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Previous Article | Next Article 
Molecular and Cellular Biology, August 2006, p. 5569-5579, Vol. 26, No. 15
0270-7306/06/$08.00+0 doi:10.1128/MCB.00405-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Isabela Cajiao,,
Jeong-Seon Kim,
,
Atsushi P. Kimura,¶
Aiwen Zhang,||
Nancy E. Cooke, and
Stephen A. Liebhaber*
Departments of Genetics and Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Received 8 March 2006/ Returned for modification 6 April 2006/ Accepted 20 May 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The hGH multigene cluster represents an informative model for the study of developmental control (17, 27). This cluster, located on chromosome 17q22-24, encompasses five growth hormone paralogs (Fig. 1A). hGH-N is expressed in pituitary somatotrope cells; the four remaining genes, hCS-L, hCS-A, hGH-V, and hCS-B, are expressed in the syncytiotrophoblast (STB) layer of the placental villi (6, 22). Activation of the pituitary and the placental genes is dependent on distinct actions of a 5'-distal locus control region (LCR). This hGH LCR is comprised of five DNase I hypersensitive sites (HS) (Fig. 1A). The pituitary cell-specific HSI and HSII, located –14.5 and –15.5 kb 5' to the cluster, are sufficient to drive high-level hGH-N expression in pituitary somatotropes (17). Selective inactivation of HSI results in a dramatic loss of hGH-N expression (15). HSIV, located at –30 kb, forms exclusively in STB chromatin, but a specific role for this HS has not yet been demonstrated. HSIII and HSV are shared by placental and pituitary chromatin. The CD79b gene, which encodes the signal transduction subunit of the B-cell antigen receptor (26), is located between the hGH cluster and the hGH LCR (Fig. 1A) (3). The CD79b protein first appears at the pre-B-cell stage, and its expression is maintained throughout all stages of B-cell differentiation (4, 14). Remarkably, CD79b mRNA is also abundant in pituitary somatotropes (5). This pituitary CD79b mRNA appears to be structurally the same as that in B cells, yet it fails to produce appreciable protein. The mechanism of this posttranscriptional control remains undefined.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To purify mouse lymphocyte cells, spleens removed from anesthetized mice were dispersed through a 30-µm nylon mesh to generate a single-cell suspension. Lymphocytes were isolated by Ficoll gradient using Ficoll-Plaque plus (Amersham Biosciences, Piscataway, NJ) by centrifugation at 650 x g for 30 min without braking. After centrifugation, the cells at the interface between Ficoll and the aqueous phase were collected and washed twice with phosphate-buffered saline (PBS).
For primary B-cell chromatin immunoprecipitation (ChIP) assays, lymphocytes were further purified by negative selection using MACS CD43 (Lys48) microbeads (Militenyi Biotec, Germany). B cells were magnetically enriched (excluding pro-B cells) using MACS LD depletion columns and Mini-MACS cell sorting kits (Militenyi Biotec, Germany) according to the manufacturer's instructions.
Transgenic constructs and generation of transgenic mouse lines.
All transgenic mouse lines used in
this study are listed in Table
1 (also see the diagram in Fig.
3A). The hGH/P1
line (87-kb hCD79b/GH transgene), –1.3CD79b
(5.6-kb hCD79b transgene), and –0.2CD79b
(4.6-kb hCD79b transgene) have been previously reported
(5,
27).
