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The Journal of Immunology, 1999, 163: 5758-5762.
Copyright © 1999 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: IFN-{gamma} Regulated Germline Transcripts Are Expressed from {gamma}2a Transgenes Independently of the Heavy Chain 3' Enhancers1

John T. Collins and Wesley A. Dunnick2

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Several results indicate that transcriptional enhancers lying 3' of the C{alpha} gene regulate RNA expression and switch recombination of heavy chain genes. To investigate this regulation we prepared transgenic mice with a 10.5-kb transgene that included the germline form of the murine {gamma}2{alpha} gene, including promoter, I, S, and C regions. RNA was expressed from these {gamma}2a transgenes with correct IFN-{gamma} regulation, in spite of the fact that they lacked the 3' enhancers. This RNA expression was independent of insertion site and dependent on copy number, indicating that the {gamma}2a gene includes locus control region-like elements. Addition of either a cassette containing 3' enhancer DNase I hypersensitive sites 1, 2, 3B, and 4 or the intronic µ enhancer increased transcription from the {gamma}2a transgene by ~75-fold in B cells. However, this increased transcription was not responsive to IFN-{gamma} treatment of the transgenic B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Several results implicate a series of five DNase I hypersensitive sites (HS1, HS2, HS3A, HS3B, and HS4)3 that lie 2–32 kb 3' of the C{alpha} gene (1, 2, 3, 4) in heavy chain gene activity. All of these hypersensitive sites have been shown to be active as enhancers in B cells by transient transfection (3, 4, 5, 6, 7, 8, 9, 10), stable transfection (2), or transgenesis (11, 12, 13), and so we shall refer to them collectively as the "3' enhancers." The 3' enhancers seem to be more active in mature B cells than in the pre B or plasma cell stages of B cell development (9, 10), and the activity of HS1 and HS2 seems to increase in activated B cells (11, 12, 13). Various combinations of the 3' enhancers are highly synergistic with one another (2, 9, 10). Madisen and Groudine (2) found that a combination of HS1, HS2, HS3B, and HS4 was expressed independently of insertion site, but in a copy number dependent way in stable transfectants. Since insertion site independence and copy number dependence define a locus control region (LCR) in transgenic mice, Madisen and Groudine (2) suggested that the 3' enhancers together have the activity of an LCR. The spontaneous deletion of all these elements (3, 14) or replacement of HS1/HS2 by a phosphoglycerate kinase promoter driving a neomycin resistance gene (15) dramatically reduced expression of a VDJ-rearranged heavy chain gene on the same chromosome. These results in cell lines further imply a function for these elements in heavy chain gene expression.

Disruption of these elements in the endogenous locus has dramatic effects on heavy chain gene function. Replacement of either HS1/HS2 or HS3A by the promoter:neo cassette discussed above dramatically inhibits expression of the {gamma}3, {gamma}2b, {gamma}2a, and {epsilon} heavy chains (16, 17, 18). These replacements inhibit both switch recombination that results in the expression of these isotypes and the germline transcription that precedes switch recombination (19, 20, 21, 22).

Although each of the studies discussed above points toward a role of the 3' enhancers in heavy chain gene function, none of them unambiguously demonstrate it. Deletion of HS1/HS2 or HS3A without the promoter:neo cassette insertion does not lead to significant reduction in either germline transcription or switch recombination (16, 17, 18), arguing against an obligatory requirement for HS3A and HS1/HS2. We decided to use a positive test for activity of the 3' enhancers in normal B cells. We chose to link the 3' enhancers to a {gamma}2a transgene, because expression of IgG2a was dramatically down-regulated by the hypersensitive site replacements (16, 17, 18), and expression of the {gamma}2a promoter is regulated by cytokines (23, 24, 25). To this end we prepared transgenic mice with a germline version of the murine {gamma}2a gene, with or without a cassette including HS1, HS2, HS3B, and HS4 (the DNA cassette hereafter referred to as "HS1234"). We investigated the ability of B cells from these transgenic mice to express transgenic germline {gamma}2a transcripts in response to appropriate stimuli.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Transgenic constructs

The 10.5-kb ISC{gamma}2a fragment was created by linking, in the order and orientation of the endogenous gene, the C{gamma}2a 4.4-kb EcoRI/BglII from MIgG2a-11 (26), the S{gamma}2a 4.5-kb EcoRI fragment from pS{gamma}2a-1 (originally derived by Ken Marcu, State University of New York, Stony Brook; Ref. 25), and the I{gamma}2a 1.6-kb EcoRI fragment from p{gamma}2a/E1.6 (25) into Bluescript KS-. The EcoRI sites 5' and 3' of I{gamma}2a were each cut with EcoRI, filled in with the large fragment of DNA polymerase, and religated, resulting in a 4-bp insertion in the I exon of the transgene. The 8.5-kb HS1234 cassette was created by cloning the 5-kb HS12 NotI fragment from pHS12 into pHS34 (2), resulting in the relative orientation of the four hypersensitive sites found in germline DNA. The 1-kb Eµ XbaI fragment (27) was cloned into Bluescript KS-.

