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Chromatin structure at the 3′-boundary of the human β-globin locus control region hypersensitive site-2
The International Journal of Biochemistry & Cell Biology 33 (2001) 1183–1192 www.elsevier.com/locate/ijbcb
Chromatin structure at the 3%-boundary of the human b-globin locus control region hypersensitive site-2 AeRi Kim, Vincent Murray * School of Biochemistry and Molecular Genetics, Uni6ersity of New South Wales, Sydney, NSW 2052, Australia Received 17 April 2001; accepted 19 June 2001
Abstract Chromatin structure was examined at the 3%-boundary region of the human b-globin locus control region hypersensitive site-2 (LCR HS-2) using several footprinting agents. Erythroid K562 cells (possessing HS-2) were damaged by the footprinting agents: hedamycin, bleomycin and four nitrogen mustard analogues. Purified DNA and non-erythroid HeLa cells (lacking HS-2) were also damaged as controls for comparison with K562 cells. The comparison between intact cells and purified DNA showed several protected regions in K562 cells. A large erythroid-specific protected region of 135 bp was found at the boundary of HS-2. The length of this protected region (135 bp) was close to that of DNA contained in a nucleosome core (146 bp). Another two protected regions were found upstream of the protected region. A 16-bp erythroid-specific footprint co-localised with a GATA-1 motif— this indicated that the GATA-1 protein could be involved in positioning the nucleosome. Further upstream, a 100-bp footprint coincided with an AT-rich region. Thus our footprinting results suggest that the 3%-boundary of LCR HS-2 is flanked by a positioned nucleosome and that an erythroid-specific protein binds to the sequence adjacent to the nucleosome and acts to position the nucleosome at the boundary of the hypersensitive site. Crown Copyright © 2001 Published by Elsevier Science Ltd. All rights reserved. Keywords: Nucleosome; Genomic footprinting; GATA-1; Bleomycin; Hedamycin; Ligation-mediated PCR
1. Introduction The fundamental unit of chromatin structure is the nucleosome which consists of a core region and a linker region. Approximately 146 bp of Abbre6iations: bp, base pair; DHS, DNase I hypersensitive site; HS-2, hypersensitive site-2; LMPCR, ligation-mediated polymerase chain reaction; LCR, locus control region. * Corresponding author. Tel.: + 61-2-9385-2028; fax: +612-9385-1483. E-mail address: [email protected] (V. Murray).
DNA interacts with the histone octamer to form a core particle. The linker region contains histone H1 and linker DNA that connects core particles [1]. Chromatin structure is thought to play a crucial role in the regulation of gene expression [2]. Transcription factors can bind to DNA in three general ways: (1) a factor will only bind in the presence of a nucleosome; (2) a bound nucleosome does not prevent binding of the factor; (3) a factor will only bind in the absence of a nucleosome. In this latter case, the absence of a
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nucleosome leads to a DNase I hypersensitive site (DHS) that appears to be a very important determinant in the control of gene expression. DNase I hypersensitive sites (DHS) are a common feature at enhancers and promoters of expressed genes [3,4]. DHSs are thought to be nucleosome-free regions that permit the binding of transcription factors to DNA and lead to the induction of gene expression [5]. The pivotal role of DHSs has led to a great deal of research effort into the processes that define these regions and the subsequent mechanism(s) of gene activation. An advance in our understanding of the subject has been the discovery of the SWI/ SWF complex [2,6– 8]. This and other complexes permit the remodelling of chromatin. Using ATP as an energy source, the structure of chromatin is altered by these complexes to allow the interaction of transcription factors with DNA. These very large protein complexes have been found in yeast, Drosophila and humans. The discovery of these complexes provides an insight into the dynamic nature of chromatin where proteins are
added or removed in order to affect the level of gene expression. This work with SWI/SWF complexes gives an insight into the creation of DHSs where chromatin remodelling factors can lead to the removal of nucleosomes. In order to maintain a nucleosome-free region at a DHS, there must be a mechanism to prevent a nucleosome from sliding into the DHS. The sliding of nucleosomes is an energy requiring process in cells [9]. A DHS is often flanked by positioned nucleosomes [10–12]. There are two ways that a positioned nucleosome can arise. First, the DNA sequence has the ability to position a nucleosome. In vitro reconstitution experiments showed that nucleosomes can be precisely positioned at the same sequence as found in vivo [13]. Certain DNA sequences appear to have the ability to translationally position a nucleosome [14]. Second, a protein factor can position nucleosomes (Fig. 1). The protein factor usually binds to the sequence adjacent to a positioned nucleosome. Nucleosome positioning by a protein factor has been observed in several genes [15,16].
Fig. 1. Map of LCR HS-2 in the human b-globin gene cluster. The five expressed b-globin genes are shown as well as promoter elements. The LCR HS regions are indicated. The expanded view of the LCR HS-2 shows a ‘positioned’ nucleosome at the 5%- and 3%-boundaries as well as ‘positioning’ proteins. The location of transcription factor binding elements is also indicated. There are two GATA-1 motifs between bp 8940 and 9000 (white and grey box). The grey box at bp 8988 is the putative nucleosome ‘positioning’ protein. The black boxes indicate a tandem NF-E2 motif while the striped boxes represent CACC motifs.
