The CD8α Gene Locus Is Regulated by the Ikaros Family of Proteins

Molecular Cell, Vol. 10, 1403–1415, December, 2002, Copyright 2002 by Cell Press

The CD8␣ Gene Locus Is Regulated by the Ikaros Family of Proteins Nicola Harker,1 Taku Naito,2 Marta Cortes,2 Arnd Hostert,1 Sandra Hirschberg,1 Mauro Tolaini,1 Kathleen Roderick,1 Katia Georgopoulos,2 and Dimitris Kioussis1,3 1 Division of Molecular Immunology National Institute for Medical Research Medical Research Council London NW7 1AA United Kingdom 2 Cutaneous Biology Research Center Massachusetts General Hospital Harvard Medical School Charlestown, Massachusetts 02129

Summary Ikaros family members are important regulatory factors in lymphocyte development. Here we show that Ikaros may play an important role in CD4 versus CD8 lineage commitment decisions by demonstrating: (1) that it binds to regulatory elements in the endogenous CD8␣ locus in vivo using thymocyte chromatin immunoprecipitations, (2) that Ikaros suppresses position effect variegation of transgenes driven by CD8 regulatory elements, and (3) that mice with reduced levels of Ikaros and Aiolos show an apparent increase in CD4 populations with immature phenotype, i.e., cells that failed to activate the CD8␣ gene locus. We propose that Ikaros family members function as activators of the CD8␣ gene locus and that their associated activities are critical for appropriate chromatin remodeling transitions during thymocyte differentiation and lineage commitment. Introduction The CD8␣:CD8␤ heterodimer is expressed during differentiation in the thymus along with CD4 to form the thymocyte subset known as double-positive (DP). Further differentiation of DP thymocytes gives rise to singlepositive (SP) CD4⫺CD8⫹ or CD4⫹CD8⫺ mature populations (Fowlkes and Pardoll, 1989; Gorman et al., 1988; Jay et al., 1982; Ledbetter and Seaman, 1982; Ledbetter et al., 1981). An 85 kb murine genomic fragment containing 2 kb upstream of the CD8␤ gene to 25 kb downstream of the CD8␣ gene, used as a transgene, directed expression of CD8 in a proper developmental and subset-specific manner (Hostert et al., 1997a). DNaseI hypersensitivity analysis of the CD8␣␤ loci revealed four clusters (CI to CIV) of hypersensitive sites (DSS) located within the 85 kb genomic region, highlighting the presence of regulatory elements (Figure 1A). Transgenic and knockout analyses of these regions have revealed a complex pattern of regulation which is governed by regulatory element redundancy, as well as by hierarchical relationships that eventually determine the expression 3

Correspondence: [email protected]

of the locus at various stages of thymocyte differentiation (Ellmeier et al., 1997, 1998; Garefalaki et al., 2002; Hostert et al., 1997b, 1998). Although much insight has been gained about the cis regulatory elements that direct CD8␣␤ gene expression, it remains unclear which trans factors contribute to their activity. Several observations have suggested that Ikaros family proteins may play a role. First, in the thymus of Ikaros-deficient animals there is a skewing toward production of CD4 SP at the expense of DP cells (Wang et al., 1996). Second, the proximity of the CD8␣ locus with Ikaros complexes in pericentromeric heterochromatin in nonexpressing cells suggested that Ikaros may silence CD8␣ gene expression by binding to and recruiting the locus in the vicinity of centromeric heterochromatin (Brown et al., 1997). Ikaros is an integral component of chromatin remodeling complexes in thymocytes and T cells (Kim et al., 1999), and therefore it is possible that it is involved in establishing, and possibly maintaining, chromatin structures that are compatible with either activation or repression of lineage-specific genes during this developmental process (Georgopoulos, 2002). In this paper we report that Ikaros is part of chromatin complexes formed in vivo on regulatory elements of the CD8␣ locus within CD8-expressing cells. In order to assess whether the observed association of Ikaros with the CD8 regulatory regions affects the expression of the CD8␣ gene, we examined the effects of reduced Ikaros levels on the activity of the CD8 CII and CIII regulatory elements. For this purpose, reporter transgenic constructs containing these elements were used for the following reasons. Taking regulatory regions out of the context of their natural chromosomal location and separating them from other elements, which may aid or mask their function, results in transgenic constructs that are vulnerable to suppressive effects from the surrounding chromatin, leading to a phenomenon known as position effect variegation or PEV (Kioussis and Festenstein, 1997). PEV is observed when a gene is transposed by chromosomal translocation, inversion, or transgenesis in the proximity of heterochromatin and is thought to be the result of stochastic silencing of a gene in a proportion of cells that would be expected normally to express it (Henikoff, 1996; Tartof et al., 1989). Since variegating transgenes are more sensitive to changes in the concentration of nuclear factors compared to their endogenous counterparts in their normal chromosomal locations (Festenstein et al., 1999), we assessed the possible involvement of Ikaros in CD8␣ regulation by comparing the extent of variegation of transgenes under the control of CD8 regulatory regions in genetic backgrounds that are either haploid or diploid for Ikaros. Finally, we examined the pattern of CD8 expression in developing thymocytes in mice deficient in both Ikaros and Aiolos gene expression. Taken together, the data presented here argue strongly that Ikaros family proteins function as activators of the CD8␣ gene locus possibly by participating in the chromatin remodeling occurring