Transgenic mice were produced by injection of SalI/SstII fragments (vector-free) into fertilized (SJL x C57BL/6)F2 eggs (28). Transgenic lines were established and maintained by breeding to (SJL x C57BL/6)F1 males or females.

RNA expression analysis

T cell-depleted or RBC-lysed splenocytes from transgenic mice or nontransgenic littermates were cultured for 3 days in growth medium containing combinations of 20 µg/ml LPS, 100 U/ml IFN-{gamma}, and CD40 ligand (CD40L)-expressing Sf9 cells (29). RNA was extracted from cell cultures and tissues by the single-step method (30). RT-PCR products (25) were radiolabeled by the addition of [32P]dATP to the reactions and fractionated by electrophoresis on 6% acrylamide gels. cDNAs representing both endogenous and transgenic {gamma}2a-processed transcripts were amplified using the primers I{gamma}2a-4 and C{gamma}2a-5 (25) for 29 cycles (95oC, 62oC, and 72oC, each for 1 min). Hypoxanthine phosphoribosyltransferase (HPRT) cDNA was amplified for 24 cycles as described (31). I{gamma}2a-containing germline transcripts were detected with the JCD226 probe, and actin transcripts were detected with the JC85 probe, using S1 nuclease protection (25). For S1 nuclease analysis of the start sites of {gamma}2a transcripts, a 233-bp AlwNI/PvuII fragment, which includes residues 921-1154 relative to the EcoRI site 5' of I{gamma}2a (GenBank accession number L08600), was cloned into phage M13 and designated JCA233.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Expression from {gamma}2a transgenes

To study the expression of a heavy chain gene whose activity is apparently influenced by the 3' enhancers, we prepared several independent lines of transgenic mice with a 10.5-kb {gamma}2a gene. This construct begins at -900 bp relative to the 5' most start site for germline transcription and extends through the complete I, S, and C regions 2 kb 3' of C{gamma}2a (Fig. 1GoA). Splenic B cells were prepared from such transgenic mice and cultured with LPS or LPS + IFN-{gamma} for 3 days. Transgenic, endogenous {gamma}2a and HPRT RNA from these cultures was detected by semiquantitative RT-PCR. All PCRs crossed introns, and therefore detected only processed RNA. We had inserted 4 bp at the EcoRI site in the I exon of the transgene; thus PCR products from transgenic transcripts were 4 bp longer than those from the endogenous gene (403 vs 399 bp).



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  		FIGURE 1. Structure and copy number analysis of transgenes. A, Schematic diagram of the murine heavy chain locus and the DNA fragments that were cloned for transgenic preparation. Regulatory elements are depicted as ovals, exons are dark hatched rectangles, and switch regions as light hatched rectangles. B, BamHI; S1, SalI; SII, SstII; E, EcoRI; P, PstI. B, Representative Southern blot used in the verification of transgene insertion and the determination of transgene copy number. BamHI-digested tail DNA was analyzed using the BamHI/SstII fragment from the 3' end of the ISC{gamma}2a cloned fragment. Head-to-tail insertions of the ISC{gamma}2a fragment are revealed by hybridization as a 5.2-kb BamHI fragment (4.7 kb from the tail + 0.5 kb from the head). Alternating, head-to-tail insertions of the ISC{gamma}2a + either HS1234 or Eµ are revealed as a 4.7-kb BamHI fragment (the BamHI site is in the polylinker at the head of the HS1234 or Eµ fragments). Copy number was determined by quantitation on a PhosphorImager, with the endogenous band ("Endg.") as a standard, 2 gene copies.

 
Shown in Fig. 2Go is a survey of transgenic mice ranging in copy number from 2 to ~11. Expression of the transgene is dependent on copy number. Using expression from the endogenous {gamma}2a gene as a standard, the most transgenic RNA is detected from LPS + IFN-{gamma}-treated B cells with the most copies of the transgene (lines 4606 and 730). Under the conditions of PCR and exposure for the experiment in Fig. 2Go, transgenic RNA is not detectable in lines with two copies of the transgene (however, see below). In some cases significant endogenous {gamma}2a germline transcripts were detected in LPS-treated splenic B cells, probably due to in vivo exposure to IFN-{gamma} (our mouse colony is infected with mouse hepatitis virus). Nevertheless, even in the presence of some {gamma}2a induction in vivo, we detect IFN-{gamma}-dependent increases in {gamma}2a germline transcripts in vitro. Transgenic RNA expression parallels that of the endogenous gene; in the higher copy number mice, transgenic RNA is also increased by IFN-{gamma} treatment of the T-depleted splenocytes.



