Human High Mobility Group Box 1 Affects Thymocyte Development and Transgene Variegation

This information is current as Belaïd Sekkali, Ewa Szabat, Eleni Ktistaki, Mauro Tolaini, of September 28, 2021. Kathleen Roderick, Nicky Harker, Amisha Patel, Keith Williams, Trisha Norton and Dimitris Kioussis J Immunol 2005; 175:5203-5212; ; doi: 10.4049/jimmunol.175.8.5203

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Human High Mobility Group Box Transcription Factor 1 Affects Thymocyte Development and Transgene Variegation1

Belaı¨d Sekkali,2 Ewa Szabat,3 Eleni Ktistaki,3 Mauro Tolaini, Kathleen Roderick, Nicky Harker, Amisha Patel, Keith Williams, Trisha Norton, and Dimitris Kioussis4

It has been shown previously that a human CD2 (hCD2) disabled control region (LCR) transgene is unable to establish an open chromatin configuration in all the T cells, and this leads to position effect variegation of the transgene. In this study we show that thymus-specific overexpression of human high mobility group box transcription factor 1 (HBP1), a transcription factor that binds a specific sequence within the hCD2 LCR, affects thymus cellularity as well as the number of CD8؉ thymocytes in two independent transgenic mouse lines and increases the proportion of T cells that fully activate the transgenic locus in hCD2 variegating mice in a sequence-specific dependent manner. This finding suggests that overexpression of HBP1 can affect lineage commitment and can relieve the suppressive influence of heterochromatin, allowing thymocytes to express the variegating target Downloaded from locus more efficiently. These effects could be the result of direct HBP1 action on LCR activity. Alternatively, the extra HBP1 molecules may sequester repressive elements away from the LCR, thus allowing transcription permissive states to form on the transgene locus. The Journal of Immunology, 2005, 175: 5203–5212.

ransgenic mouse research on expression has led to Deletion analysis has shown that the 2-kb 3Ј-flanking region of

