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Genomics 89 (2007) 354–361 www.elsevier.com/locate/ygeno

A map of nuclear matrix attachment regions within the breast cancer loss-of-heterozygosity region on human 16q22.1

Sergey A. Shaposhnikov a,b, Sergey B. Akopov c, Igor P. Chernov c, Preben D. Thomsen d, ⁎ ⁎ Claus Joergensen d, Andrew R. Collins a, Eirik Frengen b,e, , Lev G. Nikolaev c,

a Department of Nutrition, Faculty of Medicine, University of Oslo, PB 1046 Blindern, 0316 Oslo, Norway b Biotechnology Center of Oslo, University of Oslo, PB 1125 Blindern, 0317 Oslo, Norway c Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia d The Royal Veterinary and Agricultural University, Gronnegardsvej 7, Frederiksberg C, Denmark e Department of Genetics, Ullevål University Hospital and Faculty of Medicine, University of Oslo, 0310 Oslo, Norway Received 6 July 2006; accepted 1 November 2006 Available online 22 December 2006

Abstract

There is abundant evidence that the DNA in eukaryotic cells is organized into loop domains that represent basic structural and functional units of chromatin packaging. To explore the DNA domain organization of the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1, we have identified a significant portion of the scaffold/matrix attachment regions (S/MARs) within this region. Forty independent putative S/MAR elements were assigned within the 16q22.1 locus. More than 90% of these S/MARs are AT rich, with GC contents as low as 27% in 2 cases. Thirty-nine (98%) of the S/MARs are located within and 36 (90%) in introns, of which 15 are in first introns of different genes. The clear tendency of S/MARs from this region to be located within the introns suggests their regulatory role. The S/MAR resource constructed may contribute to an understanding of how the genes in the region are regulated and of how the structural architecture and functional organization of the DNA are related. © 2006 Elsevier Inc. All rights reserved.

Keywords: Functional genomics; Gene regulation; Physical mapping; Nuclear matrix; Scaffold/matrix attachment regions

The organization of DNA into the highly condensed between the topological organization of DNA within nuclei and structure of eukaryotic plays an important role the organization of functional units, such as replicons and in the regulation of , DNA synthesis, recombi- transcriptons, in the genome [3–6]. Matrix attachment regions nation, and repair by modulating accessibility of DNA. (MARs) are usually defined as short (100–1000 bp) DNA Packaging of eukaryotic chromosomal DNA into several sequences capable of binding specifically to the isolated nuclear hierarchical levels of organization appears to involve partition- matrix in vitro [7,8]. Similarly defined scaffold attachment ing of the 30-nm chromatin fiber into a series of discrete loop regions (SARs) [9,10] are most probably very similar if not domains by attachment to the nuclear scaffold or matrix [1]. The identical to MARs [11]. Experimental evidence indicates that S/ average size of the loops (25 to 200 kb) seems to be the same in MARs may physically separate neighboring chromatin loops or interphase nuclei and metaphase chromosomes [2]. It has been domains that differ structurally and/or functionally, e.g., by their suggested that with such a size, there could be a correlation transcriptional activity and/or topological state [12,13].An attractive hypothesis ascribes to S/MARs an involvement in ⁎ large-scale regulation of genome activity; it appears that a Corresponding authors. E. Frengen is to be contacted at Biotechnology specific fraction of them can bind nuclear matrix under certain Center of Oslo, University of Oslo, PB 1125 Blindern, 0317 Oslo, Norway. Fax: +47 22119899. L.G. Nikolaev, fax: +7 495 330 6538. conditions and form chromatin domains depending on cell or E-mail addresses: [email protected] (E. Frengen), tissue type [14,15]. Experimental S/MAR detection is usually [email protected] (L.G. Nikolaev). carried out by matrix-reassociation assays. In the present study,

