The L2a element is a mouse CD8 silencer that interacts with MAR-binding proteins SATB1 and CDP

Abstract

Previous transgenic-reporter and targeted-deletion studies indicate that the subset-specific expression of CD8αβ heterodimers is controlled by multiple enhancer activities, since no silencer elements had been found within the locus. We have identified such a silencer as L2a, a previously characterized ∼ 220 bp nuclear matrix associating region (MAR) located ∼ 4.5 kb upstream of CD8α. L2a transgenes driven by the E8(I) enhancer showed no reporter expression in thymic subsets or T cells in splenic, inguinal and mesenteric lymph node peripheral T cells. Deletion of L2a resulted in significant reporter de-repression, even in the CD4(+)CD8(+) double positive (DP) thymocyte population. L2a contains binding sites for two MAR-interacting proteins, SATB1 and CDP. We found that that binding of these factors was markedly influenced by the content and spacing of L2a sub-motifs (L and S) and that SATB1 binds preferentially to the L motif both in vitro and in vivo. A small fraction of the transgenic CD8 single positive (SP) thymocytes and peripheral CD8(+) T cells bypassed L2a-silencing to give rise to variegated expression of the transgenic reporter. Crossing the L2a-containing transgene onto a SATB1 knockdown background enhanced variegated expression, suggesting that SATB1 is critical in overcoming L2a-silenced transcription.

Figure 1. The L2a element

(A) Schematic of the murine CD8 locus and intergenic region with expanded details of L2a. Positions of established exons (red boxes), enhancer regions (blue lines) and DNAse 1 hypersensitivity sites (blue arrows) shown to be critical for CD8 transcriptional regulation are indicated. A third cluster of two hypersensitive sites (Cluster I, not shown) resides 3’ to CD8a but is yet to be implicated in its regulation. The L2a element (green) is located ~4.5 kb upstream of the mouse CD8a gene. The 270 bp AccI/SstI fragment spanning L2a is expanded to show the L, S, and INTER-LS regions identified in footprinting studies. A 12 bp palindromic sequence is found located at the end of the INTER-LS region close to the S region. (B) Schematic of the transgenic constructs. Both wildtype (WT) and L2a deleted (L2aD) constructs were derived from a human CD2 reporter gene construct (Sawada et al., 1994) and a CD8 intergenic construct (Tg-a) modified by Ellmeier et al. (1997). For L2aWT, a 4.3 kb HindIII/HindIII DH Cluster II fragment containing L2a was introduced upstream of the CD8α promoter, and a 7.6 kb BamHI/BamHI fragment containing the E8_I enhancer was subcloned upstream of DH cluster II fragment. Components of the constructs are highlighted. The L2aD deletion mutant was generated by deletion of a 200 bp AccI/BstXI fragment spanning L2a. This fragment served as the WT200 L+S probe in EMSA experiments.

Figure 2. L2a silences CD8 reporter expression on thymic, lymph node and intraepithelial (IEL) T cells

Representative fluorescent activated cytometric analyses of T cells isolated from one (L2aWT5) of seven independent L2aWT transgenic mice and one (L2aD5) of five independent L2aD transgenic lines. Transgenic thymic (A) and lymph node (B) were stained with anti-CD4, anti-CD8α and anti-hCD2. Transgenic intestinal IELs (C) were stained with anti-CD8α, anti-CD8β and anti-hCD2. Expression of the hCD2 reporter gene on gated populations (circled in red) were analyzed and results are represented in histograms. Percentages and MFIs of hCD2 positive (>1.0%) subsets are shown.

