(Received for publication, October 24, 1995; and in revised form, December 28, 1995)
From the
The nature of chromatin organization over Alu repetitive
elements is of interest with respect to the maintenance of their
transcriptional silencing as well as their potential to influence local
chromatin structure. We previously demonstrated that the pattern of
nucleosomal organization over Alu elements in native chromatin is
specific and similar to the pattern observed with an in vitro reconstituted Alu template. This pattern, distinguished by a
nucleosome centered over the 5`-end of the Alu element, is associated
with repression of polymerase III-dependent transcription in vitro (Englander, E. W., Wolffe, A. P., and Howard, B. H.(1993) J.
Biol. Chem. 268, 19565-19573; Englander, E. W., and Howard,
B. H.(1995) J. Biol. Chem. 270, 10091-10096). In the
current study, additional templates representing both evolutionarily
old and young Alu subfamilies were found to direct a similar pattern of
nucleosome assembly, consistent with the view that nucleosome
positioning in vitro is shared by a majority of Alus. We
discovered however, that the specific nucleosome positioning pattern
was disrupted over one member of a young Alu subfamily, which recently
transposed immediately downstream to a TA
sequence in the neurofibromatosis type 1 locus (Wallace, M. R.,
Andersen, L. B., Saulino, A. M., Gregory, P. E., Glover, T. W., and
Collins, F. S.(1991) Nature 353, 864-866). Upon removal
of this sequence motif, the expected pattern of assembly was restored
to the neurofibromatosis type 1-Alu template. This finding indicates
that, at least in vitro, certain sequences can override the
propensity for positioning nucleosomes that is inherent to Alu
elements. The finding also raises the possibility that a similar
situation may occur in vivo, with potential implications for
understanding mechanisms by which certain Alu elements may evade
chromatin-mediated transcriptional silencing.
The Alu class of mammalian short interspersed elements (SINEs)
is the most abundant family of interspersed DNA repeats in primates. It
encompasses more than 500,000 copies of a 300-bp ()dimeric
sequence comprising 5-10% of human genomic DNA (reviewed in (1) and (2) ). Since Alu elements carry internal RNA
polymerase III promoter motifs and form efficient pol III-dependent
transcription templates in
vitro(3, 4, 5) , a subject of continuing
interest is how these elements are regulated by the host cell. This
issue has been a subject of several lines of investigation, initially
leading to the hypothesis that most Alu promoters are mutated and
inherently weak and therefore require specialized 5`-flanking sequences
for expression(6, 7, 8) . More recently it
was demonstrated, however, that the normally very low level of Alu
transcription in vivo can be selectively increased (i.e. relative to other pol III-transcribed genes) in response to viral
infection(9, 10, 11, 12, 13) ,
inhibition of translation(14) , or demethylation of internal
Alu CpG dinucleotide residues(15) . In addition, template
availability experiments demonstrated that only a minor fraction of the
transcriptional potential of Alu elements in naked genomic DNA is
realized in either native chromatin or chromatin from which histone H1
was removed(16) . Taken in combination, this evidence appears
to favor the view that Alu elements are subject to one or more levels
of transcriptional repression in human cells.
A potentially
attractive mode of gene silencing, especially for dispersed repeat
elements, is packaging of transcription control regions into
nucleosomes. Accordingly, the nucleosomal organization over Alu
elements in vitro and in vivo has been the focus of
ongoing studies in our laboratory. Analysis of nucleosome assembly in vitro over a naturally occurring Alu element that
transposed into the human -fetoprotein gene (AFP-Alu) (17) revealed that this element directs precise rotational and
translational nucleosome positioning(18) . Similar
translational nucleosome positioning over this Alu was demonstrated in
native human chromatin, and examination of human Alu elements as a
family revealed that a significant fraction of repeats is associated
with rotationally positioned nucleosomes(19) . These results
strongly suggested that Alu elements carry inherent signals that direct
nucleosomes to center over their 5`-ends, which correspond to their
potential transcription start site. Moreover, assembly of a nucleosome
in this position resulted in total repression of Alu promoter-mediated
pol III-dependent transcription(18) , leading us to propose
that the transcriptional silencing of Alu elements observed in vivo is likely to have a chromatin-mediated component. As noted above,
Alu elements are not significantly derepressed by simple removal of
histone H1 from chromatin(16) . This contrasts with other pol
III-transcribed genes (20, 21, 22, 23) and suggests that
the unusually high degree of transcriptional repression of Alu repeats
may be related to their propensity to direct a non-random pattern of
nucleosome assembly.
