Model proposing that Rtt106 mediates the cell cycle recruitment of SWI/SNF and RSC chromatin remodeling complexes to the HIR-dependent histone genes.

<p>Based on our results demonstrating the role of Rtt106 in the recruitment of the SWI/SNF and RSC complexes, we propose the following model. Rtt106 is shown to be associated with HIR and Asf1 at the <i>HTA1-HTB1</i> promoter sequence throughout the cell cycle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021113#pone.0021113-Fillingham1" target="_blank">[25]</a>. The indicated NEG sequence region, which is essential for HIR-mediated repression, is believed to be bound by a yet unidentified factor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021113#pone.0021113-Moran1" target="_blank">[14]</a> that tethers the HIR complex and its associated factors to the promoter of the histone genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021113#pone.0021113-Osley3" target="_blank">[9]</a>. We propose that Rtt106 is the key factor of an Rtt106/HIR/Asf1 complex that mediates the cell-cycle regulated recruitment of the RSC complex outside of S phase, in G2/M and early G1 phases when histone genes are repressed and according to the previously published cell cycle regulated localization of RSC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021113#pone.0021113-Ng1" target="_blank">[23]</a>. As shown by our SWI/SNF occupancy data, in late G1 phase, we propose that cell cycle specific signals trigger a switch which allows Rtt106 to recruit SWI/SNF in late G1 phase and which displace RSC from the histone gene promoter. In late G1 and S phases, SWI/SNF is shown bound to the histone gene promoter through its interaction with Rtt106 allowing transcriptional activation of the histone genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021113#pone.0021113-Dimova1" target="_blank">[22]</a>.</p

Rtt106 is essential for recruitment of RSC to the HIR-dependent histone genes.

<p>(A) Chromatin immunoprecipitation of the RSC complex were carried out as described in Material and Methods. An antibody directed against the TAP tag of the Rsc8-TAP subunit in wild-type and <i>rtt106Δ</i> strains was used. Presence of the indicated histone gene loci promoter sequence was monitored by qPCR. A non-transcribed region of chromosome V (InterV) was used as an internal background control. Error bars show the range between biological duplicates. (B) 25 µg of whole cell extract prepared from the <i>RSC8-TAP</i> (wild-type background) and <i>RSC8-TAP rtt106Δ</i> strains were loaded per lane. Protein levels of two representative RSC subunits, Rsc8-TAP and Sth1, were compared between the <i>RSC8-TAP</i> (lane 1) and <i>RSC8-TAP rtt106Δ</i> strain (lane 2) by western blots using an antibody targeting the TAP-tag in Rsc8-TAP, or an antibody against endogenous Sth1, which is the catalytic subunit of the RSC complex. Actin was used as a loading control. (C) ChIP assays were performed as in A to monitor the presence of the Rsc8-TAP tagged subunit at the <i>HTA1-HTB1</i> promoter in wild-type (WT) strain and strains deleted of <i>RTT106 (rtt106Δ)</i>, <i>ASF1 (asf1Δ)</i>, or <i>HPC2 (hpc2Δ)</i>. Enrichment of <i>HTA1-HTB1</i> promoter sequence in immunoprecipitated material was monitored by qPCR and normalized to the control region, InterV. The graph shows the average of duplicate IP reactions, and the error bars show the range between individual IPs.</p

Rtt106 is required for SWI/SNF localization to <i>HTA1-HTB1</i> loci.

<p>(A) Chromatin immunoprecipitation was carried out as described in Material and Methods. Antibodies specific for the N-terminus or C-terminus of the endogenous Swi2/Snf2 subunit were used for immunoprecipitation of the SWI/SNF complex in cross-linked chromatin extracts from the wild-type strain and strains deleted of <i>RTT106, ASF1</i>, or <i>HPC2</i>. The amount of <i>HTA1-HTB1</i> promoter (black bars) precipitated was monitored by qPCR and inter V region (grey bars) was used as an internal background control from a non-transcribed region on chromosome V. Data is expressed as percentage of ChIP/Input and error bars show the range of repeated IPs. (B) 25 µg of whole cell extracts prepared from the untagged wild-type and <i>rtt106Δ</i> strains were loaded per lane. Protein levels of four SWI/SNF subunits, Swi2/Snf2, Swi1, Snf5 and Swi3, were compared between the wild-type strain (lane 1) and the <i>rtt106Δ</i> strain (lane 2) by western blot analysis using specific antibodies against each individual protein. Actin was included as a loading control.</p

SWI/SNF recruitment to the <i>HTA1-HTB1</i> promoter is cell cycle regulated and dependent on Rtt106.

<p>(A) SWI/SNF binding to the <i>HTA1-HTB1</i> promoter in synchronized cultures of wild-type (blue line) and <i>rtt106Δ</i> (red line) strains was studied by chromatin immunoprecipitation (ChIP) assay on samples taken at the indicated time-points after release from G1 arrest induced by α-factor treatment. Samples for ChIP were cross-linked for 1h and SWI/SNF in chromatin extracts was immunoprecipitated using a specific antibody targeting the N-terminal end of Swi2/Snf2 subunit. Enrichment of <i>HTA1-HTB1</i> promoter relative to a control region (InterV) was quantified by real-time PCR. (B) Expression levels of mRNA from cell cycle regulated genes <i>CLN2</i> (solid line) and <i>CLB2</i> (dashed line) were monitored by RT-qPCR in <i>rtt106Δ</i> (red lines) and wild-type strain (blue lines) as a control for cell cycle progression and were normalized to expression of <i>ACT1</i>. Total RNA was purified from samples taken at the indicated time-points from the ChIP culture used in (A). (C) <i>HTA1</i> mRNA levels were monitored by RT-qPCR in wild-type (blue line) and <i>rtt106Δ</i> (red line) strains using the same samples used in (B) and were normalized to <i>ACT1</i> expression.</p

Rtt106 physically associates with RSC and SWI/SNF <i>in vivo</i>.

