Effect of deletion of the DNase I hypersensitive sites on the transcription of chicken Ig-b gene and on the maintenance of active chromatin state in the Ig-b locus Hiroki Matsudo1, Kyoichi Osano1, Hiroshi Arakawa2 and Masao Ono1 Department of Life Science, and Frontier Project ‘Life’s Adaptation Strategies to Environmental Changes’, Rikkyo University, College of Science, Toshima-ku, Tokyo, Japan GSF, Institute for Molecular Radiobiology, Neuherberg-Munich, Germany Keywords chicken Ig-b gene; DNase I hypersensitive sites; DT40; histone acetylation; transcription Correspondence M Ono, Department of Life Science, College of Science, Rikkyo University, Toshima-ku, Tokyo 171-8501, Japan Fax ⁄ Tel: +81 339852387 E-mail: mono@rikkyo.ac.jp (Received 11 September 2004, revised November 2004, accepted 15 November 2004) doi:10.1111/j.1742-4658.2004.04482.x The role of DNase I hypersensitive sites (DHSs) in transcription of the B cell-specific Ig-b gene and in maintenance of active chromatin state in the Ig-b locus were examined A total of 10 DHSs were divided into four regions, and each region was deleted separately in chicken B lymphocytederived DT40 cells Deletion of three DHSs located between the Ig-b promoter and its upstream Na channel gene, resulted in the absence of Ig-b mRNA Three regions except the region in the Na channel gene were involved in the transcription of Ig-b gene The enhancing activity of DHSs as determined by transient transfection assays did not always correlate with the effect of DHS deletion on the expression level of Ig-b mRNA In each deletion, cells contained the same DHSs as observed in the predeletion cells, indicating that deleted DHSs did not participate in the maintenance of DT40-specific DHSs Enhanced acetylation of H3 and H4 histones at the Ig-b promoter and at DT40-specific DHSs was observed in cells in which DHSs between the Na channel gene and Ig-b promoter were deleted; therefore, these DHSs are prerequisite for transcription of the Ig-b gene but not required for the maintenance of active chromatin state in the Ig-b locus Thus, epigenetic factors required for the maintenance of the active chromatin state are suggested to reside in other regions than those deleted in this study In vertebrate cells, chromatin of active or potentially active genes and flanking regions are characterized by (a) sensitivity to DNase I [1–6]; (b) the presence of cell type-specific DNase I hypersensitive sites (DHSs) [7,8]; and (c) core histone modifications such as acetylation and methylation specific for active chromatin [9–11] These characteristics have been reported specifically for loci such as b-globin [1,3,12,13], and Ig-b ⁄ growth hormone (GH) [14–16] Thus, it is possible to differentiate between active, or potentially active, and inactive chromatin states by comparing differences in chromatin sensitivity to nuclease and histone modifications However, the mechanism by which the chromatin structure is modified in order to initiate cell type-specific gene expression as well as the maintenance of this active state in differentiated cells remains to be elucidated [17–19] Along with membrane immunoglobulin and Ig-a ⁄ mb1, Ig-b is a component of the antigen receptor complex and belongs to the immunoglobulin superfamily [20,21] The Ig-b gene is expressed early in B cell development [22–24] The mechanism of B cellspecific expression of mouse and human Ig-b genes has been studied mainly in the proximal promoter region, and several cis-elements and transacting factors have Abbreviations ChIP, chromatin immunoprecipitation; DHS, DNase I hypersensitive site; GH, growth hormone; LCR, locus control region; R-PCR, real-time polymerase chain reaction 422 FEBS Journal 272 (2005) 422–432 ª 2004 FEBS H Matsudo et al been identified [25–27] However, these studies have not taken the state of chromatin into consideration Cell type-specific DHSs not only correspond to promoters and enhancers but also are likely to participate in the establishment and maintenance of an active chromatin state [18,28,29] In addition to their presence in and adjacent to active or potentially active genes, cell type-specific DHSs are found in regions situated far upstream or downstream of a gene [1,7,8] To identify novel regions participating in cell