10 research outputs found
The majority of the Sp2 binding sites are also bound by the heterotrimeric transcription factor Nf-y.
<p>Binding sites of the trimeric transcription factor Nf-y were determined by ChIP-seq of the Nf-ya, Nf-yb and Nf-yc subunits using wt and <i>Sp2ko</i> MEFs. (A) Venn diagrams showing the overlap of high-confidence Nf-ya, Nf-yb and Nf-yc binding sites in wild type and in <i>Sp2ko</i> MEFs. (B) Venn diagram showing the overlap of Nf-y and Sp2 binding sites. (C) The strength of Nf-y binding correlates with the strength of Sp2 binding at shared sites. Normalized ChIP-seq tag counts at individual Nf-ya and Sp2 peaks were plotted against each other. (D) Representative genome browser snapshots of promoters bound by Sp2 as well as by Nf-y (<i>Mcm3</i> and <i>Pan2</i>), only by Nf-y (<i>Wapal</i> and <i>Atxn3</i>) or only by Sp2 (<i>Fanci</i> and <i>Taf1c</i>). (E) Sequence motifs enriched at Sp2 sites also bound by Nf-y (left), at sites only bound by Nf-y (middle) or only bound by Sp2 (right). The numbers next to the logos indicate the occurrence of the motifs, and the statistical significance (<i>E</i>-value) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.ref048" target="_blank">48</a>]. (F) Central motif enrichment analysis of the motifs shown in Fig. 7E. (G) Venn diagram showing the overlap of Sp1/3, Nf-y and Sp2 binding sites. (H) Left, sequence motifs at sites that are bound by Sp1/3 but not by Sp2 and Nf-y. Right, sequence motifs at sites that are bound by Sp2 and Sp1/3 but not by Nf-y.</p
The N-terminal domain of Sp2 can rescue target gene expression.
<p>(A) The N-terminal domain of Sp2 has the capacity to activate transcription. The Gal4 DNA-binding domain (Gal4), and Gal4-Sp1NT and Gal4-Sp2NT fusions were transfected into HEK293 cells along with a 5xUAS-luciferase reporter construct. Fold activation by Gal4-Sp1NT and Gal4-Sp2NT is expressed relative to the Gal4 DNA-binding domain set to 1. (B, C) Relative expression of the <i>Nlk</i> (B), <i>Grb2</i> and <i>Oxr1</i> (C) genes in wt MEFs, <i>Sp2ko</i> MEFs and in <i>Sp2ko</i> MEFs re-expressing full-length Sp2 (Sp2FL) or the Sp2NT and Sp2ZF mutants. (C) Schematic representation of the exon-intron structures of the <i>Grb2</i> and <i>Oxr1</i> genes and the promoters bound by either Sp1/Sp3 or by Sp2. The primer pairs P1-P2 detect specifically transcripts derived from the Sp2-bound promoters. The primer pairs P3-P4 detect all transcripts. Transcript levels in wt MEFs determined by qPCR were set to 1. <i>Gapdh</i> mRNA levels were used for normalization. Data are presented as the average of three independent experiments +/-SD.</p
Different binding site selection of Sp1/Sp3 and Sp2 in MEFs.
<p>Binding sites of Sp1 and Sp3 in MEFs were determined by ChIP-seq. (A) Venn diagrams showing the overlap of Sp1 or Sp3 peaks obtained with two different antibodies for each factor. (B) Venn diagrams showing the overlap of Sp1 and Sp3 peaks. 3597 overlapping peaks were obtained with all four antibodies. (C) Distribution of Sp1/Sp3 binding sites in MEFs relative to annotated genes (TSS, +/- 500 bp). (D) Overlap of high-confidence Sp1/Sp3 and Sp2 binding sites. (E) Representative genome browser snapshots of promoters bound by all three Sp factors (left), by Sp1 and Sp3 but not by Sp2 (middle), or exclusively by Sp2 (right) in wild type (wt), <i>Sp2ko</i> and <i>Sp3ko</i> MEFs. (F) ChIP-qPCR validation experiments using Sp1_Ab1, Sp2_Ab1 and Sp3_Ab1. Percent of input values represent the mean of at least three independent experiments +/- SD.</p
Sequence motifs at Sp1/Sp3 and Sp2 binding sites in MEFs.