0888-7543/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2006.11.003 S.A. Shaposhnikov et al. / Genomics 89 (2007) 354–361 355 such a technique allowing mapping of S/MAR elements in vitro homology to the locus 16q22.1 (21 clones). The 74 clones [16,17] has been used to explore the DNA domain organization containing chromosome 16q22.1 sequence were included in the within a region of human chromosome 16q22.1. This region has further analysis. been shown to be a candidate region for containing a tumor The 74 clones selected from the library of putative S/MARs suppressor gene [18–20]. We have previously generated a high- were compared to one another, yielding 40 independent resolution integrated map encompassing this candidate region sequences. To estimate the number of independent clones in [21], which functioned as a platform to reveal the DNA domain the library, we assumed that the frequency of occurrence of organization. cloned sequences in library samples fits the Poisson distribu- tion. Accordingly, the Poisson curve was adjusted by the least- Results and discussion squares method to fit the data obtained and used to calculate the library size as the number of selected clones divided by q, S/MAR library construction and analysis where q is a parameter of the Poisson distribution. The estimated number was found to be about 80. Therefore, 40 S/ S/MARs were selected from the pool of short fragments MARs found here may represent about half of the potential covering the 16q22.1 region with the use of the in vitro binding elements of this region identifiable in HeLa cells. procedure [16,17] and nuclear matrix prepared from HeLa S3 As was shown previously [16,17], virtually all DNA cells by the LIS extraction method [10] described under fragments from the selected by this procedure Materials and methods. Five successive rounds of binding to bind nuclear matrix (scaffold) preferentially. The S/MAR nuclear matrix were performed to select for sequences that properties were verified for eight randomly selected clones. attach to the matrix. The resulting patterns of DNA fragments To this end, eight DNA fragments were radioactively labeled after each binding round are shown in Fig. 1. The initial short- and their abilities to bind nuclear matrix were compared to those fragments library gave a smear of fragments. The successive of negative control fragments of lambda and T7 phage DNA as matrix binding-purification rounds gradually replaced the smear described previously [16,17]. As is evident from the results in with a ladder of a limited number of fragments, indicating Fig. 2 and Table 1, all eight S/MARs do bind the nuclear matrix preferential selection of a putative matrix-binding fraction of the much more strongly than the negative controls. Four of the initial DNA. The selection process was repeated until satura- clones analyzed (1B1, 1B7, 3A8 and 2F9) show a stronger tion, i.e., until patterns of fragments obtained after two binding than the positive control (Table 1). In addition, it was successive selection rounds became indistinguishable (cf. recently demonstrated that four S/MAR sequences selected patterns after rounds 4 and 5 in Fig. 1). The resulting mixture using the above procedure [17] actually form a loop domain and of selected DNA fragments was cloned and 208 randomly expression of genes located within this domain depends on selected bacterial colonies were picked into 96-well plates and domain conformation [30]. sequenced. The sequences were compared to the GenBank database, revealing 74 sequences that belong to the region of Analysis of S/MAR sequences human chromosome 16q22.1. The rest of the sequences were either derived from Escherichia coli DNA, most likely due to Forty sequences containing S/MARs were identified with coisolation of bacterial genomic DNA with the plasmid DNA corresponding genome sequences and their general properties (109 clones), or from vector DNA (4 clones) or showed no were analyzed using the PC/Gene package. The results are presented in Table 2. It is noticeable that most (36 of 40 or >90%) of the S/MARs identified are AT-rich; the GC content was as low as 27% in the case of S/MARs 1B1 and 3A3. Only 4 sequences contain more than 45% of GC. Similarly, 38 of 40 of the S/MARs identified in this work contain prolonged AT-rich stretches and were rich in inverted repeats. In this respect they correspond to “classical” S/MAR-elements as defined in Refs. [8] and [31]. This is in contrast to our previous finding for the FXYD5-COX7A1 human chromosome 19 locus [17], where AT-rich S/MARs constituted the minority of these elements. This difference in the classes of S/MARs belonging to different human genome loci could reflect differences in the organization and/or regulation of these loci. Only 8 of 40 identified S/MARs contain more than 25% repeated sequences; therefore S/MARs are considerably depleted in repeats compared with genomic DNA (∼50% Fig. 1. Selection of DNA fragments binding preferentially to nuclear matrix. A repeated sequences). In most cases they contain sequences 1% agarose gel electrophoresis of the initial short fragment library of the region of human chromosome 16q22.1 (lane 0) and fragments after rounds 1–5 of the belonging to long interspersed elements. This fact was observed selection is shown. M, DNA size marker. Note that the initial smear gradually by us and others earlier (reviewed in [13]) and may have transforms into a ladder of distinct bands. functional significance. 356 S.A. Shaposhnikov et al. / Genomics 89 (2007) 354–361