–0.5CD79b contains a 4.9-kb genomic fragment generated
by BspMI and EcoRI digestions of a 5.6-kb hCD79b genomic DNA
fragment (5). This
fragment was gel isolated, extracted with a QIAquick gel extraction
column (QIAGEN, Germany), purified through an Elutip D column
(Schleicher & Schuell, Germany), and adjusted to a concentration of
2 ng/µl in TE buffer (10 mM Tris-HCl [pH 7.6], 0.1 mM
Na2EDTA) prior to microinjections into fertilized mouse
oocytes (C57BL/6 x SJL). A BAC genome clone encompassing the
hCD79b/GH locus was screened by Invitrogen Life Technologies
using colony hybridization with TCAM and SCNex13 probes corresponding
to TCAM1 and SCN4A genes, respectively. A clone of
148.3 kb was identified (clone #535D15). A 123-kb insert was released
by NotI digestion. The NotI sites are present in exon 1 of the
SCN4A gene and between TCAM1 and SMARCD2
genes. The resulting released BAC (named CD/hGH BAC) insert
was separated by pulsed-field gel electrophoresis (Bio-Rad, Hercules,
CA) and purified from an excised slice of the agarose gel by digestion
with 1 U/100 µl ß-agarase I (New England Biolabs,
Beverly, MA) followed by phenol-chloroform extraction. This purified
123-kb DNA fragment was used for microinjection as described above. All
transgenic mouse founders were generated by routine procedures by the
University of Pennsylvania Transgenic and Chimeric Mouse Core Facility.
Positive founders were identified by Southern blot analyses of
NcoI-digested tail DNA. Transgene integrity was intact in all reported
lines. Transgene copy numbers were quantified by PhosphorImager (Storm
840 PhosphorImager; Molecular Dynamics, Sunnyvale, CA) analyses of
Southern blots. The hCD79b transgene signal was normalized to
the hybridization signal from the endogenous mouse
-globin gene on the same
filter.
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Isolation of intact nuclei from B-cell lines and DNase I hypersensitive site mapping. Cells from the various B-cell lines were collected, washed with PBS, and resuspended at 108 cells/ml in ice-cold sucrose buffer I (0.32 M sucrose, 3 mM CaCl2, 2 mM magnesium acetate, 10 mM Tris-HCl [pH 8.0], 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 0.05% Triton X-100). The cell suspensions were centrifuged at 500 x g for 5 min at 4°C and washed with RB buffer (100 mM NaCl, 50 mM Tris-HCl [pH 8.0], 3 mM MgCl2, 0.1 mM PMSF, 5 mM sodium butyrate). For DNase I mapping, the nuclei were resuspended again at 108 cells/ml of RB buffer, and a baseline (at 0 s) aliquot was withdrawn. RB buffer containing 3 mM CaCl2 and 300 U of DNase I (Invitrogen, Carlsbad, CA) per ml was added to yield final concentrations of 1 mM CaCl2 and 100 U of DNase I per ml. The sample was incubated at 37°C. Aliquots were withdrawn at the indicated times and EDTA was added to 50 mM, followed by digestion of the nuclei with 120 µg of proteinase K per ml in 0.8 M NaCl, 0.5% SDS, extraction with phenol and chloroform, and a final ethanol precipitation terminating each reaction. DNase I-digested DNA samples were subsequently digested with restriction enzymes and transferred to Zetaprobe membranes (Bio-Rad, Hercules, CA) for hybridization. Hybridization probes were synthesized by PCR amplification using primer sets (Prb1 and Prb2) from human genomic DNA (Table 2).
