6J SJL F2 hybrid eggs of mice. This fragment contained the linear arrayed human PRM1, PRM2 protamine genes and the TNP2 transition protein gene. This gene cluster is juxtaposed by 14 kb of non-coding sequence 5` to the proximal PRM1 gene and 11 kb of non-coding sequence 3` of the distal TNP2 gene nestled among 42 Alu elements. Six founding transgenic animals including two females were produced. Gene fingerprint analysis from each resulting integration event did not reveal any gross rearrangement of this cluster or its members within the founding members or offspring (data not shown). The fidelity of this segment of the human genome containing these numerous repetitive elements was thus maintained, when introduced into and propagated in the transgenic state.

Five founding animals showed germ line transmission of the integrated loci. The number of loci integrated within the different lines varied from 4 to 12 copies. FISH analysis, presented in Fig. 1, revealed that animals 892-34 and 884-10 had integrated the h PRM1h PRM2h TNP2 gene cluster into mouse chromosome 2, whereas animals 882-21 and 883-00 had integrated the h PRM1h PRM2h TNP2 gene cluster into mouse chromosomes 6 and X, respectively. Animals from lines 882, 892, 884, and 885 were then bred further, whereas animal line 883 was excluded from breeding since the human PRM1 PRM2 TNP2 locus had integrated into the mouse X chromosome and was subject to X-inactivation. Animal 885-00 lost the h PRM1h PRM2h TNP2 locus during the Fgeneration and was excluded from further analysis.

Expression of the PRM1, PRM2, and TNP2 Genes in Human Testis and the Transgenic Mouse Testis

Comparative transcript analysis was undertaken to assess whether this fragment of the human genome from cosmid hP3.1 contained the necessary information to express the human PRM1, PRM2, and TNP2 transgenes in the appropriate context. As shown in Fig. 2 A, the h PRM2 transcript was the most abundant of the spermatid-specific h PRM1, h PRM2, and h TNP2 transcripts in human testis. Comparatively, h PRM1 was present at a level of 50% of that of h PRM2, and h TNP2 was present at a level of 3% of that of h PRM2. The relative proportions of each of the human PRM2, PRM1, and TNP2 mRNAs present in transgenic mouse testis shown in Fig. 2 B were similar to that observed in human testis shown in Fig. 2A. There was no evidence of any significant cross-hybridization or lack of specificity among the mouse or human probes. Furthermore, transgenic ovary, brain, lung, heart, kidney, and liver RNAs did not hybridize to any of the human or mouse probes (data not shown). The appropriate spatial expression of each member of the human PRM1 PRM2 TNP2 transgenic gene cluster was independent of the cognate mouse cluster. One can conclude that this segment of the human genome contains the requisite information for tissue-specific regulation and expression of each of its members in a manner similar if not identical to that observed in vivo.


Figure 2: Expression of PRM1, PRM2, and TNP2 mRNAs. A, expression of PRM1, PRM2, and TNP2 mRNAs in normal human testis. Total RNA was prepared from normal human testis, then equal aliquots were electrophoretically resolved, then transferred to positively charged nylon membranes. Each membrane was hybridized overnight at 42 °C in the hybridization solution containing the corresponding radiolabeled fragment probe to each transcribed region. Following hybridization the membranes were washed at various levels of stringency to remove background, then autoradiographed at -70 °C for 12 h to visualize the PRM1 and PRM2 mRNAs and 4 days to visualize the TNP2 mRNAs. B, relative expression of human and mouse PRM1, PRM2, and TNP2 mRNAs in the normal and transgenic mouse. Total RNA was prepared from mouse testis, and then equal aliquots were electrophoretically resolved prior to transfer to a positively charged nylon membrane. The membranes were hybridized overnight at 42 °C in the hybridization solution containing the corresponding radiolabeled fragment probe for each of the human PRM1, PRM2, or TNP2 sequences, or the corresponding radiolabeled oligonucleotide probe for each of the mouse Prm1, Prm2, or Tnp2 sequences. Following hybridization the membranes were washed, then autoradiographed at -70 °C. The PRM1 mRNA was visualized after 6 h. PRM2 was visualized after 1.5 h, TNP2 was visualized after 4 days. The mouse sequences were visualized after 14 days. The human probes did not cross-react with the mouse mRNAs, nor did the mouse probes cross-react with the human mRNAs. The relative expression of the mouse Prm1: Prm2: Tnp2 mRNAs in the transgenic and nontransgenic states were essentially identical.