To the extent that Alu elements have strong
inherent signals for positioning nucleosomes, they provide a system for
investigating the nature of sequence motifs that may override such
signals. In this context, we considered the available experimental and
theoretical data showing that homopolymeric tracts of adenine residues
are not energetically favorable for wrapping around the histone core
and accordingly are excluded from the central regions of nucleosomal
DNA (24, 25, 26) . Here, we examine a
naturally occurring sequence of this type
(TA
) juxtaposed immediately 5` to the start
site of an Alu element that recently transposed into the
neurofibromatosis type 1 (NF1) locus, causing that
disorder(27) . We analyzed the pattern of in vitro nucleosome assembly over this Alu element within its natural
genomic context and also compared it to the pattern obtained in the
context of prokaryotic DNA. It was found that the adjacent region
consisting of T
A
interferes with nucleosome
assembly over the start site of the NF1-Alu. As expected, replacement
of this sequence with non-homopolymeric prokaryotic DNA restored the
pattern of nucleosome formation characteristic of Alu templates as well
as the in vitro transcriptional repression associated with
nucleosome positioning on these elements.
Figure 1:
Schematic representation of Alu
templates. The AluK, AFP-Alu, NF1-Alu, and the NF1/d-Alu templates are
shown (see ``Materials and Methods''). Their assignment to
the main human Alu subfamilies is indicated as Major (AluK), Precise
(AFP-Alu), and PV (NF1)(2) . The structural features of the
dimeric Alu element are depicted. Open bars represent the left
(120-bp) and the right (
150-bp) monomers. Solid boxes represent pol III promoter motifs, the A box and the B box; A represents the
20-bp A/T-rich spacer, and AA marks
the
35-bp poly(A) tail. Note that the synthetic AluK has no
poly(A) tail adjacent to its right monomer. The solid bar represents the T
A
sequence flanking the
NF1-Alu. Straight lines represent prokaryotic vector
sequences, whereas wavy lines represent segments of human genomic
DNA.
The NF1-Alu/d (Fig. 1) template was generated by digesting the NF1-Alu fragment with EaeI at position -1 relative to the Alu transcription start site and at position 297 within the vector. The ends were filled in with Klenow, and the fragment was cut with MluI at position 310 within the polylinker to generate a MluI/blunt end fragment, which was then subcloned into pUC21 cut with EcoRV and MluI. The NF1/d-Alu template was generated by cutting this construct with PvuII and EcoRI at positions 54 and 323, respectively, within the vector.
The AluK/pUC19 (Fig. 1) construct was synthesized as
a consensus Alu sequence of 285 bp(28) . The 490-bp
fragment used as template for nucleosome reconstitution was prepared by
cutting the construct with PvuII and EcoRI within the
vector. The resulting fragment consisted of
200 bp of pUC19 DNA
and
285 bp of Alu sequence.
To confirm that nucleosome positioning in vitro is a
general property of Alu repeats, a 285-bp synthetic consensus Alu
element (AluK) (Fig. 1) inserted into a prokaryotic vector (28) was tested as a template for nucleosome assembly. This
consensus element was designed to conform to the Alu major family
sequence(33) , which represents 85% of Alu repeats that
currently reside in human DNA. In contrast, the AFP-Alu (Fig. 1)
element, whose nucleosomal assembly pattern was analyzed previously,
represents a more evolutionary recent family designated
precise(34) , which comprises 10-15% of Alu elements. The
NF1-Alu represents a young family that is currently transposing in the
human genome. Although the consensus sequences representing the
different families exhibit
95% similarity, the individual members
of the older subfamilies retain on average only
85% sequence
identity (reviewed in Refs. 2 and 35).