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Cell- or tissue-specifically positioned nucleosomes have been observed in mammalian genes [12,17,18]. There are at least four DHSs in the 5%-locus control region (LCR) of the human b-globin gene cluster (Fig. 1). The LCR acts as an enhancer and is responsible for erythroid-specific expression of the b-globin gene cluster [5,19]. Hypersensitive site-2 (HS-2) is a nucleosome-free region that is approximately 600 bp in length [20]. The binding sequences for erythroid-restricted and ubiquitous transcription factors are present in HS-2 [21– 25]. The NF-E2, GATA-1 and CACC binding motifs have all been shown to be occupied in erythroid cells as determined by LMPCR footprinting. The NF-E2 and GATA-1 motifs bind the erythroid-restricted NF-E2 and GATA-1 transcription factors, respectively. The CACC, GT-I and GT-II motifs are thought to bind different members of the ubiquitous Sp1 family of transcription factors. The transcription factor NF-E2 can displace a nucleosome from reconstituted chromatin in the HS-2 sequence [26]. In this study, we investigated the chromatin structure at the 3%-boundary region of HS-2 in the human b-globin gene cluster. Various DNA damaging agents (nitrogen mustard analogues, hedamycin and bleomycin) were used to investigate the chromatin structure of the HS-2 boundary. The ability of these compounds to footprint transcription factors has been demonstrated in the b-globin gene cluster [23– 25]. Hedamycin and bleomycin can also detect the presence of a positioned nucleosome since they preferentially damage DNA in the linker region of nucleosomes [18,27– 29]. This property of bleomycin and hedamycin is useful in the study of chromatin structure at the DHS boundary because a positioned nucleosome is expected at this position. Four nitrogen mustard analogues were used in this study. Chlorambucil is used as an anti-tumour drug. The other three are ‘DNA-directed’ nitrogen mustard analogues that have an attached DNA-affinic moiety, either acridine or amsacrine [25]. The four nitrogen mustards preferentially damage at guanine and, to a lesser extent, adenine bases [25,30]. Bleomycin cleaves mainly at GT, GC and GA dinucleotides [30– 33] while
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hedamycin has a preference for guanine bases [24,29,30,34]. Furthermore, all damaging agents chosen for this study can enter intact human cells without any pre-treatment— this is a major advantage of these compounds. Two kinds of human cell line, K562 and HeLa, were used to examine chromatin structure at the 3%-boundary of HS-2. The erythroid K562 cell line displays the LCR HS-2 and expresses o- and g-globin genes [35]. The non-erythroid HeLa cell line lacks the LCR HS-2, does not express globin genes, and was utilised as a control for comparison with K562 cells. Damaged cellular DNA was analysed by the ligation-mediated polymerase chain reaction (LMPCR) procedure at base pair resolution [36,37]. We have previously examined chromatin structure at the 5%-end of the human b-globin LCR HS-2 and found a large footprint consistent with the presence of a positioned nucleosome [18]. An erythroid-specific ‘positioning’ protein was thought to be responsible for maintaining the nucleosome at this position (Fig. 1). This study investigated chromatin structure at the 3%boundary region of the human b-globin LCR HS-2 in intact human cells and attempted to answer the question, ‘What molecular structure is present at the boundary of a mammalian DNase I hypersensitive site?’ This study provides evidence that a nucleosome is positioned at the 3%boundary in erythroid K562 cells by erythroidspecific ‘positioning’ protein(s). 2. Materials and methods
2.1. Cell culture K562 cells were grown in RPMI 1640 medium (with no antibiotics) containing 10% foetal calf serum. Hemin was added to the medium (50 mM) for 4 days to induce b-globin expression [38]. The harvest of uninduced and induced K562 cells was carried out at a density of 106 cells/ml. HeLa cells were grown in RPMI 1640 medium containing 2.5% newborn calf serum and 2.5% foetal calf serum. HeLa cells at 95% confluence were harvested after washing with phosphate buffered saline.
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2.2. Cell damage Approximately 107 cells were washed with phosphate buffered saline and treated with an appropriate concentration of nitrogen mustard analogue, hedamycin or bleomycin at 37 °C [23 – 25]. The treatment with a nitrogen mustard analogue or hedamycin was performed in the dark. Cells were incubated for 3 h with a nitrogen mustard analogue and for 1 h with hedamycin or bleomycin. The purification of DNA from the treated cells was carried out as described previously by Murray and Martin [39].
2.3. Purified DNA damage Purified human DNA was damaged with appropriate concentrations of nitrogen mustard analogues, hedamycin or bleomycin [23– 25]. The damage reaction was performed at 37 °C for 1 h with bleomycin or the nitrogen mustard analogues and for 30 min with hedamycin. For the bleomycin reaction, Fe2 + SO4 was included at the equivalent bleomycin concentration. Damaged DNA was purified by ethanol precipitation.
2.4. LMPCR primer sequences Human b-globin LCR HS-2 sequence specific primers were designed for analysis of the 3%boundary. The primers for the coding strand were 5%-GTATGTGAGCATGTGTCCTCTAACA-3% (first strand synthesis) and 5%-GAGCATGTGTCCTCTAACAGCACAG-3% (PCR). The primers for the non-coding strand were 5%-ATGTTCTCAGCCTAGAGTGATGACT-3% (first strand synthesis) and 5%-AGCCTAGAGTGATGACTCCTATCTG-3% (PCR).
2.5. LMPCR LMPCR was performed to investigate the sites and intensity of damage in genomic DNA treated with nitrogen mustard analogues, hedamycin and bleomycin at base pair resolution. The LMPCR was performed with the modifications described by Cairns and Murray [23]. Damaged DNA was amplified with two sets of sequence-specific
primers and the linker primers. The amplified fragments were analysed on a 6% (w/v) polyacrylamide sequencing gel. A G+ A Maxam/Gilbert chemical sequencing ladder (amplified by the same primers) was also included on the gel.
3. Results The 3%-boundary region of the human b-globin LCR HS-2 was examined with various footprinting agents in intact cells and purified DNA. The damaged DNA was amplified using a set of primers for the coding strand and non-coding strand, respectively and analysed at base pair resolution. The coding strand was examined from bp 9130 to 8950 and on the non-coding strand from bp 8750 to 9150. The intensity of damage was compared in intact cells and purified genomic DNA (Figs. 2–4). Protection occurred where the damage intensity was reduced in cells compared to purified DNA and conversely enhancement occurred where damage was increased in cells compared to purified DNA. Undamaged control DNA was also amplified to check the background level of DNA damage. G+ A sequencing fragments were included to determine the exact DNA sequence at the damage site. In a comparison between uninduced K562 cells and induced K562 cells, no significant differences were found for any of the six damaging agents as found previously [23,25,40].
