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The Journal of Immunology, 1999, 163: 4637-4641.
Copyright © 1999 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Ig Heavy Chain 3' HS1–4 Directs Correct Spatial Position-Independent Expression of a Linked Transgene to B Lineage Cells1

Christine Chauveau2,*, Emmelie Å. Jansson2,{dagger}, Susanne Müller{dagger}, Michel Cogné* and Sven Pettersson3,{dagger}

* Laboratoire d’Immunologie Génétique, Faculté de Médecine, Limoges, France; and {dagger} Center for Genomics Research, Karolinska Institute, Stockholm, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Ig H chain locus is regulated by a set of cis-acting elements. Hypersensitive sites (HS) located 3' of the IgH, HS1–4, has been suggested to act as a locus control region (LCR) in cell lines. To assess the proposed role of HS1–4 acting as an LCR, we generated transgenic mice harboring a VH promoter-ß-globin reporter gene linked to the Ig H chain HS1–4 3'regulatory sequences. Transgene expression is strictly confined to B lymphocytes, with no detectable expression outside the B cell lineage in all transgenic founder lines. Furthermore, reporter gene activity is integration independent but not copy number dependent. Thus, additional sequences are required to allow the HS1–4 regulatory region to act as a classical LCR in mice. Our data are discussed in the context of tissue-specific gene expression in B lineage cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Regulation of transcription and rearrangement in the Ig H chain (IgH)4 locus is tuned by a complex interplay of multiple regulatory elements. Germline transcription of the VH and Cµ region and initiation of VDJ rearrangements are regulated by upstream elements including the VH promoter, the DQ52 promoter/enhancer, and the Eµ enhancer (1). However, the problems to direct Ig-gene expression in a correct spatial and temporal fashion have focused the attention on additional regulatory elements located within the 3'end of the IgH locus (2). One of these enhancer elements, the IgH 3' enhancer (3, 4, 5, 6) (Fig. 1Go), has been shown to be active in late B cell development and can be activated in resting B cells in a ligand-receptor-dependent fashion (7, 8).



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  		FIGURE 1. Schematic representation of the endogenous H chain locus and the pVH-LCR construct used to generate the transgenic mice. Enhancers are denoted E, with their individual name indicated above; the H chain promoter is denoted pVH. The constant region genes are indicated by filled boxes whereas the VDJ sequences are marked in gray. Shown is also the size of the 3' regulatory region.

 
DNase I hypersensitivity assays and functional assays have revealed three additional transcriptional active enhancers (HS3a, HS3b, and HS4) in addition to the 3'enhancer (HS1,2) in the 3'end of the IgH locus (9, 10, 11) (Fig. 1Go). The overall structure of the IgH 3' region encompassing the HS3a123b4 enhancer elements (referred to as HS1–4) consists of an over 20-kb long palindrome. Thus, HS3a and HS3b elements are part of large inverted repeats flanking the HS1,2 enhancer (12).

A number of Ig-transgenic lines, linked to VH promoter-IgH-Eµ, have been generated with high levels of expression in B lymphocytes but always with aberrant expression outside B lineage cells. Similar data have also been observed with the HS1,2 enhancer-driven transgenes (7, 13).

A DNA fragment containing all 3' HS sites except HS3a has previously been shown to direct position-independent and copy-dependent expression of a linked c-myc gene that was integrated as a stable transfectant in a plasmacytoma cell line. The authors suggested that the 3'end of the IgH locus might act as a locus control region (LCR) (11). An LCR, as originally identified in the ß-globin locus, is defined functionally by its ability to direct tissue-restricted expression of a linked gene in a position-independent, but copy number-dependent, manner (14, 15, 16, 17).

To assess the purported role of the IgH 3' region, acting as an LCR, transgenic mice carrying all the HS1–4 3' IgH enhancers were generated. Whereas this region directs correct B cell-specific expression of a linked VH promoter-dependent reporter gene, no strict copy-dependent expression is observed. Thus, the mechanism of action of the 3' regulatory region on the IgH locus, and IgH gene expression, is more complex than previously anticipated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of plasmid for microinjection

The pVH-LCR vector contains all four 3' IgH enhancers inserted downstream of the reporter gene. The HS1,2 is a 0.6-kb StuI-EcoRV DNA fragment (3, 4). HS3a and HS3b are duplicated enhancers flanking the HS1,2 enhancer but orientated in opposite directions on the chromosome. Two 2.1-kb EcoRI-HindIII genomic fragments, HS3a and HS3b, respectively, were prepared and inserted on both sides of the HS1,2 enhancer, thus mimicking the endogenous configuration (10, 18). HS4 is the 1.38-kb PstI-HindIII DNA fragment (11). The pVH promoter is a 0.2-kb HindIII fragment derived from a rearranged murine VH segment.