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-32P]ATP
and T4 polynucleotide kinase. This primer set spans intron 5 to
distinguish cDNA from amplified genomic DNA. PCR products at 25 cycles
were digested; HinfI specifically cleaves mCD79b cDNA, and
SfcI specifically cleaves hCD79b cDNA. Fragments were
separated on 6% polyacrylamide gels, and bands were quantified by
PhosphorImager analyses. The ratios of hCD79b to
mCD79b cDNA products were normalized to the transgene copy
numbers, and the final hCD79b mRNA expression value was
calculated as a percentage of the expression from a single endogenous
mCD79b gene. All PCR results were confirmed to be within the
linear range of amplification. Chromatin immunoprecipitation (ChIP) assay for modified histones. ChIP studies were performed with human B-cell lines (1484 and U266), a human erythroid cell line (K562), and tissues isolated from hGH/P1 mice (line 811D) (27). Preparation of unfixed chromatin and the ChIP assay were carried out as described previously (8). Briefly, 0.3 mg nuclei was digested with 25 U of micrococcal nuclease at 37°C for 6 min in 1 ml 50 mM NaCl, 20 mM Tris-HCl (pH 7.5), 3 mM MgCl2, 1 mM CaCl2, 10 mM sodium butyrate, 0.1 mM PMSF. The reaction was stopped by the addition of Na2EDTA to a final concentration of 0.5 mM, and salt-soluble chromatin was isolated as described previously (13). Soluble chromatin was concentrated using a Microcon centrifugal filter (Amicon Inc., Bedford, PA), and 250 µg of this chromatin input was incubated with 10 µl each of the antibodies to acetylated H3 and H4 (Upstate Biotechnologies, Lake Placid, NY), or in the absence of antibody, in a total volume of 500 µl. Protein A-Sepharose (Amersham Pharmacia Biosciences, Piscataway, NJ) precipitates were generated and washed, and DNA was purified from the pellets (bound) as described previously (23). DNA samples from the input and bound fractions were amplified by PCR using various primer sets listed in Table 2. Parallel dilutions of 1/300, 1/600, and 1/1,200 were made from 15% of the input sample and from the entire immunoprecipitated sample pellet. Aliquots containing a final concentration of 0.16%, 0.08%, and 0.04% of each immunoprecipitated sample were analyzed by PCR to confirm that each assay was within the linear range of amplification. Each product was analyzed by electrophoresis on 1% agarose gels followed by Southern blotting. The blots were hybridized with 1 x 106 to 2 x 106 cpm/ml random primer-labeled probe at 65°C overnight. The membranes were subsequently washed at 60°C in 0.1% SDS and 0.5x SSC. Signals were quantified by PhosphorImager analysis. Each ratio was normalized to the comparable signal detected at the ubiquitously expressed GAPDH locus. For the histone H3 acetylation and H4 acetylation ChIP assays, two independent assays were performed from different preparations of nuclear extracts. For the histone H3 K4 dimethylation ChIP assay, a single experiment was performed.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HS mapping surveyed two adjacent EcoRI restriction fragments spanning the CD79b gene and 31 kb of 5'-flanking sequences (Fig. 2). HS mapping of the 12.5-kb EcoRI fragment containing the human CD79b gene, the contiguous 8.0 kb of 5'-flanking sequences, and 850 bp of 3'-flanking sequences revealed two HS (HSB1 and HSa; Fig. 2A). HSa mapped within CD79b intron 1 and was detected in all cells studied, irrespective of cell type or CD79b expression. HSB1 mapped to the CD79b promoter and formed specifically in B-cell chromatin. DNase I analysis of the adjacent 5' EcoRI fragment, 23.1 kb in length, identified three HS (Fig. 2B). Two of these HS corresponded to the previously reported HSIII and HSV of the hGH LCR (17). HSIII was detected in all cell types tested, including pituitary, placenta, B, and erythroid cells, whereas HSV was observed in B-cell lines and pituitary, placenta, and JEG3 cells but not in K562 cells (Fig. 2B and data not shown). A novel HSB2 was also identified in this region. This HS was exclusive to B cells and mapped 14 kb 5' to the CD79b promoter. HSB2 intensity roughly paralleled the levels of CD79b mRNA in the various B-cell lines. In summary, the DNase I mapping identified three novel HS at the human CD79b/GH locus; a constitutive HSa within the CD79b gene, a B-cell-specific HS mapping to the CD79b promoter, and a second B-cell-specific HS located 14 kb 5' of the CD79b gene (Fig. 2C).