Analysis of DNaseI
footprinting patterns of the reconstituted AluK template revealed a
pattern of 10-bp periodic nicking that is characteristic of
nucleosomal DNA. The pattern revealed defined rotational positioning
around the start of the Alu consensus sequence (Fig. 2, arrows), consistent with nucleosome positioning observed
earlier for the in vitro reconstituted AFP-Alu element within
the context of its native genomic sequence(18) . This
observation further supports our proposal (19) that nucleosome
positioning signals are inherent to the Alu sequence and confer the
capacity to direct nucleosome positioning to a significant fraction of
human Alu repeats.
Figure 2:
DNaseI
footprinting pattern over the AluK template assembled with histone
octamers. End-labeled 496-bp DNA fragment containing the Alu gene
5`-flanked by 200 bp of pUC21 sequence was used for analyzing the
top strand. Digestions were carried out with DNaseI at 0.75 µg/ml
for 1, 2, and 4 min on ice. Digestion reactions for the free DNA
templates were supplemented with carrier DNA and bovine serum albumin.
Digestion products were resolved in 6% sequencing gel, and a photograph
of an autoradiogram is shown. The end-labeled template is drawn to
scale, and the structural features of the dimeric Alu element are
indicated. G+A is the sequence ladder, and M is the end-labeled
molecular weight marker (pBR322/MspI). Horizontal arrows mark periodic DNaseI nicking with the reconstituted (Rec)
template.
Previously, others have observed that certain
sequences are relatively refractory to nucleosome
assembly(24, 36, 37) . We observed that one
such sequence, a short homopolymeric tract,
TA
, occurred immediately 5` to the recently
transposed NF1-Alu and, accordingly, decided to examine its effect on
the assembly of a nucleosome predicted to center over the transcription
start site of the adjacent NF1-Alu element. The NF1-Alu element located
centrally in a
600-bp fragment (Fig. 1) was generated and
reconstituted with purified histones by salt gradient dialysis. The
disappearance of free DNA confirmed the complete association of the
template with histones (Fig. 3a, lane 3). The
DNaseI footprint, however, failed to generate periodic preferential
nicking indicative of nucleosomal DNA (Fig. 3b);
instead, the DNaseI pattern corresponding to the Alu sequence was
similar for the reconstituted and the free templates. Nonetheless, a
marked difference in the footprinting pattern between the reconstituted
and the free DNA was detected outside of the Alu sequence, at position
-60 relative to the transcription start site (arrow). This suggested that a nucleosome or a nucleosome-like
particle that would account for the formation of the observed bandshift
was associated with the proximal portion of this template. To rule out
a situation in which the observed bandshift was due to a nucleosome
associated with the right Alu monomer, the original template was
truncated to remove its distal portion. The resulting
330-bp
fragment (NF1/t-Alu), consisting of
170 bp of a 5`-upstream
region, the left Alu monomer, and the intermonomeric linker, was
reconstituted with purified histones, and its complete assembly was
confirmed by gel retardation assay (Fig. 3a, lane
7). Footprinting differences between DNaseI patterns in the region
corresponding to the Alu sequence or the immediately adjacent
5`-flanking sequence T
A
were likewise absent
in this case (Fig. 3c). Differences in the footprinting
pattern suggesting the presence of nucleosome-associated DNA that could
account for the observed bandshift were apparent only in the proximal
portion of the template, i.e. upstream of position -60
(arrows). These results are in striking contrast to those observed with
other Alu templates, including AluK (Fig. 2), in which the
10-bp periodic nicking pattern is most readily seen immediately
upstream of the Alu start site. Since this region is occupied by
T
A
, the data suggest that this tract is
responsible for exclusion of a nucleosome from its otherwise preferred
position.
Figure 3:
Gel retardation and DNaseI footprinting
analyses. a, the extent of reconstitution for end-labeled
NF1-Alu-derived templates was monitored by gel retardation assay (0.7%
agarose gel). NF1 is the 600-bp parent fragment (lane 2),
NF1/d is modified to eliminate the T
A
5`-flanking sequence (lane 4), and the NF1/t (lane
6) is the parent template lacking the right monomer (see
``Materials and Methods''). Reconstitutes are denoted by
+ and free templates by -. The marker (M) is
pBR/MspI digest. DNaseI footprinting patterns are as follows:
the 5`-end-labeled NF1-Alu (600 bp) (b) and the NF1/t-Alu (330
bp) (c) fragments were subjected to DNaseI digestion. The
assays were carried out as described in Fig. 2. Digestion
products were resolved in a 6% sequencing gel, and a photograph of an
autoradiogram is shown. The landmarks of the Alu gene are indicated; in
the case of the
330-bp template, a major portion of the right
monomer has been truncated. Solid bars mark the position of
the T
A
sequence, and arrows denote
DNaseI cleavage sites uniquely enhanced in reconstituted templates. The
M lane is PBR/MspI digest, and the products of A+G
sequencing reaction are shown for sequence
alignment.