Generation of transgenic mice

A purified pVH-LCR DNA fragment was used to generate transgenic founder lines (7). Positive founders were identified by PCR and Southern blot analysis. F2 animals were analyzed for expression and used in all subsequent experiments. The sequences of the oligonucleotides used as primers in the PCR were: CAG GTG CAC CAT GGT GTC (including the NcoI site of the ß-globin gene) and AAG CTT GAA AAC CTC AGA GGA (including the HindIII site of pVH).

RNA extraction and ribonuclease protection assay

Total RNA from different organs was extracted and analyzed by ribonuclease protection assay (RPA) (3). The riboprobe, which spans the VH promoter and 52 bp from the ß-globin gene, was PCR amplified from the pVH-LCR vector and cloned into a topo-cloning vector (Invitrogen, San Diego, CA). A 390-bp nonprotected radioactive riboprobe was achieved by in vitro transcription of an EcoRV cut vector with SP6 polymerase (Promega, Madison, WI) in the presence of [{alpha}-32P]UTP (Amersham, Arlington Heights, IL). This probe generated a 160-bp protected fragment when hybridized to a correctly initiated transcript. The heat shock protein, HSP70, used as internal standard, as well as the Cµ riboprobe, has been described previously (19).

Enrichment of B and T lymphocytes

Isolation of B and T lymphocyte populations was made from single cell suspensions of whole spleen. Splenic T cells were incubated with anti-Thy1.2-coated magnetic beads and isolated on a MiniMACS column according to instructions (Miltenyi Biotech, Auburn, CA). The B cell population were separated on a MiniMACS column following incubation with mouse anti-B220. The isolated cell populations were analyzed for enrichment on a FACScan.

RT-PCR analysis

First strand syntheses were performed on RNA from the different cell populations using the Ready To Go Kit (Pharmacia Biotech, Uppsala, Sweden) as described in the manual. The following primers were used for the PCR amplification: ß-globin transgenic, upper, 5'-TGGTGGTCTACCCTTGG-3'; ß-globin transgenic, lower, 5'-AAGAAAGCGAGCTTAGTGAT-3'; Btk, 5'-CTGGAGAGCATCTTTCTGAA-3'; Btk, 5'-CTTCTCGGAATCTGTCTTTC-3'; and GAPDH (Clontech, Palo Alto, CA). The PCR reactions were performed under the following conditions for 30 cycles: 1 min denaturation at 94°C, 1 min annealing at 54°C, and 2 min at 72°C.

Determination of copy number

Tail DNA (10 µg) was digested with EcoRI and probed with a 2.3-kb fragment, covering a large portion of the ß-globin gene and the VH promoter, generated from an EcoRI digest of the -128 3' E plasmid (7). To determine the copy number of the individual founder animals, the intensity of the bands, quantified by PhosphorImager (Molecular Dynamics, Sunnyvale, CA), was compared with an internal standard. To ensure that the amount of DNA was equivalent, the same blot was subsequently probed with a probe specific for HS4. The primers used to generate the HS4 probe were as follows: HindIII site, 5'-AGGTTGGGTTGGTCACCAGATTCT-3'; PstI site, 5'-CTGCAGACTC ACTGTTCACCATG-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of transgenic mice

To assess whether the enhancers in the 3'end of the IgH locus could act as an LCR, we generated transgenic mice harboring a natural VH promoter-ß-globin reporter gene potentiated by HS1–4 inserted 3' of the reporter gene. The construct (pVH-LCR) thereby mimics the endogenous locus in orientation and relative order (Fig. 1Go). Six independent founder lines, denoted I-VI, were established and analyzed in detail. Expression of the ß-globin reporter gene was tested by RPA. The riboprobe generated a 160-bp protected fragment appearing as a double band (Fig. 2Go). The upper band represented the specific transcript whereas the lower represented a cryptic transcript. As shown in Fig. 2Go, all of the six founder lines were found to express the correct transcript of the reporter gene in RNA prepared from spleen, although at different levels.



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  		FIGURE 2. All founders express the correct transcript. RPA with splenic RNA showing the correct transcript (ß) marked by an arrow and internal standard HSP70. Indicated above is the number of the founder line as well as a negative mouse line. The unspecific transcript is marked by an asterisk.