type-specific transcription of the rat Ig-b gene, wide range examination was carried out to find DHSs specific for Ig-b producing cells The transcriptional enhancing activities of the DHSs were examined by transient transfection [30] Three regions having transcriptional enhancing activity were found in the intergenic region between the Ig-b and GH genes A member of the OCT family transcription factors appeared to be involved in the transactivation of the region which had the highest enhancing activity of the three DHSs The state of acetylation in H3 and H4 histones and dimethylation in the H3 histone Lys4 residue were examined by chromatin immunoprecipitation (ChIP) [31] The active promoter and cell type-specific DHS with enhancing activity showed active histone modifications The sensitivity of chromatin to DNase I was measured by real-time PCR (R-PCR) [16] The regions with active histone modifications were again sensitive to DNase I To determine the in vivo role of DHSs in B cell-specific transcription of the Ig-b gene, genetic studies such as transgenics and gene targeting are required Chicken B lymphocyte-derived DT40 cells are particularly useful for gene targeting because of the high rate of homologous recombination [32,33] Chicken Ig-b gene is expressed in DT40 cells [34] Previously, the location of DHSs was surveyed in a 40 kb region between 19 kb upstream and 21 kb downstream from the transcriptional start site of the Ig-b gene [15] Twelve DT40-specific DHSs were found: three in the upstream Na channel gene, two between the Na channel and Ig-b genes, one at the transcriptional start site, one in the first intron of the Ig-b gene, four between Ig-b and its downstream growth hormone (GH) genes, and one in the downstream region of the GH gene Transcriptional enhancing activity, as determined by transient transfection, was associated with DHSs in the Na channel gene and in the first intron of the Ig-b gene Furthermore, the acetylation status of H3 and H4 histones was examined [15] In the present study, 10 DT40-specific DHSs found in the Ig-b locus were divided into four groups based on location For each group, a deletion construct was introduced into DT40 cells Ig-b gene expression, acetylation of H3 and H4 FEBS Journal 272 (2005) 422–432 ª 2004 FEBS Deletion of hypersensitive sites in chicken Ig-b locus histones, and the remaining DHSs in the deleted cells were examined to determine the roles of these DHSs on the transcription of Ig-b gene and on the maintenance of the active chromatin state Results Generation of DT40/Cre cells with a long deletion in one allele of the Ig-b locus To genetically examine the mechanism of B cell-specific transcription of the Ig-b gene, we generated DT40 ⁄ Cre cells with a 16 kb deletion (16 kb Del) in one allele of the Ig-b locus (Fig 1) Introduction of the 16 kb Del construct into DT40 ⁄ Cre cells resulted in two deletion clones In one of these two clones, the Bsr gene between the loxP sequences was excised by Cre recombinase Following digestion of the genomic DNA with HindIII, homologous recombination and the excision of Bsr gene were confirmed by Southern hybridization using 0.9 kb DNA ()10.1 kb to )9.2 kb) as the probe (Fig 2) The sizes of the resulting bands for each clone (Fig 2B) were the same as expected from the restriction map (Fig 2A), and thus homologous recombination followed by excision of the Bsr gene was achieved Establishment of DT40/Cre cells expressing small Ig-b mRNA Initially, Ig-b protein appeared essential for the proliferation of DT40 cells To compensate for the absence of Ig-b mRNA, a chicken b-actin promoter-driven Ig-b gene whose transcript (1.5 kb) is shorter than wild type (1.7 kb) due to deletion of the 3¢ untranslated region, was introduced into 16 kb Del cells From several puromycin resistant clones, one clone containing wild type and mutant Ig-b mRNA at a : ratio was selected and used for subsequent studies (Fig 2C) Generation of DT40/Cre clones deleted in DT40-specific DHSs Ten DT40-specific DHSs are present in the 16 kb region of the Ig-b locus (Fig 1) [15] These DHSs were divided into the following four groups (Fig 1): region I, having transcriptional enhancing activity [15] in the Na channel gene ()7.8 kb to )6.0 kb; DHSs, )7.3 kb and )6.5 kb); region