the discovery of a novel class of cis-acting regulatory el- the hCD2 locus, which contains these clusters of HSSs, is sufficient http://www.jimmunol.org/ T ements, locus control region (LCR),5 which acts in cis to for hCD2 LCR function (2, 5). Transgenic lines carrying the hCD2 ensure that active transcriptional units are established in all cells of minigene, in which the HSS3 is either partially deleted or omitted, a given tissue (1). LCRs can direct the expression of transgenes in are subject to position effect variegation (PEV) when the transgene a tissue-specific, position-independent, and copy number-depen- is integrated in transcriptionally repressive regions, such as those dent manner. Studies of the human CD2 (hCD2) and ␤-globin found in centromeres. PEV is a phenomenon observed with LCRs have led to the proposal that LCRs are involved in the es- that are in close proximity to repressive heterochromatin locations. tablishment and/or maintenance of an open chromatin configura- In such cases heterochromatin is thought to spread to a varying tion by overcoming heterochromatin-mediated silencing (1–4). degree in different cells of the same lineage. Consequently, in by guest on September 28, 2021 The hCD2 gene is expressed in all T cells and thymocytes. Stud- some of the cells the gene is spared heterochromatinization, and ies using transgenic mice have established that a 28.5-kb fragment therefore it is expressed, whereas in cells in which the gene has Ј containing the human CD2 gene and 5 kb of 5 -flanking and 9 kb been engulfed by heterochromatin it is silenced. This effect in our Ј of 3 -flanking sequences carries all the necessary information for PEV model is apparently transgene copy number and orientation correct tissue-specific expression (5). Within this fragment, a pro- independent (2). Furthermore, it appears that the decision on Ј moter together with a classical enhancer that is located 3 to the whether to express a hCD2 transgene with a disabled LCR is a gene have been identified (6). The enhancer coincides with a stochastic one, and once made, it is maintained through subsequent strong DNase I-hypersensitive site (HSS) called HSS1. Two ad- cell divisions (4). Very similar observations were made in exper- ditional HSSs (HSS2 and HSS3) with no enhancer activity were iments involving other disabled LCRs (3, 7–9). also identified within the 2-kb 3Ј-flanking region (2). Disabled LCRs reveal their sensitivity to position effects when integrated into heterochromatic regions of the mouse genome (4, National Institute for Medical Research, Division of Molecular Immunology, London, 9). Such chromosomal locations induce a mosaic expression pat- United Kingdom tern, which bears striking similarity to PEV as described in Dro- Received for publication September 20, 2004. Accepted for publication July 27, 2005. sophila and yeast (10). It has been reported that repeats of trans- The costs of publication of this article were defrayed in part by the payment of page genes in Drosophila can induce heterochromatinization and charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. subsequently PEV (11). Furthermore, multiple copies of a lacZ ␤ 1 This work was supported by the Medical Research Council, United Kingdom. B.S. transgene reporter under the control of the human -globin LCR was supported by Grant PL970203 from the European Community. E.K. was sup- also are subject to PEV. However, the latter result should be seen ported by Marie Curie Individual Fellowship HPMF-CT-2000-00805 from the Euro- in the light of numerous studies showing that lacZ gene sequences pean Community. can have an adverse effect on the transcriptional activity of the 2 Current address: Department for Molecular Biomedical Research VIB, Ghent Uni- versity, Ghent, B-9052 Belgium. transgenic locus (12–15). We have shown previously that multiple 3 E.S. and E.K. contributed equally to this work. copies of a CD2 transgene with a full LCR integrated in the cen- tromere do not variegate, whereas low copy (two to three) inte- 4 Address correspondence and reprint requests to Dr. Dimitris Kioussis, National Institute for Medical Research, Division of Molecular Immunology, The Ridgeway, grants of constructs with disabled LCR in the centromere can be Mill Hill, London, U.K. NW7 1AA. E-mail address: [email protected] severely affected by the surrounding heterochromatin. Conversely, 5 Abbreviations used in this paper: LCR, locus control region; DN, double negative; multiple copies of hCD2 with disabled LCR integrated in a favor- GH, growth hormone; h, human; HMG, high mobility group; HBP1, human HMG box transcription factor 1; HSS, DNase I-hypersensitive site; PEV, position effect able chromosomal position do not exhibit variegation (2). Re- variegation; SP, single positive; LEF, lymphoid enhancer factor. cently, using Cre-loxP technology in our laboratory, we have