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Figure 2. Diagrams of the CD8␣␤ Loci and the Transgenic Constructs Used in This Study Map of the genomic mouse CD8␣␤ gene loci, showing the location of the CD8␣ and CD8␤ genes and the four clusters of hypersensitivity sites (labeled CI-CIV; Hostert et al., 1997a). Transgene A: Map of the (CIII-2)-CD8␣ transgenic construct, showing cluster III site 2 of the CD8␣␤ gene loci upstream of the CD8␣ gene (exons indicated by filled boxes). Transgene B: Map of the CD2-(CIII-1,2,3)-(CII-2) transgenic construct, showing the hCD2 minigene upstream of cluster III and cluster II site 2 of the CD8␣ gene locus (exons indicated by filled boxes). Transgene C: Map of the CD2-(CIII-1,2,3)-(CII-1,2*) transgenic construct, showing the hCD2 minigene upstream of cluster III, cluster II site 1, and truncated cluster II site 2 of the CD8␣ gene locus (exons indicated by filled boxes). Transgene D: Map of the CD2⌬LCR transgenic construct, showing the hCD2 minigene with its partial LCR (exons indicated by filled boxes). The pattern of expression of each transgene is indicated on the right-hand side of the figure.

at the early stages of initiation of CD8␣ gene transcription and also at later stages. Results Binding of Ikaros to the CD8 Regulatory Elements In Vivo In order to assess the extent of physical association of Ikaros with the endogenous CD8␣ gene locus se-

quences, we performed chromatin immunoprecipitation (ChIP) combined with polymerase chain reaction (PCR) (Figure 1). Chromatin prepared from nuclei of wild-type thymocytes was subjected to immunoprecipitation with antibodies to Ikaros (I) or an immunoglobulin (IgG)-isotype control (C). Fold enrichment was calculated as the ratio of normalized values obtained from the Ikarosand Ig-control chromatin immunoprecipitations. Significantly, Ikaros enrichment was reproducibly seen with

Figure 1. Mapping of Ikaros Protein Associations at the CII and CIII Regulatory Regions of the CD8␣␤ Loci in Thymocytes (A) A physical map of the CD8␣␤ loci with clusters of tissue-specific DHS sites involved in its regulation are shown. (B and C) Diagrams of CII and CIII clusters are presented in the upper panels. Solid arrows indicate the position of DSS. The locations of 14 and 19 PCR amplicons that span the CII and CIII clusters, respectively, examined by ChIP are shown. The lower panel shows the results of the dual primer PCR performed for each subregion. Lane C is the control ChIP using whole mouse IgG, and I is the anti-Ikaros ChIP. The lower band is the normalizing standard (G6PD promoter), and the upper band is from the CD8 region examined for Ikaros targeting. The signal intensity of the control and test amplicons shown in the middle panel was measured with Phosphoimager, and the relative fold enrichment was calculated as described. (D) Chromatin immunoprecipitations using extracts from Ikaros wild-type and Ikaros null thymocytes.