II, promoter and upstream region of Ig-b gene ()2.7 kb to +0.05 kb; DHSs, )2.1 kb, )1.0 kb, and kb); region III, in the first intron of the Ig-b gene (+0.6 kb to +1.1 kb; DHS, +0.9 kb); region IV, between Ig-b and GH genes (+4.5 kb to +6.7 kb; DHSs, +4.8 kb, +5.2 kb, +5.8 kb, and +6.1 kb) Dele423 Deletion of hypersensitive sites in chicken Ig-b locus H Matsudo et al Fig Organization of the chicken Ig-b locus and deleted regions Exons are indicated by rectangles, and intron and intergenic regions by solid lines Horizontal arrows represent transcriptional orientations Exon numbers are indicated The hypothesized organization of a portion of the Na channel gene for which the nucleotide sequence is unknown is represented by a dotted rectangle Sizes of the intergenic regions are shown in kb Upward arrows indicate DNase I hypersensitive sites (DHSs), distances from the transcriptional start site of the Ig-b gene in kb are indicated underneath Black arrow, DT40-specific; gray arrow, LMH-specific; white arrow, common in both DT40 and LMH DHS arrows with enhancing activity are enclosed by rectangles Positions of the eight targets for real-time PCR (R-PCR) are shown in the middle [15]: 1, DHS )7.3 kb; 2, DHS )6.5 kb; 3, Na channel exon 24; 4, Ig-b promoter; 5, DHS +0.9 kb; 6, DHS +5.2 kb; 7, DHS +6.1 kb; 8, GH exon At the bottom, positions of the arm sequences used for the targeting constructs are shown by black bars and regions deleted by dotted lines Numbers above the 5¢- and 3¢- ends of the region deleted are the distances from the transcriptional start site of the Ig-b gene tion constructs for regions I, II, and III were introduced into the 16 kb Del cells with the extra Ig-b gene, and between two and eight of the resulting clones were found to have deletions for each of these regions as judged by PCR To remove the Bsr gene from these clones by Cre recombinase, tentative recombinants were treated with tamoxifen and then blasticidin sensitive clones were obtained In the course of the study, Ig-b protein was found nonessential for the proliferation of DT40 cells (data not shown), and therefore deletion of region IV was carried out by the introduction of the region IV deletion construct into 16 kb Del cells Genomic Southern hybridization was performed to confirm deletions (Fig 3) For region I (Fig 3A), genomic DNA was digested with HindIII, and hybridized with a 0.9 kb probe for the Na channel gene; for regions II (Fig 3B) and III (Fig 3C), DNA was cut with EcoRI, and hybridized with Ig-b cDNA; for region IV (Fig 3D), SacI-digested DNA was hybridized with a probe for the GH gene In each clone, the sizes of the resulting bands that hybridized with the probe coincided with that estimated from the restric424 tion map, and thus establishment of clones lacking region I to IV was confirmed A 1.3 kb band in Fig 3A, and a 3.1 kb band in Fig 3D are derived from the 16 kb Del allele, while the 6.6 kb band in Fig 3B,C is from the extra Ig-b gene Expression of Ig-b mRNA in DT40/Cre clones deleted in DT40-specific DHSs The level of Ig-b mRNA in cells with a deletion in region I was the same as that in 16 kb Del cells (Fig 4A), and thus this region was shown to be nonessential for the transcription of Ig-b gene in DT40 cells Because region I enhances transcriptional activity when linked to the Ig-b promoter and introduced into DT 40 cells [15], activity of this region in vivo was demonstrated not to correlate with that observed in transient transfection assays Absence of Ig-b mRNA in cells with a deletion in region II (Fig 4A) demonstrated the essential role of this region in the transcription of the Ig-b gene Deletion of region III decreased the expression of Ig-b mRNA to 40% of the expression in predeletion cells (Fig 4A) Region III was found to enhance FEBS Journal 272 (2005) 422–432 ª 2004 FEBS H Matsudo et al Deletion of hypersensitive sites in chicken Ig-b locus A B C and contains four DHSs within a 1.3 kb region The level of Ig-b mRNA in deletion cells was reduced to 56% of that in 16 kb Del cells (Fig 4B) The proximal part of this region (+4.6 kb to +5.5 kb containing +4.8 kb and +5.2 kb DHSs) has no transcriptional enhancing activity in transient transfection assays, while the distal part (+5.6 kb to +6.4 kb including +5.8 kb and +6.1 kb