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Prior analyses had revealed that the hGH/P1 transgene supports robust hCD79b expression in mouse B cells at levels of 25 to 42% of a single endogenous mCD79b gene (5). To extend this baseline, we established five lines carrying a 123-kb BAC transgene encompassing CD79b with more extensive flanking regions (details will be reported separately) (Fig. 3A). CD79b mRNA was expressed from this CD/hGH BAC transgene at levels similar to that observed in the hGH/P1 lines (10 to 53% of the endogenous mCD79b locus; Fig. 3C). With these two large transgenes as standards, we next assessed expression from a set of transgenes that isolated CD79b from its remote sequences. The first transgene, –8.0CD79b, contained the CD79b gene along with 8 kb of 5'-flanking sequences. This transgene specifically excluded HSV, HSIII, and HSB2 as well as much of the 3'-flanking region (Fig. 3A). hCD79b was expressed in the splenic B cells of all five –8.0CD79b lines. The mean expression was indistinguishable from that of the larger transgenes, although there was slightly more prominent variation in expression values (6% to 49% of the endogenous mouse locus) (Fig. 3D and H). Two additional hCD79b transgenes were generated that shared a 3' end with –8.0CD79b (Fig. 3A). The –1.3CD79b transgene excluded the pituitary cell-specific HSI and HSII, and the –0.5CD79b transgene further excluded a B-cell-specific hypomethylated CpG island (Fig. 3A and unpublished data). The –1.3CD79b transgene was robustly expressed in all five lines. Its expression per copy was 10 to 30% of that of the endogenous mCD79b gene (Fig. 3E and H). The –0.5CD79b transgene maintained a similar level of expression, ranging from 16 to 19% of that of the endogenous gene (Fig. 3F and H). Thus, the five transgenes, CD/hGH BAC, hGH/P1, –8.0CD79b, –1.3CD79b, and –0.5CD79b, expressed mean levels of CD79b mRNA that were within a twofold range (17 to 27%). These data indicated that a complement of cis-acting elements necessary for full activation and expression of CD79b is in close juxtaposition to the structural gene.
A final hCD79b transgene was generated that contained only a 200-bp minimal promoter region (Fig. 3A). It should be noted that the CD79b gene utilizes a GC-rich, TATA-less promoter, and for this reason it has a cluster of multiple transcription initiation sites (5, 28). The expression of the –0.2CD79b transgene (Fig. 3G and H) was remarkable in three respects. First, its level of expression, 4.6% relative to that of the endogenous mCD79b gene, was substantially below that of the other five transgenes. Second, despite its weak expression, the –0.2CD79b transgene retained site-of-integration independence and copy number-dependent expression, with four of five lines demonstrating essentially identical levels (4%) of CD79b mRNA per gene copy. Finally, this transgene retained B-cell specificity (Fig. 4). Thus, determinants sufficient to establish an autonomous and B-cell-specific chromatin locus are encompassed within the CD79 gene or in its immediate 200 bp of 5'-flanking sequences. An additional element(s) necessary for enhancement of expression to full levels is located between –200 and –500 of the transcription start site.
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Levels of H3K4me2 were determined by ChIP analysis at the hCD79b/GH locus (Fig. 6). A prominent peak of H3K4me2 was observed at the CD79b promoter (probes CDP and P3) in the high-expressing 1484 cell line (Fig. 6B). A low level of modification also appeared to be present between CD79b and HSV in these cells. In contrast, the region 3' of the gene remained unmodified. In the U266 cells, which contain only trace levels of CD79b mRNA, a modest peak of modification was identified at the CD79b promoter and at HSV (Fig. 6C). K562 cell chromatin lacked modification throughout the surveyed region (Fig. 6D). The primary B cells from an hGH/P1 transgenic mouse had a strong H3K4me2 peak at the CD79b promoter (probes CDP and P3) and minimal enrichment at HSV (Fig. 6E). Hepatocytes from the same line had low levels of modification at the CD79b promoter (probe CDP) and lacked significant enrichment elsewhere. As was the case in the H3 and H4 acetylation ChIP study, there was no evidence for 3' extension of H3K4me2 modification within the hGH cluster. The peak of H3K4me2 modification at two adjacent sites in the two expressing cell sources (1484 and transgenic B cells) were mutually confirming. Comparisons among the high- and low-expressing B-cell lines and the nonexpressing K562 cells revealed a direct relationship between gene activation and H3K4 methylation at the CD79b promoter. Analysis of primary B cells and hepatocytes from the hGH/P1 mouse extended these observations. The significance of the H4 acetylation and H3K4 dimethylation at the CD79b promoter in the hepatocytes remains unclear. Overall, these results reveal that histone modifications associated with chromatin activation in B cells are limited to the immediate vicinity of the hCD79b gene and fully exclude the adjacent hGH cluster.