A Med15 - Hrp1 complex associates with fission yeast Mediator

The multiprotein Mediator complex is an important regulator of RNA polymerase II-dependent genes in eukaryotic cell. 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 now demonstrate that Med15 exists in a protein complex together with Hrp1, an ATP-dependent chromatin remodeling protein. The Med15/Hrp1 subcomplex is not a component of the core Mediator complex, but can interact with the repressive L-Mediator conformation. Deletion of MED15 and HRP1 cause similar effects on global steady-state levels of mRNA, but only MED15 is required for galactose-dependent activation of the inv1 gene. Hrp1 has been found in complex with other proteins and genome-wide analysis demonstrates that Med15 only associates with a distinct subset of Hrp1-bound gene promoters. Global analysis reveals that Hrp1-binding normally is associated with increased histone H3 density, but at promoters also bound by Med15, histone H3 density is instead increased. Our findings reveal that Med15 functions as a separate entity in fission yeast and indicate that the function and organization of the Mediator complex may differ significantly between eukaryotes. Keywords: ChIP-chip Genome-wide binding of the Mediator subunit Med15 was analysed using a ChIP-on-chip approach. a-myc ChIP DNA purified from a Med15-myc strain was amplified, labeled and hybridised onto Eurogentec IGR+ORF arrays together with input DNA from the same sample. Similarly, global H3 density was analysed using an anti-H3 antibody for ChIP-on-chip. The analysis of histone density was carried out in three strains: wildtype (711) and knockout strains lacking either Med15 or Hrp1. Furthermore, expression analysis of wildtype, delta-Med15 and delta-Hrp1 strains was carried out using Affymetrix arrays. In each case, 2-3 biological replicates were analysed, and for the Eurogentec experiments dye-swap was always performed.