DHSs) decreases the transcriptional activity of the Ig-b promoter to 50% of the activity observed with the promoter alone Thus, like region I, in vivo activity of region IV did not correlate with the activity observed in transient transfection assays These observations demonstrate that regions II, III, and IV are involved in the transcription of the Ig-b gene in DT40 cells, while region I is not Furthermore, the enhancing activity of DHSs as determined by transient transfection assays does not always correlate with the effect of DHS deletion on the expression level of Ig-b mRNA Presence of DHSs in the Ig-b locus in cells deleted in regions I–IV Fig Construction of DT40 ⁄ Cre cells lacking the 16 kb region in one Ig-b allele and expressing extra Ig-b mRNA shorter than the wild type (A) Organization of the Ig-b locus in DT40 ⁄ Cre cells lacking the 16 kb region between )9.0 kb and +6.7 kb 1, wild type allele; 2, 16 kb deletion (Del) allele with blasticidin resistance (Bsr) gene; 3, 16 kb Del allele without Bsr gene Position of the probe (0.9 kb; )10.1 kb HindIII to )9.2 kb AccI fragment) for hybridization is indicated by a black rectangle Size and position of the HindIII fragment hybridizable with the probe is shown below the map Exons and introns are indicated as in Fig (B) Genomic Southern hybridization HindIII digested DNA (2.5 lg) was separated by 0.75% agarose gel-electrophoresis and hybridized with the probe shown in (A) 1, DNA from wild type DT40 ⁄ Cre cells; 2, 16 kb Del allele with Bsr gene; 3, 16 kb Del allele without Bsr gene Restriction fragment size (given in kb) was determined by kHindIII Positions of the 1.3 kb, 2.7 kb, and 6.2 kb bands are shown in the right margin (C) Detection of the extra Ig-b mRNA by Northern hybridization Total RNA (3.0 lg) was prepared from cells containing the expression vector for extra Ig-b mRNA and Northern hybridized with Ig-b cDNA probe (nucleotides 312–1542; PmaCI ⁄ EcoRI) [34] 1, 16 kb Del allele without Bsr gene; 2, 16 kb Del allele without Bsr gene and with extra Ig-b construct Positions of 1.7 kb endogenous and 1.5 kb extra Ig-b mRNA are shown in the right margin transcriptional activity in transient transfection fourfold in DT40 cells [15], and thus, in contrast to region I, in vivo activity of this region correlated with the enhancing activity in transient transfection assays Region IV is located between the Ig-b and GH genes FEBS Journal 272 (2005) 422–432 ª 2004 FEBS DHSs in the Ig-b locus were examined by genomic Southern hybridization in cells with region II deletion (Fig 5) DHSs located at )13 kb, )7.3 kb, and )6.5 kb in the Na channel gene were present in the region II deletion cells, similar to the parent cells (Fig 5A) DHSs located between the Ig-b and GH genes, and DHSs at +10.3 kb and +13 kb positioned downstream of the GH gene were both present in region II deletion cells as well as in the predeletion cells (Fig 5C,D) Because the sequence from )2.7 kb to +0.05 kb was lost in region II deletion cells, no band corresponding to DHSs at )2.1 kb, )1.0 kb, and kb was observed, while a DHS at +0.9 kb was present (Fig 5B) In summary, deletion of DHSs in region II did not affect the presence of DHSs in the Ig-b locus DHSs in region I, III, and IV deletion cells were also examined DHSs detected between )13 kb and +13 kb in the Ig-b locus [15] were again present in all three types of deletions although DHSs located at the deletion region disappeared (data not shown), and thus regions I, III, and IV were shown not to participate in the maintenance of DT40-specific DHSs in the Ig-b locus Acetylation of H3 and H4 histones in cells deleted in region II Acetylation of H3 and H4 histones is enhanced in and around potentially active and transcribed genes 425 Deletion of hypersensitive sites in chicken Ig-b locus H Matsudo et al A B C D Fig Confirmation of DHS deletions by Southern hybridization Homologous recombination and the excision of Bsr gene by tamoxifen treatment was first examined by PCR and then confirmed by Southern hybridization Deleted regions: (A), I ()7.8 kb to )6.0 kb); (B), II ()2.7 kb to +0.05 kb); (C), III (+0.6 kb to +1.1 kb); (D), IV (+4.5 kb to +6.7 kb) Left, gene organization; right, autoradiogram of the Southern hybridization Position of the probes, the size