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The hCD79b/GH locus presents an informative model for analysis of chromatin structures and their developmental regulation. This locus contains six closely packed genes with three distinct tissue specificities: pituitary, placenta, and B cell. In addition, the striated muscle-specific SCN4A gene is juxtaposed 5' of these genes, and a testis-specific TCAM gene is juxtaposed 3' of the locus (Fig. 6). HS formation and histone modifications at this locus have been previously mapped in pituitary and placental chromatin (7, 15, 17, 19). In the pituitary tissue, the hGH LCR establishes a 32-kb acetylated chromatin domain that directly links the LCR with the target hGH-N gene promoter (15). In the placenta tissue, the LCR determinants HSIII-HSV and the target placental genes are each individually acetylated and are separated by a 28-kb region of unmodified chromatin (8, 19). Of note, the CD79b gene is located between the hGH cluster and its LCR. As such, it is situated in the "activated" domain in pituitary chromatin and in a region of hypoacetylated histones in placental chromatin. Significantly, CD79b is robustly transcribed in the pituitary tissue and is inactive in the placenta (5). The "bystander" activation of CD79b in the pituitary tissue is independent of B-cell-specific transcription factors, and as such it appears to represent a mechanistically independent and epigenetically driven pathway of gene activation (5). Thus, the specific configurations of chromatin modifications at the hCD79b/GH locus in the pituitary and placenta tissues predict divergent pathways of LCR-mediated transcriptional activation and have distinct effects on the adjacent CD79b gene.
To further explore transcriptional interactions at the hCD79b/GH locus, we have mapped its transcriptional determinants and chromatin structures in B cells. HS mapping, which has the capability to scan for critical chromatin determinants (10, 12), identified a set of B-cell-restricted sites, HSB1 and HSB2, and a constitutive site, HSa (Fig. 2). The B-cell specificity of HSB1 and HSB2 suggested that they play a role in CD79b expression. The functional importance of these sites was tested in a series of transgenic mouse lines. The initial studies assessed the expression of hCD79b from transgenes that encompass the entire locus on 123-kb or 87-kb genomic fragments. These levels were then compared to a series of smaller transgenes that isolate the CD79b gene with limited flanking regions (Fig. 3). Three major observations resulted from these studies. First, CD79b expression was maintained within a twofold mean range for all but the smallest transgene, –0.2CD79b. These comparisons led us to conclude that the region between –200 bp and –500 bp upstream from the CD79b gene contains one or more determinants that enhance CD79b expression. Second, the copy number dependence and site-of-integration independence of CD79b expression was maintained in all transgenes, including –0.2CD79b. Whereas we did observe as much as an eightfold variation of expression in some of the transgenes, the remarkable consistency of expression for the –0.5CD79b and –0.2CD79b transgenes and the maintenance of B-cell specificity in the –0.2CD79b transgene (Fig. 4) indicated that an element(s) sufficient to establish an autonomous chromatin domain in B cells is located within or in very close juxtaposition to the hCD79b gene itself. Third, these studies effectively eliminated HSB2 as a critical determinant of CD79b expression. Exclusion of this HS from the CD79b transgene had no appreciable impact on expression levels, and the copy number dependence and B-cell specificity was maintained in its absence. This data set leads us to conclude that control over CD79b is local as opposed to long range, and that the two HS retained in the –0.2CD79b transgene, the B-cell-specific HSB1 and the constitutive HSa, may be sufficient to establish an autonomous B-cell chromatin domain.