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 5204 HBP1 IN THYMIC DEVELOPMENT AND VARIEGATION shown that reducing the transgene copy number in a variegating Northern blot analysis mouse increased PEV (A. Williams, manuscript in preparation). Total thymocyte RNA was extracted using TRIzol reagent (Invitrogen Life Thus, there is strong evidence that variegation of hCD2 transgenes Technologies) according to the manufacturer’s instructions. RNA mole- is not related to the number of integrated copies. cules were resolved by agarose gel electrophoresis and transferred onto Recently, high mobility group (HMG) box transcription factor 1 Hybond-N membranes (Amersham Biosciences) in 20ϫ SSC, 3 M NaCl, (HBP1), a member of the HMG box family that is expressed highly and 0.3 M sodium citrate; pH 7.0. Membranes were hybridized to a probe containing the open reading frame of human HBP1 in 3ϫ SSC, 0.1% SDS, in the thymus, was shown by us to bind a region in the hCD2 LCR. 10ϫ Denhardt’s solution, 10% dextran sulfate, and 250 ␮g/ml denatured Deletion of the HBP1 binding site (FT1) from the LCR leads to salmon sperm DNA at 65°C for 16 h and washed to a final stringency of PEV when the transgene is integrated in heterochromatin regions 0.2ϫ SSC/0.1% SDS at 65°C. As a loading control, the membranes were (9). We proposed at the time that HBP1 might play a direct or hybridized to a GAPDH probe as described above. indirect role in chromatin opening and in establishing an active FACS analysis locus. A similar role has been attributed to the erythroid-specific erythroid Kruppel-like factor (16), which is essential for adult T cells from thymus were stained with saturating concentrations of FITC-, ␤ PE-, PerCP-, or Tricolor-conjugated Abs in PBS plus 2% FCS-1% BSA at -globin (17, 18) and is involved, via SWI/SNF 4°C for 30 min, then washed with PBS and subsequently analyzed using family members (16), in the remodeling of the ␤-globin locus into BD Biosciences FACSCalibur and CellQuest programs. The following Abs an active structure (19). were used in this study: FITC-anti-mCD8␣ (YTS169.4), PE-anti-human HBP1 is a member of the HMG family capable of in- CD2 (BD Pharmingen), FITC anti-mTCR ␤-chain (Caltag Laboratories), PerCP-anti-mCD8␣ (Ly-2; 1/200; BD Pharmingen), allophycocyanin-anti- teracting with specific DNA sequences. The DNA specificity is mCD4 (1/400; BD Pharmingen), and Tricolor-anti-mCD4 (Caltag Labora- variable among the members, and based on methylation interfer- tories). T cells from the hCD2 nonvariegating line Mg4 and CBA or B10 Downloaded from ence assays performed for lymphoid enhancer factor (LEF)-1/T have been used as positive and negative controls for hCD2 expression, cell factor-1 factor, a consensus heptamer-binding site for se- respectively. The experiments shown in Figs. 5 and 6A were performed quence-specific HMG boxes (A/T)(A/T)CAAAG was originally with a different batch of hCD2 Ab from that used in the rest of the exper- iments; as a result, the average values from different experiments may proposed (20). Subsequent studies of LEF-1, SRY, SOX-4, and appear different for the same line. The comparisons presented in this study SOX-5 were in agreement with this idea (21–24). However, dif- are made within the same experiment. ferent binding sites sharing a TCAT tetranucleotide have been re- Double-negative (DN) thymocytes were selected by incubating the cells http://www.jimmunol.org/ ported for HBP1 (25, 26). Among sites that bind HBP1, the with biotinylated anti-CD8␣ and anti-CD3⑀ Abs, followed by streptavidin MicroBeads (Miltenyi Biotec). The cells were stained with PE-conjugated TTCATTCATTCA binding site, found in the hCD2-LCR, appears anti-CD8␤, anti-TCR␤, anti-Mac1, anti-Ter119, anti-CD19, anti-DX5, and to have the highest affinity (9). streptavidin (all Abs were from BD Biosciences), and PE-negative cells In this study we investigated the effects of increased levels of were sorted according to CD44 and CD25 expression on MoFlo sorter HBP1 in overcoming heterochromatin-mediated transgene silenc- (DakoCytomation). ing and in thymocyte development by generating transgenic mice overexpressing HBP1 in thymocytes. Such mice exhibit lower thy- mus cellularity and have increased numbers of mature CD8 single- positive (SP) cells in the thymus. Using two independent hCD2 by guest on September 28, 2021 variegating transgenic reporter lines, it was found that thymus- specific overexpression of HBP1 in two independent HBP1 trans- genic effector lines can lead to an increase in the proportion of thymocytes that fully express the transgenic hCD2 locus, provided the HBP1 binding site is present on the reporter construct.

Materials and Methods Transgenic mouse generation ϫ Mice (CBA/Ca and C57BL/10 and (CBA/Ca C57BL/10) F1) were bred at the National Institute for Medical Research in London. Transgenic mice were generated by injecting DNA constructs into ((C57BL/10/CBA) ϫ

(C57BL/10/CBA)) F2 fertilized mouse eggs. The human HBP1 cDNA (9) was put under the control of the p56lck proximal promoter by cloning it by blunt end into the BamHI site of the p1017 vector (gift from Dr. R. Perl- mutter, Amgen, Thousand Oaks, CA). The hCD2-1.5 construct has been described previously (2). Transgenic founder animals were identified by Southern blot analysis of DNA from tail biopsies and bred to CBA mice to establish the transgenic lines. Each transgenic line was backcrossed to CBA at least four times. All animals were maintained in specific pathogen- free facilities at the National Institute for Medical Research. Mouse han- dling and experimental procedures were conducted in accordance with na- tional and institutional guidelines for animal care and use.

