From the
Alu sequences are interspersed throughout the genomes of primate
cells, occurring singly and in clusters around RNA polymerase
II-transcribed genes. Because these repeat elements are capable of
positioning nucleosomes in in vitro reconstitutes (Englander,
E. W., Wolffe, A. P., and Howard, B. H. (1993) J. Biol. Chem. 268, 19565-19573), we investigated whether they also
[Abstract]
influence in vivo chromatin structure. When assayed
collectively using consensus sequence probes and native chromatin as
template, Alu family members were found to confer rotational
positioning on nucleosomes or nucleosome-like particles. In particular,
a 10-base pair pattern of DNase I nicking that spanned the RNA
polymerase III box A promoter motif extended upstream to cover diverse
5`-flanking sequences, suggesting that Alu repeats may influence
patterns of nucleosome formation over neighboring regions.
Computational analysis of a set of naturally occurring Alu sequences
indicated that nucleosome positioning information is intrinsic to these
elements. Inasmuch as local chromatin organization influences gene
expression, the capacity of Alu sequences to affect chromatin structure
as demonstrated here may help to clarify some features of these
elements.
RNA polymerase III (pol III)
When considering the high
degree to which the Alu family has been amplified, one question that
must be addressed is how the host cell deals with the large potential
sink for transcription factors that these pol III promoter-containing
elements represent. This is a significant issue, since, unlike
satellite sequences which tend to be masked in large heterochromatic
domains, Alu and other pol III short interspersed elements
(14) are largely located within introns and flanking regions of
pol II transcription units
(11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23) .
It has been argued that the vast majority of Alu repeats lack
appropriate 5`-flanking signals and thus, even if unmasked from
chromatin proteins, would be unable to recruit basal pol III
transcription factors efficiently
(24, 25) . Multiple
lines of evidence
(26, 27, 28) ,
In principle, there are far-reaching
implications to the finding that Alu elements can position nucleosomes;
yet because in vitro reconstitution studies do not necessarily
predict in vivo chromatin structure
(29, 30, 31, 32, 33, 34) ,
a critical question has remained as to whether Alu sequences also
possess the capacity to position nucleosomes within native chromatin.
To address this issue we examined Alu repeats as a family and at a
single previously studied locus. Our results suggest that a readily
detectable fraction of Alu repeats is associated with rotationally
positioned nucleosomes in native chromatin. Examination of a single Alu
previously shown to direct translational positioning of nucleosomes
in vitro revealed a similarly phased array in chromatin.
The lower strand of the left monomer was
examined through extension of a primer spanning residues 8
The DNase I
cleavage pattern for the right monomer was determined for the upper
strand by extension of a primer spanning Alu positions 262
The results presented in this study support the idea that a
significant fraction of human Alu repeats is associated with
rotationally positioned nucleosomes. Such positioning is consistent
with previous in vitro experiments in which nucleosomes were
found to assemble on the 5` ends of Alu left monomer units in a
nonrandom rotational phase. Remarkably, the observed in vivo positioning extends over 5`-genomic flanking sequences which for
Alu sequences are known to be diverse in sequence structure
(2, 50) . This is the first instance to our knowledge in
which a nonsatellite repetitive element appears to impose rotational
positioning on sequences that do not comprise an integral part of their
repeat unit. We cannot at present exclude the possibility that
nonrepetitive flanking sequences contribute to the nucleosomal
organization evident in our data. However, it was previously
demonstrated that nucleosome positioning signals were contained within
the Alu sequence
(1) . This is consistent with theoretical
bending and flexibility predictions of Alu sequences which suggest that
the observed rotational positioning could be accounted for by
structural features inherent to these elements. We do not know at
present what fraction of Alu repeats conforms to the pattern detected
here, as some 5` sequences may have structural determinants that cannot
be overridden by the Alu sequence; in addition, rotational signals may
be lost due to random mutational drift. In this regard, we note that
Alu-specific primers used to detect positioning were designed to match
the consensus sequence of majority of human Alus and might not detect
sequences that have been mutated significantly. In any case, the data
presented here and previously
(1) suggest that rotational
positioning of nucleosomes can be dictated by these elements.
