The CD8α Gene Locus Is Regulated by the Ikaros Family of Proteins Nicola Harker, Taku Naito, Marta Cortes, Arnd Hostert, Sandra Hirschberg, Mauro Tolaini, Kathleen Roderick, Katia Georgopoulos, Dimitris Kioussis  Molecular Cell  Volume 10, Issue 6, Pages 1403-1415 (December 2002) DOI: 10.1016/S1097-2765(02)00711-6

Figure 1 Mapping of Ikaros Protein Associations at the CII and CIII Regulatory Regions of the CD8αβ Loci in Thymocytes (A) A physical map of the CD8αβ 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. Molecular Cell 2002 10, 1403-1415DOI: (10.1016/S1097-2765(02)00711-6)

Figure 2 Diagrams of the CD8αβ Loci and the Transgenic Constructs Used in This Study Map of the genomic mouse CD8αβ gene loci, showing the location of the CD8α and CD8β genes and the four clusters of hypersensitivity sites (labeled CI-CIV; Hostert et al., 1997a). Transgene A: Map of the (CIII-2)-CD8α transgenic construct, showing cluster III site 2 of the CD8αβ gene loci upstream of the CD8α 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 CD8α 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 CD8α gene locus (exons indicated by filled boxes). Transgene D: Map of the CD2ΔLCR 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. Molecular Cell 2002 10, 1403-1415DOI: (10.1016/S1097-2765(02)00711-6)

Figure 3 A Reduction in Levels of Ikaros Results in Increased PEV of a CD8α 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 CD8.2α (CD8α/Lyt-2.2) antibodies. CD4+ and CD8+ populations were identified by plotting α-CD4 against α-panCD8, and the resulting CD8+ SP T cell populations were gated and analyzed for the expression of the transgenic CD8.2α (CD8α/Lyt-2.2) allele (shown in the histograms). The percentage of CD8+ SP cells expressing the transgene is indicated. (B) Graph showing the percentage of CD8.2α (CD8α/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. Molecular Cell 2002 10, 1403-1415DOI: (10.1016/S1097-2765(02)00711-6)

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 α-CD4 against α-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. Molecular Cell 2002 10, 1403-1415DOI: (10.1016/S1097-2765(02)00711-6)

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 α-CD4 against α-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. Molecular Cell 2002 10, 1403-1415DOI: (10.1016/S1097-2765(02)00711-6)

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 α-CD4 against α-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+/+/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 2002 10, 1403-1415DOI: (10.1016/S1097-2765(02)00711-6)

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−/−. Molecular Cell 2002 10, 1403-1415DOI: (10.1016/S1097-2765(02)00711-6)

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. Molecular Cell 2002 10, 1403-1415DOI: (10.1016/S1097-2765(02)00711-6)