©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nucleosome Positioning by Human Alu Elements in Chromatin (*)

Ella W. Englander , Bruce H. Howard (§)

From the (1) Laboratory of Molecular Growth Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

RNA polymerase III (pol III)() -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) .

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) ,() 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.

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.


MATERIALS AND METHODS

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 410cpm/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.


RESULTS

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.

The lower strand of the left monomer was examined through extension of a primer spanning residues 8 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.

The DNase I cleavage pattern for the right monomer was determined for the upper strand by extension of a primer spanning Alu positions 262 278 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.




DISCUSSION

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 -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.

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,() 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.

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 301-496-9038; Fax: 301-480-9354.

The abbreviations used are: pol, polymerase; bp, base pair(s); kb, kilobase(s); MNase, micrococcal nuclease; AFP, -fetoprotein.

G. Humphrey and B. H. Howard, unpublished results.

G. Humphrey, R. Vorce, E. W. Englander, and B. H. Howard, unpublished results.

V. Russanova and B. H. Howard, unpublished results.


ACKNOWLEDGEMENTS

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.


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