3.1. Coding strand The sequencing gel in Fig. 2 shows DNA damage by the nitrogen mustard analogue, C3-AA, on the coding strand of purified DNA and intact cells. A comparison between purified DNA and intact cells revealed a large protected region in uninduced and induced K562 cells. A region of 104 bp between bp 9045 and bp 9148 was protected from C3-AA damage in K562 cells (compared to purified DNA) except for a small region from bp 9064 to 9069. For chlorambucil in K562 cells, the intensity of damage was reduced in the region from bp 9045 to 9152 covering 108 bp except for the region from bp 9064 to 9069. The
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The sequencing gel in Fig. 3 shows damage by hedamycin on the coding strand of purified DNA and intact cells. A comparison of hedamycin damage in purified DNA and intact cells showed significant differences. In K562 cells a large region spanning 117 bp from bp 9019 to 9135 was protected with respect to purified DNA. Upstream of the protected region, enhanced hedamycin damage was seen with K562 cells centred at bp 8997. Protection was also observed in K562 cells dam-
Fig. 2. Phosphorimage of a DNA sequencing gel comparing nitrogen mustard analogue damage on the coding strand at the boundary of LCR HS-2 in four different environments. Lanes control P, H, U and I were undamaged controls for purified human DNA, HeLa cells, uninduced K562 cells, and induced K562 cells, respectively. C3-AA damage lanes P, H, U and I were derived from purified human DNA (15 mM), HeLa cells (120 mM), uninduced K562 cells (120 mM), induced K562 cells (120 mM), respectively. Chlorambucil damage lanes P, H, U and I were derived from purified human DNA (300 mM), HeLa cells (1 mM), uninduced K562 cells (200 mM), induced K562 cells (400 mM), respectively. Lane GA was a G + A sequencing reaction with purified human DNA.
protected regions in K562 cells were the same for C3-AA and chlorambucil damage. Upstream of the large protected region, there were areas representing enhancement and protection of damage in K562 cells. The two regions centred at bp 8983 and bp 9018 exhibited enhancement of damage with both C3-AA and chlorambucil. The region between the two enhanced damage regions bp 8985 and 9000 was protected in cells compared to purified DNA. This latter sequence contains a GATA-1 binding motif.
Fig. 3. Phosphorimage of a DNA sequencing gel comparing hedamycin damage on the coding strand at the boundary of LCR HS-2 in four different environments. Lanes control P, H, U and I were undamaged controls for purified human DNA, HeLa cells, uninduced K562 cells, and induced K562 cells, respectively. Hedamycin damage lanes P, H, U and I were derived from purified human DNA (4 mM), HeLa cells (20 mM), uninduced K562 cells (20 mM), induced K562 cells (20 mM), respectively. Lane GA was a G +A sequencing reaction with purified human DNA.
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Fig. 4. Phosphorimage of a DNA sequencing gel comparing nitrogen mustard analogue damage on the non-coding strand at the boundary of LCR HS-2 in four different environments. Lanes control P, H, U and I were undamaged controls for purified human DNA, HeLa cells, uninduced K562 cells, and induced K562 cells, respectively. C3-AA damage lanes P, H, U and I were derived from purified human DNA (20 mM), HeLa cells (120 mM), uninduced K562 cells (120 mM), induced K562 cells (120 mM), respectively. Chlorambucil damage lanes P, H, U and I were derived from purified human DNA (150 mM), HeLa cells (1 mM), uninduced K562 cells (200 mM), induced K562 cells (400 mM), respectively. Lane GA was a G + A sequencing reaction with purified human DNA.
aged by bleomycin (data not shown)— a region of 123 bp, between bp 9018 and 9140, was protected in K562 cells compared to purified DNA. A summary of the data obtained with all the DNA damaging agents is shown in Fig. 5. No relative protection was found with HeLa cells (compared to purified DNA) damaged by hedamycin, bleomycin, C3-AA and chlorambucil for the whole analysed region on the coding strand.
3.2. Non-coding strand Damage by the nitrogen mustard analogues, C3-AA and chlorambucil, on the non-coding
strand revealed two areas of protection (Fig. 4). A large protected region was observed in K562 cells damaged by C3-AA and chlorambucil between bp 8865 and 8940 in K562 cells. There was another small protected region at bp 8845–8850 in K562 cells. The non-coding strand was also investigated with bleomycin. Three protected regions were observed in K562 cells: a large region of 91 bp between bp 8850 and 8940; and two smaller regions between bp 8790 and 8791 and bp 8810 and 8817 (Fig. 5). The latter two regions coincided with GT-I and GT-II motifs, respectively. Analysis of the damage by the nitrogen mustard analogues, C50-AMSA and C20-AMSA, on the non-coding strand of purified DNA and intact cells showed protected regions in K562 cells (relative to purified DNA). Four regions, bp 8751– 8757, bp 8817–8827, bp 8841–8940, and bp 9000–9100 in intact cells were protected from damage (Fig. 5). The large 100-bp protected region, bp 8841–8940, was at a similar sequence to that found with bleomycin, C3-AA and chlorambucil. The large 101-bp protected region from bp 9000 to 9100 was also observed on the other strand with hedamycin, bleomycin, C3-AA and chlorambucil. All of the protected regions observed in K562 cells (see above), were absent in HeLa cells except for protected regions bp 8860–8900, 8817–8827 and 8751–8757 that were observed with C50AMSA and C20-AMSA (data not shown).
4. Discussion Chromatin structure at the 3%-boundary region of the b-globin LCR HS-2 was examined in intact human cells (Fig. 5). Approximately 450 bp was examined in this region. The footprints were mapped at base pair resolution. Three major footprints were observed: (i) a large 135-bp region from bp 9018 to 9152; (ii) a 16-bp region from bp 8985 to 9000; a 100-bp region from bp 8841 to 8940. We postulate that the large 135-bp footprint is caused by a positioned nucleosome core, and the 16-bp footprint by a nucleosome positioning protein (GATA-1), while the 100-bp footprint
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contains an AT-rich region. Each footprint was confirmed by use of more than one footprinting agent. The size of these footprints has been determined by combining results from several DNA damaging agents. The largest footprint was a region, up to 135 bp in length, located at the 3%-boundary from bp 9018 to 9152. In this region, DNA in erythroid K562 cells was protected from damage by the footprinting agents compared to purified genomic DNA. The protection was confirmed by the use of four footprinting agents, C3-AA, chlorambucil, hedamycin and bleomycin, that have different mechanisms of action. The protection could be due to a positioned nucleosome at the 3%boundary of HS-2. The size of the protected region, 135 bp, is close to the length of DNA in a nucleosome core, 146 bp [1]. Two agents, hedamycin and bleomycin, have selective activity in the linker region of a nucleosome [18,27–
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29,41]. In addition to the size of the protected region and properties of footprinting agents used, the location of protected region is at the boundary of the DHS. It is expected that a positioned nucleosome exists at the boundary of a DHS as is found in the yeast PHO5 and PHO3 genes [10], the promoter of Drosophila hsp 26 gene [11], the enhancer of rat prolactin gene [12] and the 5%-boundary region of the human b-globin LCR HS-2 [18]. Non-erythroid HeLa cells showed a different damage pattern to erythroid K562 cells. The damage pattern for HeLa cells was similar to purified DNA in the 3%-boundary region of HS-2 from bp 8950 to 9170. In this putative positioned nucleosome region, HeLa cells were damaged similarly to purified DNA. This implies that in HeLa cells, there are no positioned nucleosomes in this region. The DNA in this sequence appears to be present as a random array of nucleosomes. A
Fig. 5. Map of footprints at the 3%-boundary of LCR HS-2. The footprints observed with various footprinting agents are indicated as rectangles with the beginning and end of the footprint in base pairs. NPP is the putative nucleosome positioning protein. The location of primers designed for LMPCR are shown as arrows.