 
B cell-specific expression of the transgene

To examine whether the transgene was expressed in a tissue-specific manner, RNA was prepared from different organs (spleen, heart, liver, kidney, brain, and thymus) and was determined by RPA. All six founders displayed tissue-specific expression of the transgene; high levels of transgene expression were observed in splenic cells. No expression was detected in the nonlymphoid tissues. Fig. 3Go shows the RPA analysis of three of the founder lines. In addition, a weak signal was observed in thymus. To further examine the expression observed in thymus, we included a Cµ riboprobe, which detects endogenous Cµ gene expression in B lymphocytes, and hence indirectly the number of B cells (Fig. 4GoA). All the thymus RNAs examined contained relatively high levels of the Cµ transcript (Fig. 4GoA), and, since T cells express only marginal levels of Cµ (20), we concluded that the Cµ expression observed originates mainly from activated B cells contaminating the thymus preparations. In addition, RT-PCR analysis was performed, using MACS-sorted splenic B cells as well as T lymphocytes isolated from spleen and thymus originating from the high copy number founder V. The purity of the sorted cells was determined by FACS analysis and found to be 98% for B lymphocytes, 60% for the T cells isolated from spleen, and 99% for thymus (data not shown). To further select for a pure T cell population, Thy1-enriched splenic T cells were stimulated with anti-CD3 for 48 h in vitro. As a control for B cell contamination, we used the expression of the cytoplasmic tyrosine kinase Btk, known to be expressed in B cells but not in T cells (21). GAPDH was used as control for the amount of cDNA. As shown in Fig. 4Go, expression of the transgene was exclusively found in B lymphocytes or in T cell preparations containing contaminating B lymphocytes, as indicated by the positive signal from the Btk control. Moreover, no transgene expression was detected in the anti-CD3-activated pure T lymphocyte population or in the purified T cells from the thymus, where also no detectable amount of B cell contamination was observed, determined by the absence of a positive Btk signal (Fig. 4Go). In conclusion, these data show that the pVH-driven transgene under the control of HS1–4 is expressed in a stringent tissue-specific manner in B cells.



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  		FIGURE 3. Expression of the transgene is tissue restricted. Transgenic lines were examined for expression in various organs. RPA showing the expression pattern of the transgene of the three representative transgenic lines. H, heart; L, liver; K, kidney; B, brain; T, thymus; and S, spleen. Arrows show the migration of the specific transcript (ß), the cryptic transcript (*), and the internal standard (HSP).

 


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  		FIGURE 4. Expression of the transgene is restricted to B lineage cells. A, RPA with RNA prepared from spleen (S) and thymus (T) from different founder lines as marked above. Indicated by arrows are the specific transcripts for the Cµ (Cµ) and HSP70 controls (HSP), as well as that of the transgene (ß). B, RT-PCR analysis from MACS-sorted B and T cells. Indicated are the PCR-amplified ß-globin product, the Btk product, and the GAPDH product.

 
3' region and copy-dependent transgene expression

To firmly evaluate whether the HS1–4 enhancers possessed LCR properties, as described in cell lines (11), we examined the expression of the transgene in relation to the copy number. Copy numbers were determined by Southern blot analysis using a hybridization probe covering the ß-globin gene to the EcoRI site and the complete VH promoter. The same blot was subsequently hybridized with a probe specific for HS4 as internal control (data not shown). Data representing the corrected copy number were plotted (Fig. 5GoA) and shown to range from 2–3 (founder I) to 80–82 (founder V) copies. Transgene expression levels were analyzed by RPA and compared with an internal standard (HSP70). Fig. 5GoB shows the relative expression of the ß-globin reporter gene corrected by the expression of HSP70 and represents the mean value of three independent experiments. Although these experiments show an overall tendency for copy dependence, there is no strict correlation. Particularly, two founder lines (II and IV) with high copy numbers gave rise to only low levels of expression. Furthermore, even though the expression of the transgene was increased in all founders proportionally, upon stimulation of splenic cells with LPS for 72 h in vitro, we did not obtain copy number-dependent expression (data not shown).