In
considering chromatin organization at the local level, translational
positioning constitutes a separate parameter associated with nucleosome
assembly. Results obtained with limited MNase digestion confirm that
translational positioning of nucleosomes at a single locus, namely the
AFP-Alu repeat within the fourth intron of the
There are at least two reasons that nucleosome interactions with Alu
repetitive elements in the human genome merit close examination. The
first relates to Alu transcriptional regulation: a satisfactory
understanding has been lacking as to why these elements are expressed
at extremely low levels in normal somatic cells. In contradiction to an
early proposal that Alu pol III promoters are defective, it has been
shown that some Alu elements are strongly transcribed both in vivo and in vitro (27, 28, 51, 52, 53) .
Since Alu repeats are heavily methylated at CpG dinucleotides in
vivo (54, 55) , methylation may partially account
for transcriptional silencing. While complete CpG methylation is
associated with a modest 2-3-fold decrease in Alu template
activity in transient expression assays,
A second reason for characterizing the chromatin
organization associated with Alu repeats relates to their potential
effect on expression of adjacent genes
(57, 58, 59, 60) . To the extent that
Alu repeats confer rotational positioning over 5`-flanking sequences,
their presence may influence access of regulatory proteins to cognate
binding motifs within such sequences. While this notion is purely
speculative, characterization of Alu-nucleosome interactions in
vivo might be an important issue in studying the potential role of
these sequences with regard to gene regulation. In situations where the
presence of Alu repeats next to promoter/enhancer elements alters gene
expression, it might be useful to consider the possibility that this
effect is mediated at least in part by specific nucleosomal
organization.
We thank Drs. R. Simpson, R. Maraia, A. Wolffe, R.
Reeves, J. Hayes, and Y. Nakatani for critical comments, H. R. Drew for
providing computer algorithms, and M. Lanigan for preparation of the
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
-transcribed
interspersed repetitive elements are nearly ubiquitous in the genomes
of higher eukaryotic cells, yet have remained enigmatic with respect to
the impact that they may have on their host organisms
(2, 3, 4, 5, 6, 7, 8, 9) .
Intriguingly, during evolution each species, and to greater extent each
order, appears to have acquired a unique complement of these repetitive
elements. A marked example of this divergence is provided by primates,
which differ from other mammalian orders in possessing a particularly
abundant set of
300 bp of RNA pol III-transcribed repeats known as
the Alu family
(10, 11, 12) . In the human
haploid genome, an estimated 500,000 Alu copies comprise about five
percent of chromosomal DNA
(13) .
(
)
contradict this view, however, leading to the alternative
proposal that Alu elements are subject to specific repression.
Recently, one mechanism has been identified that has the potential to
mediate selective Alu silencing
(1) . Englander et al. (1) reported that Alu sequences possess the capacity to fix the
rotational and translational positions of tetramer or octamer particles
reconstituted in vitro. In that study the reconstitution of an
Alu element with octamers of core histones was shown to result in the
complete abrogation of in vitro pol III-dependent template
activity. It was further shown that transcription could be completely
blocked when a CpG-methylated Alu template was reconstituted with
(H3/H4)
tetramers.
Isolation of Nuclei and Genomic DNA
Nuclei were
isolated from HeLa cells by modification of established methods.