and position of restriction fragments hybridizable with the probe, and exon ⁄ introns are shown as in Fig 2A Positions of the DT40 specific-DHSs and the exon number are indicated as in Fig 1, wild type DT40 ⁄ Cre allele; 2, each DHS deletion allele with Bsr gene; 3, each DHS deletion allele without Bsr gene a, DNA from wild type DT40 ⁄ Cre cells; b, DNA from cells with 16 kb Del allele (with extra Ig-b gene); c, DNA from cells with 16 kb Del allele (with extra Ig-b gene) and each DHS deletion allele with Bsr gene; d, DNA from cells with 16 kb Del allele (with extra Ig-b gene) and each DHS deletion allele without Bsr gene Probes: (A), fragment from Na channel gene ()10.1 kb to )9.2 kb; HindIII ⁄ AccI); (B) and (C), Ig-b cDNA (nucleotides 312–1542; PmaCI ⁄ EcoRI) [34]; (D), GH gene (+6.6 kb to +7.4 kb; NcoI ⁄ NcoI) [46] Restriction enzyme digestion: A, HindIII; B and C, EcoRI; D, SacI Restriction fragment size (given in kb) was determined by kHindIII Size of the bands observed in each autoradiogram is indicated at the right side The 6.6 kb band detected in lanes b, c, and d in autoradiogram (B), and lanes c and d in (C) is derived from the extra Ig-b gene The 3.1 kb band found in lanes b, c, and d in autoradiogram (D) is derived from 16 kb Del allele 426 FEBS Journal 272 (2005) 422–432 ª 2004 FEBS H Matsudo et al Deletion of hypersensitive sites in chicken Ig-b locus Fig Level of Ig-b mRNA in DT40 ⁄ Cre cells lacking DT40-specific DHSs Using total RNA (3.0 lg) prepared from the deletion clones, the level of Ig-b mRNA was examined by Northern hybridization with the Ig-b cDNA probe (nucleotides 312–1542; PmaCI ⁄ EcoRI) [34] Intensity of the bands was compared using TYPHOON 9210 and IMAGE QUANT (A) Positions of 1.7 kb endogenous and 1.5 kb extra Ig-b mRNA are shown at the left and right, respectively Lanes: RNA prepared from: 1, wild type DT40 ⁄ Cre cells (Wild type); 2, 16 kb Del allele (with extra Ig-b gene) (Control 1); 3, 16 kb Del allele (with extra Ig-b gene) and the region I deletion allele (I Del); and 5, 16 kb Del allele (with extra Ig-b gene) and the region II deletion allele (II Del); 6, 16 kb Del allele (with extra Ig-b gene) and the region III deletion allele (III Del) (B) Lanes: and 2, 16 kb Del allele (without extra Ig-b gene) (Control 2); and 4,16 kb Del allele (without extra Ig-b gene) and the region IV deletion allele (IV Del) Probes: and 3, human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA; and 4, Ig-b cDNA [1,31,35–37] Enhanced acetylation is mainly observed at DHSs and their surroundings, although low level of acetylation has been reported at DHSs such as HS-85 and 3¢ HS1 in the mouse b-globin locus [1] Using ChIP followed by R-PCR, the acetylation status of H3 and H4 histones in the chicken Ig-b locus has been reported [15] In DT40 cells, acetylation of H3 and H4 histones is enhanced at the DHSs ()7.3 kb, )6.5 kb, kb, +0.9 kb, +5.2 kb, and +6.1 kb) deleted in this study [15] The acetylation status in region II deletion cells was compared with that of predeletion cells (Fig 6) A prominent acetylation of both histones detected close to the transcriptional initiation site (target 4) of the Ig-b gene in wild type DT40 cells [15] was observed at the same position both in predeletion cells and region II deletion cells Similar to wild type DT40 cells [15], enhanced acetylation was demonstrated at the targets closely positioned to DT40-specific DHSs in the Na channel gene (close to DHSs at )7.3 kb and )6.5 kb; targets and 2), in the Ig-b first intron (DHS at +0.9 kb; target 5), and between the Ig-b and GH genes (DHSs at +5.2 kb and +6.1 kb; targets and 7) At these targets (targets 1, 2, 5, 6, and 7) in the region II deletion cells, acetylation levels of both histones were the same as that of the predeletion cells In chicken liver-derived LMH cells where no Ig-b mRNA is detected [15], and thus used as the negative control, no enhanced acetylation of either histone was demonstrated FEBS Journal 272 (2005) 422–432 ª 2004 FEBS at the targets located close to the Ig-b promoter and DT40-specific DHSs Acetylation levels of both histones at the targets in the last exon of the Na channel gene (target 3) and in the fourth exon of the GH gene (target 8) was shown to be the same as in the cells before and after region