(Received for publication, March 1, 1996; and in revised form, March 25, 1996)
From the
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,
-globin, and
-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.
For many years the nuclear matrix received little attention, as
it was thought to act merely as a structural element(1) . It
has now been suggested that the nuclear matrix may play a key role in
genome organization and gene potentiation(2) . 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) . These loops differ from their somatic
counterparts with respect to the packaging of their DNA (4) and
their average size. Loops within the sperm nucleus are 27 kb (
)in size (5) compared with
60 kb in all other
types of cells studied to date(6) . We have termed these sperm
nuclear matrix attachment regions (SMARs) (7) . The somatic
nuclear matrix has come under intense study, as actively transcribed
genes have been shown to be associated with the nuclear
matrix(8) . Somatic nuclear matrix attachment regions (MARs)
have been identified in or near introns(9) ,
enhancers(10) , origins of replication(11) , and sites
of transcription initiation(12) , as well as other regulatory
elements(9) . MARs have also been identified at the ends of the
DNase I-sensitive domain in numerous loci (13, 14) and
shown to facilitate position-independent gene activity(15) .
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) . 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) . 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.
Physical characterization of each of the candidate MARs
employed nuclei prepared from frozen sperm essentially as
described(19) . Nuclei were resuspended in 50 mM HEPES, pH 7.5, buffer containing 10 mM NaCl, 5 mM MgOAc, and 25% glycerol, at 1
10
/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) . 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 MgCl
. 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. (
)PCR was maintained within the linear range of
amplification. DNA halos were prepared from HeLa cells essentially as
described(21) , digested to completion with HindIII,
and then treated as described above for sperm halos.
To 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, 22) and then expressed as unique sequence patterns
as described(18) . 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) and were used to guide their physical identification.
Figure 1:
Computational MAR
analysis of the human PRM1 PRM2
TNP2 locus. The PRM1
PRM2
TNP2 biological locus was subjected to MAR motif analysis
implemented on a SUN UNIX workstation. Potential regions of nuclear
matrix association are represented as peaks above the statistically
weighted likelihood of 50%. The likelihood of a region being a MAR was
calculated as a function of the localization and frequency of a number
of characteristic MAR motifs, as described(18) . Regions of
strong contact with the nuclear matrix are predicted to be centered at
positions 8,175 and 34,100 (nucleotide coordinates along the domain
according to (16) ).
DNA ``halos'' were prepared by extracting sperm nuclei with a high ionic strength reducing buffer(5) . 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.
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. Both
fractions were purified and then subjected to PCR amplification using
unique sets of primers targeted to discrete regions of the
haploid-expressed PRM1 PRM2
TNP2 locus and the somatic expressed
-globin locus (Fig. 3). The distribution of each amplicon showed one of three
patterns. The majority, i.e. at least 80% of the
non-matrix-associated loop DNA, partitioned to the supernatant.
Similarly, greater than 80% of the matrix-attached DNA partitioned with
the nuclear pellet. In contrast, intermediately matrix-associated DNA
partitioned into both the supernatant and pellet (30-70%).
Regions that lay outside the DNase I-sensitive domain localized
consistently to the non-matrix-associated loop fraction, as did the
DNase I-insensitive
-globin locus. Regions within the domain were
intermediately associated with the nuclear matrix. This intermediate
affinity for the sperm nuclear matrix is similar to that observed for
the human
-interferon locus(23) . The region surrounding
and including the potentiated
-interferon gene shows weak nuclear
matrix association and is bounded by points of stronger contact 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 appear to be localized in a manner
similar to the MARs of the chicken lysozyme (13) and human
apolipoprotein B (14) loci. The 5` region of attachment to the
sperm nuclear matrix was bounded by positions 8,818-9,760, while
the corresponding 3` region was bounded by positions
32,586-33,536. The strong attachment to the nuclear matrix of
these
950-bp regions at the ends of the PRM1
PRM2
TNP2 DNase I-sensitive domain suggests
the presence of a sequence-dependent MAR-like element. However, these
regions do not share extensive similarity.
Figure 3:
Loop and matrix-associated segments of the PRM1 PRM2
TNP2 domain. The
relative DNase I-sensitive profile that defined the human PRM1
PRM2
TNP2 domain is shown (adapted
from (17) ). The PRM1, PRM2, and TNP2 genes are indicated as hatched boxes positioned along the
corresponding sequence of human chromosome 16p13.13(20) . DNA
halos were prepared, digested with various restriction endonucleases,
separated into their loop (supernatant) and nuclear matrix-bound
(pellet) fractions, and purified. PCR primer pairs that span specific
regions of the PRM1
PRM2
TNP2 domain are shown below the ruler as small black
boxes.