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similar nucleosome array was found in a tissuespecific enhancer of the rat prolactin gene [12]. There are alternative explanations for the large footprint at the 3%-end of HS-2. First, the footprint might not be caused by a nucleosome core but might consist of a large protein complex at the boundary of the HS site. Second, the footprint could be unconnected with the structure of the HS site and could have a separate function. The former explanation is a variation on our preferred hypothesis and at present there are no likely protein candidates. The latter explanation is possible but appears unlikely given the location of the footprint. There was a small footprint of 16 bp at bp 8985–9000 with chlorambucil and C3-AA. This region includes a binding motif for the erythroid-specific factor GATA-1. Because protection was only observed in erythroid K562 cells and not in non-erythroid HeLa cells, it is presumed that the erythroid-specific GATA-1 (or GATA-2) protein is bound at this region. How is the nucleosome translationally positioned at the 3%-boundary of the HS-2 sequence in K562 cells? The DNA sequence might be responsible for positioning the nucleosome. However, the DNA sequence itself is not thought to be sufficient because the nucleosome footprint is not found in HeLa cells. The more likely possibility is the binding of an erythroid-specific protein bound in the region. A candidate for this role is the erythroid-specific GATA-1 protein. There is a GATA-1 binding motif next to the positioned nucleosome and a footprint is found at this motif only in erythroid K562 cells. In addition it has been shown that the GATA-1 protein is capable of displacing a nucleosome [42]. Thus it is probable that the GATA-1 protein plays a crucial role in this process. In cells, nucleosomes can slide along the DNA— a process that requires ATP [9]. In order to maintain a nucleosome-free HS site, a mechanism must exist to prevent sliding. A likely candidate for this role would be a protein (GATA-1) that binds to DNA at the HS site boundary and prevents nucleosome sliding (Fig. 5). Another possibility for positioning is the 100bp footprint (bp 8841– 8940) observed at the
AT-rich region. The region was protected from damage with the four different nitrogen mustard analogues and bleomycin in K562 cells. It is thought that a large protein or protein complex is bound at this region. Although it has not been identified in Matrix Attachment Region (MAR) scans of the b-globin gene cluster, it does show sequence similarity to other MARs in the b-globin cluster. There are AT-rich regions in each of the other b-globin LCR HSs. The involvement of a MAR in the maintenance of a nucleosome-free hypersensitive site is highly attractive because of the connection between chromatin loops and the control of gene expression [5]. It is possible that a MAR complex could mark the boundary of the HS and serve as a barrier to the sliding of nucleosomes. An alternative explanation for the 100-bp footprint is the binding of a nucleosome core. However, this is unlikely because the footprint is significantly smaller than the 146 bp expected from the binding of the histone octamer. A further interpretation of the results is that histone octamers could be present in the DHS of HS-2 but are modified by SWI/SNF complexes or histone acetylation. In this model the HS-2 would consist of a modified, positioned nucleosome array that can bind transcription factors [43,44]. The GATA-1 protein could define the 3%-end of modified chromatin in HS-2. In addition the GATA-1 protein could recruit the SWI/SNF complexes or histone acetylases to the HS-2 to create the DHS. In conclusion, the chromatin structure at the 3%-end of HS-2 was investigated utilising various DNA damaging agents. The evidence suggests that a nucleosome is positioned at the 3%boundary in erythroid K562 cells and the erythroid-specific GATA-1 protein is bound at sequences adjacent to the nucleosome. It is proposed that GATA-1 positions the nucleosome by preventing movement of nucleosome into the HS-2. Thus, in this manner, a DHS is maintained as a nucleosome-free region where transcription factors can access the DNA and stimulate transcription.
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Acknowledgements This work was supported by the Australian Research Council, the NHMRC, and by an Overseas Postgraduate Research Scholarship to AK.
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[31] J. Kross, W.D. Henner, S.M. Hecht, W.A. Haseltine, Specificity of deoxyribonucleic acid cleavage by bleomycin, phleomycin, and tallysomycin, Biochemistry 21 (1982) 4310 – 4318. [32] V. Murray, R.F. Martin, Comparison of the sequence specificity of bleomycin cleavage in two slightly different DNA sequences, Nucleic Acids Res. 13 (1985) 1467 – 1481. [33] V. Murray, L. Tan, J. Matthews, R.F. Martin, The sequence specificity of bleomycin damage in three cloned DNA sequences that differ by a small number of base substitutions, J. Biol. Chem. 263 (1988) 12854 – 12859. [34] A.S. Prakash, A.G. Moore, V. Murray, C. Matias, W.D. McFadyen, G. Wickham, Comparison of the sequence selectivity of the DNA-alkylating pluramycin antitumour antibiotics DC92-B and hedamycin, Chem. Biol. Interact. 95 (1995) 17 – 28. [35] E.J. Benz, M.J. Murnane, B.L. Tonkonow, B.W. Berman, E.M. Mazur, C. Cavallesco, T. Jenko, E.L. Snyder, B.G. Forget, R. Hoffman, Embryonic-fetal erythroid characteristics of a human leukemic cell line, Proc. Natl. Acad. Sci. USA 77 (1980) 3509 –3513. [36] P.R. Mueller, B. Wold, In vivo footprinting of a muscle specific enhancer by ligation mediated PCR, Science 246 (1989) 780 – 786. [37] G.P. Pfeifer, S.D. Steigerwald, P.R. Mueller, B. Wold, A.D. Riggs, Genomic sequencing and methylation analysis by ligation mediated PCR, Science 246 (1989) 810 – 813.