Briefly, cells were incubated for 30 min on ice in a hypotonic buffer
(1 mM Tris, pH 8, 0.1 mM EDTA, 1 mM
polymethylsulfonyl fluoride, 1 mM dithiothreitol)
(35) and Dounce homogenized (20 strokes) in the presence of 3
mM MgCland 0.1% Nonidet P-40. Nuclei were
recovered by centrifugation through a 0.25 M and subsequently
a 1 M sucrose layer in a swinging bucket rotor and resuspended
in a storage buffer (0.25 M sucrose, 10 mM Tris, pH
8, 3 mM MgCl
, 0.4 mM polymethylsulfonyl
fluoride, 1 mM dithiothreitol, 50% glycerol). The quality of
various nuclei preparations was monitored by microscopy. Genomic DNA
was purified following lysis of HeLa cells at 37 °C for 3 h in the
presence of 0.5% SDS and 0.2 mg/ml proteinase K. DNA was extracted
several times with phenol:chloroform and precipitated by spooling from
70% ethanol.
Linear Polymerase Chain Reaction-mediated DNase I
Footprinting Analysis
Primers were designed using the major Alu
family consensus sequence which is expected to represent 65% of
Alu repeats
(36) . The primers were selected to minimize
cross-hybridization due to homology between the left and the right
monomer. Accordingly, the primer used to extend in the sense
orientation on the lower strand of the left monomer spans residues
8-24 (5`-GCGGTGGCTCACGCCT-3`) and harbors a 3-bp mismatch with
the corresponding region in the right monomer. Primer used for
extension in the antisense orientation, on the upper strand of the left
monomer, spans residues 96-122
(5`-TTAGTAGAGA(C/G)GGGGTTTCACCATG-3`)
(27) and is expected to
extend through the full length of the left monomer. The upper strand of
the right monomer was analyzed using an antisense primer comprising
residues 262-278 (5`-GGAGTCTCGCTCTGTCG-3`) within the right
monomer. Template DNA was purified following DNase I (Worthington)
digest in nuclei; a typical digest was at 25 °C with 0.04
unit/µg DNA for 0, 2, 4, and 6 min or with naked DNA at 0.001
unit/µg for 0, 15, 30, and 60 s. To confirm the integrity of DNA in
a given preparation, samples were incubated without DNase I for 6 min
or 60 s for nuclei and genomic DNA, respectively, and examined by
primer extension. Linear amplification reactions were carried out with
end-labeled primers
(37) : subsequently to extensive
denaturation, template DNA (50-100 ng) was amplified by 22 cycles
of Vent polymerase (New England Biolabs)-mediated linear polymerase
chain reaction. Cycles were as follows: 1 min at 90 °C, 1 min at
52-56 °C and 30 s at 72 °C. Products were resolved in 6%
polyacrylamide, 7 M urea gels. Scans of gels were generated
with a PhosphorImager using the ImageQuant software (Molecular
Dynamics).
Micrococcal Nuclease Digest and Indirect End-labeling
Hybridization
Nuclei were digested with MNase at 0.3 unit/µg
DNA for 3, 6, 9, or 12 min at 25 °C. Nuclei incubated for 12 min
without MNase served as a control for endogenous nuclease activity.
Naked DNA was digested with 0.03 unit/µg DNA for 15, 30, 60, or 90
s; the control for endogenous nuclease activity was naked DNA incubated
without MNase for 90 s. Samples were made 12.5 mM EDTA and
0.5% SDS to terminate the reaction and then incubated with 0.2 mg/ml
proteinase K for 16 h at 37 °C, phenol:chloroform extracted, and
ethanol-precipitated. Purified DNA was dissolved in a large volume of
Tris-EDTA and digested for 24 h with an excess of BamHI.
Following precipitation digestion was carried out with KpnI
under similar conditions. Digested DNA was resolved in a 10-cm 1.3%
agarose gel and transferred to a Gene ScreenPlus hybridization
membrane. DNA was UV-cross-linked to the membrane (Stratalinker) and
baked for 2 h at 80 °C. The hybridization probe was the
BamHI/PflMI fragment comprising the 350 bp most upstream
region in the AFP-Alu locus (corresponding to the
BamHI/ KpnI fragment, spanning residues
466 to 789 relative to the start of the Alu repeat)
(38) . The probe was used at 4
10
cpm/ml.
