Different domains mediate recruitment of Sp2 and Sp3 to promoters in vivo.

Flag-tagged full-length Sp2 (Sp2FL) and Sp3 (Sp3li or Sp3si, long or short isoforms) and deletion mutants encompassing either the N-terminal domains (Sp2NT and Sp3NT) or the C-terminal zinc finger domains (Sp2ZF and Sp3ZF) were re-expressed in corresponding Sp2ko or Sp3ko MEFs. (A, B) Top panels, schematic representation of the Sp2 and Sp3 mutants. The Flag-epitope at the N-termini (grey), the Sp box (yellow), the glutamine-rich domains (Q-rich, red), the Btd box (blue) and the zinc fingers (ZF, black bars) are indicated. Bottom panels, expression of the Sp2 and Sp3 mutants was monitored by immunoblotting using anti-Sp2, anti-Flag and anti-Sp3 antibodies as indicated. Re-probing for tubulin controlled loading of Sp2-containing extracts. The non-specific band indicated by an asterisk served as loading control for Sp3-containing extracts. Of note, the anti-Sp3 antibody does not recognize the Sp3NT fragment because it binds to a C-terminal epitope. (C, D) Binding of Sp2 and Sp3 mutants to selected target promoters was analyzed by ChIP-qPCR. Anti-Flag antibodies were used for ChIP. The percent of input values are mean +/- SD (n = 3).