Fig. 2. Binding of eight identified S/MARs by isolated nuclear matrix. A 6% denaturing polyacrylamide gel is shown. The DNA fragments were labeled, incubated with nuclear matrix, and then separated into pellet (matrix-bound) and supernatant fractions. The S/MARs are found preferentially in the pellet fraction. S, supernatant; P, pellet. pUCMAR10, positive control. L and T, fragments of phage DNA used as negative controls (see Materials and methods). S/MARs are indicated according to Table 1 and designated above the corresponding lanes.

Computer prediction of S/MARs obtained with the use of the Human Genome Browser (http:// genome.ucsc.edu/cgi-bin/hgGateway?db = hg12), May 2004 To compare positions of the S/MAR identified in this study assembly, and an annotation file in the supplementary material to positions of S/MARs predicted in silico, we used the MAR- (Table S2). As is seen from Fig. 3, 39 of 40 (98%) of the Wiz, MARSCAN, and SMARTEST computer programs. The 16q22.1 S/MARs were found within genes, while genes (with results of computer analysis are summarized in Table 3 and introns) occupied 74% of the locus. Of intragenic S/MARs, 36 presented in the metric map (Fig. 3). It could be seen that all were found within introns, 2 within 3′-UTRs, and 1 within a programs predict many more S/MARs in the locus than were protein-coding exon (Table 2). It could be noted that the single detected in the current selection experiments. The mean length exonic S/MAR that was found (2A8) is the only non-AT-rich of domains delimited by the predicted S/MARs is 13–28 kb, one and does not contain any AT-enriched regions. Thus, it is i.e., much less than was estimated previously on the basis of clear that S/MARs within the human chromosome 16q22.1 are different experimental approaches [32–34]. It should be noted, associated with genes. These data distinguish the 16q22.1 locus however, that not all S/MARs are utilized as loop anchors in a from the region within19q13.12, where 50% of S/MARs were given cell at a given time [35,36]. At the same time, the three located between genes even though the genes cover 50% of the algorithms gave significantly different distributions of S/MARs locus [17]. To our knowledge, this is the first example of such a within the locus (Fig. 3). Comparing with experimentally large-scale correlation of S/MARs and gene introns within the defined positions of S/MARs, we can conclude that the relative genome so far reported. predictive power (as defined in Table 3)ishighestfor Another interesting feature of this locus is that 15 of 36 SMARTEST and lowest for MARSCAN (see Table 3). Our intronic S/MARs were found in the first introns of genes. It is results indicate that in silico prediction is not yet able to not a general property of S/MAR distribution, as indicated by substitute for experimental approaches and show the importance our earlier data from the 1-Mb region within human chromo- of critical analysis of the biocomputing data. some 19q13.12, where only 8 of 16 identified S/MARs were intronic, and only 1 of them was located in the first intron [17]. Map of S/MARs within the human chromosome 16q22.1 region The first intron is a favorable place for intragenic regulatory regions [37], bringing them close to the promoter region. The The resulting map of the S/MAR elements detected within promoter–S/MAR distance could be an important factor in the the region of human 16q22.1 is shown in Fig. 3. An interactive correct functioning of the border elements [38,39]. map of all in silico identified and predicted S/MARs can be In contrast to the 19q13.12 region [17], S/MARs of the 16q22.1 locus are distributed nonuniformly, making distinct clusters of these elements. The largest cluster of S/MARs, Table 1 consisting of seven elements within a 45 kb fragment, was Nuclear matrix binding efficiency of eight randomly selected S/MARs from the found within the CBFP gene. At the same time, there are region of human chromosome 16q22.1 extensive regions lacking S/MARs, the longest (>400 kb) being S/MAR 1B1 1B7 3A8 1E8 2A8 1A4 2F9 1G7 located between S/MARs 3D6 and 1B9. It cannot be excluded, Binding efficiency, % a 590 313 177 70 68 8.1 167 32 however, that these regions do contain S/MARs that were not a Relative to the positive control, see Ref. [17]. detected in this study. Three main clusters of S/MARs were S.A. Shaposhnikov et al. / Genomics 89 (2007) 354–361 357