Copy Number-dependent, Position-independent Expression of the Human PRM1, PRM2, and TNP2 Transgenes

The tenet that this human genomic fragment of 40 kb containing the h PRM1h PRM2h TNP2 gene cluster acts as a single locus, i.e. domain, similar to that of the -globin locus (21) was supported by three observations. FISH analysis demonstrated that every transgenic mouse bearing the h PRM1h PRM2h TNP2 locus integrated this multigenic human domain into the mouse genome in a position-independent manner. All transgenic animals expressed the h PRM1, h PRM2, and h TNP2 genes in the correct spatial manner. The relative proportion of each member of the transgenic h PRM1h PRM2h TNP2 cluster was similar to that observed in vivo. However, this did not demonstrate the hallmark of a genic locus, i.e. copy number-dependent expression of the independently integrated loci. To address this remaining issue, the relative expression of each member of the cluster as a function of the number of copies of the integrated locus was determined. For example, Fmouse 892-00 containing nine copies of the human PRM1 PRM2 TNP2 locus showed an 2-fold higher level of the corresponding human mRNAs than founder 890-00, which only contained four copies. The relative expression of each member of the human PRM1 PRM2 TNP2 locus within each of the Fgeneration transgenic mice analyzed is shown in Table I. Transgenic animals from lines 884, 892, 882 harbored 6, 9, and 12 copies of the locus, respectively. As expected, the relative level of each of the three gene products of the human PRM1 PRM2 TNP2 locus was directly proportional, i.e. 1:1.5:2, to the number of copies, i.e. 6:9:12, of the integrated locus. Furthermore, within each animal the relative level of each gene product was similar to that observed in normal human testis. As expected, their coordinate proportional expression remained dependent upon the number of copies of the integrated loci. The creation of this series of transgenic animals that demonstrate position-independent copy number-dependent expression of each of the members of the human PRM1 PRM2 TNP2 cluster, confirms that the entire biological locus is contained within this 40,255-bp NotI fragment of the human genome. Accordingly, this region must contain all elements that ensure locus activation in order to permit correct temporal and spatial expression. This region must also demarcate the boundaries of the genic domain.

Definition of the h PRM1h PRM2h TNP2 DNase I-sensitive Domain

To assess whether this biologically defined locus demarcated the physical domain, individual aliquots of permeabilized human sperm were treated with DNase I for various periods of time. The digested DNAs were then analyzed by slot blot analysis using six unique genomic DNA fragments designated as probes A-F, that encompass the h PRM1h PRM2h TNP2 transgenic locus as shown in Fig. 3. Relative hybridization to human -actin, which is constitutively expressed, provided a DNase I-sensitive control possessing a t= 13.3 min, whereas human -globin, which is expressed only in cells of erythroid lineage, served as a DNase I-insensitive control, possessing a t= 34 min in human sperm. As shown in Fig. 3, fragments B-E displayed varying degrees of enhanced DNase I sensitivity. Their tvalues were 11.7, 10.6, 12, and 16 min, respectively, similar to that of the -actin DNase I-sensitive control. In comparison, fragments A ( t= 48 min) and F ( t= 46 min) were markedly less sensitive, similar to that of the -globin DNase I-insensitive control.


Figure 3: DNase I sensitivity of the region of human chromosome 16p13.2 encompassing the PRM1, PRM2, and TNP2 locus. The relative DNase I sensitivity of selected sequences, A-F, that span the 40-kb NotI fragment of human cosmid hP3.1 was assessed. Equivalent amounts of human sperm were treated with 250 units of DNase I for 0, 5, 15, 30, and 60 min. The DNA was then isolated and slot blotted onto nylon membranes. The membranes were hybridized using -P-labeled fragments A-F in addition to -actin and -globin that served as controls. Autoradiographic images were quantitated by densitometric analysis, and the mean percent hybridization was plotted as a function of time. Relative DNase I sensitivity was defined as the time required to reduce the amount of detectable hybridization product by 50% and is indicated by the tvalue. The relative DNase I sensitivity of each of each fragment was compared to the DNase I-sensitive -actin of 13.3 min and the erythroid-specific DNase I-insensitive -globin of 34 min. Fragments B, C, D, and E displayed varying degrees of DNase I sensitivity with tvalues of 11.7, 10.6, 12, and 16 min, respectively. Fragments A and F were markedly less sensitive. Their tvalues were 48 and 46 min, respectively.