Hybridization was at 65 °C for 40 h, and washes were with 2
SSC, 0.5% SDS at 25° and 40 °C and with 0.2
SSC, 0.1%
SDS at 65 °C. Exposure was 8 days at 80 °C.
Computer Analysis
Computer algorithm developed to
predict the phase and the strength of rotational positioning signals
(39) was used to analyze a random set of Alu repeats within
their native genomic DNA context. For a DNA sequence window of
specified length, this algorithm calculates an energy function ()
related to bending the DNA along a nucleosomal surface (the major
groove at the center of the DNA is assumed to face the histone core).
When the window is moved along a longer input DNA sequence to be
analyzed, the output from the program is a series of
values. If
rotational positioning information is present, these values form a
sinusoidal pattern with a 10-bp periodicity.
Intranuclear DNase I Footprinting for Alu
Repeats
To investigate whether the capacity to position
nucleosomes is a general property of Alu repeats, we first asked if a
defined nucleosomal rotational phase could be detected in association
with these elements. The approach employed was intranuclear DNase I
footprinting in combination with linear primer extension
(32, 37) . Alu consensus sequence primers
(9) were designed with two objectives: to permit detection of
the majority of Alu repeats and to minimize cross-hybridization due to
homology between the left and right monomer moieties within the dimeric
Alu structure (Fig. 1 B). Using three appropriately
positioned primers, the positions of DNase I cleavage were mapped for
the upper (Fig. 1) and the lower strand (Fig. 2 A)
of the left monomer as well as the upper strand of the right monomer
(Fig. 2 B).
Figure 1:
Intranuclear footprinting
pattern for the upper strand of the left monomer. HeLa cell nuclei were
treated with DNase I, and the partially cleaved DNA was used as
template for extension with primers corresponding to consensus Alu
sequence. A, autoradiogram showing extension products for the
upper strand of the left monomer; the primer spanned residues 96
122 (27). Relative position of Alu repeat is indicated. Increasing
incubation times with DNase I are from left to right, positions of
enhanced DNase I nicking are indicated by horizontal arrows.
Marker is the end-labeled pBR322/ MspI digest ( M).
B, schematic representation of primers aligned with the Alu
sequence. Alu left and right monomers are denoted by bars, and
pol III A and B box promoter elements are indicated as solid
rectangles. Putative nucleosome positions are denoted by
ovals. C, densitometric scan of DNase I footprinting
patterns in nuclei and in naked DNA. Relative position of Alu repeat is
indicated. Arrows mark peaks of cleavage in
nuclei.
Figure 2:
Intranuclear footprinting pattern for Alu
repeats. A, autoradiogram showing extension products
corresponding to the lower strand of the left monomer. The end-labeled
primer spanned residues 8 24 of the Alu major subfamily
consensus sequence. A schematic representation of the relevant portion
of Alu repeat is shown. Increasing incubation time points with DNase I
are from left to right. An arrowhead denotes
a hypersensitive site within the B box, and an arrow marks a
hypersensitive site at position
55. The vertical bar denotes the portion of the inter-monomer spacer which is protected
in nuclei. B, autoradiogram showing extension products for the
upper strand of the right monomer. The primer spans residues 262 to 278
in the right monomer. Increasing incubation time with DNase I in nuclei
and naked DNA is from left to right. The
inter-monomer spacer protected in nuclei and hypersensitive in naked
DNA is denoted by a vertical bar.