Primer pairs delimit the corresponding
amplicons within the loop and nuclear matrix fractions. A PCR primer
set directed to the
-globin locus was used as a
non-matrix-associated control. This same region of the
-globin
locus contains a somatic MAR, as shown in HeLa nuclei. Nuclear
matrix-bound restriction fragments in which greater than 80% of the
amplicon partitioned with the matrix-bound fraction are identified as black boxes. Nuclear matrix-associated fragments are indicated
by gray boxes for those amplicons that partitioned
(30-70%) within both fractions. Non-matrix-associated fragments
are demarcated by open boxes for those amplicons that comprised from 0
to 20% of the matrix fraction. Large restriction fragments that contain
the SMARs often showed sterically reduced localization to the matrix
fraction. Sites of attachment to the nuclear matrix are denoted as stars. Matrix association for the StyI-digested
sample could not be ascertained for the
-globin locus, as there is
a StyI site between the
-globin
primers.
Regions of intermediate
nuclear matrix association are likely to reflect local differences in
the organization of sperm chromatin. It has been shown that
approximately 15% of the chromatin in human sperm remains histone-bound
rather than undergoing protamine replacement (24) . The
intermediate association that is observed (Fig. 3) may reflect
differential affinity of the sperm nuclear matrix for histone-bound
chromatin as compared with protamine-bound chromatin. This is
consistent with previous DNase I-sensitivity data of the PRM1 PRM2
TNP2 domain in human
sperm(17) , reflecting increased accessibility of this segment
of the genome to the exogenous nuclease. Accordingly, DNase I
sensitivity may be correlated with the degree of interaction of each
gene with the sperm nuclear matrix. To test this hypothesis, mature
spermatozoa loop and matrix-bound DNAs were subjected to PCR analysis
using primer sets directed toward numerous well characterized loci
throughout the human genome. As shown in Fig. 4, PCR analysis of
the regions containing the PRM1, PRM2, and TNP2 genes showed that these genes were associated with the nuclear
matrix. Similarly, amplification of regions of the
-globin HBA2
and
-actin genes, which are also DNase I-sensitive in mature
spermatozoa, (
)also showed an association with the sperm
nuclear matrix. In contrast, the
-globin, acrosin, and PGK-1 and PGK-2 genes, all of which are DNase I-insensitive in
terminally differentiated mature spermatozoa,
showed no
association with the sperm nuclear matrix. In somatic nuclei, DNase I
sensitivity has been shown to correlate directly with the potentiation
of genes for transcription. While there does not appear to be any
transcription in mature sperm, the intermediate association of the
DNase I-sensitive regions with the sperm nuclear matrix may represent a
means by which the paternal genome is imprinted for activation and/or
templated for postfertilization protamine replacement. Both processes
are necessary for the formation of a viable male pronucleus.
Figure 4:
DNase I sensitivity and nuclear matrix
association are coincident in the male haploid genome. BstXI-digested halos were separated into their loop
(supernatant) and nuclear matrix-bound (pellet) fractions and then
purified. Amplicon localization is characterized by the degree of
localization to the loop or matrix fraction, represented by the percent
scale. The DNase I-insensitive -globin, PGK-1, PGK-2, and
acrosin genes showed no association with the sperm nuclear matrix (open boxes). The DNase I-sensitive PRM1 gene, PRM2 gene, and TNP2 gene as well as the
-actin
and
-globin genes were predominantly associated with the nuclear
matrix fraction (gray boxes). Only
-globin and
-actin showed an interaction with the HeLa nuclear matrix (data
not shown).
It is
clear that MARs and SMARs share only limited sequence and
organizational characteristics. For example, the mouse -globin
locus has been shown to contain a MAR that functions independently of
the type of somatic cell(29) . It always anchors that region of
the genome to the somatic nuclear matrix. As shown for HeLa nuclei in Fig. 3, this property is shared with the human
-globin
locus. However, unlike the organization within the somatic nucleus,
this region clearly does not interact with the mature haploid sperm
nuclear matrix. This is in corollary with that observed for the haploid
sperm-specific PRM1
PRM2
TNP2 locus.