II deletion In region II deletion cells, no Ig-b mRNA was detected but the acetylation status of both histones before and after deletion was demonstrated to be the same, indicating that region II is essential for B cell-specific transcription of the Ig-b gene but unnecessary for the maintenance of the active chromatin state in the Ig-b locus In transient transfection assays, the DNA fragment containing a DHS at )2.1 kb shows no enhancing activity of the Ig-b promoter in DT40 cells, while the DHS at )1.0 kb decreases promoter activity to 50% [15] It would be interesting to learn whether the deletion of DHSs at )2.1 kb and )1.0 kb has any effect on the level of Ig-b mRNA; however, deletion of these DHSs has been unsuccessful so far (data not shown) Discussion Ten Ig-b-producing cell-specific DHSs were grouped into four regions, and each of these regions was deleted in DT40 cells The roles of the DHSs on Ig-b gene expression and the maintenance of active chromatin state in the Ig-b locus were examined in the deletion cells Establishment and maintenance of active 427 Deletion of hypersensitive sites in chicken Ig-b locus A C H Matsudo et al B D Fig DNase I hypersensitive sites in the DT40 ⁄ Cre cells lacking region II Isolated nuclei were treated with DNase I for at 20 °C Concentration of DNase I for treatment of nuclei from left to right: 75, 50, 25, mL)1 The DNA was purified from nuclei and digested with EcoRI (A), ScaI (B), EcoRV (C) or EcoT22I (D) The digests were Southern hybridized with 0.9 kb ScaI ⁄ KpnI DNA ()4.6 kb to )3.7 kb; nucleotides 849–1751 in accession number AB066568) (A,B) [34], 1.2 kb Ig-b cDNA (nucleotides 312–1542; PmaCI ⁄ EcoRI) [34] (C) or 0.2 kb GH exon (nucleotides 3515–3744 in D10484) (D) [46] Restriction fragment size (given in kb) was determined by kHindIII Analyzed region, DHS, and position of the probe are shown at the bottom The 2.5 kb band observed in (C) is derived from the extra Ig-b gene Autoradiograms: left, wild type DT40 ⁄ Cre cells having 16 kb Del allele (with extra Ig-b gene); right, wild type DT40 ⁄ Cre cells having 16 kb Del allele (with extra Ig-b gene) plus the region II deletion allele (mutant) 428 FEBS Journal 272 (2005) 422–432 ª 2004 FEBS H Matsudo et al Fig H3 and H4 acetylation of the Ig-b locus in the region II deletion cells The acetylation of wild type DT40 ⁄ Cre cells with 16 kb Del allele (with extra Ig-b gene), wild type DT40 ⁄ Cre cells with 16 kb Del allele (with extra Ig-b gene) plus the region II deletion allele, and LMH cells is indicated by the black, gray and white bars, respectively (Top) H3 acetylation (Middle) H4 acetylation Acetylation values are shown by fold enrichment compared with untreated control DNA as reported previously [15,31] Three amplifications were performed for each target The values of the ng control DNA equivalents were estimated using standard calibration, and fold enrichment was determined from these values [31] Values are mean ± SE An over-scaled bar is shown by double wavy lines, and its mean value is shown at the left Bottom; the positions of eight targets are shown Exon ⁄ introns are shown as in Fig Arrows, DHSs; +, transcriptional start site chromatin is essential for transcription of cell type-specific genes Active chromatin is characterized by an open chromatin structure that is generally sensitive to DNaseI, presence of cell type-specific DHSs [7,8], and active-type modification of core histone tails [9–11] From the earliest stage of the study [6], DNase I general sensitivity has been demonstrated to be a good parameter to determine chromatin state [1–5] Distinct differences are often obtained between positive and negative controls in DHS and histone modification analyses; in contrast, these differences are often much smaller in DNase I general sensitivity analysis Although DNase I general sensitivity analysis of the FEBS Journal 272 (2005) 422–432 ª 2004 FEBS Deletion of hypersensitive sites in chicken Ig-b locus chicken Ig-b locus has not been finished because of the technical difficulty of this type of assay, there may be no difference in the range and extent of general sensitivity to DNase I with or without DHSs deleted in this study because the positions of DHSs remain the same In the chicken b-globin locus, the DNase I sensitive