Analysis of the upper strand for the left
monomer was carried out using a primer that spans residues from
position 96 to 122 (Fig. 1 B) within the consensus
sequence of the major Alu subfamily
(27, 36) . When
extension products spanning the left monomer in an antisense
orientation were resolved by denaturing polyacrylamide gel
electrophoresis, a 10-bp periodicity in DNase I cleavage,
suggestive of rotationally positioned nucleosomes, was observed. This
pattern (Fig. 1 A, arrows), starts at position
55 within the left monomer and extends upstream beyond the start
site of Alu repeats, consistent with the in vitro footprinting
results
(1) . Densitometric analysis of the footprint confirmed
that the preferential nicking occurs at intervals consistent with a
10-bp helical phase (Fig. 1 C). Control reactions
using naked DNA as substrate for DNase I demonstrated that naked Alu
DNA is cleaved in a pattern that is clearly distinct from that observed
for chromatin. Importantly, the footprinting pattern observed with
native chromatin (Fig. 1 A) shows periodic DNase I
cleavage sites located beyond the start of the Alu repeat. These
findings strongly suggest that diverse 5`-flanking regions assume
rotationally fixed nucleosomal positions dictated by the properties of
adjacent Alu sequences.
24
(Fig. 2 A). To the extent that the translational phasing
of nucleosomes agrees with previous in vitro results
(1) , the region visualized with this primer should comprise the
3` boundary of a nucleosome and a
70 bp inter-nucleosomal linker
that spans the Alu pol III B box element, the 3` end of the left
monomer, and the A/T-rich inter-monomer spacer (see Fig. 1B).
Consistent with those earlier results, an extended ladder was not
observed, i.e. at most one or two DNase I cleavage sites
matching the predicted 10-bp periodicity were evident. A hypersensitive
site detected in nuclei at about position 55, which is absent in the
naked DNA control (Fig. 2 A, arrow), might
correspond to a periodic cleavage site near the 3` boundary of the
nucleosome; alternative explanations that do not involve nucleosomes
are, however, also likely. Inspection of this region revealed two
additional features: (i) enhancement of cleavage within the B box at
position
78 (Fig. 2 A, arrowhead) that is
weak in naked DNA and (ii) partial protection over the proximal portion
of the nuclease sensitive A/T-rich spacer between the left and the
right monomers (Fig. 2 A, vertical bar). Thus,
although these results indicate differences between chromatin and naked
DNA, the patterns observed do not support the interpretation that a
nucleosome resides beyond the B box of the left monomer.
278
within the right monomer (Fig. 1 B). The pattern in
nuclei again differed significantly from that obtained with naked DNA.
The most conspicuous disparity was the greatly diminished
susceptibility to DNase I of the inter-monomer spacer
(Fig. 2 B, vertical bar). This was observed
previously, although to a lesser degree, for the lower strand
(Fig. 2 A, vertical bar). No 10-bp ladder was
evident within the right monomer to suggest rotational positioning,
consistent with our failure to detect such a pattern earlier in an
in vitro reconstitution system
(1) . As noted
previously
(1) , the right monomer may be involved primarily in
translational positioning, since it encompasses a nucleosome core-sized
146-bp sequence flanked on both sides by poly(A) tracts. Such poly(A)
tracts are significantly underrepresented within the central
120 bp
of MNase-resistant nucleosome core particles
(40, 41) .
Alu Sequence Analysis
We next asked whether the
observed rotational positioning of nucleosome-like particles over the
left monomer region could be explained on the basis of Alu DNA sequence
features. On the basis of in vitro and in vivo studies
(29, 39, 42, 43, 44, 45, 46) ,
local bending and flexural properties of DNA have been identified in
some cases as primary determinants for this type of positioning. These
properties can be calculated by a computer algorithm that predicts the
phase and approximate relative strength of positioning information
(39) . The signature of a rotational signal in the output from
this program is a sinusoidal curve with a 10-bp periodicity (no
information of predictive value is provided on translational phasing).
As shown in Fig. 3, inspection of a set of full-length Alu
sequences in this manner reveals that a strong rotational signal is
centered in the conserved Alu region
(47) between the A and B
box pol III promoter elements.
