From the
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
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
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
In contrast to the well
characterized in vivo and in vitro
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
Mouse Cot-1 DNA and the Bio-Nick labeling system were purchased
from Life Technologies, Inc. CENTRI-SEP
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
The
slides containing the various preparations of transgenic metaphase
chromosomes were prewarmed in 2
The corresponding human
sequences and the mouse
The corresponding mouse Prm1, Prm2, and
Tnp2 mRNAs were detected using a series of
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
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 PRM1
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
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.
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 corresponding genic domain resides within this
segment of human chromosome 16p13.2 as an
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 PRM1
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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.
-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.
-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.
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 PRM1
h PRM2
h 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.
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 10
cpm/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.
spin
columns. The eluted biotinylated DNA probe was then brought to a final
volume of 100 µl with TE for storage at -20 °C.
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.
-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 10
cpm/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.
-
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.
-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 PRM1
Approximately 20 PRM2
TNP2
Locus
10
permeabilized
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.
Creation of Transgenic Mouse Model of the Human
PRM1
A 40,255-bp NotI
fragment of cloned human genomic DNA from cosmid hP3.1 was
microinjected into the pronucleus of fertilized C57BL/6J PRM2
TNP2 Locus
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.
h PRM2
h TNP2 gene cluster
into mouse chromosome 2, whereas animals 882-21 and 883-00 had
integrated the h PRM1
h PRM2
h 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 PRM1
h PRM2
h TNP2 locus during the
F
generation 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 PRM1
h PRM2
h 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 PRM1
h PRM2
h 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 PRM1
h PRM2
h 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, F
mouse
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 F
generation 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 PRM1
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 PRM2
h TNP2 DNase I-sensitive
Domain
h PRM2
h 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 t
values 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 t
value. 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 t
values of 11.7, 10.6, 12, and 16 min, respectively. Fragments A
and F were markedly less sensitive. Their t
values 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.
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) .
Expression of the Human PRM1
Fidelity of expression of the transgenic
h PRM1, h PRM2 and h TNP2 genes indicates that
this PRM2
TNP2 Locus in
Transgenic Mice
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.
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 PRM2
h 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 PRM1
h PRM2
h 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.
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 PRM1
h PRM2
h 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 PRM2
h 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
PRM2
TNP2 cluster in the
three F
animals 884-11:892-34:882-19 was
1:1.5:2.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.