Recruitment of Fkh1 to replication origins requires precisely positioned Fkh1/2 binding sites and concurrent assembly of the pre-replicative complex

<div><p>In budding yeast, activation of many DNA replication origins is regulated by their chromatin environment, whereas others fire in early S phase regardless of their chromosomal location. Several location-independent origins contain at least two divergently oriented binding sites for Forkhead (Fkh) transcription factors in close proximity to their ARS consensus sequence. To explore whether recruitment of Forkhead proteins to replication origins is dependent on the spatial arrangement of Fkh1/2 binding sites, we changed the spacing and orientation of the sites in early replication origins <i>ARS305</i> and <i>ARS607</i>. We followed recruitment of the Fkh1 protein to origins by chromatin immunoprecipitation and tested the ability of these origins to fire in early S phase. Our results demonstrate that precise spatial and directional arrangement of Fkh1/2 sites is crucial for efficient binding of the Fkh1 protein and for early firing of the origins. We also show that recruitment of Fkh1 to the origins depends on formation of the pre-replicative complex (pre-RC) and loading of the Mcm2-7 helicase, indicating that the origins are regulated by cooperative action of Fkh1 and the pre-RC. These results reveal that DNA binding of Forkhead factors does not depend merely on the presence of its binding sites but on their precise arrangement and is strongly influenced by other protein complexes in the vicinity.</p></div

Alterations of distance between the Fkh1/2 binding sites in <i>ARS607</i>.

<p><b>(A)</b> Schematic representation of <i>ARS607</i> origins inserted into the <i>GAL</i>-<i>VPS13</i> locus. Approximate locations of Fkh1/2 consensus binding sites (blue boxes) and the ACS (pink box) are indicated. In all constructs except wt <i>ARS607</i> the ACS-distal (3’) Fkh1/2 binding site was mutated (red X) and a new Fkh1/2 site was introduced at various distances from the ACS-proximal (5’) site. <b>(B)</b> Depiction of modified <i>ARS607</i> origins with small insertions and deletions between Fkh1/2 binding sites. Approximate locations of Fkh1/2 consensus binding sites (blue boxes), the ACS (pink box), poly-A track and sites of nucleotide insertions or deletions are indicated. Detailed sequences of all modified origin loci are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006588#pgen.1006588.s004" target="_blank">S4 Fig</a>. <b>(C)</b> Fkh1 binding to <i>ARS607</i> with altered distances between Fkh1/2 sites as determined by ChIP assay. Fkh1 occupancy at mutant <i>ARS607</i> is shown relative to its binding to the native <i>ARS607</i> locus in the same strain. A strain with no ARS in the <i>VPS13</i> locus and the native late-replicating origin <i>ARS522</i> that contains no Fkh1/2 binding sites are shown as controls (<i>VPS13</i> and <i>ARS522</i>, respectively). <b>(D)</b> Relative copy number of <i>VPS13</i>-<i>ARS607</i> DNA in HU-arrested cells. Cells were arrested in G1 with α-factor and then released into HU-containing media for 45 and 75 minutes. Graphs show the ratio of <i>VPS13</i>-<i>ARS607</i> and late-replicating <i>ARS522</i> loci, the ratio in G1-arrested cells was set as 1. Relative copy number of the native <i>ARS607</i> locus is shown as control. Full data for each strain is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006588#pgen.1006588.s001" target="_blank">S1 Fig</a>. <b>(E)</b> Mcm4 binding to <i>ARS607</i> loci with altered distances between Fkh1/2 sites as determined by ChIP assay. Mcm4 occupancy at mutant <i>ARS607</i> is shown relative to its binding to the native <i>ARS607</i> origin in the same strain. A strain with no ARS in the <i>VPS13</i> locus and the native origin <i>ARS522</i> are shown as controls (<i>VPS13</i> and <i>ARS522</i>, respectively).</p

Analysis of Fkh1/2 binding sites at replication origins.

<p><b>(A)</b> Double Fkh1/2 binding sites (RYMAAYA) with different spacing and orientation were located throughout the yeast genome. The sites were then mapped onto a genome-wide early DNA replication initiation profile [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006588#pgen.1006588.ref008" target="_blank">8</a>]. Percentage of double Fkh1/2 sites that co-localize with early-replicating loci is shown. Black dashed line indicates the background and is calculated as average frequency of overlap between scrambled Fkh1/2 double consensus sites (in all orientations; with 50–100 bp gap) and early origins. <b>(B)</b> Distribution of double Fkh1/2 sites at late replication origins was analysed as in <b>A</b>. <b>(C)</b> The general pattern of sequence elements in early replication origins with divergent Fkh1/2 binding sites. Two Fkh1/2 sites (blue arrows) are separated by a stretch of 71–79 bp linker DNA that contains a poly-A track. Location of the ACS (pink ellipse) varies, but is typically found in close proximity to or overlapping with one Fkh1/2 site. The ACS-proximal Fkh1/2 consensus sequence is located on the DNA strand complementary to that containing the ACS. The model is based on alignment of 20 early origins containing divergent Fkh1/2 binding sites (see text and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006588#pgen.1006588.s003" target="_blank">S3 Fig</a> for details).</p

Alterations of Fkh1/2 site orientations in <i>ARS305</i>.