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different chromatin preparations in the vicinity of the first (CII-1) and third (CII-3) DSS of cluster II (Figure 1B). A similar analysis of chromatin-bound Ikaros in an 11 kb genomic fragment containing the CIII cluster revealed significant and reproducible enrichment of sequences present in all three of the DSS (Figure 1C). All of the areas that were enriched in the Ikaros chromatin immunoprecipitations contained a combination of several high- and low-affinity binding sites for Ikaros as revealed by in vitro binding assays (data not shown). No significant enrichment was detected for other sequences within the CII and CIII regions even though they contained several putative high-affinity binding sites for Ikaros. Furthermore, the specificity of the Ikaros-ChIP for the CII and CIII regions was further verified by chromatin immunoprecipitations using extracts from Ikaros null thymocytes, which showed no enrichment of the areas containing the Ikaros-associating DSS (Figure 1D). Ikaros binding is specific to the CD8␣ locus, as Ikaros proteins were not found in general associated with thymocyte-specific DHS sites within other genes such as the Ikaros gene itself (data not shown). Taken together, these studies show that Ikaros associates in vivo with regions of the locus whose chromatin is specifically accessible (DNaseI hypersensitive clusters II and III) only in CD8-expressing cells and not in nonexpressing populations, i.e., SP-CD4 or other cell types (N.H., unpublished data). Reduction in Ikaros Levels Increases PEV of a CD8(CIII-2) Transgene that Is Active in CD8 SP Cells As explained in the introduction, transgenes are usually more sensitive than the endogenous gene loci to the concentration of factors that affect their expression. Thus, CD8 CIII and CII transgenic mice (Figure 2) were bred to mice heterozygous for a germ line deletion that inactivates Ikaros (Wang et al., 1996). In the first instance, we examined mice transgenic for a CD8 regulatory cassette that expresses in CD8 SP cells. This transgene, consisting of the CD8.2␣ allele downstream of a region that encompasses the second DSS of CIII (Figure 3A, transgene A; TgA) (Hostert et al., 1997b), exhibits PEV indicating integration within restrictive chromatin (Figure 3A). TgA mice expressed the transgene in 17.8% of CD8 SP thymocytes when Ikaros was present at wildtype level (Ik⫹/⫹/TgA), whereas, when Ikaros was reduced, CD8 cells expressing the transgene fell to 5.5% (Ik⫹/⫺/TgA) (Figure 3A). This was mirrored in lymph node mature T cells where the proportion of CD8 T cells expressing the transgene was reduced from 25.5% in wildtype Ikaros mice to 8.4% in Ikaros heterozygotes. Figure 3B summarizes the analyses from 24–30 mice, and it shows that the percentage of CD8 SP T cells expressing

the transgenic CD8.2␣ in the thymus, spleen, and lymph node in Ik⫹/⫺/TgA is significantly reduced relative to Ik⫹/⫹/TgA mice. We conclude from these data that Ikaros functions as an activator of CD8 transgenes expressed in mature CD8 T cells. Ikaros Levels Modify PEV of Transgenes with CD8 Regulatory Elements that Are Active in Immature Thymocytes Two more transgenic lines carrying a reporter gene under the control of CD8 regulatory elements and exhibiting variegated expression in immature thymocytes (DP), as well as in mature CD8 (SP) cells in the thymus and periphery, were analyzed in a similar manner. These expression cassettes contain a hCD2 reporter minigene upstream of the whole cluster III along with either DSS-2 of cluster II (Figure 4A; transgene B; TgB) or DSS-1 and DSS-2 of cluster II (Figure 5A; transgene C; TgC). In mice with wild-type levels of Ikaros, 6.1% of DP, 42.8% of CD8 (SP) thymocytes, and 68.1% of lymph node CD8 T cells were TgB positive thymocytes. In mice with haploid levels of Ikaros, these percentages fell to 3.4%, 31.2%, and 48.4%, respectively (Figure 4A). Similarly, the proportion of TgC positive DP, CD8 SP thymocytes and lymph node CD8 T cells fell from 7.5%, 14.2%, and 20.9%, respectively, in mice with wild-type levels of Ikaros to 0.6%, 2.3%, and 5.3% of the respective populations in mice with haploid levels of Ikaros (Figure 5A). A summary of the expression of TgB and TgC in the DP and CD8 SP T cells in the thymus, spleen, and lymph node in a number of wild-type and Ikaros heterozygous mice is shown in Figures 4B and 5B, respectively. Consistent with the effects described in previous sections, the PEV seen with these two transgenic constructs is increased upon Ikaros reduction, both at the immature and mature stages of T cell development. Taken together, these data indicate that Ikaros can function as a suppressor of PEV of transgenic constructs containing CIII and CII regulatory regions from the CD8␣ gene locus when these are integrated in restrictive chromatin environments. Furthermore, Ikaros appears to enable the activity of transgenic CD8 regulatory elements not only within CD8 SP thymocytes and peripheral CD8 T cells but also at an earlier stage of differentiation. The Ikaros Effect on PEV Is Specific for the CD8 Regulatory Elements Given Ikaros’ association with heterochomatin, we tested whether the effect of reduced Ikaros levels on PEV was specific to the CD8 regulatory regions or to their heterochromatic location. For this purpose, we examined the effects of Ikaros reduction on the expression of other variegating transgenes containing a hCD2 mini-