Although the –0.2CD79b transgene contained elements sufficient to establish an autonomous chromatin domain, its level of expression was significantly below that of the transgenes with more extensive 5'-flanking regions. As noted above, this comparison suggests that one or more determinants residing in the region between the 5' termini of –0.2CD79b and –0.5CD79b have transcription-enhancing activity. Prior analyses by others using in vitro and cell transfection approaches (28) have identified a putative B-cell-specific activation element, BCS (for "B29 conserved sequence"), in this region. This BCS can bind to the multifunctional transcription factor Ying Yang 1 (YY1) in vitro (25). YY1, a zinc finger, GL1-Kruppel family member, can function as either a silencer or an enhancer through interaction with a variety of chromatin-modifying proteins (1, 9, 20). However, the role of this factor in CD79b gene expression has not been further evaluated. Previous studies using cell transfection models have also suggested that the minimal promoter of the hCD79b gene, extending to –0.2 kb, exhibits B-cell-specific activity. This minimal promoter region contains EBF, Oct1-2, PU.1 (Ets family), Ikaros, and Sp1 transcription factor motifs (28). Ikaros is essential for normal development and proliferation of lymphoid cells and exists as an integral component of chromatin-remodeling complexes (18, 24). PU.1, a member of the Ets family, is critical for B-lymphocyte development and interacts with the chromatin-modifying enzymes CBP and p300 (29). The activity of these factors may correlate with HSB1 formation and function and may contribute to the autonomous chromatin domain established by the –0.2CD79b transgene. However, these studies in transfected B cells also suggested that the minimal promoter (–193 bp) has full transcriptional activity, and extension of the 5'-flanking region to –469 bp resulted in a prominent loss of activity (28). These data, which led the authors to propose that repressor determinants are located between –193 and –496, are inconsistent with our transgenic studies. Indeed, we detect enhancer activity in this region based on comparison of the –0.2CD79b versus –0.5CD79b transgenes. Thus, the preceding cell transfection studies, while useful in identifying potential cis-trans interactions at the CD79b promoter, may reflect functional activities that are not in operation during in vivo B-cell differentiation in the intact animal.
The analysis of
histone modifications at the hCD79b/GH cluster in B cells
(Fig. 5 and
6) revealed that histone
H3 and H4 acetylation and H3K4 methylation are predominantly limited to
the immediate vicinity of the CD79b promoter. This stands in
marked contrast to the distribution of histone modifications at this
locus in pituitary and placental chromatin. Therefore, three distinct
patterns of chromatin modification are established at the
hCD79b/GH locus (Fig.
7). These patterns correspond to three specific gene expression profiles.
In the pituitary cell, an extensive domain of histone acetylation links the LCR with the target hGH-N promoter. This pituitary domain, which may be established by linear "spreading" of histone acetyltransferase complexes, encompasses the intervening
CD79b gene and results in bystander gene activation. Consistent with this model of bystander activation and its dependence
on HSI action, hCD79b is robustly transcribed in hGH/P1 and –8.0CD79b transgenic mouse
pituitary glands but not in the –1.3CD79b, –0.2CD79b, and hGH/P1(
HSI)
transgenic mouse pituitary glands (5). In the placenta,
histone acetylation is limited to the LCR and to the placental genes. These modified regions are separated by an extensive segment
of unmodified chromatin containing the pituitary cell-specific LCR determinants HSI and HSIII, the CD79b gene, and the
hGH-N gene. Previous studies have demonstrated that hCD79b, which is excluded from the modified region, is not
expressed in the placenta of humans and transgenic mice (5). The discontinuous
pattern of histone modification in the placenta predicts a long-distance interaction, or "looping," between the
LCR and the four placental genes to activate expression. Finally, the activating modifications in B-cell chromatin are tightly restricted to
the CD79b gene; these modifications exclude all adjacent genes in the region. These data, taken together, reveal a remarkable
complexity of chromatin structures at a single locus. These structures impact the expression of neighboring genes in an asymmetric and
tissue-specific fashion. How common these sorts of interactions are and how well they represent other multispecificity loci can now be further
explored.
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ACKNOWLEDGMENTS |
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This work was supported by NIH R01 HD25147 and NIH HD046737 (N.E.C. and S.A.L.) and by postdoctoral fellowship 5013-03 from the Leukemia and Lymphoma Society (I.C.).
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FOOTNOTES |
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These authors contributed equally to this work.
Present address: Children's Hospital of Pittsburgh, Pittsburgh, Pa.
Present address: Pennsylvania State University, University Park, Pa.
¶ Present address: Hokkaido University, Sapporo, Japan.
|| Present address: Ohio State University, Columbus, Ohio.
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