Sp2 potentiates binding of Nf-y at shared sites.

<p>(A, B, C, D) Nf-y binding is attenuated in <i>Sp2ko</i> MEFs. (A) Western blot analysis of Sp2, Nf-ya and Nf-yb in wild type and in <i>Sp2ko</i> MEFs. The asterisk in the Sp2 blot marks a non-specific band. The anti-Tubulin blot served as a loading control. (B) Binding of Nf-y at sites that are also bound by Sp2 is weakened in <i>Sp2ko</i> MEFs. Individual Nf-y binding sites were plotted against the ratio (wt/<i>Sp2ko</i> MEFs) of normalized Nf-yc tag counts. Top panel, blue dots: High confidence Nf-y binding sites that are co-bound by Sp2 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.g007" target="_blank">Fig. 7B</a>). Bottom panel, red dots: High confidence Nf-y binding sites that are not co-bound by Sp2. For clarity, a subset of promoters (<i>Nrxn2</i>, <i>Osbp</i>, <i>Sp2</i>, <i>Amd1</i>, <i>Atxn3</i> and <i>Wapal</i>) is indicated. (C) Representative binding profiles of Sp1, Sp2, Sp3, Nf-ya, Nf-yb and Nf-yc in wild type and in <i>Sp2ko</i> MEFs at a promoter bound by all three Sp factors and Nf-y (<i>Osbp</i>), at a promoter bound by Sp2 and Nf-y but not by Sp1/Sp3 (<i>Nrxn2</i>), and at a promoter bound by Sp1/Sp3 and Nf-y but not by Sp2 (<i>Atxn3</i>). Additional genome browser snapshots are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.s005" target="_blank">S5</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.s006" target="_blank">S6</a> Figs. (D) ChIP-qPCR validations of Nf-y occupancy at selected target promoters in wild type and in <i>Sp2ko</i> MEFs (Nf-ya, Nf-yb and Nf-yc ChIPs). The <i>Sp2</i>, <i>Sp1</i>, <i>Osbp</i>, <i>Amd1</i> and <i>Dctn4</i> promoters are bound by all three Sp factors and by Nf-y; the <i>Oxr1</i> and <i>Plcl1</i> promoters are bound by Sp2 and Nf-y but not by Sp1/3; the <i>Atxn3</i> promoter is bound by Sp1/3 and Nf-y but not by Sp2; the <i>Fanci</i> promoter is bound by all three Sp factors but not by Nf-y; and the <i>Raf1</i> promoter is bound by Sp1/Sp3 but neither by Sp2 nor by Nf-y. Data are mean of at least three independent experiments +/- SD. (E) Re-expression of Sp2FL or Sp2NT in <i>Sp2ko</i> MEFs potentiates Nf-y binding. Left, Western blot analysis of Flag-Sp2 mutants, Nf-ya and Nf-yb in rescued <i>Sp2ko</i> MEFs. The anti-Flag blot was split in an upper and a lower part for space reasons. Right, ChIP-qPCR analysis of Flag-Sp2 and Nf-yb binding to selected promoters in rescued MEFs.