Fluorescence in situ hybridization Metaphase spreads were obtained from transgenic mouse spleen cells cul- tured for 2 days after LPS stimulation (Sigma-Aldrich; final concentration, FIGURE 1. Northern blot analysis of HBP1 expression in the thymus of 20 mg/ml). The hCD2 DNA probe was labeled and hybridized with the HBP1-overexpressing and age-matched CBA mice. RNA was isolated metaphase spreads following procedures previously described (2). Slides were then mounted in Antifade (Vector Laboratories), counterstained with from adult mouse thymus and hybridized with an open reading frame of 4Ј,6-diamidino-2-phenolindole, and subsequently examined using a Delta hHBP1. The lower panel shows the hybridization with GAPDH probe as a Vision fluorescence microscope. Images were collected with cooled CCD loading control. At the top of the figure, a schematic representation of camera (Photometrics) using capture software (Soft WoRx). HBP1 domains and the construct used for transgenesis are depicted. The Journal of Immunology 5205

RT-PCR analysis of RNA (9). We proposed that HBP1 might play a role in chromatin open- For the HBP1.C line, total RNA from sorted DN subpopulations was pre- ing and/or remodeling activities by binding to the LCR. To inves- pared with TRIzol (Invitrogen Life Technologies) following the manufac- tigate further the effect of HBP1 on chromatin configuration, and turer’s instructions. For the HBP1.A line, we sorted 50 cells from each DN consequently on hCD2 LCR function, a cDNA encoding the hu- subpopulation per sample using the MoFlo (DakoCytomation) sorter. In man HBP1 protein was cloned in the plck expression vector (29) to both cases, the RNA was reverse transcribed, and the cDNA was amplified by PCR as previously described (27, 28). Human growth hormone (hGH) generate HBP1 transgenic mice (Fig. 1). The plck vector contains primer was used for both the RT reaction and the first PCR. Human GH the p56lck proximal promoter that is active almost exclusively in (nested) was used for the second PCR. HBP1 primer was used in both thymocytes (30) and has been used to express transgenes specifi- PCRs. The primers for the hprt gene that was used as an internal control cally in the thymus (29, 31, 32). Three transgenic mouse lines have been described previously (27, 28): HBP1, 5Ј-GCAAAGGCTTTG GCTGAAGA-3Ј; hGH, 5Ј-AGGTCTGCTTGAAGATCTGC-3Ј; and hGH (HBP1A, HBP1B, and HBP1C) with multiple copies of the trans- (nested), 5Ј-AAGGCACTGCCCTCTTGAAGC-3Ј. gene (n ϭ 45–60) at pericentromeric or telomeric chromosomal sites were generated. We did not detect any rearrangement of the Results transgenic construct, indicating that most, if not all, integrated cop- Generation of HBP1 transgenic mice ies were intact. We could not detect any expression of the trans- We have shown previously in transgenic mice that a deletion of the gene in line HBP1B, and it was therefore discontinued. Expression HBP1-binding sequences in the hCD2-LCR construct leads to of the transgene was confirmed by Northern blot analysis of total PEV when the transgene is integrated in a heterochromatic region RNA (Fig. 1) and RT-PCR (not shown). The transgene gives rise Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. A, Thymic cellularity in neonates and 1-, 3-, 6-, and 9-wk- old HBP1.A homozygous and age- matched CBA male mice. B, Thymic cellularity in neonates and 1-, 3-, 6-, and 9-wk-old HBP1.C heterozygous mice and their nontransgenic litter- mates. Each point represents a single mouse. The black lines indicate mean values of the population; numbers in parentheses indicate the number of mice within each group. 5206 HBP1 IN THYMIC DEVELOPMENT AND VARIEGATION to higher m.w. transcripts compared with the endogenous gene due matched CBA nontransgenic mice. To study the kinetics of this to the additional exons present in the expression cassette that are phenomenon, transgenic mice were compared with sex- and age- spliced onto the HBP1 mRNA. matched nontransgenic mice at 1 day postpartum and at 1, 3, 6, and 9 wk of age. Fig. 2A shows that newborn transgenic mice had Effects of overexpression of HBP1 in thymocyte development similar cellularity as nontransgenic counterparts. As early as 1 wk Examination of the thymus in HBP1A transgenic mice indicated later, however, differences became apparent, and these were more that the cellularity of this organ was lower than that in age- noticeable at the ages of 3 and 6 wk. By 9 wk of age, thymus Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. A, Flow cytometric analysis of thymocytes from HBP1.A homozygous and age-matched CBA male mice. Thymocytes were stained with CD4 and CD8 Abs. The percentages of CD4 SP, CD8 SP, and double-positive thymocytes are indicated. Plots show the percentage (B) and absolute numbers (C) of CD8ϩTCRhigh T cells. Each point represents a single mouse. The black lines indicate mean values of the population; numbers in parentheses indicate the number of mice within each group. The Journal of Immunology 5207 cellularity was the same in both groups, possibly because normal 3). No increase in CD8 SP T cell number was observed in the involution in wild-type mice had reduced the organ to a similar periphery. Taken together these results indicate that HBP1 size. When homozygous transgenic mice were compared with het- overexpression leads to overproduction of CD8 SP thymocytes erozygous littermates, it was found that the former had an even despite a decrease in the overall cellularity