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  		FIGURE 5. Expression of the transgene is not copy dependent. a, Copy numbers of the transgenic founder lines. Copy numbers of the individual founder animals were calculated from the intensity of the bands, quantified by PhosphorImager. Copy numbers were corrected by an internal control and plotted against the number of the founder line. b, Expression levels of the transgene measured in spleen. The intensity of the specific transcript (measured by PhosphorImager quantification) of the transgene was divided by the intensity measured from the HSP70 transcript and plotted as relative expression against the number of the founder line. Data represent the mean values of three individual experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we show that a VH promoter-ß-globin reporter gene linked to the HS 1–4 of the IgH locus exhibits B cell-specific expression, thus reproducing the endogenous expression pattern of IgH genes. Using RT-PCR analysis, we show that the transgene is expressed only in splenic B cells or impure T cell fractions that contained residual amounts of B cells, but not in pure T lymphocyte preparations from the thymus or anti-CD3-stimulated splenic T lymphocytes. This is the first time that the endogenous expression pattern of the IgH genes is fully reproduced in a transgenic mouse model. Studies on single enhancer elements in transgenic animals have failed to reconstruct the endogenous expression pattern of the Ig genes, and, in all instances, aberrant expression in other organs have been observed additionally to the spleen. Transgenic mice harboring a VH promoter/intronic Eµ enhancer-linked reporter gene will be predominantly expressed in lymphoid cells (B and T) but with detectable expression in other organs (20, 22). This expression pattern was found even in the presence of the matrix attachment regions flanking the Eµ enhancer, which have been shown to be important for enhancer activity as well as chromatin accessibility (23, 24). Similarly, a VH/HS1,2 linked-reporter gene can direct preferential expression to both B and T lineage cells (13). Interestingly, addition of HS3a, HS3b, and HS4 enhancers in the pVH-LCR construct abrogates the aberrant expression in thymocytes. At present, we do not know the underlying molecular mechanism that directs correct B cell-specific gene expression. One possibility may be that different transcription factors will bind to the enhancer elements forming, a closed structure that could prevent a nonwarranted interaction with other proteins. In the situation with single enhancer elements, such elements would be more accessible to be trans-activated by additional transcription factors and perhaps interact with other cis-acting elements. Alternatively, active repression could be responsible for the down-regulation of expression in T lymphocytes. Recently, a protein binding to a conserved site in the HS3a and HS3b has been described that forms a complex with small Maf proteins. Bach2 is exclusively expressed at early stages of B cell differentiation and has been suggested to act as a repressor on the IgH 3' enhancer region (25). A similar protein could account for the repression of the IgH genes in thymocytes.

In a study employing a stably transfected human B cell line the integration of a c-myc gene under the control of HS1,2,3b,4 was shown to result in a copy number-dependent and position-independent expression (11). To further assess this issue, we have analyzed the ß-globin transgene under control of the HS1–4 in transgenic animals. Although high levels of integration-independent transgene expression in all founders were observed, there was not a strict copy number-dependent expression. We can, of course, not exclude that transgene copies, in the high copy number animals, are transcriptionally inactive. However, fluorescence in situ hybridization (FISH) analysis demonstrated that none of the founders had the transgene integrated in an area of heterochromatin (data not shown). This discrepancy between previous data in cell lines (11) and our study may be explained by the fact that the stably transfected clones are drug selected. Only clones with a certain expression level of the selection marker will be chosen and thereby bias the analysis. On the contrary, no selection pressure is installed on transgenic mice, and all founder lines were analyzed. Similar observations have also been made in the analysis of the ß-globin locus or the Eµ enhancer (26, 27). The construct in the plasmacytoma study did not contain HS3a (11), but it appears unlikely that this enhancer alone would account for the difference observed in the present study. The data presented here do not give support for the proposed model of the 6-kb minilocus of the 3' HS1–4 acting as an LCR in a strict sense. Additional elements may be missing in our construct for the completeness of the LCR. The full palindromic structure of the 30-kb endogenous locus centered at the HS1,2 was not reproduced in our animals; in particular, the inverted repeats flanking HS1,2, which were shown to significantly increase the activity of HS1,2 in plasma cells, were incomplete (28) and may contain elements necessary for a copy number-dependent expression. Alternatively, but not mutually exclusively, it is tempting to speculate on a model of a split LCR composed of the Eµ enhancer and the 3' HS1–4 that surround the Ig genes (see Fig. 1Go). In the endogenous locus, a cooperation between the intronic and 3' enhancer elements with the VH promoter ensures the correct spatial and temporal expression of Ig genes. The absence of Eµ and its flanking matrix attachment regions may have influenced the LCR function in our animals. Such a model is further supported by the finding that Eµ together with HS4 is active in early B cell development. Both 5' and 3' elements may therefore be necessary to border and insulate the Ig locus, keeping it in an open chromatin conformation. The pVH-LCR construct described here has the unique ability to direct B lymphocyte-specific expression, which indicates that the 3' regulatory region certainly plays a role in the control of Ig gene expression. The proposed model of a split LCR guiding IgH gene expression is very important to test since such experiments will be instrumental not only to learn about Ig-gene expression but also to further our understanding on the mechanisms of LCR.


   Acknowledgments
 
We thank Anna Ridderstad for excellent technical assistance.


   Footnotes
 
1 This work was supported by Cancerfonden, Sweden. Back

2 The first two authors contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Sven Pettersson, Center for Genomic Research, Karolinska Institute, S-17177 Stockholm, Sweden. E-mail address: Back

4 Abbreviations used in this paper: IgH, Ig H chain; LCR, locus control region; HS, hypersensitive site; HSP, heat shock protein; RPA, ribonuclease protection assay. Back

Received for publication June 29, 1999. Accepted for publication August 24, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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