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  		FIGURE 2. The ISC{gamma}2a transgene is insertion site independent and copy number dependent. RT-PCR was performed with RNA extracted from T-depleted splenocytes harvested from various transgenic mice (copy numbers as indicated) after 3-day cultures in the presence of LPS, with or without IFN-{gamma}. "Relative expression" is the ratio of the amount of transgenic PCR product to the amount of endogenous PCR product, as determined by densitometry of the x-ray film. The asterisked values were too small to accurately determine. The relative expression of 0.60 in line 4606 is anomolously high due to false signal on the left-hand side of the lane.

 
Transgenic RNA expression is independent of insertion site of the transgene. All founder lines with copy number >3 express significant transgenic transcripts. Even though expression in the B cells with two copies of the transgene (722, 733, and 4601) is very low, it is apparently detected after longer exposures of x-ray films (Fig. 3Go). Moreover, the transgene exhibits at least some of the cell type specificity of the endogenous gene; neither the endogenous nor the transgene are expressed in thymocytes (Fig. 3GoA, lanes 3, 6, and 11). To ensure that our PCRs were within a semiquantitative range, we performed PCRs from various cDNA dilutions for almost all the samples shown in Figs. 2Go and 3GoA. As examples, the semiquantitative nature of the PCR and IFN-{gamma} inducibility of the transgene is demonstrated for two transgenic lines in Fig. 3GoB. Five-fold dilutions of cDNA result in a 3- to 5-fold reduction in PCR signal for both the {gamma}2a and the HPRT reactions.



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  		FIGURE 3. The ISC{gamma}2a transgene is expressed in a cytokine-inducible and cell-type specific manner. A, RNA from 3-day cultures of T cell-depleted splenocytes or fresh thymocytes was analyzed by RT-PCR. B, Semiquantitative radiolabeled PCR. Three (4601 samples) or four (131 samples) 5-fold dilutions of cDNA were amplified for the indicated mRNAs from T-depleted splenocytes treated as indicated. The relative expression values, as defined in the legend for Fig. 2Go, for the 4601 LPS + IFN{gamma} samples were 0.020–0.026.

 
Hence, because RNA expression from {gamma}2a transgenes is insertion site independent and copy number dependent, the {gamma}2a gene includes elements that resemble an LCR. Surprisingly, expression of the {gamma}2a gene does not depend strictly on the 3' enhancers. RNA from {gamma}2a genes can be expressed, with cytokine inducibility, in many different DNA sites in the absence of other regulatory elements from the heavy chain locus.

Expression of {gamma}2a transgenes linked to enhancers

On a per gene basis, RNA expression from the ISC{gamma}2a transgenes is roughly 10% that of the endogenous gene. We hypothesized that the 3' enhancers might increase this RNA expression to be equal to that of the endogenous gene. We prepared three lines of transgenic mice by coinjection of the ISC{gamma}2a transgene and a cassette first derived by Madisen and Groudine (2) that includes HS1, HS2, HS3B, and HS4. The "head" of the ISC{gamma}2a transgene and the "tail" of the HS1234 cassette shared the same restriction site (SalI) and the tail of the ISC{gamma}2a and the head of the HS1234 cassette shared the same restriction site (SstII, Fig. 1GoA). Analysis of genomic DNA in Southern hybridization with both {gamma}2a and HS3/HS4 probes demonstrated that virtually all transgenic copies inserted head to tail, alternating the {gamma}2a and the HS1234 pieces of DNA (Fig. 1GoB). We presume that DNA repair activities in the fertilized eggs ligated like restriction sites to one another before the insertion process.