Table 2 Properties of S/MAR sequences S/MAR Position a Length (bp) Density of AT-rich regions (%) b Density of inverted repeats (%) c G+C (%) Position relative to genes No. d 1A9 65159219–65160818 1600 70 61 33 CMTM1, 1st intron 1 1H6 65206980–65208326 1347 36 46 45 CMTM4, 3′-UTR 1 1G7 65379677–65379908 232 85 10 28 CCDC79, 4th intron 2 3A3 65382265–65382871 607 92 59 27 CCDC79, 2nd intron 4 1H5 65387511–65388099 489 44 18 36 CCDC79, 1st intron 1 1G3 65542072–65542482 411 75 27 32 Intergenic, no genes 1 2F9 65630048–65630448 401 77 60 31 CBFB, 3rd intron 2 2D7 65635622–65636090 469 91 44 28 CBFB, 3rd intron 1 3D8 65644278–65644724 447 51 N/A e 38 CBFB, 3rd intron 1 1H2 65652417–65653035 619 80 40 29 CBFB, 3rd intron 5 2H2 65655156–65655741 586 62 36 34 CBFB, 3rd intron 1 2B2 65668077–65668982 906 84 36 29 CBFB, 4th intron 1 2A5 65673905–65674754 850 76 60 32 CBFB, 5th intron 1 2E2 65929190–65930108 818 76 38 33 LRRC36, 1st intron 1 1H7 65938910–65939185 276 42 22 37 LRRC36, 4th intron 1 3D6 66068693–66069143 451 84 30 30 ATP6V0D1, 1st intron 1 1B9 66503599–66504000 402 15 21 47 PSKH1, 2nd intron 1 2D4 66616015–66616290 276 24 11 40 DUS2L, 1st intron 1 1C5 66678442–66679087 646 80 30 30 NFATC3, 1st intron 3 1A4 66678787–66679155 369 71 21 33 NFATC3, 1st intron 2 3C5 66681203–66682061 859 73 41 30 NFATC3, 1st intron 2 2C8 66730932–66731260 329 53 15 35 NFATC3, 3rd intron 1 1B8 66746071–66746819 749 26 20 42 NFATC3, 3rd intron 1 2D11 66770329–66770633 305 7 8 44 NFATC3, 6th intron 1 1H3 66803614–66804260 647 52 25 35 NFATC3, 9th intron 1 2F6 66820179–66820536 358 24 11 46 RBM35B, 3′-UTR 1 1B6 66892739–66893120 382 44 18 40 SLC7A6OS, 4th intron 1 1E8 66898656–66899077 422 76 52 29 SLC7A6OS, 2nd intron 2 2H12 67032260–67033232 973 64 56 32 SMPD3, 1st intron 1 2H7 67138490–67139110 621 71 25 31 ZFP90, 1st intron 1 2A8 67519138–67519423 286 0 3 49 TMCO7, 10th exon 2 3A8 67532196–67532635 440 89 42 29 TMCO7, 12th intron 10 3D2 67546100–67546439 340 72 22 31 TMCO7, 12th intron 1 3B2 67580767–67581083 317 78 14 31 TMCO7, 13th intron 1 2D12 67599287–67600094 782 58 37 36 TMCO7, 13th intron 1 1B7 67602127–67602546 420 68 28 32 TMCO7, 13th intron 3 1H12 67719555–67719918 364 38 11 43 DERPC, 1st intron 1 2F10 67781362–67781606 245 75 20 32 SNTB2, 1st intron 1 3B8 67786383–67786729 347 83 37 30 SNTB2, 1st intron 1 1B1 67788844–67789275 432 82 38 27 SNTB2, 1st intron 9 a From Human Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway), May 2004 assembly. b Percentage of continuous AT-rich (≥75% A+T) regions 20 bp or longer in length. c Percentage of perfect inverted repeats longer than 6 bp (including palindromes). d Occurrence in the sequenced part of the S/MARs library, times. e The sequence contains long poly(A) and poly(T) stretches and cannot be correctly evaluated by the software used. found within introns of three genes—seven S/MARs are areas with a high density of genes, such as the LCAT gene clustered in the CBFB gene (core-binding factor, β subunit), cluster area, contain not more than one S/MAR element per and clusters of six S/MARs are located in the NFATC3 gene 100 kb; whereas the most “S/MAR-dense” region with seven S/ (nuclear factor of activated T cells) and in the TMCO7 gene, MAR elements per 100 kb contains only one gene, CBFB. coding for a hypothetical protein with unknown function. At the same time, only 20% of the genes from our map Hypothetical chromatin domains within the breast cancer loss contain one or more S/MAR elements (16 of 81 genes). To of heterozygosity region of human chromosome 16q22.1 compare the density of genes with the density of S/MAR elements within the region of human chromosome 16q22.1, we Based on the positions of the S/MARs detected and an counted the number of genes and S/MAR elements per 100 kb estimated 80 S/MARs in the region within human chromosome of the map (see Table S1 in the supplementary material). The 16q22.1, the mean length of hypothetical chromatin domains results are summarized in Fig. 4. We found that the genes and can be estimated to be (2900/80) ∼36 kb. This length is short S/MAR elements within the area of human chromosome compared to the average DNA loop size in HeLa cells, which 16q22.1 show an inversely correlated distribution. Thus, the was previously estimated at 86 kb [40], and compared to our 358 S.A. Shaposhnikov et al. / Genomics 89 (2007) 354–361