The comparative DNase I-sensitivities of fragments A-F that span this region of human chromosome 16p13.2 establish the physical limits of the DNase I-sensitive domain. As shown in Figs. 3 and 4, fragments A and F mark the boundaries of an 28.5-kb region of enhanced DNase I-sensitivity. Bounding these end fragments at nucleotide positions 8,375-8,413 and 33,881-33,914 are short A-rich regions, often associated with ends of domains (32, 33, 34) . As shown in Fig. 4 , a cluster of putative topoisomerase II sites within each region are also present near the ends of the domain. These sites are located at positions 9,154, 9,203, 9,374, 32,901, 33,022, and 33,095. They share at least 86% identity to the topoisomerase II consensus sequence of the chicken lysozyme gene GTNWAYATTNATNNR (32) . They are also located at similar positions with respect to the DNase I-insensitive ends of the domain. This supports the view that these end regions may serve as attachment points to the nuclear matrix.


Figure 4: The human PRM1 PRM2 TNP2 multigenic domain. The comparative DNase I sensitivities of selected fragments that are denoted by the filled diamonds along an 40-kb region of chromosome 16p13.2 was assessed. The location of the PRM1, PRM2, and TNP2 genes are indicated by the filled boxes. The hollowed stars demarcate the position of the 5` and 3` topoisomerase II sites. The DNase I-insensitive ends mark the boundaries of the 28-kb DNase I-sensitive domain of the PRM1 PRM2 TNP2 locus.




DISCUSSION

In vitro and in vivo strategies have yielded a basic understanding of the mechanism controlling the temporal and spatial expression of genes. It is now generally accepted that relatively large regions of chromatin encompassing a single gene locus or multigenic locus assume a DNase I-sensitive conformation within somatic cells destined to express that gene (17, 18, 19) . Recent advances toward defining the elements controlling this event, have identified what are now termed LCRs, i.e. locus control regions ( cf. Ref. 35). These cis-elements are contained as part of the boundary regions of single independent regulatory units (36) , demarcated as large DNase I-sensitive chromatin domains, that in somatic cell nuclei average 60 kb in size (37) . Our understanding of these regions within the genome of the male gamete is rudimentary, although it is known that structural chromatin loop domains of an average size of 27 kb are present within the human sperm nucleus (38) .

Operationally, a domain contains LCR-``like'' elements that impart copy number-dependent and position-independent expression onto a transgene that has integrated into the host genome (21) . To begin to define those elements that control haploid-specific gene expression, transgenic animals harboring gene fusion constructs of segments of the mouse Prm1 and mouse Prm2 genes linked to various reporter genes have been produced (12, 13, 14, 15, 39, 40) . This revealed that relatively small regions within each regulatory unit assumed a fundamental role controlling the temporal and spatial expression of these genes.

However, the PRM1, PRM2, and TNP2 genes exist as a coordinately regulated cluster, not as independently regulatory domains. It is also apparent that this multigenic sperm-specific locus is of similar size as the chromatin loop domains observed in sperm. It was necessary to resolve whether this gene cluster existed as a domain within the sperm nucleus, before the potentiative mechanism that ultimately controls the coordinate expression of these three genes could be understood.

Expression of the Human PRM1PRM2TNP2 Locus in Transgenic Mice

Fidelity of expression of the transgenic h PRM1, h PRM2 and h TNP2 genes indicates that this 40-kb segment of human chromosome 16p13.2 contains all the necessary information to ensure appropriate regulation of this suite of genes. That is, spatial specificity was maintained and the relative proportion of each of the transgenic PRM1: PRM2: TNP2 mRNAs was similar to that observed in vivo.