<p>(A) Sequence motifs enriched at Sp1/Sp3 and Sp2 binding sites. Logos were obtained by running MEME-ChIP [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.ref048" target="_blank">48</a>] with 300 bp summits of the top 600 Sp1/Sp3, Sp2, Sp1/Sp3-specific and Sp2-specific ChIP-seq peaks. The numbers next to the logos indicate the occurrence of the motifs, and the statistical significance (<i>E</i>-value). (B) Central motif enrichment analysis of the motifs shown in Fig. 2A. (C) Occurrence of the canonical “CCAAT” sequence at the top 600 Sp1/Sp3, Sp2, Sp1/Sp3-specific and Sp2-specific ChIP-seq peaks. (D) Pairwise distribution of the canonical “CCAAT” sequence at the top 600 Sp1/Sp3, Sp2, Sp1/Sp3-specific and Sp2-specific ChIP-seq peaks. Each short blue line indicates a CCAAT motif. Black lines connect two CCAAT motifs in a promoter if they are located within a distance of 30 to 50 nucleotides.</p
Different domains mediate recruitment of Sp2 and Sp3 to promoters <i>in vivo</i>.
<p>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 <i>Sp2ko</i> or <i>Sp3ko</i> 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).</p
The <i>bona fide</i> DNA-binding domain of Sp2 is dispensable for genomic binding.
<p><i>Sp2ko</i> MEFs re-expressing Flag-tagged full-length Sp2 (Flag-Sp2FL), the N-terminal domain (Flag-Sp2NT) or the C-terminal zinc finger domains (Flag-Sp2ZF) were subjected to ChIP-seq analysis. (A) Venn diagram showing the overlap of sites bound by Flag-Sp2FL, Flag-Sp2NT and Flag-Sp2ZF with sites bound by endogenous Sp2 in wild type MEFs. (B) Full-length Sp2 and the N-terminal region of Sp2 have similar chromatin binding efficiencies. ChIP-seq tag counts (normalized to 20x10<sup>6</sup> reads) at individual Flag-Sp2NT and Flag-Sp2FL peaks found in both samples were plotted against each other. (C) Sites bound by Flag-Sp2NT represent high affinity binding sites of native Sp2. Individual native Sp2 peaks [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.ref018" target="_blank">18</a>] were plotted against their normalized tag counts. Those sites that were also detected by ChIP-seq in <i>Sp2ko</i> MEFs expressing the N-terminal domain of Sp2 (Flag-Sp2NT mutant) were overlaid with red dots. (D) Representative binding profiles of Sp2 in wild type MEFs (Sp2 / wt), and of Flag-Sp2FL, Flag-Sp2NT and Flag-Sp2ZF expressed in <i>Sp2ko</i> MEFs. (E) Schematic representation of the genomic binding features of Sp1/Sp3 and Sp2 based on the results shown in Figs. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.g001" target="_blank">1</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005102#pgen.1005102.g004" target="_blank">4</a>.</p
Zinc Finger Independent Genome-Wide Binding of Sp2 Potentiates Recruitment of Histone-Fold Protein Nf-y Distinguishing It from Sp1 and Sp3
<div><p>Transcription factors are grouped into families based on sequence similarity within functional domains, particularly DNA-binding domains. The <u>S</u>pecificity <u>p</u>roteins Sp1, Sp2 and Sp3 are paradigmatic of closely related transcription factors. They share amino-terminal glutamine-rich regions and a conserved carboxy-terminal zinc finger domain that can bind to GC rich motifs <i>in vitro</i>. All three Sp proteins are ubiquitously expressed; yet they carry out unique functions <i>in vivo</i> raising the question of how specificity is achieved. Crucially, it is unknown whether they bind to distinct genomic sites and, if so, how binding site selection is