of the thymus in lower cellularity, suggesting that the phenomenon was dose de- these mice. pendent (data not shown). Similar lower cellularity was observed in heterozygous mice of line HBP1C, but the phenomenon was less An hCD2 transgene with a disabled LCR exhibits chromosomal pronounced (Fig. 2B). PEV To study the effects of HBP1 overexpression on their devel- We have shown previously that hCD2 transgenes with only 1.5 opment, thymocytes from 6-wk-old HBP1A transgenic mice kb (CD2-1.5) of the minimum 2-kb LCR sequences (Fig. 4A) were isolated and stained for CD4, CD8, and TCR. Results exhibit variegating expression (2). This deletion leaves behind shown in Fig. 3 indicate an increase in the proportion and ab- a region called FT1, which is recognized by the DNA-binding solute numbers of CD8 SP thymocytes with high levels of TCR, protein HBP1 (9). We also have shown previously that deletion suggesting that they are postpositive selection CD8 cells. This of a 30-bp sequence including the FT1 region resulted in trans- effect was seen in all groups of mice regardless of sex or age genes that were susceptible to PEV, suggesting that HBP1 (data not shown). Interestingly, a mild, but opposite, effect was might be involved in the protection of the hCD2 transgene from observed for the absolute numbers of CD4 SP thymocytes. Ex- PEV (9). amination of the DP population showed that HBP1 transgenic To investigate directly the involvement of the HBP1 protein in mice had significantly lower numbers than their nontransgenic modulating PEV, three new different transgenic mouse lines car- Downloaded from counterparts, suggesting that effects on this population account rying the CD2-1.5 construct at different positions in their genome mainly for the lower thymus cellularity seen in these mice (Fig. were generated (Fig. 4B). Both CD2-1.5A and CD2-1.5E mice http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Analysis of CD2-1.5 transgenic mice. A, The hCD2–1.5 construct contains 5 kb of the promoter region, the human CD2 gene with all but the first intron deleted, and 1.5 kb of the 2-kb LCR. The 1.5-kb LCR contains a classical enhancer (HSS1) and the 5Ј se- quences of a partially deleted HSS3. B, The transgenic hCD2 expression pattern is integration site dependent. Flow cytometric analysis of CD2 expression in CD2-1.5 lines A (35 copies), E (2 copies), and F (20 copies) is represented. Fluorescence in situ hybridization image of metaphase , with the transgene high- lighted in red (black arrow), shows the integration site in the chromosomal arm (i and ii) or in the centromere (iii). 5208 HBP1 IN THYMIC DEVELOPMENT AND VARIEGATION carry the transgene in the long arm of the , whereas genic mice by flow cytometry (Fig. 5). Single-transgenic CD2- CD2-1.5F carries the transgene in a pericentromeric region. 1.5A mice have, on the average, 12% CD2high-expressing The variegating mice did not exhibit discreet expressing and thymocytes (Fig. 6A). In double transgenics, an increase to an nonexpressing populations; instead, a wide range of expression average of 26.4% was observed in the proportion of CD2high- levels was observed, with only a proportion expressing as high as expressing thymocytes (Fig. 6A). The CD2-1.5F variegating expected from the number of copies integrated, suggesting that mouse shows an average of 10% CD2high-expressing thymo- PEV in these mice is not an all-or-none phenomenon involving all cytes (Fig. 6A). In double transgenics, the increase in HBP1 integrated copies. resulted in an increase in the proportion of CD2high-expressing thymocytes to an average of 21% (Fig. 6A). A similar effect on high Overexpression of HBP1 increases the proportion of CD2 - the extent of variegation was observed with the HBP1C line expressing T cells in variegating mice (Fig. 6B). These results show clearly that HBP1 influences PEV It is possible that PEV reflects incomplete silencing by neighbor- in CD2 variegating mice that carry a CD2-1.5 construct with a ing heterochromatin, which may be the result, at least in part, of disabled LCR. The CD2-1.5E nonvariegating line was also the competition between suppressive and activating chromatin pro- crossed to the HBP1A line, and their analysis showed that over- teins whose competitive efficiency may depend on their relative expression of HBP1 had no effect on the pattern or level of CD2 concentrations (33). In support of this hypothesis, experiments in expression (Fig. 5). transgenic mice indicated that overexpression of HP1␤ (M31) in To assess whether the effect of HBP1 overexpression is se- hCD2 variegating transgenic mice results in an increase in the quence specific, the hCD2-1.3B variegating line (that does not proportion of T cells that take the decision to silence the transgene contain the HBP1 binding site) was crossed to the HBP1A trans- Downloaded from (34). Using a similar strategy, the HBP1 factor, which binds to the genic line. No increase in the proportion of CD2high-expressing FT1 region of the LCR, was tested directly for its ability to mod- thymocytes was found when HBP1 was overexpressed (71.4 vs ulate the transgenic hCD2 locus expression in CD2-1.5A and CD2- 72.2%; see Fig. 6C for the average). To confirm that the relief of 1.5F transgenic mouse lines as well as in the nonvariegating PEV in CD2-1.5 transgenic mice is dependent on HBP1 binding, CD2-1.5E mouse line. As a control, we used a variegating line we examined the extent of variegation in single-transgenic mice