In view of these results, we wished to confirm the
possibility that the presence of the TA
sequence interferes with nucleosome formation over the start of
the Alu element. To accomplish this, the T
A
tract was removed (see ``Materials and Methods''). The
resulting construct was used to generate a
600-bp PvuII-EcoRI fragment termed NF1/d-Alu, carrying
190 bp of prokaryotic sequence immediately flanking the 5`-end of
the NF1-Alu element. The sequence replacing the T
A
is comprised of
50% G+C and has no homopolymeric tracts
(not shown). Complete reconstitution of this fragment with purified
histones was confirmed by the gel retardation assay (Fig. 3a, lane 5). In contrast to its parent
templates, DNaseI footprinting analysis did reveal differences in the
patterns on comparison of the reconstituted and free templates over the
region corresponding to the proximal portion of the Alu left monomer
and the flanking upstream sequence (Fig. 4, arrows).
Thus, the footprinting pattern obtained with the modified template
suggested that formation of nucleosomal DNA over the start site of the
Alu element was dependent on the removal of the T
A
sequence.
Figure 4:
DNaseI footprinting pattern for the
NF1/d-Alu template. The NF1-Alu fragment was modified to eliminate the
TA
5`-flanking sequence. The resulting
NF1/d-Alu template was assembled with histone octamers and subjected to
DNaseI digestion. The assay was conducted as described above, and the
digestion products were analyzed in a 6% sequencing gel. A schematic
structure of the Alu gene is aligned with the footprint; left and right
monomers are depicted by open bars and promoter motifs by A and B. Horizontal arrows denote
modifications of the footprinting pattern for the reconstituted (Rec) template.
Since we previously found that an octamer centered
over the start of the AFP-Alu element repressed pol III-mediated
transcription(18) , we next examined the ability of the NF1
constructs to direct transcription. For this analysis, we used
nucleosome-free and fully reconstituted templates (Fig. 3a). Control experiments demonstrated that
replacement of the TA
sequence had no
apparent effect on the ability to direct transcription by the free
template (Fig. 5, a (lanes 2 and 3)
and b (lane 1)). Interestingly, in these reactions it
was found that while the natural NF1-Alu template was transcriptionally
active when associated with histones (Fig. 5a, lanes 4-6), the NF1/d-Alu template lacking the
5`-T
A
tract was reproducibly repressed by the
assembly of nucleosomes (Fig. 5b, lanes 2 and 3). As shown in Fig. 4, the pattern of assembly in the
latter case indicated that the NF1/d-Alu was associated with a histone
octamer located over the transcription start site of this template.
These results further confirm that the ability of an Alu element to
position a nucleosome over its start site corresponds closely to its
transcriptional repression when reconstituted in vitro with
core histones.
Figure 5:
Analysis of in vitro transcription from the NF1-Alu and the NF1/d-Alu templates. The
templates were assembled with histone octamers and tested for in
vitro transcription. Transcription reactions were resolved in 6%
sequencing gels and visualized by autoradiography. Transcription
reactions were assembled in a final volume of 25 µl with HeLa
nuclear extract and 100 ng of template free, or assembled with histone
octamers at the ratio of 1:0.9 DNA mass/histone mass, as follows. a, the NF1-Alu template; in lanes 2 and 3,
the transcript resulting from the free unlabeled template is visualized
at 350 nucleotide (RNA), and in lanes 4 and 5 Alu RNA
is transcribed from the reconstituted end-labeled template visualized
at 600 bp (DNA). In lane 6 is a control that confirms the
integrity of the assembled template when the
[
-
P]GTP is omitted from the assay. M is the
pBR322/MspI digest. b, transcription from the
NF1/d-Alu template; lane 1, the expected transcript (350 bp)
from the naked unlabeled template (RNA). Lanes 2 and 3, no transcript is detected when the end-labeled NF1/d-Alu
template (DNA) is assembled with histone octamers. In lane 4 is a control reaction in which the
[
-
P]GTP was omitted to confirm the
integrity of the reconstituted template.