In accord with the data presented above and that of others, there must be more than one type of association with the nuclear matrix. It is reasonable to assume that there are at least four classes of nuclear matrix association, i.e. regulatory element-associated MARs, somatic boundary elements, haploid boundary elements, and structurally associated elements. Class 1 regulatory element-associated MARs possess an innate ability to be bound by the nuclear matrix as they can be identified by an in vitro competition assay(26) . These MARs are not typically situated at the ends of the DNase I-sensitive domain. They have been localized to regions containing enhancers(10) , origins of replication(11) , and other regulatory elements (9) and may also represent regions where transcriptionally generated supercoiling is relaxed(2) . Class 1 MARs likely contain specific consensus sequences recognized by cell-specific nuclear matrix proteins. In fact, the nuclear matrix protein NMP-1, which binds to specific sequences within the histone H4 gene, has recently been shown to be the transcription factor YY1(27) . However, most of these MARs probably do not act as promoters or enhancers themselves. Instead, proximity of the regulatory element to the matrix-associated region and the nuclear matrix may concentrate all of the diverse elements necessary for transcription. In light of the locus-specific regulatory sequence motifs and the array of cell-specific proteins within the nuclear matrix(28) , it is possible that no single consensus sequence for the class 1 MAR will be identified.
Class 2 somatic boundary element MARs are localized to the ends of DNase I-sensitive domains and act as boundary elements in somatic nuclei. They may shield loci against inappropriate potentiation and silencing in multiple types of cells. The MARs of the chicken lysozyme locus that delimit the DNase I-sensitive domain have been shown to mediate position-independent expression(15) . It has been suggested that end region MARs may regulate transcription by inducing negative superhelical torsional stress across the domains that they limit(2) . A universal consensus sequence for this second class of MAR should become clear as more are identified and sequenced, since many loci possess cell type-independent end region MARs. The AT-rich MAR may be representative of this class.
The regions of matrix association described above for
the haploid-specific PRM1 PRM2
TNP2 domain are representative of class 3 haploid boundary element
nuclear matrix attachment regions, i.e. SMARs. This report is
the first identification of a haploid-specific MAR. Like the class 2
MARs, SMARs seem to act as boundary elements, attaching the ends of
chromatin domains to the sperm nuclear matrix. The validation of the
computational model suggests that SMAR sequences resemble those of the
class 2 somatic boundary element MARs. However, MARs and SMARs are not
identical. Unlike class 3 SMARs, class 2 somatic MARs have been shown
to be cell type-independent. For example, MARs of three developmentally
regulated Drosophila melanogaster genes have been shown to
exhibit identical binding profiles regardless of tissue type or
developmental stage(10) . Further, MARs from the
-globin
locus remain constant throughout the induction of terminal
differentiation of the erythroid progenitors(29) , while MARs
of the chicken histone genes have been shown to be retained throughout
the cell cycle(30) . These differences among the class 3 SMARs
and the class 1 and class 2 MARs are highlighted in Fig. 3and Fig. 4. Genes like
-globin that contain a somatic cell
type-independent MAR (29) do not partition with the sperm
nuclear matrix, and SMARs of the haploid-expressed PRM1
PRM2
TNP2 domain do not attach to the HeLa
nuclear matrix (Fig. 3). As with the class 2 MARs,
identification of a consensus sequence for SMARs will depend upon the
identification and sequencing of multiple SMARs.
The intermediate
association with the sperm nuclear matrix of those genes that exhibit
DNase I sensitivity in mature spermatozoa can be considered to
exemplify a class 4 structurally mediated nuclear matrix association.
This intermediate affinity for the sperm nuclear matrix may be similar
to that observed for the somatic expressed -interferon gene.
However, in haploid cells, this cannot be identified using an in
vitro competition assay, and it appears that it is not dependent
on the presence of a consensus sequence. It is not known if this type
of association reflects a structural parameter specific to sperm
chromatin.
While the function of MARs has been discussed
extensively, the biological role for nuclear matrix attachment and
nuclear matrix association within the male haploid genome remains to be
clarified. The class 3 end region SMARs, like the class 2 end region
MARs discussed above, appear to act as boundary elements. It is not
clear whether they shield from position effects, as has been shown for
some class 2 MARs (15) . Three independent lines of transgenic
animals containing SMARs from the PRM1 PRM2
TNP2 locus have been shown to yield copy
number-dependent, site of integration-independent
expression(17) . However, such expression can also be achieved
by a locus control region, as exemplified by the
-globin
locus(25) . Whether the human PRM1
PRM2
TNP2 locus contains an locus control region and/or
utilizes the SMARs as a means of locus control remains uncertain.
The haploid-specific SMARs and the intermediately associated regions described above represent two of at least four classes of nuclear matrix-associated regions. Further clarification of the classes and functions of various nuclear matrix-associated regions will prove both interesting and enlightening toward the study of the mechanisms of gene potentiation and paternal genome templating.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U15422[GenBank].