Volume 10, Issue 5, Pages 1083-1096 (November 2002) Evidence for Distinct CD4 Silencer Functions at Different Stages of Thymocyte Differentiation  Ichiro Taniuchi, Mary Jean Sunshine, Richard Festenstein, Dan R. Littman  Molecular Cell  Volume 10, Issue 5, Pages 1083-1096 (November 2002) DOI: 10.1016/S1097-2765(02)00735-9

Figure 1 Identification of Functional Elements within the CD4 Silencer by Transient Transfection of 1200M Cells (A) Schematic structure of the CAT reporter plasmids. Three copies of different CD4 silencer fragments (S) were inserted upstream of the CD4 enhancer (E)/CD4 promoter (P) in the pCD4/CD4ECAT vector. (B) Analysis of end deletions of a core fragment (nucleotides 131-265) derived from the previously described 434 bp silencer (Sawada et al., 1994). Results are the average from three separate experiments. (C) Effect on transcriptional repression function of specific mutations within site 1 (nucleotides165-185) in the core silencer fragment. Alignment of nucleotide sequences from the mouse and human CD4 silencers is shown. Underlined sequences represent a palindrome-like structure. Results from three experiments were averaged with standard deviations. Molecular Cell 2002 10, 1083-1096DOI: (10.1016/S1097-2765(02)00735-9)

Figure 2 Analysis of CD4 Silencer Site 1 Function in Transgenic Reporter Mice (A) Schematic structure of the reporter transgene. The human CD2 cDNA and the SV40 polyadenylation signal (PolyA) were inserted in exon II of a CD4 minigene (Sawada et al., 1994). Black boxes and the solid line represent exons and part of the first intron in the mouse CD4 gene, respectively. The wild-type silencer fragment comprising nucleotides 132-434 or the site 1 mutant (1-Mut.1 in Figure 1B) was inserted within the first intron. (B) Expression of hCD2 on splenic T cells from transgenic mice. Splenocytes were stained with FITC-anti-hCD2, PE-anti-mCD8, and TC-anti-mCD4 and were analyzed by flow cytometry. Expression of hCD2 on CD4+ or CD8+ T cells from each transgenic line (solid line) or from a nontransgenic littermate (dotted line) is shown. Below the histograms is a summary of hCD2 expression in each transgenic mouse line. The percentage of hCD2 expressing cells in either CD4 single-positive (CD4+) or CD8 single-positive (CD8+) peripheral lymphocytes in each transgenic mouse line is shown. Molecular Cell 2002 10, 1083-1096DOI: (10.1016/S1097-2765(02)00735-9)

Figure 3 CD4 Expression in Mice with Targeted Mutations in the Silencer (A) Strategy for introducing deletions or mutations into the silencer region within the CD4 locus. The targeting vectors consist of a 1.8 kb 5′-homology region, a Neomycin resistance gene (Neo) flanked with loxP sites (arrow heads), a segment containing wild-type or mutant silencer, and 5 kb of 3′-homology sequence. Mutant sequences at sites 1-3 in constructs I, J, and K are shown in red at the bottom. The restriction sites shown are BamHI (B), KpnI (K), SacI (S), BglII (Bg), NotI (N), and XbaI (Xb). (B) Strategy for Cre-mediated removal of the Neo gene in ES clones with targeted silencer mutations. (C) Flow cytometric analysis and CD4 and CD8 expression on ES cell-derived peripheral blood lymphocytes from chimeric mice. ES cell-derived lymphocytes were identified by gating for surface expression of Ly9.1. Molecular Cell 2002 10, 1083-1096DOI: (10.1016/S1097-2765(02)00735-9)

Figure 4 Stochastic Derepression of CD4 and Stable Maintenance of Silencing in CD8 Lineage T Cells from Mice with Silencer Mutations (A) CD4 and CD8 expression in peripheral T lymphocytes and CD3high thymocytes from wild-type and heterozygous (+/−) or homozygous (−/−) mutant mice carrying deletion of the silencer (nucleotides 1-429) or the site1 mutation. Cells were stained with FITC-anti-mCD3, PE-anti-mCD8, and TC-anti-mCD4 antibodies. CD4 and CD8 expression in CD3-positive peripheral lymphocytes or CD3high thymocytes is shown. In site 1 mutant mice, the percentage of CD8+ cells expressing CD4 was 36.05 ± 7.2% (n = 5) in heterozygotes and 64.3 ± 6.4% (n = 3) in homozygotes. (B) Maintenance of CD4 silencing in T cells from mice with mutations in site 1 or site 3. CD4−CD8+ T cells were sorted from spleens of site 1 or site 3 mutant mice and were stimulated with immobilized anti-mCD3 and anti-mCD28 antibodies in the presence of IL-2. Molecular Cell 2002 10, 1083-1096DOI: (10.1016/S1097-2765(02)00735-9)

Figure 5 Distinct Patterns of CD4 Derepression in Double-Negative Thymocytes and in Mature CD8 Lineage T Lymphocytes from Mice with Silencer Mutations CD8−, CD3−, γδTCR−, Thy1+ thymocytes, which correspond to CD4−CD8− double-negative cells, were gated and analyzed for CD4 expression (left panels). The panels on the right display CD4 expression on lymph node CD8+ T cells. The red and black lines correspond to CD4 expression in mutant mice and wild-type mice, respectively. Mean fluorescence of the level of CD4 in the total population of DN thymocytes and in the derepressed population of CD8+ T cells is indicated in each panel. The staining patterns are representative of results from three experiments. Molecular Cell 2002 10, 1083-1096DOI: (10.1016/S1097-2765(02)00735-9)

Figure 6 Effect of HP-1 on Variegated CD4 Expression in CD8+ T Cells in Mice with a Mutant Silencer Mice with the mutation in site 3 of the silencer were crossed with an HP-1β transgenic mouse strain. Expression of CD4 in immature thymocytes and in CD8+ peripheral blood T cells was analyzed in mice heterozygous for the silencer mutation with or without the HP-1β transgene. Representative histograms of CD4 expression in immature versus mature T cells, shown on the left, are from the same mice. Results with individual mice are shown on the right. Molecular Cell 2002 10, 1083-1096DOI: (10.1016/S1097-2765(02)00735-9)

Figure 7 A Model for Different Mechanisms of Repression of CD4 Transcription during Thymocyte Development (A) In double-negative thymocytes (left), a complex of CD4 silencer binding factors would recruit corepressor molecules (rectangular box), resulting in active repression (arrow represents blocked transcription) that is reversed in double-positive thymocytes (green arrow for transcription). At the transitional stage from DP thymocytes to cytotoxic, CD8 lineage T lymphocytes, lineage-specific modifications of chromatin structure (small orange circles), resulting in a mark for epigenetic maintenance, are established. CD4 silencer binding factors that are the same as or different from those required in DN thymocytes would recruit distinct machinery (ellipse) that establishes epigenetic silencing. (B) When site1 is not functional, there is partial, uniform derepression of CD4 in double-negative thymocytes, reflecting some leaky transcription (dashed arrow) at the locus. During transition from the double-positive stage to CD8 SP thymocytes, the epigenetic mark is established in a fraction of the CD8 lineage T lymphocytes, but not completely in the rest of these cells. HP-1 contributes to the successful establishment of the epigenetic mark. Molecular Cell 2002 10, 1083-1096DOI: (10.1016/S1097-2765(02)00735-9)