[38] T.R. Rutherford, D.J. Weatherall, Deficient heme synthesis as the cause of noninducibility of hemoglobin synthesis in a friend erythroleukemia cell line, Cell 16 (1979) 415 – 423. [39] V. Murray, R.F. Martin, The sequence specificity of bleomycin-induced DNA damage in intact cells, J. Biol. Chem. 260 (1985) 10389 – 10391. [40] M.D. Temple, M.J. Cairns, A. Kim, V. Murray, Protein – DNA footprinting of the human epsilon-globin promoter in intact human cells using nitrogen mustard analogues and other DNA-damaging agents, Biochim. Biophys. Acta 1445 (1999) 245 – 256. [41] B.L. Smith, G.B. Bauer, L.F. Povirk, DNA damage induced by bleomycin, neocarzinostatin, and melphalan in a precisely positioned nucleosome, J. Biol. Chem. 269 (1994) 30587 – 30594. [42] J. Boyes, J. Omichinski, D. Clark, M. Pikaart, G. Felsenfeld, Perturbation of nucleosome structure by the erythroid transcription factor GATA-1, J. Mol. Biol. 279 (1998) 529 – 544. [43] M. Truss, J. Bartsch, A. Schelbert, R.J. Hache, M. Beato, Hormone induces binding of receptors and transcription factors to a rearranged nucleosome on the MMTV promoter in vivo, EMBO J. 14 (1995) 1737 – 1751. [44] R. Candau, S. Chavez, M. Beato, The hormone responsive region of mouse mammary tumor virus positions a nucleosome and precludes access of nuclear factor I to the promoter, J. Steroid Biochem. Mol. Biol. 57 (1996) 19 – 31.
Chromatin structure at the 3%-boundary of the human b-globin locus control region hypersensitive site-2 AeRi Kim, Vincent Murray * School of Biochemistry and Molecular Genetics, Uni6ersity of New South Wales, Sydney, NSW 2052, Australia Received 17 April 2001; accepted 19 June 2001
Abstract Chromatin structure was examined at the 3%-boundary region of the human b-globin locus control region hypersensitive site-2 (LCR HS-2) using several footprinting agents. Erythroid K562 cells (possessing HS-2) were damaged by the footprinting agents: hedamycin, bleomycin and four nitrogen mustard analogues. Purified DNA and non-erythroid HeLa cells (lacking HS-2) were also damaged as controls for comparison with K562 cells. The comparison between intact cells and purified DNA showed several protected regions in K562 cells. A large erythroid-specific protected region of 135 bp was found at the boundary of HS-2. The length of this protected region (135 bp) was close to that of DNA contained in a nucleosome core (146 bp). Another two protected regions were found upstream of the protected region. A 16-bp erythroid-specific footprint co-localised with a GATA-1 motif— this indicated that the GATA-1 protein could be involved in positioning the nucleosome. Further upstream, a 100-bp footprint coincided with an AT-rich region. Thus our footprinting results suggest that the 3%-boundary of LCR HS-2 is flanked by a positioned nucleosome and that an erythroid-specific protein binds to the sequence adjacent to the nucleosome and acts to position the nucleosome at the boundary of the hypersensitive site. Crown Copyright © 2001 Published by Elsevier Science Ltd. All rights reserved. Keywords: Nucleosome; Genomic footprinting; GATA-1; Bleomycin; Hedamycin; Ligation-mediated PCR
1. Introduction The fundamental unit of chromatin structure is the nucleosome which consists of a core region and a linker region. Approximately 146 bp of Abbre6iations: bp, base pair; DHS, DNase I hypersensitive site; HS-2, hypersensitive site-2; LMPCR, ligation-mediated polymerase chain reaction; LCR, locus control region. * Corresponding author. Tel.: + 61-2-9385-2028; fax: +612-9385-1483. E-mail address: [email protected] (V. Murray).
DNA interacts with the histone octamer to form a core particle. The linker region contains histone H1 and linker DNA that connects core particles [1]. Chromatin structure is thought to play a crucial role in the regulation of gene expression [2]. Transcription factors can bind to DNA in three general ways: (1) a factor will only bind in the presence of a nucleosome; (2) a bound nucleosome does not prevent binding of the factor; (3) a factor will only bind in the absence of a nucleosome. In this latter case, the absence of a
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nucleosome leads to a DNase I hypersensitive site (DHS) that appears to be a very important determinant in the control of gene expression. DNase I hypersensitive sites (DHS) are a common feature at enhancers and promoters of expressed genes [3,4]. DHSs are thought to be nucleosome-free regions that permit the binding of transcription factors to DNA and lead to the induction of gene expression [5]. The pivotal role of DHSs has led to a great deal of research effort into the processes that define these regions and the subsequent mechanism(s) of gene activation. An advance in our understanding of the subject has been the discovery of the SWI/ SWF complex [2,6– 8]. This and other complexes permit the remodelling of chromatin. Using ATP as an energy source, the structure of chromatin is altered by these complexes to allow the interaction of transcription factors with DNA. These very large protein complexes have been found in yeast, Drosophila and humans. The discovery of these complexes provides an insight into the dynamic nature of chromatin where proteins are
added or removed in order to affect the level of gene expression. This work with SWI/SWF complexes gives an insight into the creation of DHSs where chromatin remodelling factors can lead to the removal of nucleosomes. In order to maintain a nucleosome-free region at a DHS, there must be a mechanism to prevent a nucleosome from sliding into the DHS. The sliding of nucleosomes is an energy requiring process in cells [9]. A DHS is often flanked by positioned nucleosomes [10–12]. There are two ways that a positioned nucleosome can arise. First, the DNA sequence has the ability to position a nucleosome. In vitro reconstitution experiments showed that nucleosomes can be precisely positioned at the same sequence as found in vivo [13]. Certain DNA sequences appear to have the ability to translationally position a nucleosome [14]. Second, a protein factor can position nucleosomes (Fig. 1). The protein factor usually binds to the sequence adjacent to a positioned nucleosome. Nucleosome positioning by a protein factor has been observed in several genes [15,16].