Splenocytes from ISC{gamma}2a + HS1234 transgenic mice were cultured in LPS with or without IFN-{gamma}. Transgenic RNA expression was up-regulated dramatically (about 75-fold) compared with ISC{gamma}2a-only transgenes, and therefore we could detect it by S1 nuclease protection (Fig. 4GoA). Transgenic and endogenous transcripts are not distinguished using this probe; both protect the same 226-bp fragment. However, endogenous {gamma}2a expression is only a few percent of the total {gamma}2a RNA expression, as demonstrated by the fact that {gamma}2a RNA expression is almost undetectable in nontransgenic B cells treated with LPS + IFN-{gamma} (Fig. 4GoA, lane 1) or in line 4606 (ISC{gamma}2a) transgenic cells (lane 2, about one-half of the RNA expression is from the transgene; Fig. 2Go). However, this high level of stable RNA expression driven by the HS1234 cassette is not responsive to IFN-{gamma} treatment, taking into account total RNA recovery as estimated by actin protection (Fig. 4GoA, lanes 9–14; Fig. 4GoB, lanes 1–3). We observed at most a 2- to 3-fold effect of cell activation by LPS or CD40L (for example, fresh splenocytes in Fig. 4GoA, lane 11, vs LPS + IFN-{gamma}-treated T-depleted splenocytes in lane 12; Fig. 4GoB, lanes 1 and 2). The HS1234 effect seemed to be restricted to B cells. Transgenic RNA was not detected in thymocytes or various nonlymphoid tissues (Fig. 4GoB, lane 4 and data not shown).



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  		FIGURE 4. Expression of ISC{gamma}2a transgenes is increased 25- to 75-fold when linked to enhancers from the murine heavy chain locus. I{gamma}2a and actin transcripts were detected by an S1 nuclease protection assay. A, RNA was prepared from total splenocytes (RBC-lysed) cultured for 3 days with the indicated additions. The RNA analyzed in lane 11, however, was prepared from fresh (not cultured) splenocytes. Transgenic donors of the splenocytes all carried the ISC{gamma}2a transgene linked to Eµ or HS1234 (as indicated) and are identified by founder number (275, etc.). Transgene copy numbers are indicated. B, RNA was prepared from fresh total splenocytes, fresh thymocytes, or RBC-lysed splenocytes which had been cultured for 3 days with LPS or LPS + IFN-{gamma}. All cell samples are from line 4443 (ISC{gamma}2a + HS1234). C, Transcription start site analysis with S1 nuclease. A total of 40 µg nontransgenic or 10 µg transgenic RNA was hybridized to JCA233 for S1 nuclease analysis. The 5'-most start site in the nontransgenic sample (lane 1) was defined as "+1" and other start sites are indicated by nucleotide distance relative to it (left side of the panel). The probe extends 69 bp 5' and 164 bp 3' of +1, and the approximate migration position of full length protection of the 233-bp insert is indicated ("FL"). +1 and +51 correspond to the two major transcription start sites in 18.81A20 cells (25 ). The material at the top of each lane is undigested probe, or probe that has been "nibbled" into the M13 polylinker portion.

 
To determine whether the extraordinary enhancement of the {gamma}2a germline promoter was a unique ability of the HS1234 cassette, we also prepared mice with the ISC{gamma}2a transgene coinjected with the Eµ element. We constructed the Eµ fragment so that its ends shared SstII and SalI restriction sites with the ISC{gamma}2a fragment, and again observed mostly head-to-tail insertions, alternating ISC{gamma}2a and Eµ elements. Because Eµ is active in either orientation (27), we inserted it backwards relative to ISC{gamma}2a. Thus, transcripts initiating from the germline promoter in this element (32) would proceed backwards through the {gamma}2a gene, and would be less likely to be scored as spliced {gamma}2a germline transcripts. We found that Eµ is also able to interact with the {gamma}2a germline promoter. Very high levels of transgenic {gamma}2a transcripts were observed in splenic B cells from these mice (Fig. 4GoA, lanes 3–8). The levels were slightly less than those from ISC{gamma}2a + HS1234 B cells, but on a per gene basis approached them. As with the ISC{gamma}2a + HS1234 transgenic B cells, the transgenic transcripts were not regulated by IFN-{gamma} (Fig. 4GoA, lanes 3–8). One way in which the ISC{gamma}2a + Eµ transgenic mice differed from the ISC{gamma}2a + HS1234 transgenic mice was that the former had abundant transcripts in thymocytes (data not shown), as predicted by many other studies of this element (33, 34).

