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. 2010 Sep 24;285(39):29729-37.
doi: 10.1074/jbc.M110.153858. Epub 2010 Jul 9.

A chromatin-remodeling protein is a component of fission yeast mediator

Olga Khorosjutina  1 , Paulina H Wanrooij, Julian Walfridsson, Zsolt Szilagyi, Xuefeng Zhu, Vera Baraznenok, Karl Ekwall, Claes M Gustafsson
Affiliations

A chromatin-remodeling protein is a component of fission yeast mediator

Olga Khorosjutina et al. J Biol Chem. .

Abstract

The multiprotein Mediator complex is an important regulator of RNA polymerase II-dependent genes in eukaryotic cells. In contrast to the situation in many other eukaryotes, the conserved Med15 protein is not a stable component of Mediator isolated from fission yeast. We here demonstrate that Med15 exists in a protein complex together with Hrp1, a CHD1 ATP-dependent chromatin-remodeling protein. The Med15-Hrp1 subcomplex is not a component of the core Mediator complex but can interact with the L-Mediator conformation. Deletion of med15(+) and hrp1(+) causes very similar effects on global steady-state levels of mRNA, and genome-wide analyses demonstrate that Med15 associates with a distinct subset of Hrp1-bound gene promoters. Our findings therefore indicate that Mediator may directly influence histone density at regulated promoters.

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Figures

FIGURE 1.
FIGURE 1.
Med15 interacts with chromatin modifier Hrp1. A, L-Mediator IgG purified from TAP-Med13 cells polypeptides was resolved on 10% SDS-PAGE, stained with Coomassie Blue, and identified by fingerprint MALDI-TOF. The high molecular mass region is presented. B, extract prepared from FLAG-Med15 cells was purified over M2-agarose. Proteins are separated on 10% SDS-PAGE and stained with Coomassie Blue. C, untagged and FLAG-Med15 cell extracts were purified over M2-agarose. Fractions of input (Inp), flow-through (Ft), wash, and eluate (Elu) were separated on 10% SDS-PAGE and immunoblotted with antibodies against Hrp1. D, extract from FLAG-Med15 cells was incubated with anti-Hrp1 beads or empty beads as a control (ctr). Immunoblot of bounded fractions with anti-FLAG antibody is presented.
FIGURE 2.
FIGURE 2.
Med15 and Hrp1 proteins are components of L-Mediator and functionally related. A, Mediator complexes were IgG-purified, resolved on 10% SDS-PAGE, and immunoblotted with antibodies against indicated proteins. B, FLAG-tagged Med15 remains associated with L-Mediator in Δhrp1 cells. L-Mediator was analyzed as in panel A. C, Med15 and Hrp1 targets form an extensively overlapping set. A Venn diagram depicting the overlap of Med15- and Hrp1-dependent genes from the microarray expression analysis is shown. Target gene expression increased at least 2-fold in Δmed15 and Δhrp1.
FIGURE 3.
FIGURE 3.
Inv1+ induction. A, the inv1+ transcript has a 5′-UTR of 892 nucleotides. The Scr1 binding site is located about 170 bp upstream of the transcription start site. Positions of primers used for ChIP analysis are indicated. B, total RNA was isolated upon induction of inv1+ expression. inv1+ mRNA levels were assessed by quantitative RT-PCR and normalized to the act1+ mRNA levels at each time point. C, changes in mRNA levels in WT, Δhrp1, and Δmed15 cells. The mRNA levels were quantified as in panel B. D, ChIP analysis reveals enrichment of Hrp1, Med7 and Med15 under derepressing conditions. E, deletion of med15+ impairs glucose repression of inv1+ gene transcription. Levels of inv1+ mRNA levels in WT, Δhrp1, and Δmed15 cells in the presence of glucose are shown. Error bars in panels D and E indicate S.D.
FIGURE 4.
FIGURE 4.
A, changes in H3 density in response to sucrose induction and subsequent glucose repression of the inv1+ gene. H3 density was assessed by ChIP analysis. Error bars indicate S.D. B, Med15 and Hrp1 bind to the same genomic regions. The Venn diagram presents the overlap in binding of Med15 and Hrp1 to intergenic regions.
FIGURE 5.
FIGURE 5.
Med15 (A) and Hrp1 (B) occupancy correlates with genome-wide histone H3 density. A moving average window size of 100 was used.
FIGURE 6.
FIGURE 6.
Deletion of med15+ leads to increased Hrp1 occupancy at a number of Med15 target promoters. The error bars represent one standard deviation.
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