(CD2-1.3B) that carried an hCD2 transgene that differs from the carrying an hCD2 construct with the full LCR minus 30 bp con- http://www.jimmunol.org/ constructs present in lines CD2-1.5A, -E, and -F by only a 200-bp taining the HBP1 binding site (hCD2-⌬FT1) and that exhibit var- deletion containing the HBP1 binding site (2). These reporter lines iegation (9). We compared these mice with double-transgenic mice were crossed to the HBP1A and HBP1C lines, and the expression (HBP1A/hCD2-⌬FT1) and observed no difference in the extent of pattern of the hCD2 locus in double-positive thymocytes of dou- variegation (Fig. 6D), supporting the idea that the relief of varie- ble-transgenic mice was compared with that of single hCD2 trans- gation shown in Fig. 5 depends on the presence of an intact HBP1 by guest on September 28, 2021

FIGURE 5. Overexpression of HBP1 suppresses PEV specifically in CD2 transgenic mice carrying a CD2-1.5-LCR construct. Flow cytometric analysis of CD2 expression on thymocytes from single CD2-1.5 transgenic mice lines and double-transgenic littermates overexpressing HBP1. The percentages represent the proportion of CD2high-expressing T cells before and af- ter overexpression of HBP1. The mean fluorescence in- tensity (MFI) reflects the CD2 expression level in the intermediate population. Each histogram shows a repre- sentative result, from five to 11 mice, of human CD2 expression. The Journal of Immunology 5209 Downloaded from