Table 3 are in accord with a concept of flexible status-dependent higher Comparison of different algorithms for predictions of the S/MARs level DNA loop organization. This concept is consistent with Software the view that S/MARs can be subdivided into two groups MARSCAN SMARTEST MAR–Wiz [15,43], so-called “stable/structural” and “dynamic”. The last Total No. of predicted S/MARs 229 103 109 are also called function dependent, or tissue specific, and are Total No. of positives a 5165 thought to be transient depending on the stage of the cell cycle No. of random positives b 1.72 0.77 0.82 and activity of the genes, whereas the structural S/MARs are Relative predictive power c 2.9 20.8 6.1 believed to be more stable and common for all cell types or – – 1A4 + tissues. It is not possible to conclude if the S/MARs defined in 1A9 – + – 1B1 – + – this study belong to the first or the second category. In that view 1B6 –– – it would be interesting to construct similar S/MAR maps of the 1B7 + –– region using other types of cells. The genomic interval mapped 1B8 –– – in the current work has been identified as a possible region –– – 1B9 harboring tumor suppressor genes based on the high frequency 1C5 – + – 1E8 – ++ of allele loss in both invasive ductal and invasive lobular breast 1G3 –– + carcinomas [20,44–46]. The E-cadherin gene, which is located 1G7 –– – within this region, has been shown to be mutated in lobular 1H2 –– – breast carcinomas, resulting in loss of E-cadherin expression –– 1H3 + [44]. However, in most cases of ductal carcinoma, which is the 1H5 –– – 1H6 – + – major mammary cancer type, E-cadherin is normally expressed 1H7 –– – [44], suggesting that other genes within chromosome 16q22.1 1H12 –– – may be involved in the development of this tumor subtype. The 2A5 + + – construction of comprehensive S/MAR maps of the region –– – 2A8 using a panel of breast cancer cell lines may provide 2B2 –– – 2C8 –– – information on relevance for the etiology of breast carcinomas. 2D4 –– – Changes of positions of regulatory elements such as S/MAR 2D7 –– – within the region may allow an understanding of how the genes 2D11 –– – in the region are regulated and how the structural architecture is – – 2D12 + related to the functional organization of the DNA. 2E2 – ++ 2F6 –– – 2F9 – + – Concluding remarks 2F10 –– – 2H2 + –– The distribution of S/MARs identified in the 16q22.1 – 2H7 ++ region has some characteristics different from that of the 2H12 – ++ 3A3 – + – 19q13.12 locus studied previously. First, a great majority of 3A8 – + – 16q22.1 S/MARs are localized inside genes, preferably in their 3B2 –– – first introns. Second, 16q22.1 S/MARs are distributed within 3B8 + + – the locus nonuniformly, making up several clusters consisting – – 3C5 + of up to seven of these elements. These findings indicate that 3D2 –– – 3D6 –– – different regions of the genome can be characterized by 3D8 –– – properties of their domain organization which, in turn, can a Coincidences with experimentally identified S/MARs. reflect specific function and/or regulation. b Total length of experimentally identified S/MARs (21,800 bp) divided by the total length of the locus (2,900,000 bp) and then multiplied by the total Materials and methods number of predicted S/MARs. c Total number of positives divided by the number of random positives. Basic protocols

