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. 2010 Aug 9;30(20):4922–4939. doi: 10.1128/MCB.00354-10

FIG. 7.

FIG. 7.

Cross-reactive, pro-T-cell-specific complexes formed at the CE4A and CE4B regions. CE4B can nucleate a pro-T-cell-specific complex in vitro and can cross-compete with the T-lineage-specific A1 complex formed by the CE4A region probe. (A) The sequence requirements for formation of different T-lineage complexes on probe CE4A-P3 can be separated. A homologous probe from the CE4A region cannot compete for complex A1 if the Runx sites are mutated (compare lanes 2 and 3), indicating that a Runx site is needed for A1 formation. Complexes A2 and A3 are competed by the homologous CE4A-P3 probe, but not when the GGAA core of the Ets site is mutated (lane 4). A probe from the CE4B region competes for complex A1 but not for complexes A2 or A3 (lane 5), and the ability to compete for A1 depends on a Runx site here as well (lanes 6 and 7). Complex A1 may also contain Ikaros, as it can be shifted by an anti-Ikaros antibody (lane 9). (B) The CE4B-P3 probe nucleates a pro-T-cell-specific complex, B1 (lane 1; “B”) and a myeloid cell-specific complex, B3. Complex B1 does not depend on the STAT site previously shown to contribute to PU.1 expression (lanes 3 and 4). In contrast, the myeloid complex B3 and the weaker T-lineage complex B2 do require the STAT site for formation of complexes (lanes 3 and 4 and lanes 10 and 11). (C) Complex B1 depends on the sequences spanning nonconsensus Runx and Ikaros sites (lanes 2 and 3). Probe CE4A-P3 can be used in competition to eliminate complex B1 formation, and the ability to compete requires the Runx site (lanes 4 and 5). (D) Like A1, complex B1 can be supershifted by an anti-Ikaros antibody (lane 3). (E) Interaction of Runx factors with complexes at multiple sites in CE4A and CE4B. A pan-Runx antibody inhibits complex formation on multiple probes by Adh.2C2 pro-T-cell nuclear extracts (red arrows, lanes 2, 5, and 15). Anti-Ets2 antibody had no effect on any complex. (F) The sequence of probe CE4B-P3 is shown, with asterisks indicating mutated nucleotides. TRANSFAC predicted transcription factor binding sites are shown with their matrix similarity scores in parentheses.