FIGURE 6. Flow cytometric anal- ysis of CD2high-expressing T cells in the presence or the absence of HBP1 overexpression. A, Plots showing the high proportion of CD2 -expressing http://www.jimmunol.org/ double-positive thymocytes in the single CD2 transgenic and double- transgenic mice overexpressing HBP1 line A. B, Plots showing the proportion of CD2high-expressing thymocytes in the single CD2 trans- genic and double-transgenic mice overexpressing HBP1 line C. C, Plots showing the proportion of CD2high- expressing thymocytes in the single by guest on September 28, 2021 CD2-1.3B and double-transgenic overexpressing HBP1. D, Plots showing the proportion of CD2high- expressing thymocytes in the single hCD2-⌬FT1 and double-transgenic overexpressing HBP1. Each point represents a single mouse. The black lines indicate the mean values of the population. n indicates the number of mice, and s.d. represents the SD. 5210 HBP1 IN THYMIC DEVELOPMENT AND VARIEGATION

does not suggest a developmental block as that seen in other transgenic and knockout mice. Despite the overall decrease in thymus cellularity, it was sur- prising to find that deregulation of HBP1 expression led to an increase in the proportion and absolute numbers of CD8 SP mature thymocytes. Increased numbers of CD8 cells have been observed in several other transgenic mice. For example, Bcl2- or TOX-over- expressing transgenic mice show an accumulation of mature CD8 thymocytes for different reasons (36, 37). It is possible that extra HBP1 molecules accelerate the rate of commitment toward the CD8 lineage at the expense of CD4 SP cell production. Inter- estingly, a phenotype similar to that described in this study has been seen in mice overexpressing a stabilized ␤-catenin trans- gene. These mice show enhanced thymic involution and en- hanced generation of CD8 SP thymocytes (38). It is therefore possible that overexpression of HBP1, which has been shown to be involved in the Wnt-␤-catenin pathway, deregulates this sig- FIGURE 7. PCR analysis of sorted thymocyte populations. RNA from naling cascade, resulting in faster involution and increased CD8 DN1, DN2, DN3, and DN4 thymocyte populations of the HBP1.A and SP thymocyte production. We hope to be able to address this Downloaded from HBP1.C lines were reverse transcribed and subsequently analyzed by PCR question in the future. for the presence of the HBP1 transcript. HBP1 is expressed as early as the DN1 stage in HBP1.A mice and only at the DN3 stage in the HBP1.C line. Cell-to-cell variations in heterochromatin spreading have been In every case, the hprt gene was used as an internal control of the RT and proposed to explain the mosaic silencing seen in PEV, with inac- PCRs. tivation or full expression indicating whether a heterochromatic structure has encompassed a gene (39). Furthermore, it has been