Transcript analysis also revealed that the relative proportion of the mouse Prm1: Prm2: Tnp2 mRNAs differed from that of human mRNAs, but remained constant whether they existed in the normal or transgenic state. Both the mouse and human PRM2 mRNAs were the most abundant of the three gene products. In contrast, the relative proportion of h TNP2 mRNA was reduced by 12-fold when compared to the corresponding mouse Tnp2 mRNA. This is believed to reflect the absence of a conserved 3`-untranslated region octanucleotide, the deletion of which decreases the stability of the human mRNA (41) . The transgenic analysis described in this paper, which shows comparatively low levels of the corresponding human mRNA, supports the notion that the absence of this octanucleotide adversely affects the quantity of the message. This is consistent with the mechanism governing sperm mRNA levels being conserved through evolution. The relative proportion of the mouse Prm1 mRNA appeared less than the human PRM1 mRNA. The biological significance of this latter observation is not yet clear since both species produce fertile gametes. The fluctuating levels of the PRM1 and TNP2 mRNAs among species may reflect natural variation.

The 40,255-bp Fragment of 16p13.2 Is an Independent Genetic Locus

The hP3.1 NotI fragment from human chromosome 16p13.2 behaves as a copy number-dependent, position-independent regulatory unit. The appropriate biological expression of the transgenic h PRM1h PRM2h TNP2 gene cluster demonstrated that this segment of the human genome contains all necessary cis-element information to regulate this locus. The level of the endogenous mouse Prm1, Prm2, and Tnp2 transcripts in any of the transgenic animals was not altered. This was even apparent within the animals that incorporated up to 12 copies of the h PRM1h PRM2h TNP2 locus. It can be concluded that sufficient factors are present within the round spermatid to support the even larger load placed on the transgenic transcription machinery by these human genes. For the first time this biologically demarcates the extent of a male-specific-haploid-expressed multigenic locus.

The corresponding genic domain resides within this segment of human chromosome 16p13.2 as an 28.5-kb DNase I-sensitive region extending from nucleotide positions 7,083-35,565 in human sperm. This is consistent with this segment being contained within the 15% of the human sperm genome that remains histone associated (6) . The issue of whether the ends of the DNase I-sensitive domain share sequence similarity with those known to attach to the nuclear matrix was thus raised. Extensive computer analysis failed to reveal extended similarity between the sequences at the ends of the DNase I-sensitive domain and most of those typically associated with SARs or MARs (33, 34) . However, like SARs and MARs, both ends are flanked by a cluster of topoisomerase II sites that are again flanked by A-rich regions located equidistantly from each topoisomerase II cluster. In turn, these regions are flanked by a series of homologous Alu repetitive elements. Their significance is not yet clear, since this entire region of chromosome 16p13.2 contains numerous Alu elements at a frequency as great as 1.5/kb (9) . As suggested by others (42, 43) , perhaps these elements act as modulators of gene expression. To date this is the most compact of any multigenic domain known and is of similar size as the single 24-kb chicken lysozyme gene (44) . It is tempting to speculate that like the locus of the -globin gene cluster or the locus of the single lysozyme gene, the h PRM1h PRM2h TNP2 locus that contains all elements to ensure correct coordinate expression, is framed within this 28.5-kb DNase I-sensitive domain.