Fig. 1. Map of LCR HS-2 in the human b-globin gene cluster. The five expressed b-globin genes are shown as well as promoter elements. The LCR HS regions are indicated. The expanded view of the LCR HS-2 shows a ‘positioned’ nucleosome at the 5%- and 3%-boundaries as well as ‘positioning’ proteins. The location of transcription factor binding elements is also indicated. There are two GATA-1 motifs between bp 8940 and 9000 (white and grey box). The grey box at bp 8988 is the putative nucleosome ‘positioning’ protein. The black boxes indicate a tandem NF-E2 motif while the striped boxes represent CACC motifs.
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Cell- or tissue-specifically positioned nucleosomes have been observed in mammalian genes [12,17,18]. There are at least four DHSs in the 5%-locus control region (LCR) of the human b-globin gene cluster (Fig. 1). The LCR acts as an enhancer and is responsible for erythroid-specific expression of the b-globin gene cluster [5,19]. Hypersensitive site-2 (HS-2) is a nucleosome-free region that is approximately 600 bp in length [20]. The binding sequences for erythroid-restricted and ubiquitous transcription factors are present in HS-2 [21– 25]. The NF-E2, GATA-1 and CACC binding motifs have all been shown to be occupied in erythroid cells as determined by LMPCR footprinting. The NF-E2 and GATA-1 motifs bind the erythroid-restricted NF-E2 and GATA-1 transcription factors, respectively. The CACC, GT-I and GT-II motifs are thought to bind different members of the ubiquitous Sp1 family of transcription factors. The transcription factor NF-E2 can displace a nucleosome from reconstituted chromatin in the HS-2 sequence [26]. In this study, we investigated the chromatin structure at the 3%-boundary region of HS-2 in the human b-globin gene cluster. Various DNA damaging agents (nitrogen mustard analogues, hedamycin and bleomycin) were used to investigate the chromatin structure of the HS-2 boundary. The ability of these compounds to footprint transcription factors has been demonstrated in the b-globin gene cluster [23– 25]. Hedamycin and bleomycin can also detect the presence of a positioned nucleosome since they preferentially damage DNA in the linker region of nucleosomes [18,27– 29]. This property of bleomycin and hedamycin is useful in the study of chromatin structure at the DHS boundary because a positioned nucleosome is expected at this position. Four nitrogen mustard analogues were used in this study. Chlorambucil is used as an anti-tumour drug. The other three are ‘DNA-directed’ nitrogen mustard analogues that have an attached DNA-affinic moiety, either acridine or amsacrine [25]. The four nitrogen mustards preferentially damage at guanine and, to a lesser extent, adenine bases [25,30]. Bleomycin cleaves mainly at GT, GC and GA dinucleotides [30– 33] while
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hedamycin has a preference for guanine bases [24,29,30,34]. Furthermore, all damaging agents chosen for this study can enter intact human cells without any pre-treatment— this is a major advantage of these compounds. Two kinds of human cell line, K562 and HeLa, were used to examine chromatin structure at the 3%-boundary of HS-2. The erythroid K562 cell line displays the LCR HS-2 and expresses o- and g-globin genes [35]. The non-erythroid HeLa cell line lacks the LCR HS-2, does not express globin genes, and was utilised as a control for comparison with K562 cells. Damaged cellular DNA was analysed by the ligation-mediated polymerase chain reaction (LMPCR) procedure at base pair resolution [36,37]. We have previously examined chromatin structure at the 5%-end of the human b-globin LCR HS-2 and found a large footprint consistent with the presence of a positioned nucleosome [18]. An erythroid-specific ‘positioning’ protein was thought to be responsible for maintaining the nucleosome at this position (Fig. 1). This study investigated chromatin structure at the 3%boundary region of the human b-globin LCR HS-2 in intact human cells and attempted to answer the question, ‘What molecular structure is present at the boundary of a mammalian DNase I hypersensitive site?’ This study provides evidence that a nucleosome is positioned at the 3%boundary in erythroid K562 cells by erythroidspecific ‘positioning’ protein(s). 2. Materials and methods
2.1. Cell culture K562 cells were grown in RPMI 1640 medium (with no antibiotics) containing 10% foetal calf serum. Hemin was added to the medium (50 mM) for 4 days to induce b-globin expression [38]. The harvest of uninduced and induced K562 cells was carried out at a density of 106 cells/ml. HeLa cells were grown in RPMI 1640 medium containing 2.5% newborn calf serum and 2.5% foetal calf serum. HeLa cells at 95% confluence were harvested after washing with phosphate buffered saline.
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2.2. Cell damage Approximately 107 cells were washed with phosphate buffered saline and treated with an appropriate concentration of nitrogen mustard analogue, hedamycin or bleomycin at 37 °C [23 – 25]. The treatment with a nitrogen mustard analogue or hedamycin was performed in the dark. Cells were incubated for 3 h with a nitrogen mustard analogue and for 1 h with hedamycin or bleomycin. The purification of DNA from the treated cells was carried out as described previously by Murray and Martin [39].
2.3. Purified DNA damage Purified human DNA was damaged with appropriate concentrations of nitrogen mustard analogues, hedamycin or bleomycin [23– 25]. The damage reaction was performed at 37 °C for 1 h with bleomycin or the nitrogen mustard analogues and for 30 min with hedamycin. For the bleomycin reaction, Fe2 + SO4 was included at the equivalent bleomycin concentration. Damaged DNA was purified by ethanol precipitation.
2.4. LMPCR primer sequences Human b-globin LCR HS-2 sequence specific primers were designed for analysis of the 3%boundary. The primers for the coding strand were 5%-GTATGTGAGCATGTGTCCTCTAACA-3% (first strand synthesis) and 5%-GAGCATGTGTCCTCTAACAGCACAG-3% (PCR). The primers for the non-coding strand were 5%-ATGTTCTCAGCCTAGAGTGATGACT-3% (first strand synthesis) and 5%-AGCCTAGAGTGATGACTCCTATCTG-3% (PCR).
2.5. LMPCR LMPCR was performed to investigate the sites and intensity of damage in genomic DNA treated with nitrogen mustard analogues, hedamycin and bleomycin at base pair resolution. The LMPCR was performed with the modifications described by Cairns and Murray [23]. Damaged DNA was amplified with two sets of sequence-specific
primers and the linker primers. The amplified fragments were analysed on a 6% (w/v) polyacrylamide sequencing gel. A G+ A Maxam/Gilbert chemical sequencing ladder (amplified by the same primers) was also included on the gel.