Figure 3. A Reduction in Levels of Ikaros Results in Increased PEV of a CD8␣ Transgene Driven by the (CIII-2)-HSS in CD8 SP Cells (A) FACS analysis of lymphocytes isolated from the thymus and lymph nodes of C57Bl/10, Ik⫹/⫹/TgA, and Ik⫹/⫺/TgA mice. Cells were triple stained with CD4, panCD8, and CD8.2␣ (CD8␣/Lyt-2.2) antibodies. CD4⫹ and CD8⫹ populations were identified by plotting ␣-CD4 against ␣-panCD8, and the resulting CD8⫹ SP T cell populations were gated and analyzed for the expression of the transgenic CD8.2␣ (CD8␣/Lyt2.2) allele (shown in the histograms). The percentage of CD8⫹ SP cells expressing the transgene is indicated. (B) Graph showing the percentage of CD8.2␣ (CD8␣/Lyt-2.2) positive (transgenic) CD8 SP cells in the thymus, spleen, and lymph node of Ik⫹/⫹/TgA and Ik⫹/⫺/TgA mice. The mean of each data group is shown as a bar. The size of each group (n) is indicated, and the statistical significance of the data is specified at the bottom of each panel as a p value.

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gene lacking the DSS-3 of the hCD2 LCR, a site which is essential for full position-independent expression and avoidance of PEV (Figure 6A; transgene D; TgD) (Festenstein et al., 1996; Greaves et al., 1989). Thymocytes and peripheral T cells from normal and TgD mice on a wild-type or an Ikaros heterozygous background were analyzed for hCD2 expression (Figure 6A, TgD; and Supplemental Figure S1 at http://www.molecule.org/cgi/ content/full/10/6/1403/DC1). In the thymus, 60.6% of DP and 67.0% of CD8 SP express the hCD2 transgene. When Ikaros levels were reduced, 68.5% of DP and 70.1% of CD8 SP expressed the transgene. In the periphery of these mice, a reduction in Ikaros levels also did not significantly affect PEV, with the percentages of CD8 SP T cells expressing the transgene being 80.4% in the presence of wild-type levels of Ikaros and 83.6% on an Ikaros heterozygous background (Figure 6A). Similar results were obtained with another variegating hCD2 transgenic line (see Supplemental Figure S1 at http:// www.molecule.org/cgi/content/full/10/6/1403/DC1). Figure 6B summarizes the analyses of nine Ik⫹/⫹/TgD and four Ik⫹/⫺/TgD mice, showing the percentage of hCD2 positive CD8 T cells expressing the transgene in thymus, spleen, and lymph node. Given these results, we conclude that reduced expression of Ikaros does not significantly affect PEV of a heterochromatin-associated hCD2 transgene and supports the hypothesis that Ikaros action is dependent on specific recognition of sequences in the CD8 transgene loci and not due to their association with heterochromatin. Deregulation of CD4/CD8 Expression Profiles in Immature Thymocyte Caused by Reduction in Aiolos and Ikaros Levels Ikaros and its family member Aiolos are integral components of the same chromatin regulatory network in thymocytes and mature T cells (Kim et al., 1999) with possible overlapping effects on T cell differentiation. To assess the extent to which these proteins could combine to affect CD8 gene expression, we examined mice that are homozygous for Aiolos (Wang et al., 1998) and heterozygous for the Ikaros mutations. Unlike Ikaros-deficient mice, these compound mutants do not have any early overt hematopoietic defects (consistent with the lack of Aiolos expression in early hematopoietic progenitors) but show a decrease in DP thymocytes combined with an increase in CD4⫹/CD8int and CD4⫹ SP thymocytes; a significant fraction of these populations represent thymocytes at immature stages bearing low levels of T cell receptor (TCR) and CD5, similar to those found on DP thymocytes (Figure 7). One possible interpretation of these data, consistent with the results shown in previ-