Figure 3. Differential recognition and binding modalities of SATB1 and CDP for L2a

(A) Schematic of the wildtype L2a element, WT200 (L+S), and mutants with elongated INTER-LS regions. The insert sequence (GC) is shown below the construct schematics. GC-1, 2 and 4 contained one insert, two inserts and four spacer inserts, respectively, separating the L and S sites. (B) Weakened binding of CDP to L2a mutants with elongated GC inserts between the L and S sites. EMSA analysis was performed with BW5147 nuclear extract, radiolabeled 200(L+S) probe and mutated probes. The arrow indicates CDP complexes. (C) Probes with increased distance between the L and sites are unable to compete for CDP binding. Competition EMSAs were performed with BW5147 nuclear extract, WT200(L+S) probe, and unlabeled mutated GC-1, GC-2, GC-4 competitors. 10–200 molar excess of competitors were added after the addition of radiolabeled probe. (D) Supershifted pattern of SATB1 binding to mutant probes. EMSA was performed with VL3 nuclear extract, radiolabeled 200(L+S), and mutated 200(L+S) GC probes. (E) CDP and SATB1 binding patterns in the absence of the other protein. Antibodies to SATB1 or CDP used in EMSA supershift assays in (D) were added to VL3 nuclear extracts followed by addition of radiolabeled WT200(L+S) and mutated GC probes. The arrows indicate SATB1 or CDP complexes. No shifted complexes were observed for probe alone lanes or antibody plus probe only lanes (data not shown). Please note that the ratios of the SATB1/CDP bands are higher in the absence of pre-immune sera (compare lanes of Fig. 3D to equivalent lanes in Fig 3E, pre-immune). Since pre-immune sera does not react with either of the purified proteins or with their DNA complexes, we suspect that selective degradation or non-specific interactions with other proteins in the nuclear extracts accounts for these differences.

Figure 4. SATB1 binds preferentially to L2a in vitro and in vivo

(A) In vitro EMSA binding competition by equivalent amounts of CDP and SATB1. Since purified preparations of endogenous, post-translationally modified SATB1 and CDP were unavailable, competition experiments were carried with VL3 nuclear extracts that had been immunodepleted of either protein. An equivalent amount of CDP or SATB1 was defined as the quantity in SATB1-depleted or CDP-depleted VL3 extract required to shift 90% of 1 ng of the wildtype L2a (L200 L+S) probe. Lanes 1–7 in (A) and Lanes 3–8 in (B) contain SATB1- depleted and CDP-depleted VL3 extract in amounts equivalent to CDP and SATB1 protein present singly in lanes 8 and 9 in (A), and Lanes 1 and 2 in (B). The small amount of SATB1- retarded band observed with the SATB1-depleted extract alone (A, Lane 9) reflects careful titration of the anti-SATB1 antibody used for depletion so that no free anti-SATB1 antibody remains behind to interact with SATB1 in the CDP-depleted extract following mixing. Similar precautions were taken to determine the amount of anti-CDP antibody used to deplete VL3 extract of CDP. (B) In vitro EMSA binding competition assays using CDP in 2-fold excess. Immunodepleted VL3 nuclear extract was tested essentially as described for (A) except that the CDP binding activity (SATB1-depleted extract, lanes 3–8) was in two-fold excess. Amounts of probe in (A) and (B) are as indicated above the figure. No shifted complexes were observed for probe alone lanes or antibody plus probe only lanes (data not shown). (C) In vivo recruitment of SATB1 to L2a chromatin in unfractionated, DP, and CD8SP thymocytes. Primer pairs indicated by arrows above the L2a schematic were designed (Materials and Methods) to amplify either the entire L2a region (L+S) or the sub-site (L), which was established by EMSA and footprinting experiments to be the binding region for SATB1 in vitro. The input lanes were loaded as 10% of IP lanes. Amplicon sizes are indicated on the schematic and to the right of the gel.

Figure 5. L2a-dependent variegated CD8 reporter expression requires SATB1 in vivo

L2aWT2 (top panels) and L2aD5 (lower panels) transgenic mice were crossed to SATB1 “reduced” mice that exhibit ~62% (As^+/+) or ~32% (As^+/+SATB1^+/−) of WT SATB1 levels in thymic and peripheral T cells (Nie et. al, 2005; Materials and Methods). Expression of the hCD2 reporter on thymic CD8SP and DP or from CD8⁺ lymph node T cells was analyzed and percentages and MFIs of hCD2 positive (>1.0%) subsets are shown as representative of 3 independent experiments. Under highly reduced (L2a⁺As^+/+SATB1^+/−) SATB1 conditions, variegated expression of L2aWT was reduced significantly whereas no effect of SATB1 reduction was observed on L2aD mutant mice.