Haploid-specific Gene Determination: A Model

The complex pattern of transcription and translation exhibited by each member of the h PRM1h PRM2h TNP2 locus provides a unique system to examine the mechanism that regulates the expression of this multigenic domain. This must ultimately reflect the determinative process that is revealed during cell differentiation. We have begun to address this issue by carefully unravelling the mechanism of gene potentiation as it seals cell fate. This has formed the basis of the testable model presented in Fig. 5. As originally proposed by Britten and Davidson (45) , the coordinate regulation of a suite of cellular associated genes may be determined by a cascade of cell-specific factors and general factors. Cell-specific factors would be utilized to regulate cell-specific genes, e.g. the testis-specific protamines, whereas general factors would be utilized to regulate genes that support basal cellular functions, e.g. the glycolytic glyceraldehyde-3-phosphate dehydrogenase. In turn, appropriate coordinate regulation would be mediated through the formation of cis-element-factor complex(es). With the development of tools that could distinguish active from inactive chromatin (17) , large coordinately multigenic domains like the ovalbumin, x, y (18) , and the -globin (19) locus were revealed. It is now apparent that SARs, MARs, LCRs, and their corresponding factors that are typically associated with the ends of DNase I-sensitive domains (46, 47, 48) provide at least some of the requisite components necessary to form the potentiated, i.e. readied for expression genic state. As shown in the model of the determination of haploid-specific gene expression, haploid domains like that of P gk-2, denoted by the red dot, must have assumed a potentiated state at/or, prior to the first reduction division following the 4C pachytene cell stage. As indicated by the arrow, even though P gk-2 is first transcribed in the 4C pachytene cell (49) , is not translated until, as indicated by the multiple diverging arrows, the 1C round spermatid stage. Consistent with our preliminary data()and in accord with the above, the dark blue, cyan, and green dotted PRM1 PRM2 TNP2 domain, must also have assumed its potentiated confirmation by the 4C pachytene cell stage. As we have demonstrated and modeled, these domains are maintained within the mature spermatid subsequent to extensive remodeling of the genome. In comparison, the -globin domain, indicated by the open circle, is nonpotentiated and always closed. The issue remains: when is the male gametes' genome committed to its differentiative path? Commitment should be reflected upon the assumption of the potentiated state. This may either be fixed within the self-renewing stem cell population of type A spermatogonia or during the formation of type B spermatogonia. Precedence for the latter may be derived from the globin system. In this case the differentiative pathway is marked by a multi-potentiated state, which is reflected by the DNase I-hypersensitive LCRs. The commitment of the cell to its differentiative pathway is mediated by the selection of the LCRs that are to be maintained in the hypersensitive conformation (50, 51) . Commitment would be favored during major nuclear events that restructure the genome, like replication and division, which can serve to mark the timing of their replication. In this manner, genes that are destined for transcription would shift from the late replicating chromatin to early replicating chromatin fraction (36) . This would provide one of the key elements of selection during the potentiative event. The definition of the signal triggering the potentiative cascade that ultimately determines haploid-specific gene expression is currently being pursued.

  
Table: Copy number dependent expression of the human PRM1, PRM2, and TNP2 genes in transgenic mice

Total mRNA was prepared from transgenic and non-transgenic mouse testes, then analyzed by Northern transcript analysis. Each mRNA that hybridized to the hPRM1, hPRM2, hTNP2, and mouse actin probes was quantified using the Bio-Image version 3.0 Whole Band Analysis system. The integrated intensity for each of the hybridizing transgenic mRNAs was appropriately normalized to the internal mouse actin control. The relative expression of the PRM1PRM2TNP2 cluster in the three Fanimals 884-11:892-34:882-19 was 1:1.5:2.



FOOTNOTES

*
This work was supported in part by Grant 1RO1HD285040A1 from the NICHD, Grant EDUD-US93015 from Sun Microsystems, and FMRE Grant 1064 from Wayne State University (to S. A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These authors contributed equally to this work.

Supported in part by the Dean's Postdoctoral Recruitment Award.

§§
To whom correspondence should be addressed: Dept. of Obstetrics and Gynecology, Wayne State University School of Medicine, 253 CSMC, 275 E. Hancock Ave., Detroit, MI 48201. Tel.: 313-577-6770; Fax: 313-577-8554; E-mail: steve@compbio.med.wayne.edu.

The abbreviations used are: bp, base pair(s); kb, kilobase(s); LCR, locus control region; FISH, fluorescence in situ hybridization; SAR, scaffold attachment region; MAR, matrix attachment region; h, human.

J. A. Kramer, S. M. Wykes, J. E. Nelson, and S. A. Krawetz, unpublished observations.


ACKNOWLEDGEMENTS

We gratefully acknowledge NICHD Grant NO1-HD-0-2911 (to DNX Corp.) for the microinjection service of hP3.1.40. We thank the Division of Reproductive Endocrinology and Infertility of the Department of Obstetrics and Gynecology, Wayne State University for providing the normal human semen samples. Kieth Irtenkauf of the Department of Obstetrics and Gynecology provided the much appreciated photography and vivarium support.


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