<p><b>(A)</b> Schematic representation of <i>ARS305</i> origins inserted into the <i>GAL</i>-<i>VPS13</i> locus. Approximate locations of Fkh1/2 consensus binding sites (blue arrows) and the ACS (pink box) are indicated. In 5’mut construct, the ACS-proximal Fkh1/2 site was disrupted by mutation (red X). In Fkh1/2 site reversal mutants, the sites were inverted relative to their original orientations (red arrows). <b>(B)</b> Fkh1 binding to <i>ARS305</i> loci with various orientations of Fkh1/2 sites as determined by ChIP assay. Fkh1 occupancy to <i>ARS305</i> mutants is shown relative to its binding to the native <i>ARS305</i> locus. Fkh1 binding to the native late-replicating origin <i>ARS522</i> is shown as control (<i>ARS522</i>). <b>(C)</b> Relative copy number of <i>VPS13</i>-<i>ARS305</i> DNA in HU-arrested cells. Cells were arrested in G1 with α-factor and then released into HU-containing media for 45 and 75 minutes. Graphs show the ratio of <i>VPS13</i>-<i>ARS305</i> and late-replicating <i>ARS522</i> loci, the ratio in G1-arrested cells was set as 1. Relative copy number of the native <i>ARS305</i> and origin-free <i>VPS13</i> loci are shown as controls. Full data for each strain is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006588#pgen.1006588.s002" target="_blank">S2 Fig</a>. <b>(D)</b> Mcm4 binding to <i>ARS305</i> loci with various orientations of Fkh1/2 sites as determined by ChIP assay. Mcm4 occupancy to mutant <i>ARS305</i> is shown relative to its binding to the native <i>ARS305</i> locus. The strain with no ARS in the <i>VPS13</i> locus and the native origin <i>ARS522</i> are shown as controls (<i>VPS13</i> and <i>ARS522</i>, respectively).</p

Fkh1 binds to licensed origins.

<p><b>(A)</b> Schematic representation of ACS mutants of origins <i>ARS305</i>, <i>ARS607</i> and <i>ARS737</i>. The ACS and Fkh1/2 binding sites are shown as pink and blue boxes, respectively. Wild-type and mutated ACS sequences are shown, with the mutated nucleotides indicated in red. <b>(B)</b> Fkh1 binding to ACS-mutated origins. Fkh1 binding to <i>VPS13</i>-<i>ARS</i> loci in strains carrying wt or ACS-mutated versions of origins were determined by ChIP assay. In all cases, the signals from the strains with wt origins were set to 1. <b>(C)</b> Schematic representation of the origin re-licensing assay. Cells with <i>ARS607</i> inserted into the <i>GAL</i>-<i>VPS13</i> locus together with different ts mutations of pre-RC proteins (<i>cdc6</i>, <i>mcm2</i> and <i>cdc45</i>) were grown at a permissive temperature (24°C) and arrested in G1 with α-factor. Arrested cells were transferred to a non-permissive temperature (37°C) and transcription of the <i>GAL-VPS13</i> locus was induced by switching to growth medium containing galactose. After two hours, transcription was shut down by transferring the cells into glucose-containing medium to initiate the relicensing of the <i>ARS607</i> replication origin. Samples were collected 40 minutes later and the presence of Fkh1 and Mcm4 proteins at the origin was determined by ChIP assay. Binding of Mcm4 <b>(D)</b>, or Fkh1 <b>(E)</b> to the <i>GAL</i>-<i>VPS13</i>-<i>ARS607</i> locus in <i>cdc6-ts</i>, <i>mcm2-ts</i> and <i>cdc45-ts</i> strains determined by ChIP assay. Samples were taken from G1 arrested cells before transcription induction (Raf), during active transcription (Gal) and after repression of transcription of the locus (Glc). Protein occupancy was normalized to the signal obtained from G1 arrested cells growing at permissive temperature. <b>(F)</b> Cell cycle dependent binding of Fkh1 to origins. Cells were first arrested in G1 with α-factor, released to S phase and arrested again in M with nocodazole. Fkh1 binding was determined by ChIP assay and its binding to designated loci in G1 phase was set to 1. Replication-unrelated binding of Fkh1 to <i>CLB2</i> promoter was used as a control.</p