Our initial study examined AFP-Alu, a human-specific element that is a member of the Alu precise subfamily representing 10-15% of the Alu elements in the human genome. Here, we extended the analysis using the AluK consensus element, which represents older Alu subfamilies corresponding to 85% of human Alu repeats as well as the NF1-Alu element representing the most recent Alu subfamily(38, 39, 40) . Combined, our results indicate that the ability of Alus to position nucleosomes precisely is very likely associated with many if not most Alu repeats. In addition, we demonstrated that the most recent Alu subfamily, represented here by the NF1-Alu, carries the same inherent signals to position nucleosomes. Cumulatively, these observations bolster our previous evidence that Alu elements en masse position nucleosomes centered over their 5`-ends (19) and that Alu transcriptional silencing is in part due to chromatin-mediated repression(16) .
In so far as the formation of a nucleosome over Alu transcription start sites contributes to the silencing of Alu genes in vivo in the same manner that occurs in vitro, the results presented here suggest a view of Alu regulation somewhat different from those previously proposed. In this regard, it is noteworthy that Alu transcripts originate in vivo from all subfamilies(13, 15, 41, 42) , rather than, as expected, only from the most recent subfamily members. In fact, speculations that certain Alu templates may be driven to high levels of expression due to strong upstream promoters or enhancers have not been substantiated. Instead, the available data indicate that although many (>100) distinct Alus are transcriptionally active in human cells, they are active at uniformly low levels, and no one particular sequence appears to be overrepresented relative to others(13, 15, 41, 42) . An explanation consistent with this finding could be that sequence motifs, which override the nucleosome positioning information within the left Alu monomer, account for the observed in vivo availability of Alu templates for transcription. This possibility provides an additional facet to the idea that certain Alu repeats may become transcriptionally competent as a result of their 5`-flanking regions(6, 8) . Our results suggest that some 5`-flanking sequences may exert stimulatory effects by preventing the formation of a nucleosome over the Alu transcription start site. A similar idea has been suggested previously with regard to RNA polymerase II, specifically that the presence of poly(dA-dT) stretches, in the vicinity of promoters, facilitates pol II-mediated transcription in yeast due to exclusion (43) or destabilization of nucleosomes (44) .
Some evidence indicates that poly(dA-dT) tracts of varying lengths differ from the regular B-type DNA helix in their conformation(45, 46) . These structural differences have been seen as antagonizing the tendency of DNA to fold around the nucleosomal surface, consistent with a body of experimental data which indicate that poly(dA-dT) tracts resist folding into nucleosomes(24, 36, 37) . Published studies on this topic are not entirely consistent, however, showing on the one hand that poly(dA-dT) sequences can assemble into nucleosomes under certain conditions (47, 48, 49, 50) and on the other hand that some types of poly(dA-dT) tract may always resist this type of assembly(48) .
While A+T-rich regions are known to
be preferred insertion sites for Alu
elements(51, 52, 53) , these regions vary
considerably in their nucleotide composition such that only 3% of
Alu repeats in the GenBank data base are flanked by sequences
comprising >10 consecutive A or T residues within the immediate
50-bp upstream region. (
)In less than 10% of these sequences (i.e. 0.3% of the 15,000 Alus in the data base) are there runs
of >10 consecutive A or T residues immediately adjacent to the Alu
element, bringing the total estimated number of Alu repeats in the
human genome that would be flanked by this type of sequence to
1500 or less than 1%. At the present time, with the exception of
the natural T
A
sequence examined here, it is
not known which types of A/T tract may interfere with the in vitro nucleosome assembly over the Alu start site. Nonetheless, the
notion that up to
1500 Alu elements might be accessible to low
level transcription as a result of the absence of a precisely
positioned nucleosome over their transcription start sites is
provocative and at the same time consistent with the estimated number
of Alus believed to be transcribed constitutively in human cells.