ous sections, is that the aberrant CD4⫹CD8int and CD4 SP cells detected in the Ikaros/Aiolos mutant thymuses are “DP thymocytes” that failed to initiate CD8 expression. Discussion Studies on the role of Ikaros during lymphocyte development have suggested that Ikaros has the potential to suppress or activate gene expression during this developmental process (Cortes et al., 1999; Georgopoulos, 2002; Koipally et al., 2000). A number of reports have indicated the presence of Ikaros binding sites in the promoters of a variety of lymphoid-specific genes (e.g., ␭5 and TdT [Hahm et al., 1994; Lo et al., 1991; Sabbattini et al., 2001; Trinh et al., 2001]). Ikaros can bind these sites in vitro, and their deletion from a transgene can affect the latter’s expression. In the current study we provide several lines of evidence to show that the T cellspecific gene CD8␣ is a bona fide in vivo target of Ikaros. Thus, we show that Ikaros binds CD8 regulatory elements in vivo in a sequence-specific manner in their natural chromosomal location. Two fragments, 8 and 11 kb in size, that contain clusters II and III of the CD8 DNaseI HSS were examined by chromatin immunoprecipitations using Ikaros antibodies. These studies generated an in vivo map of Ikaros-specific associations with small areas in the vicinity of the first and third HSS of cluster II and of all three HSS of cluster III. It is significant that Ikaros proteins associate with almost all of the HSS (4/5) in these two CD8 regulatory clusters, as these regions are critical for CD8 expression in DP thymocytes and SP-CD8 T cells (Hostert et al., 1998). Thus, Ikaros may contribute to the local chromatin structure of the CII and CIII regulatory units and thereby potentiate expression of the CD8␣ gene during thymocyte development. There is ample evidence that Ikaros can affect gene expression directly in a positive as well as in a negative manner (DiFronzo et al., 2002; Georgopoulos, 2002; Ito et al., 2002; Lopez et al., 2002). It has also been proposed that Ikaros may regulate negatively the expression of genes with which it associates through its ability to localize into heterochromatic regions (Brown et al., 1997, 1999; Cobb et al., 2000). These studies also raised the possibility that Ikaros’ localization into heterochromatin may affect those genes that are present in this compartment in a nonspecific fashion (Brown et al., 1999). To distinguish among these possibilities, but also to examine the effects of Ikaros on CD8␣ expression, we exploited PEV of “CD8” transgenes (i.e., transgenes under the control of CD8 regulatory regions).

Figure 4. A Reduction in Levels of Ikaros Increases PEV of a CD2 Transgene Driven by the CD8(CIII-1,2,3)-(CII-2) Regulatory Elements in DP and SP Stages of T Cell Differentiation (A) FACS analysis of lymphocytes isolated from thymus and lymph node of C57Bl/10, Ik⫹/⫹/TgB, and Ik⫹/⫺/TgB mice. Cells were triple stained with CD4, panCD8, and hCD2 antibodies. CD4⫹ and CD8⫹ populations were identified by plotting ␣-CD4 against ␣-panCD8 and the resulting DP, and CD8⫹ SP T cell populations were gated and analyzed for expression of hCD2 (shown in the histograms). The percentage of CD8⫹ SP cells expressing the transgene is indicated. (B) Graph showing the percentage of hCD2 positive (transgenic) DP and CD8 SP cells in the thymus, spleen, and lymph node of Ik⫹/⫹/TgB and Ik⫹/⫺/TgB mice. The mean of each data group is shown as a bar. The size of each group (n) is indicated, and the statistical significance of the data is specified at the bottom of each panel as a p value.