Growth and transformation of E. coli, preparation of plasmid DNA, agarose previous data for 19q13.12 region (88 kb) [17]. However, it is gel electrophoresis, and other standard manipulations were performed as important to emphasize that this hypothetical domain size, described in [22]. calculated as a mean distance between S/MAR elements detected within the area by our experimental procedure, does Construction of a short fragment library of the region of human not necessarily comply with sizes of DNA loops present in a chromosome 16q22.1 living cell. The dynamic nature of chromatin loops depending on the gene activity and replicative status of the cell has been A pool of overlapping P1-derived artificial chromosome (PAC [23]) clones from the previously constructed map covering 2.8 Mb within the region of reported [41]. Just a fraction of S/MARs serve as anchors at any human chromosome 16q22.1 [21] was used as a source of DNA for construction given time and additional cellular factors are necessary for of the small fragments library. The following clones were selected: 6-131G17, binding of an S/MAR to the nuclear matrix [36,42]. These data 6-71C24, 6-159B22, 6-42D11, 6-132O4, 6-73B1, 1042E2, 6-133B6, 6-100O24, ..Saohio ta./Gnmc 9(07 354 (2007) 89 Genomics / al. et Shaposhnikov S.A. – 361

Fig. 3. A map of the region of human chromosome 16q22.1 with positions of the 40 S/MARs detected in the current study indicated by vertical arrows. Known genes and their directions of transcription are indicated by horizontal arrows. The positions of S/MARs predicted by the MARSCAN, SMARTEST, and MAR-Wiz programs are indicated by black squares. 359 360 S.A. Shaposhnikov et al. / Genomics 89 (2007) 354–361

Acknowledgments

We thank Gaute Brede, Hans Prydz, and Eugene Sverdlov for fruitful discussions. The work was supported by the Scientific School Program of the Russian Federation President (Project NSh 2006.2003.4), the Program of the Russian Academy of Sciences in Molecular and Cellular Biology, the Norwegian-Russian Medical Center (University of Oslo), and a fellowship from “Odd Fellow Medisinsk-Vitenskapelig Forskningsfond.” E.F. is supported by grants from The Research Council of Norway's Functional Genomics Program Fig. 4. Distribution of genes (dashed line) and S/MARs (solid line) along the human chromosome 16q22.1 region. The genes and S/MAR elements show an (FUGE, Grant 151882). inversely correlated distribution. Appendix A. Supplementary data 6-191B24, 6-85K16, 6-155L14, 6-234L8, 160E12, 6-32H24, 179N7, 138C8, 1096M19, 954E14, 6-167M3, 1050C9, 1038A12, 6-100O11, 6-187B3, 992D5, Supplementary data associated with this article can be found, 6-86F1, 1136M7, 6-237I1. 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