Figure 3:
Nucleosome positioning information in Alu
sequences. Full-length Alu elements from the conserved subfamily were
selected from the data base collected by Jurka and Smith (61); the
corresponding genomic 5`-flanking regions were obtained from
GenBank.Those elements which occurred in tandem with an
immediately upstream Alu repeat were excluded. The remaining combined
(Alu and 5`-flanking) sequences were aligned to position 42 in the Alu
conserved subfamily consensus sequence (61-63). Input for the
computer program (39) for each combined sequence was a 240-bp region
extending 163 bp upstream and 77 bp downstream from this alignment
point (corresponding to
120 bp of upstream 5`-flanking region and
the entire left monomer). Parameters settings were: window, 62 steps;
helix repeat, 10.2 bp; local weights, 1.0, and local phase shifts, 0.0,
at all steps; output for these settings spans position
90 to
position
+88. The axis value,
, is a measure of the
energy required to bend a DNA region in a path corresponding to a
nucleosome surface. The averaged output for the five sequences is
shown, together with superimposed data from individual sequences in the
inset. Alignment with Alu landmarks is indicated below.
Arrows denote experimentally determined DNase I digestion
peaks for the AFP-Alu element following in vitro reconstitution with octamer particles (1).
Rotational Positioning: Comparison of
Results
Three lines of evidence, two experimental (this study
and earlier in vitro results)
(1) and one theoretical,
indicate that Alu left monomer elements are capable of setting the
rotational phase of nucleosomes or nucleosome-like particles. An
obvious question is whether the determined/predicted rotational phases
revealed by these approaches are in agreement. Alignment of the
rotational phases determined/predicted by these approaches is shown in
Fig. 4
. Within the uncertainties associated with such a
comparison, it can be seen that consistent results are obtained in all
three cases. From this analysis we conclude that rotational positioning
for Alu elements as a group reflects DNA sequence features intrinsic to
these repeats.
Figure 4:
Summary of
DNase I cleavage sites over Alu repeats in native chromatin aligned
with sites detected with the in vitro assembled AFP-Alu. Major
Alu family consensus sequence is aligned with the sequence of the
AFP-Alu. The diverse upstream sequences are represented by a
bar, whereas the AFP-Alu flanking sequence is shown in
lower case letters. Arrows mark approximate DNase I cleavage
sites in chromatin or sites detected for the in vitro assembled AFP-Alu, as indicated. Empty arrowheads indicate cleavage predicted by a computer algorithm. Note that
primer extension using the antisense primer corresponding to Alu
positions 96 to 122 (Fig. 1 B) measured DNase I nicking of the
upper sense strand. Likewise, earlier results obtained with in
vitro nucleosome reconstitutes measured DNase I nicking of the
sense strand (although in that case nicking sites were detected by
5`-end labeling of nucleosomal templates). The theoretical analysis
shows minima at positions where the minor groove faces directly away
from the histone core surface, i.e. positions corresponding to
maximum cleavage by DNase I. Since DNase I nicking sites on
complementary strands exhibit a 2-3-bp stagger to the 3` side,
the two experimental approaches should be roughly in agreement and
should be displaced 1-2 bp 3` to the minima determined by the
computer program.
Translational Positioning at the AFP-Alu Repeat
Locus
We next addressed whether nucleosomes associated with Alu
repeats assume a defined translational position in vivo.