accomplished. In this study, we have examined the genomic binding patterns of Sp1, Sp2 and Sp3 in mouse embryonic fibroblasts by ChIP-seq. Sp1 and Sp3 essentially occupy the same promoters and localize to GC boxes. The genomic binding pattern of Sp2 is different; Sp2 primarily localizes at CCAAT motifs. Consistently, re-expression of Sp2 and Sp3 mutants in corresponding knockout MEFs revealed strikingly different modes of genomic binding site selection. Most significantly, while the zinc fingers dictate genomic binding of Sp3, they are completely dispensable for binding of Sp2. Instead, the glutamine-rich amino-terminal region is sufficient for recruitment of Sp2 to its target promoters <i>in vivo</i>. We have identified the trimeric histone-fold CCAAT box binding transcription factor Nf-y as the major partner for Sp2-chromatin interaction. Nf-y is critical for recruitment of Sp2 to co-occupied regulatory elements. Equally, Sp2 potentiates binding of Nf-y to shared sites indicating the existence of an extensive Sp2-Nf-y interaction network. Our results unveil strikingly different recruitment mechanisms of Sp1/Sp2/Sp3 transcription factor members uncovering an unexpected layer of complexity in their binding to chromatin <i>in vivo</i>.</p></div
Nf-y is necessary for recruitment of Sp2 to shared sites.
<p>(A) Sp2 and Nf-y are bound simultaneously to their target sites. Sp2 and Nf-y occupancy at selected target promoters was analyzed by sequential ChIP (re-ChIP) using anti-Sp2 and anti-Nf-yb antibodies as indicated. The <i>Sp2</i>, <i>Osbp</i>, <i>Sp1</i>, <i>Amd1</i>, <i>Nxt1</i> and <i>Nipal3</i> promoters are co-occupied by Nf-y and Sp2; the <i>Fanci</i> promoter is only bound by Sp2 but not by Nf-y; the <i>Atxn3</i> promoter is only bound by Nf-y but not by Sp2, and the <i>Raf1</i> promoter is neither bound by Sp2 nor by Nf-y. The percent of input values are mean +/- SD (n = 3). (B, C) MEFs were treated with a control siRNA (sicontrol) or siRNAs targeting <i>Nf-ya</i>, <i>Nf-yb</i> or <i>Nf-yc</i>. (B) Top panel, immunoblot analysis of Nf-ya and Nf-yb showing the knockdown efficiency. Bottom panel, knockdown of Nf-yc was controlled by RT-qPCR due to the poor performance of the Nf-yc antibody in immunoblots. (C) Binding of Nf-y subunits and Sp2 to selected promoters after siRNA treatment was analyzed by ChIP-qPCR. The <i>Sp1</i>, <i>Osbp</i>, <i>Amd1</i> and <i>Dctn4</i> promoters are co-occupied by Nf-y and all three Sp factors; the <i>Oxr1</i> and <i>Plcl1</i> promoters are bound by Nf-y and Sp2 but not by Sp1 and Sp3; the <i>Fanci</i> and <i>Taf1c</i> promoters are bound by all three Sp factors but not by Nf-y; and the <i>Raf1</i> promoter is bound by Sp1 and Sp3 but neither by Sp2 nor by Nf-y. The percent of input values are mean +/- SD (n = 3).</p
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.</p
Model depicting the recruitment of Sp1/Sp3 and Sp2 to their target promoters <i>in vivo</i>.
<p>Consistent with <i>in vitro</i> binding data, Sp1 and Sp3 are recruited to GC boxes by their zinc finger domain. The majority of Sp2 is recruited to CCAAT motifs (often arranged in tandem) that are co-bound by Nf-y. Binding of Sp2 to these sites is by its N-terminal region, and is independent of the zinc finger domain. The strength of binding to these sites and the presence of positively charged amino acids in the N-terminal region of Sp2 might indicate that protein-protein as well as unspecific protein-DNA interactions are involved.</p