3. Results The 3%-boundary region of the human b-globin LCR HS-2 was examined with various footprinting agents in intact cells and purified DNA. The damaged DNA was amplified using a set of primers for the coding strand and non-coding strand, respectively and analysed at base pair resolution. The coding strand was examined from bp 9130 to 8950 and on the non-coding strand from bp 8750 to 9150. The intensity of damage was compared in intact cells and purified genomic DNA (Figs. 2–4). Protection occurred where the damage intensity was reduced in cells compared to purified DNA and conversely enhancement occurred where damage was increased in cells compared to purified DNA. Undamaged control DNA was also amplified to check the background level of DNA damage. G+ A sequencing fragments were included to determine the exact DNA sequence at the damage site. In a comparison between uninduced K562 cells and induced K562 cells, no significant differences were found for any of the six damaging agents as found previously [23,25,40].
3.1. Coding strand The sequencing gel in Fig. 2 shows DNA damage by the nitrogen mustard analogue, C3-AA, on the coding strand of purified DNA and intact cells. A comparison between purified DNA and intact cells revealed a large protected region in uninduced and induced K562 cells. A region of 104 bp between bp 9045 and bp 9148 was protected from C3-AA damage in K562 cells (compared to purified DNA) except for a small region from bp 9064 to 9069. For chlorambucil in K562 cells, the intensity of damage was reduced in the region from bp 9045 to 9152 covering 108 bp except for the region from bp 9064 to 9069. The
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The sequencing gel in Fig. 3 shows damage by hedamycin on the coding strand of purified DNA and intact cells. A comparison of hedamycin damage in purified DNA and intact cells showed significant differences. In K562 cells a large region spanning 117 bp from bp 9019 to 9135 was protected with respect to purified DNA. Upstream of the protected region, enhanced hedamycin damage was seen with K562 cells centred at bp 8997. Protection was also observed in K562 cells dam-
Fig. 2. Phosphorimage of a DNA sequencing gel comparing nitrogen mustard analogue damage on the coding strand at the boundary of LCR HS-2 in four different environments. Lanes control P, H, U and I were undamaged controls for purified human DNA, HeLa cells, uninduced K562 cells, and induced K562 cells, respectively. C3-AA damage lanes P, H, U and I were derived from purified human DNA (15 mM), HeLa cells (120 mM), uninduced K562 cells (120 mM), induced K562 cells (120 mM), respectively. Chlorambucil damage lanes P, H, U and I were derived from purified human DNA (300 mM), HeLa cells (1 mM), uninduced K562 cells (200 mM), induced K562 cells (400 mM), respectively. Lane GA was a G + A sequencing reaction with purified human DNA.
protected regions in K562 cells were the same for C3-AA and chlorambucil damage. Upstream of the large protected region, there were areas representing enhancement and protection of damage in K562 cells. The two regions centred at bp 8983 and bp 9018 exhibited enhancement of damage with both C3-AA and chlorambucil. The region between the two enhanced damage regions bp 8985 and 9000 was protected in cells compared to purified DNA. This latter sequence contains a GATA-1 binding motif.
Fig. 3. Phosphorimage of a DNA sequencing gel comparing hedamycin damage on the coding strand at the boundary of LCR HS-2 in four different environments. Lanes control P, H, U and I were undamaged controls for purified human DNA, HeLa cells, uninduced K562 cells, and induced K562 cells, respectively. Hedamycin damage lanes P, H, U and I were derived from purified human DNA (4 mM), HeLa cells (20 mM), uninduced K562 cells (20 mM), induced K562 cells (20 mM), respectively. Lane GA was a G +A sequencing reaction with purified human DNA.
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Fig. 4. Phosphorimage of a DNA sequencing gel comparing nitrogen mustard analogue damage on the non-coding strand at the boundary of LCR HS-2 in four different environments. Lanes control P, H, U and I were undamaged controls for purified human DNA, HeLa cells, uninduced K562 cells, and induced K562 cells, respectively. C3-AA damage lanes P, H, U and I were derived from purified human DNA (20 mM), HeLa cells (120 mM), uninduced K562 cells (120 mM), induced K562 cells (120 mM), respectively. Chlorambucil damage lanes P, H, U and I were derived from purified human DNA (150 mM), HeLa cells (1 mM), uninduced K562 cells (200 mM), induced K562 cells (400 mM), respectively. Lane GA was a G + A sequencing reaction with purified human DNA.
aged by bleomycin (data not shown)— a region of 123 bp, between bp 9018 and 9140, was protected in K562 cells compared to purified DNA. A summary of the data obtained with all the DNA damaging agents is shown in Fig. 5. No relative protection was found with HeLa cells (compared to purified DNA) damaged by hedamycin, bleomycin, C3-AA and chlorambucil for the whole analysed region on the coding strand.
3.2. Non-coding strand Damage by the nitrogen mustard analogues, C3-AA and chlorambucil, on the non-coding
strand revealed two areas of protection (Fig. 4). A large protected region was observed in K562 cells damaged by C3-AA and chlorambucil between bp 8865 and 8940 in K562 cells. There was another small protected region at bp 8845–8850 in K562 cells. The non-coding strand was also investigated with bleomycin. Three protected regions were observed in K562 cells: a large region of 91 bp between bp 8850 and 8940; and two smaller regions between bp 8790 and 8791 and bp 8810 and 8817 (Fig. 5). The latter two regions coincided with GT-I and GT-II motifs, respectively. Analysis of the damage by the nitrogen mustard analogues, C50-AMSA and C20-AMSA, on the non-coding strand of purified DNA and intact cells showed protected regions in K562 cells (relative to purified DNA). Four regions, bp 8751– 8757, bp 8817–8827, bp 8841–8940, and bp 9000–9100 in intact cells were protected from damage (Fig. 5). The large 100-bp protected region, bp 8841–8940, was at a similar sequence to that found with bleomycin, C3-AA and chlorambucil. The large 101-bp protected region from bp 9000 to 9100 was also observed on the other strand with hedamycin, bleomycin, C3-AA and chlorambucil. All of the protected regions observed in K562 cells (see above), were absent in HeLa cells except for protected regions bp 8860–8900, 8817–8827 and 8751–8757 that were observed with C50AMSA and C20-AMSA (data not shown).