shown previously that variegation in yeast and in mammalian cells http://www.jimmunol.org/ can be modified by activating regulatory cis-acting elements, such binding site within the LCR. These data demonstrate that HBP1 as promoters or enhancers (40–43). The results of these studies probably influences PEV of the CD2 locus via the FT1 region suggest that increased transcriptional activity either helps to pre- localized in the LCR-HSS3. All data of flow cytometric analyses vent the establishment of silencing at the locus or increases the are shown as scatter plots in Fig. 6. probability that the promoter/enhancer will eventually escape si- lencing through mitosis (44). Time variegation of the onset of expression of the lck-HBP1 The studies presented in this report indicate that the concentra- transgenic constructs tion of HBP1 can influence variegation of the target hCD2 locus. We were puzzled by the observation that the effects of HBP1 over- It has been reported that HBP1 can act as transcriptional activator by guest on September 28, 2021 expression (reduced thymus cellularity, CD8 SP thymocyte over- (45, 46) or repressor (25, 26, 35). Although the precise mechanism production, and relief of hCD2 transgene PEV) were more pro- by which HBP1 deregulation leads to the phenotype we describe is nounced in the HBP1-A line despite the fact that it expressed less not known, several explanations can be envisaged. One possibility HBP1 RNA in total thymus preparations. We hypothesized that the is that HBP1 relieves variegation by helping to enhance expression explanation may lie in the fact that lck transgenic constructs of the CD2 transgenic locus directly. Such a role has been sug- show variability at the onset of their expression (3, 32). We gested for other HMG box , such as LEF-1/T cell factor-1, addressed the issue by sorting DN subsets based on their ex- in the regulation of the adenosine deaminase enhancer-LCR (47). pression of CD25 and CD44 surface molecules. RT-PCR on It is also possible that HBP1 may be able to disrupt higher order RNA isolated from DN1, DN2, DN3, and DN4 populations was chromatin structure due to its ability to bend DNA via its HMG analyzed for the expression of transgenic HBP1. As shown in box domain. DNA bending is a common (but not universal) char- Fig. 7, in the HBP1A line, transcripts appear as early as the acteristic of transcription factors, and it is thought to play a role in DN1 stage, whereas in the HBP1.C line, we did not detect any gene regulation (47–57). However, whether HBP1 acts simply to transcripts until the cells reached the DN3 stage. From this we stabilize interactions at protein-protein interfaces (thereby serving conclude that the differences observed between the two HBP1- primarily an architectural role), to destabilize the nucleosome array overexpressing lines are due to the different times of onset of at the integration site, or to directly enhance transcription is HBP1 transgene expression. unknown. An alternative explanation for the phenomenon of PEV relief by Discussion overexpression of HBP1 could be that normally HBP1 interacts The results presented in this paper show that mice overexpress- and recruits the LCR-repressive activities that are present in rate- ing HBP1 exhibit altered thymocyte development. The de- limiting levels in the nucleus and which the disabled LCR cannot creased thymus cellularity seen in these mice could be the result counteract. In this scenario, extra molecules of HBP1 might act in of increased apoptosis or decreased proliferation. HBP1 has a dominant-negative fashion by sequestering the repressor proteins been shown to affect the cell cycle either positively, by down- away from the LCR. regulating cyclin regulator p21 (26), or negatively by blocking It was unexpected that the effects seen in the HBP1.A line are the Wnt pathway (35) or by down-regulating the expression of stronger than those seen with line HBP1.C given that the levels of genes such as n- (25). We were not able to show any change transgenic mRNA are higher in the latter. In this study we show in apoptosis rates either in vitro or in vivo in these mice (not that this may be due to the variable onset of the transgene expres- shown), nor were we able to show any change in the proportion sion. It has been shown that lck-based vectors exhibit a time var- of proliferating cells in transgenic thymuses. Furthermore, anal- iegation (3, 32), and the transgenes in different lines can start ex- ysis of early stages of thymocyte development (DN1 to DN4) pressing at variable time points during thymocyte development The Journal of Immunology 5211 within the DN stages. We believe that the differences in the mag- 19. McMorrow, T., A. van den Wijngaard, A. Wollenschlaeger, M. van de Corput, nitude of the effects reflect differences in the time of onset of ex- K. Monkhorst, T. Trimborn, P. Fraser, M. van Lohuizen, T. Jenuwein, M. Djabali, et al. 2000. Activation of the ␤ globin locus by transcription factors pression of the lck-HBP1 transgene between lines HBP1.A (onset and chromatin modifiers. EMBO J. 19: 4986–4996. at DN1) and HBP1.C (onset at DN3). 20. van de Wetering, M., M. Oosterwegel, D. Dooijes, and H. Clevers. 1991. Iden- tification and cloning of TCF-1, a T lymphocyte-specific transcription factor con- The results of this study confirm that HBP1 plays an important taining a sequence-specific HMG box. EMBO J. 10: 123–132. role in the activation of CD2 transgenic loci by showing that over- 21. Giese, K., A. Amsterdam, and R. Grosschedl. 1991. DNA-binding properties of expression of HBP1 is related to the proportion of cells that switch the HMG domain of the lymphoid-specific transcriptional regulator LEF-1. Genes Dev. 5: 2567–2578. on the locus. It will be interesting to assess whether HBP1 medi- 22. Denny, P., S. Swift, F. Connor, and A. Ashworth. 1992. An SRY-related gene ates this effect at least in part by bending the DNA and/or by expressed during spermatogenesis in the mouse encodes a sequence-specific interacting with chromatin remodeling complexes with activation DNA-binding protein. EMBO J. 11: 3705–3712. 23. Connor, F., P. D. Cary, C. M. Read, N. S. Preston, P. C. Driscoll, P. 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