Initially, we carried out linear primer extension experiments
(37) with MNase-digested mononucleosome-sized fragments and Alu
consensus primers. Although in several experiments preferential nicking
was detected at about the position expected for the upstream border of
the Alu left monomer-associated nucleosomes, variability in the data
obtained with different preparations of nuclei precluded a confident
interpretation (data not shown). We therefore narrowed the analysis to
a single Alu locus (AFP-Alu) that had been demonstrated previously to
confer both translational and rotational nucleosome positioning in an
in vitro reconstitution system
(1) . Chromatin
structure at the AFP-Alu locus was characterized by indirect
end-labeling, a technique that allows detection of sites susceptible to
MNase cleavage relative to a common restriction site
(48, 49) . HeLa cell nuclei were subjected to limited
digestion with MNase, and the partial nature of cleavage was confirmed
by the appearance of a nucleosome ladder. Following purification, DNA
was subjected to extensive double restriction to allow release of a
1.2-kb Alu-containing fragment from the AFP locus (Fig. 5 A and D). The Alu repeat is located in the center of this
1.2-kb fragment, so a 350-bp upstream region was chosen as the probe
for indirect end-labeling (Fig. 5 D). As shown in
Fig. 5B, a signal corresponding to the full-length
1.2-kb fragment decreased in proportion to the extent of digestion with
MNase, shifting gradually to a ladder of bands with a 200 bp
spacing (Fig. 5 B, lanes 3-5). This
pattern, which was absent in the naked DNA control
(Fig. 5 C), suggests the existence of a translationally
positioned nucleosomal array over the AFP-Alu repeat locus. It is
noteworthy that the preferential MNase cleavage sites depicted in
Fig. 5D are consistent with the nucleosomal organization
observed previously with assembly of the 1.2-kb fragment in an in
vitro reconstitution system
(1) .
Figure 5:
Nucleosome positioning over the AFP-Alu
locus in chromatin. A, electrophoretic analysis of products
resulting from MNase cleavage in nuclei for increasing periods of time
and subsequent digest with BamHI and KpnI, resolved
in a 1.3% agarose gel and visualized by ethidium bromide staining.
B, autoradiogram showing the MNase cleavage sites in HeLa cell
chromatin downstream of the BamHI site in the AFP-Alu locus;
after photography the gel in panel A was blotted and hybridized with
the AFP-Alu locus specific probe. Lane numbers correspond to those in
A. Horizontal arrows point to regions of preferential MNase
cleavage. The marker lane is an end-labeled X174/ HaeIII
digest. C, autoradiogram of naked DNA controls processed as
described in A. D, outline of the cleavage sites over the
1.2-kb locus ( downward arrows) based on the pattern obtained
following in vitro reconstitution. The probe spanning the 350
bp downstream of the BamHI site is represented by a solid
bar.
-fetoprotein gene,
is similar for both in vitro reconstitutes and native
chromatin. Indirect evidence for translational positioning over Alu
repeats as a group is at present limited to DNase I nicking results in
which the characteristic 10-bp ladder appears to be centered over the
start of Alu left monomer. For reasons that are not yet understood,
efforts to determine translational positioning on Alu repeats as a
group using MNase digestion in nuclei have met with limited success.
The results obtained were inconclusive, possibly due to interfering
non-nucleosomal proteins and/or variable MNase cleavage of diverse
sequences near the 5` edges of the left monomer-associated nucleosomes.
Further work will be required to determine whether or not most Alu
elements assemble nucleosomes with a preferential translational phase.
(
)
this
effect may be enhanced in the context of chromatin
(1, 56) . Chromatin proteins may also repress Alu
repeats in part because the latter lack requisite sites that can
effectively compete for stimulatory transcription factors. However,
packaging by repressive chromatin proteins in a strictly random manner
appears to be unlikely, since extraction of histone H1 from nuclei does
not derepress Alu template activity.
(
)
Taken
together, these findings imply that more than one mechanism exists by
which Alu repeats are maintained in a transcriptionally silent state.
While the data presented in this study do not address Alu
transcriptional regulation, they provide the first evidence for
nucleosome positioning over Alu repeats in chromatin. As Alu repeats
are transcriptionally quiescent in HeLa cells (and nucleosomes
positioned over promoter elements are in many cases implicated in gene
repression), it is reasonable to speculate that nucleosome positioning
over the Alu A box promoter element might have a contributory role in
Alu silencing.
-fetoprotein.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.