4. Discussion Chromatin structure at the 3%-boundary region of the b-globin LCR HS-2 was examined in intact human cells (Fig. 5). Approximately 450 bp was examined in this region. The footprints were mapped at base pair resolution. Three major footprints were observed: (i) a large 135-bp region from bp 9018 to 9152; (ii) a 16-bp region from bp 8985 to 9000; a 100-bp region from bp 8841 to 8940. We postulate that the large 135-bp footprint is caused by a positioned nucleosome core, and the 16-bp footprint by a nucleosome positioning protein (GATA-1), while the 100-bp footprint
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contains an AT-rich region. Each footprint was confirmed by use of more than one footprinting agent. The size of these footprints has been determined by combining results from several DNA damaging agents. The largest footprint was a region, up to 135 bp in length, located at the 3%-boundary from bp 9018 to 9152. In this region, DNA in erythroid K562 cells was protected from damage by the footprinting agents compared to purified genomic DNA. The protection was confirmed by the use of four footprinting agents, C3-AA, chlorambucil, hedamycin and bleomycin, that have different mechanisms of action. The protection could be due to a positioned nucleosome at the 3%boundary of HS-2. The size of the protected region, 135 bp, is close to the length of DNA in a nucleosome core, 146 bp [1]. Two agents, hedamycin and bleomycin, have selective activity in the linker region of a nucleosome [18,27–
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29,41]. In addition to the size of the protected region and properties of footprinting agents used, the location of protected region is at the boundary of the DHS. It is expected that a positioned nucleosome exists at the boundary of a DHS as is found in the yeast PHO5 and PHO3 genes [10], the promoter of Drosophila hsp 26 gene [11], the enhancer of rat prolactin gene [12] and the 5%-boundary region of the human b-globin LCR HS-2 [18]. Non-erythroid HeLa cells showed a different damage pattern to erythroid K562 cells. The damage pattern for HeLa cells was similar to purified DNA in the 3%-boundary region of HS-2 from bp 8950 to 9170. In this putative positioned nucleosome region, HeLa cells were damaged similarly to purified DNA. This implies that in HeLa cells, there are no positioned nucleosomes in this region. The DNA in this sequence appears to be present as a random array of nucleosomes. A
Fig. 5. Map of footprints at the 3%-boundary of LCR HS-2. The footprints observed with various footprinting agents are indicated as rectangles with the beginning and end of the footprint in base pairs. NPP is the putative nucleosome positioning protein. The location of primers designed for LMPCR are shown as arrows.
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similar nucleosome array was found in a tissuespecific enhancer of the rat prolactin gene [12]. There are alternative explanations for the large footprint at the 3%-end of HS-2. First, the footprint might not be caused by a nucleosome core but might consist of a large protein complex at the boundary of the HS site. Second, the footprint could be unconnected with the structure of the HS site and could have a separate function. The former explanation is a variation on our preferred hypothesis and at present there are no likely protein candidates. The latter explanation is possible but appears unlikely given the location of the footprint. There was a small footprint of 16 bp at bp 8985–9000 with chlorambucil and C3-AA. This region includes a binding motif for the erythroid-specific factor GATA-1. Because protection was only observed in erythroid K562 cells and not in non-erythroid HeLa cells, it is presumed that the erythroid-specific GATA-1 (or GATA-2) protein is bound at this region. How is the nucleosome translationally positioned at the 3%-boundary of the HS-2 sequence in K562 cells? The DNA sequence might be responsible for positioning the nucleosome. However, the DNA sequence itself is not thought to be sufficient because the nucleosome footprint is not found in HeLa cells. The more likely possibility is the binding of an erythroid-specific protein bound in the region. A candidate for this role is the erythroid-specific GATA-1 protein. There is a GATA-1 binding motif next to the positioned nucleosome and a footprint is found at this motif only in erythroid K562 cells. In addition it has been shown that the GATA-1 protein is capable of displacing a nucleosome [42]. Thus it is probable that the GATA-1 protein plays a crucial role in this process. In cells, nucleosomes can slide along the DNA— a process that requires ATP [9]. In order to maintain a nucleosome-free HS site, a mechanism must exist to prevent sliding. A likely candidate for this role would be a protein (GATA-1) that binds to DNA at the HS site boundary and prevents nucleosome sliding (Fig. 5). Another possibility for positioning is the 100bp footprint (bp 8841– 8940) observed at the
AT-rich region. The region was protected from damage with the four different nitrogen mustard analogues and bleomycin in K562 cells. It is thought that a large protein or protein complex is bound at this region. Although it has not been identified in Matrix Attachment Region (MAR) scans of the b-globin gene cluster, it does show sequence similarity to other MARs in the b-globin cluster. There are AT-rich regions in each of the other b-globin LCR HSs. The involvement of a MAR in the maintenance of a nucleosome-free hypersensitive site is highly attractive because of the connection between chromatin loops and the control of gene expression [5]. It is possible that a MAR complex could mark the boundary of the HS and serve as a barrier to the sliding of nucleosomes. An alternative explanation for the 100-bp footprint is the binding of a nucleosome core. However, this is unlikely because the footprint is significantly smaller than the 146 bp expected from the binding of the histone octamer. A further interpretation of the results is that histone octamers could be present in the DHS of HS-2 but are modified by SWI/SNF complexes or histone acetylation. In this model the HS-2 would consist of a modified, positioned nucleosome array that can bind transcription factors [43,44]. The GATA-1 protein could define the 3%-end of modified chromatin in HS-2. In addition the GATA-1 protein could recruit the SWI/SNF complexes or histone acetylases to the HS-2 to create the DHS. In conclusion, the chromatin structure at the 3%-end of HS-2 was investigated utilising various DNA damaging agents. The evidence suggests that a nucleosome is positioned at the 3%boundary in erythroid K562 cells and the erythroid-specific GATA-1 protein is bound at sequences adjacent to the nucleosome. It is proposed that GATA-1 positions the nucleosome by preventing movement of nucleosome into the HS-2. Thus, in this manner, a DHS is maintained as a nucleosome-free region where transcription factors can access the DNA and stimulate transcription.
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Acknowledgements This work was supported by the Australian Research Council, the NHMRC, and by an Overseas Postgraduate Research Scholarship to AK.
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