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Transgenic constructs containing the regulatory elements from cluster III and cluster II of DNaseI hypersensitive sites (HSS) of the CD8␣ locus maintain their subset specificity but variegate, suggesting that they have integrated in heterochromatic regions of the chromosome. Three different “CD8” transgenic constructs in three different integration sites within the mouse genome responded in a similar manner to reduced levels of Ikaros protein: namely, fewer cells were able to activate the transgenic locus in DP and CD8 SP thymocytes and peripheral CD8 T cells. The effect was specific for “CD8” transgenes, as changes in the levels of Ikaros did not affect the expression pattern of another heterochromatin-associated variegating transgene under the control elements of the human CD2 gene locus. We conclude from these data that Ikaros can act as an activator of “CD8” transgenes located within restrictive chromatin environments. These Ikaros effects are likely the result of its ability to interact specifically with sequences present on the CD8␣␤ loci and not due to a global effect on the expression of heterochromatin-associated genes. Ikaros/Aiolos compound mutants display no overt hematopoietic defects and exhibit a relatively small reduction in thymus cellularity. Interestingly, however, they show a change in the DP and CD4 SP profiles (similar to that seen in Ikaros null mice, albeit less severe). The most revealing aspect in the thymuses of the compound mutant mice is the presence of CD4⫹8⫺ thymocytes, which bear an immature phenotype as judged by several maturation markers. It is possible that these cells proceed from the double-negative stage to a “double-positive wannabe” stage, which is characterized by their inability to initiate transcription from the CD8␣ locus. In an independent but relevant investigation, deletion by homologous recombination of the CII cluster from the endogenous CD8␣ locus resulted in a phenotype similar to that of Ikaros and Aiolos double-deficient mice, i.e., the thymus of the ⌬CII mice contains a large proportion of cells that have a CD4⫹8⫺ or CD4⫹8lo phenotype with characteristics of immature thymocytes. This is the result of incomplete activation of the CD8␣ locus, which leads to variegating expression of the CD8␣ gene within the DP population (Garefalaki et al., 2002). That report complements the results presented here, in that the study of the CII deletion examined the effects of removing the binding sites for the effector proteins (including Ikaros) from the endogenous CD8␣ locus, whereas in the present manuscript we explored the effects of altering the concentration levels of some of these effector proteins (Ikaros and its family members). It is reasonable to assume that in both cases, impaired interactions of Ikaros with the CD8 regulatory regions result in an inappropriate activation or silencing of the locus in a fraction of DP thymocytes.

Previous studies have shown that the great majority of Ikaros and its family members are integral components of two higher order chromatin remodeling complexes (Ikaros-NuRD and Ikaros-SWI/SNF) in immature thymocytes and mature T cells (Kim et al., 1999). Thus, Ikaros remodeling complexes can confer disparate chromatin regulating activities through the action of associated SWI/SNF and NuRD complexes (Georgopoulos, 2002). Independent studies have shown that Ikaros functions as a transcriptional potentiator rather than as a conventional activator (Koipally et al., 2002), a property that is consistent with its proposed role in chromatin remodeling. In this respect it is important to note that SWI/SNF-like complexes were recently shown by Chi et al. (2002) to regulate CD4 versus CD8 lineage commitment decisions providing support for an IkarosSWI/SNF complex involvement in the expression of the CD8 locus. In this context it should be noted that the levels of SWI/SNF components are not altered in Ikaros-deficient thymocytes (see Supplemental Figure S2 at http://www.molecule.org/cgi/content/full/10/6/ 1403/DC1). Taking together the studies on CD8␣ and Ikaros regulation, we propose that Ikaros increases chromatin accessibility in the vicinity of its cognate sites through the action of its associated remodeling activities and thereby facilitates entry of other transcription factors which, if they are activators, can lock chromatin in a more permanently accessible state through the recruitment of additional chromatin modifiers (Figure 8A). Alternatively, Ikaros recruitment to a regulatory locus may facilitate already present transcriptional regulators in propagating chromatin codes through this region (Figure 8B). The multiplicity of Ikaros association with the CD8␣ locus regulatory elements indicates that it may be critical for their activity possibly by establishing or propagating chromatin modifications within these regulatory regions. Further studies will determine which of the Ikaros-chromatin remodeling complexes are recruited to the vicinity of the CD8 regulatory regions, examine the effects that they bring to the local chromatin, and finally establish which transcriptional factors work in concert. Experimental Procedures Transgenic Constructs and Mice The generation of the transgene A ([CIII-2]-CD8␣ [named CD8␣CIII-2 in Hostert et al., 1997b]), transgene C (hCD2⌬LCR [named CD2-1.3B in Festenstein et al., 1996]), Ik⫺/⫺ (named C in Wang et al., 1996), and Aio⫺/⫺ (named Aio⌬7⫺/⫺ in Wang et al., 1998) mice is described elsewhere. To generate transgene B (CD2-[CIII-1,2,3][CII-2]), the CD2-(CIII-1,2,3) construct (described previously; Hostert et al., 1997b) was digested with SmaI. A 4 kb HindIII genomic fragment containing CD8 CII-1 and CII-2 was cut with EcoRI, and the 1.3 kb CII-2 fragment was cloned into the CD2-(CIII-1,2,3) construct.