region coincides with the region in which the core histones are hyperacetylated [3,38] In the mouse b-globin locus, hyperacetylated H3 and H4 histones are located in a much wider DNase I sensitive region [1,12] Thus, the DNase I sensitive chromatin state is not always associated with active-type modification of core histones Furthermore, a cell type-specific DHS, HS-85, in the mouse b-globin ⁄ odorant receptor locus is located in a region with a low acetylation level of the core histones and a chromatin state in the flanking region resistant to DNase I [1] In this case, the formation of cell type-specific DHS again does not occur in the context of a DNase I sensitive chromatin region, or a region with active-type histone modifications Transgenic mice with a 99 bp deletion (containing two Pit-1 binding sites) of the DHS I region which is located within the locus control region (LCR) in the human GH locus, show loss of H3 and H4 acetylation ranging from 32 kb upstream of the transcriptional start site of the GH gene to the GH gene [18] Thus, the deleted sequence is essential for the establishment and maintenance of acetylation in the GH locus Because deletion of this region has no effect on the formation of cell type-specific DHSs, the region required for H3 and H4 acetylation does not necessarily coincide with that involved in the DHS formation Deletion of DHS1–6, the entire mouse b-globin LCR, results in extraordinarily low levels of b-globin mRNA However, both the acetylation state of the promoter region of the active b-globin gene [19] and the DNase I general sensitivity in the b-globin locus are the same as the wild type control [17] Thus, there again has been no description either of the region involved in DNase I general sensitivity or of the region for the formation of DHSs, although chromatin opening activity of the LCR has been described in several loci using transgenic mouse models [29,39–42] Because the chromatin state of the cells with any deletion in the regions I–IV in the Ig-b locus, judged by DHSs as the indicator, was the same as that in predeletion cells, the region required for the maintenance of the active chromatin state is presumed to be present at the DHSs located further upstream or downstream of the DHSs examined in this study Two DT40-specific DHSs, one at )13 kb in the Na channel gene and the other at +13 kb downstream of the GH gene, have 429 Deletion of hypersensitive sites in chicken Ig-b locus already been described [15], and deletion of these DHSs should be performed in the future Transcription of the adult-type mouse b-globin gene is suggested to require the assembly of DHSs into one complex named the active chromatin hub at around the transcriptional start site This complex includes six DHSs in the LCR located 40–60 kb upstream from the active gene, DHSs present in the odorant receptor genes found 40 kb further upstream from the LCR, and those present in 20 kb downstream from the b-globin gene [43] Combined deletion of the separate DHS groups would be necessary in future studies because the deletion of a single region showed no remarkable effect on the chromatin state of the Ig-b locus The activities of DHSs in the Ig-b locus examined by transient transfection [15] did not correlate with those determined by deletion in regions I and IV Similar situations have been reported at DHSs within the human b-globin LCR [44] Although the reason for the inconsistency of the DHS activity between transient transfection and in vivo deletion is unknown, the phenotype observed in deletion cells is likely to reflect the in vivo role of the DHS because genomic DNA is deleted from its native context Another reason may be the redundancy between different elements in the Ig-b locus Materials and methods Cells Liver-derived LMH cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan) DT40 ⁄ Cre cells [45] that produce MerCreMer protein, a Cre recombinase with hormone binding domains of the mutant mouse estrogen receptor (Mer) on both ends, which localizes in the cytoplasm in the absence of estrogen derivative, 4-hydroxy tamoxifen Cells were propagated in RPMI1640 (Nissui, Tokyo, Japan) ⁄ 10% fetal bovine serum (JRH Biosciences, Lenexa, KS, USA) ⁄ 1% chicken serum (GibcoBRL, Grand Island, NY, USA) at 39.5 °C for DT40 ⁄ Cre and 37 °C for LMH DT40 ⁄ Cre cells were grown with mgỈmL)1 of geneticin (GibcoBRL) which is required for establishment of DT40 ⁄ Cre cells For selection, blasticidin (Funakoshi, Tokyo, Japan) and puromycin (Sigma-Aldrich, St Louis, MO, USA) were used at 30 lgỈmL)1 and 0.5 lgỈmL)1, respectively Preparation of targeting constructs and extra Ig-b gene expression construct pLoxBsr [45] containing the chicken b-actin promoter-driven blasticidin resistance (Bsr) gene with mutant loxP sequences at both ends was used as a targeting vector For the targeting construct, genomic DNA of about kb in size was linked on both sides with the actin 430 H Matsudo et al promoter-driven Bsr gene Positions of left and right arms of the five constructs (16 kb Del, I to IV; Fig 1) are as follows (the transcriptional start site of Ig-b gene is shown as base number one): 16 kb Del-L ()11.4 kb to )9.0 kb, SacI ⁄ MluI; 2.4 kb), 16 kb Del-R (+6703 to +8768; 2066 bp), I-L ()10.1 kb to )7.8 kb, BamHI ⁄ XhoI; 2.3 kb), I-R ()6009 to )3942; 2068 bp), II-L ()4613 to )2692; 1922 bp), II-R (+50 to +1959; 1910 bp), III-L ()1369 to +578; 1948 bp), III-R (+1079–3130; 2052 bp), IV-L (+2721 to +4524, 1804 bp), IV-R (same as 16 kb Del-R) Based on the reported nucleotide sequences [15,34,46], arm DNAs were amplified with Pyrobest polymerase (Takara Bio, Kyoto, Japan), and ligated into the vector Restriction fragments were used for the arms located in the unsequenced region The Bsr gene was inserted in the same orientation as the flanking gene DT40 ⁄ Cre cells were electroporated with linearized DNA and clones were obtained as described [47] Using cloned chicken Ig-b cDNA as the template, 0.9 kb DNA (nucleotide number 20–930) [34] was amplified by Pyrobest polymerase and cloned into HindIII ⁄ NheI sites of the pExpress vector [45] The chicken b-actin promoterdriven puromycin resistance (Puro) gene was inserted into the XhoI site of the construct Selection of homologous recombinant clones by PCR and tamoxifen treatment To determine homologous recombination, PCR was carried out as described [45] using the following primers: 5¢CGATTGAAGAACTCATTCCACTCAAATATACCC-3¢ (in Bsr gene) [45] Primers (30-mer) in the genome were set up at 10–100 bases downstream from the right arm: 5¢TAGTTTCTCAAACACTCTGTCTGAGGTGCC-3¢ (16 k Del; +8778 to +8807); 5¢-ATGGGTTCATAGGAGACC TTTGAGGGGTTG-3¢ (I: )3782 to )3811); 5¢-TAGATG CCGTTGTCCTCGTAGCTGATCCTG-3¢ (II: +2085 to +2114); 5¢-AGTGATGTCCTCGTAGGTGGCAATCTGC TC-3¢ (III: +3438 to +3467) (IV: same as 16 k Del) Clones whose DNA contained about kb predictable sequences by PCR were selected as tentative clones with homologous recombination DT40 ⁄ Cre clones with homologous recombination were treated with 0.1 mm 4-OH-tamoxifen (Sigma, St Louis, MO, USA) for 24 h and then cloned in 96 well microtiter plates Deletion of the Bsr gene was confirmed by genomic Southern hybridization DNase I digestion, hybridization, and histone acetylation analysis For DNase I hypersensitivity analysis, nuclei were prepared and treated with DNase I (Takara Bio) and DNA was prepared as described [30] Restriction enzyme digestion and FEBS Journal 272 (2005) 422–432 ª 2004 FEBS H Matsudo et al Southern hybridization were carried out as reported previously [48] Probe DNA was labeled by the random-priming methods using [32P]dCTP[aP] The following probes were used: Ig-b cDNA (nucleotides 312–1542, 1231 bp) [34], Na channel gene ()10.1 kb to )9.2 kb, HindIII ⁄ AccI; 0.9 kb), GH gene (+6560 to +7380, NcoI ⁄ NcoI; 821 bp) [34] Preparation of total RNA followed by Northern hybridization was performed as described previously [31] A human glyceraldehyde-3-phosphate dehydrogenase cDNA probe was obtained from Clontech (Palo Alto, CA, USA) The intensity of bands on the Northern 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decreased the expression of Ig-b mRNA to 40% of the. .. maintenance of active chromatin state in the Ig-b locus were examined in the deletion cells Establishment and maintenance of active 427 Deletion of hypersensitive sites in chicken Ig-b locus A C... sensitive chromatin region, or a region with active- type histone modifications Transgenic mice with a 99 bp deletion (containing two Pit-1 binding sites) of the DHS I region which is located within