<p>(A) An untagged wild-type strain and strains carrying a TAP epitope tagged Swp82, -Rsc8 or -Hir1 subunit were transformed with a plasmid expressing a HA tagged Rtt106. Whole cell extracts were made and SWI/SNF, RSC and HIR complexes were immunoprecipitated in presence of ethidium bromide, using an antibody targeting the Protein A portion of the TAP tagged subunits as indicated, followed by capture on Protein A beads. Co-immunoprecipitation of Rtt106-HA was detected by western blot, using an antibody against the HA tag. Whole cell extract corresponding to 25 µg total protein per lane was used for input samples. Western blot using an antibody against endogenous actin was included as a loading control. (B) Whole cell extracts from a strain expressing TAP-tagged Rtt106 and plasmid based Snf5-HA tagged subunit, were used for immunoprecipitation of the TAP-tagged protein as described above. Co-immunoprecipitation of SWI/SNF was detected by western blot against the C-terminal HA-tag of Snf5-HA. Input and actin loading control as described in (A).</p

Yeast strains used for this study.

<p>Yeast strains used for this study.</p

Recombinant Rtt106 interacts with SWI/SNF, RSC and HIR complexes <i>in vitro</i>.

<p>Bacterially expressed GST and GST-Rtt106 were purified and incubated with whole cell extracts from strains expressing a TAP-tagged subunit in the SWI/SNF complex (Swp82-TAP), the RSC complex (Rsc8-TAP) or the HIR complex (Hir2-TAP). Western blots to detect the TAP-tagged complexes were performed using PAP-HRP antibody.</p

Phylogenetic relationships between Tup11 and Tup12 proteins in fission yeasts.

<p>(A) Dendrogram of Tup11 and Tup12 proteins from different fission yeast species, based on ClustalW alignment of full-length translated protein sequences. <i>S. cerevisiae</i> Tup1 is used as the outgroup. (B) Cross comparison of similarity between N-terminal domains (N), middle regions (M) and C-terminal domains (C) of budding and fission yeast Tup proteins. The numbers in each box correspond to in the percentage identity of amino acid residues for each pair wise comparison of aligned protein domains. The following protein regions were compared: <i>S. cerevisiae</i> Tup1 residues 1–89 (N), 90–317 (M), 318–713 (C), <i>S. pombe</i> Tup11 residues 1–87 (N), 88–286 (M), 287–614 (C), <i>S. octosporus</i> Tup11 residues 1–87 (N), 88–271 (M), 272–601 (C), <i>S. japonicus</i> Tup11 residues 1–87 (N), 88–303 (M), 304–630 (C), <i>S. pombe</i> Tup12 residues 1–104 (N), 105–259 (M), 260–586 (C), <i>S. octosporus</i> Tup12 residues 1–88 (N), 89–228 (M) 229–555 (C), <i>S. japonicus</i> Tup12 residues 1–88 (N), 89–249 (M), 250–576 (C).</p

Ability of Tup11/12 hybrid proteins to rescue the CaCl₂ sensitivity of <i>tup11</i>Δ, <i>tup12</i>Δ in <i>S. pombe</i>.

<p>Five-fold dilutions of the wild-type strain JY741, strain FFB52 (<i>Δtup12</i>, <i>Δtup12</i>) and strain FFB52 transformed with empty vector or with plasmids expressing of intact Tup11 or Tup12 or Tup11/12 hybrid proteins, spotted on YES agar supplemented with increasing concentrations of CaCl₂. The Tup11/12 hybrid proteins contained the following amino acid residues from Tup11 and Tup12. Tup12-11-12 (Tup12 residues 1–104, Tup11 residues 88–286, Tup12 residues 260–586 followed by Gly, Ser derived from the vector); Tup12-11-11 (Tup12 residues 1–104, Tup11 residues 88–614 followed by Gly, Ser derived from the vector), Tup11-12-12 (Tup11 residues 1–87, Tup12 residues 105–586 followed by Gly, Ser derived from the vector). (B) The hybrid Tup11/12 proteins are expressed at similar levels to Tup11 and Tup12. Western blot in which Flag-tagged proteins were identified using antibodies specific for the Flag tag. The Tup proteins are the same as in A. FFB52 was used as a negative control (No Tup).</p

Ability of <i>S. octosporus</i> and <i>S. japonicus</i> Tup12 proteins to rescue the CaCl₂ sensitivity of <i>tup12Δ</i> in <i>S. pombe</i>.

<p>Five-fold dilutions of the wild-type strain JY741 and JY741 <i>Δtup12</i> transformed with empty vector or with plasmids expressing one of the fission yeast Tup12 proteins or <i>S. octosporus</i> Tup11, spotted on YES agar supplemented with CaCl₂ (0.1 M or 0.25 M, left panels) or sorbitol (1.2 M or 2.0 M, right panels).</p