Given the very high level of stable transcripts, and the lack of IFN-{gamma} regulation, we considered the possibility that the HS1234 enhancers led to transcription through the I{gamma}2a exon that was not analogous to endogenous {gamma}2a germline transcription. Our assumption was that transcripts related to normal germline transcription would initiate in the same region as the endogenous {gamma}2a transcripts, whereas inappropriate transcripts would initiate at a significant distance outside it, for example in HS1234 or in the C region. We tested the transcription start sites by S1 nuclease protection (Fig. 4GoC), again taking advantage of the fact that virtually all of the {gamma}2a transcripts derive from the transgene in ISC{gamma}2a + HS1234 B cells. Using a large amount of RNA from nontransgenic cells treated with LPS + IFN-{gamma}, we identified three predominant transcription start sites. If transcripts from the ISC{gamma}2a + HS1234 (lines 4442 and 4443) B cells initiated outside the endogenous start site region, but continued through the I{gamma}2a exon, we would have detected full-length protection of the probe used. At most, 5% of the transcripts initiate outside these DNA sequences; the vast majority of transgenic transcripts initiate in the same region as the endogenous {gamma}2a transcripts, as defined by the nontransgenic RNA (Fig. 4GoC, lanes 1–3). Some transgenic transcripts apparently use the same nucleotide start sites, and many use a different set of exact nucleotides for the start sites (Fig. 4GoC, compare lanes 2 and 3 to 1). (The exact start sites used for transgenic transcripts, and their relative abundance, was not obviously altered by B cell activation or cytokine treatment, data not shown.) Thus, the transgenic transcripts are like endogenous germline transcripts in that they initiate in the same region.

Interactions between the germline promoter and 3' enhancers

The level of RNA expression from the ISC{gamma}2a + HS1234 transgene suggests that the {gamma}2a promoter and HS1234 interact efficiently with one another. Given the tight spacing of the {gamma}2a gene and HS1234 in the transgenic array, and the tight spacing of HS1234 to each other, these interactions may be different from those in the endogenous locus. These differences in interactions are manifest in two ways. 1) Dramatically increased transcription of the {gamma}2a transgene compared with the endogenous gene. As discussed above, HS1234 increases the RNA expression of ISC{gamma}2a transgenes by about 75-fold. Per gene, about 25 times more RNA is expressed from the ISC{gamma}2a + HS1234 transgene compared with the endogenous {gamma}2a genes in an LPS + IFN-{gamma}-stimulated B cell. The difference is greater in resting B cells. 2) Loss of the regulation by cytokine and B cell activation. In addition, the use of slightly different transcription start sites is likely due to altered promoter-3' enhancer interactions, but could also be a property of the ISC{gamma}2a transgene, regardless of the 3' enhancers.

This study emphasizes the strong enhancer activity in B cells of the combined HS1234 fragments (2, 9, 10). We observed extraordinarily strong enhancement of the {gamma}2a germline promoter, many-fold over transgenes without the HS1234 enhancer and over the endogenous gene. Use of the HS1234 cassette, with an appropriate B cell promoter, may be one of the best ways to obtain high level transcription of a transgene in the B cell compartment. Contrary to the Eµ element, we did not observe transcription in thymocytes using HS1234, albeit with a single promoter tested.

An important finding from this study is that {gamma}2a transgenes include LCR-like activity, and therefore can express germline transcripts in the absence of linked 3' enhancers, or any other element from the heavy chain locus. Apparently, the 3' enhancers of the heavy chain locus do not regulate germline transcription in an absolute manner. Rather, they may make a quantitative contribution to germline transcription. We hypothesize that, in isolated ISC{gamma}2a transgenes, inducible, cell type-specific, and more general transcription factors bind to the {gamma}2a germline promoter and lead to transcription. In the endogenous locus, the transcriptional activity of the same DNA binding proteins may be enhanced by interactions with DNA proteins binding the 3' enhancers. The spatial limitations of the transgenic approach used in our study and by others (11, 12, 13) may not allow one to observe the regulated promoter-3' enhancer interactions of the endogenous locus.


   Acknowledgments
 
We thank Drs. Cheong-Hee Chang, Lathe Claflin, and John Manis for their helpful comments on the manuscript. We acknowledge Mark Berard and Maggie Van Keuren for preparation of transgenic mice and the Transgenic Animal Core of the University of Michigan Biomedical Research Core Facilities. We thank Kristine Adams for her expert assistance in these studies.


   Footnotes
 
1 This work was supported by a grant from the National Cancer Institute, CA39068. Transgenic Core support was provided by The University of Michigan Cancer Center, National Institutes of Health Grant CA46592. Back

2 Address correspondence and reprint requests to Dr. Wesley Dunnick, Room 6746 Medical Science Building II, 1301 E. Catherine, Ann Arbor, MI 48109-0620. E-mail address: Back

3 Abbreviations used in this paper: HS1, HS2, etc., DNase I hypersensitive sites 1, 2, etc. that lie 3' of C{alpha}; CD40L, CD40 ligand; Eµ, intronic enhancer associated with the Cµ gene; I exon, 5' most exon of heavy chain germline transcripts; LCR, locus control region; HPRT, hypoxanthine phosphoribosyltransferase. Back

Received for publication July 20, 1999. Accepted for publication October 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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