Figure 5. A Reduction in Levels of Ikaros Also Increases PEV of a CD2 Transgene Driven by the CD8-(CIII-1,2,3)-(CII-1,2*) Regulatory Elements (A) FACS analysis of lymphocytes isolated from thymus and lymph node of C57Bl/10, Ik⫹/⫹/TgC, and Ik⫹/⫺/TgC mice. Cells were triple stained with CD4, panCD8, and hCD2 antibodies. CD4⫹ and CD8⫹ populations were identified by plotting ␣-CD4 against ␣-panCD8, and the resulting DP and CD8⫹ SP T cell populations were gated and analyzed for expression of hCD2 (shown in the histograms). The percentage of T cells expressing the transgene is indicated. (B) Graph showing the percentage of hCD2 positive (transgenic) DP and CD8 SP cells in the thymus, spleen, and lymph node of Ik⫹/⫹/TgC and Ik⫹/⫺/TgC mice. The mean of each data group is shown as a bar. The size of each group (n) is indicated, and the statistical significance of the data is specified at the bottom of each panel as a p value.

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Figure 7. Altered Thymocyte Profiles in Ik⫹/⫺xAio⫺/⫺ and Aio⫺/⫺ Mice Thymocytes from wild-type (⫹/⫹), Aio⫺/⫺, and Ik⫹/⫺xAio⫺/⫺ mice were stained with anti-CD4CyChrome and anti-CD8FITC. Percentages of cells that fall within each boxed population are indicated. These populations (I, CD4⫹CD8⫺; II, CD4⫹CD8int; and III, CD4⫹CD8⫹) were gated and stained with either anti-TCRPE or anti-CD5PE. The percentage of each gated population that is positive and negative for TCR and CD5 is indicated. Thymic cellularities were on average 14.8 ⫻ 107 for the wild-type, 10.8 ⫻ 107 for Aio⫺/⫺, and 7.1 ⫻ 107 for the Ik⫹/⫺Aio⫺/⫺.

In order to generate transgene C, (CD2-[CIII-1,2,3]-[CII-1,2*]), a 4kb HindIII genomic fragment containing CD8 CII-1 and 2 was cut with EcoRI to generate a 2.7 kb fragment containing CII-1 and a 1.3 kb fragment containing CII-2. The CII-2 fragment was digested further with NcoI to generate a 0.8 kb NcoI-HindIII fragment containing only part of CII-2 HSS (CII-2*). The CII-2* fragment was ligated to the CII-1 fragment and then subcloned into the SmaI digested CD2-(CIII-1,2,3) construct. The transgenic constructs were separated from bacterial vector sequences by gel electrophoresis, extracted from agarose with glass beads, and further purified with Elutip columns. Transgenic mice were generated by microinjection of fertilized oocytes isolated from CBA/Ca nontransgenic mice with a solution of purified DNA at a concentration of 2–5 ␮g/ml as described previously (Greaves et al., 1989). Transgenic mice were housed in a conventional colony under specific pathogen-free conditions at NIMR. Flow Cytometric Analysis For FACS analysis of Ik/TgA, Ik/TgB, Ik/TgC, and Ik/TgD mice, 106 thymocytes or T cells were stained with the following antibodies in appropriate combinations: fluoresceine isocyanate (FITC)-conjugated anti-hCD2 (Becton Dickinson), phycoerythrin (PE)-conjugated

anti-CD4 (Becton Dickinson), tricolor (TC)-conjugated anti-CD8␣ (TCS Biologicals Ltd), biotin-conjugated anti-CD8.2␣ ([anti-CD8␣/ Lyt2.2] [2.43]). Cells stained with biotin-conjugated antibodies were subsequently stained with streptavidin (SA)-red 670 (Invitrogen Ltd). Thymocytes from wild-type, Ik⫹/⫺Aio⫺/⫺, and Aio⫺/⫺ mice were stained with FITC-conjugated anti-CD8, TC-conjugated anti-CD4, and PE-conjugated anti-T cell receptor and anti-CD5 (Pharmingen). Three-color FACS analysis was carried out on FACScan and analyzed using CellQuest software. Chromatin Immunoprecipitation (ChIP) ChIP studies were done as described by Parekh and Maniatis (1999) with modifications. Proteinase inhibitors were included in all solutions. Total thymocytes were fixed in PBS ⫹ 1% formaldehyde at RT for 10 min., followed by glycine (final 0.125 M) and further incubation at 4⬚C for 5 min. After washing twice with PBS, cells were suspended in sonication buffer (10 mM Tris-HCl [pH 7.9], 10 mM NaCl, 1 mM EDTA, 1% NP-40 and 2% sarcosyl) and sonicated to shear the chromatin to 0.5–1 kb length. Samples were dialyzed against BC0.15⬘ (20 mM HEPES [pH 7.9], 150 mM KCl, and 0.2 mM EDTA), then frozen down and stored at ⫺80⬚C until use. Control antibody (whole mouse IgG) or anti-Ikaros antibody was added to

Figure 6. A Reduction in Levels of Ikaros Does Not Affect PEV of a CD2 Transgene Driven by a Partially Deleted Locus Control Region (A) FACS analysis of lymphocytes isolated from thymus and lymph node of C57Bl/10, Ik⫹/⫹/TgD, and Ik⫹/⫺/TgD mice. Cells were triple stained with CD4, panCD8, and hCD2 antibodies. CD4⫹ and CD8⫹ populations were identified by plotting ␣-CD4 against ␣-panCD8, and the resulting DP and CD8 SP T cell populations were gated and analyzed for expression of hCD2 (shown in the histograms). The percentage of T cells expressing the transgene is indicated. (B) Graph showing the percentage of hCD2 positive (transgenic) DP and CD8ⴙ SP cells in the thymus, spleen, and lymph node of Ik⫹/⫹/TgD and Ik⫹/⫺/TgD mice. The mean of each data group is shown as a bar. The size of each group (n) is indicated, and the statistical significance of the data is specified at the bottom of each panel as a p value.

Molecular Cell 1414

Figure 8. Models on the Function of Ikaros (A) Ikaros recruits remodeling factor through binding to its cognate binding sequence. Remodeler opens up the locus and enables subsequent binding of other transcription factors, which establish specific chromatin code (depicted as flags) within the region. (B) The transcription factor binds to its recognition sequence regardless of the Ikaros binding and establishes specific chromatin code in the vicinity of it. Recruitment of remodeling activity by Ikaros facilitates the propagation of the code throughout the locus.

a 5 ⫻ 107 cells equivalent sample and incubated at 4⬚C overnight. Then ProteinG-agarose beads pretreated in BC0.15⬘ with BSA and ssDNA (200 ␮g/ml respectively) were added and incubated for 1.5 hr at 4⬚C with gentle agitation to recover immunocomplexes. The beads were washed seven times with RIPA wash buffer (10 mM Tris HCl [pH 8.0], 500 mM NaCl, 1% Triton X-100, 0.1% Na deoxycholate, and 0.1% SDS). Immunocomplexes were eluted from the beads twice with 20 mM Tris-HCl [pH 6.8] ⫹ 2% SDS, followed by proteinase-K digestion and reversal of crosslinking by incubating at 65⬚C overnight. Released DNA was phenol/chroloform extracted and was precipitated in the presence of 50 ␮g glycogen used as a carrier. Precipitated DNA was resuspended in 50 ␮l TE. PCR Analysis of ChIP DNA Multiplex PCR was carried out as described (Noma et al., 2001) in 25 ␮l volume in the presence of ␣-[32P]-dCTP with 1 ␮l ChIP DNA as a template. The PCR products were run on 8% PAGE, and the amount of PCR product was quantified using Phosphoimager. To account for background in the ChIP assay and for differences in DNA loading, the relative enrichment of Ikaros in the CII and CIII regions was normalized to the G6PD promoter region estimated within the same PCR reaction. Fold enrichment was calculated as [target/G6PD promoter signal ratio in anti-Ikaros precipitated sample]/[target/G6PD promoter signal ratio in mock precipitated sample] for each target region. The lowest fold enrichment within the given HSS cluster was arbitrarily set as 1, and the relative fold enrichment was calculated as the ratio to that set base line for fold enrichment. Acknowledgments The work described in this paper was supported by the MRC UK and by EU grants BIO4-CT97-70203 and QLRT-1999-30345. Support for K.G. was provided by NIH-RO1 AI380342. T.N. is supported by a long-term fellowship from the Human Frontier Science Program. We would like to thank members of Molecular Immunology, NIMR, K.G. lab and Joseph Koipally for critical review of the manuscript. Received: April 3, 2002 Revised: September 26, 2002 Published online: December 2, 2002 References Brown, K.E., Guest, S.S., Smale, S.T., Hahm, K., Merkenschlager, M., and Fisher, A.G. (1997). Association of transcriptionally silent

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