Figure 3.
Overexpression of MED14 acts as a growth enhancer in strains bearing point mutations in SCC2
(A) Identification of a genetic interaction between Scc2 and Mediator. A genomic tiling vector screen identified MED14 and SCC4 as possible suppressors of scc2R787G and scc2G1242V mutations. MED14 and SCC4 were cloned into a LEU2 selectable galactose overexpression plasmid and transformed into the scc2R787G and scc2G1242V plasmid shuffle strains. 10-fold serial dilutions of the indicated strains were spotted on Gal-HIS-LEU-URA and Gal-HIS-LEU+5FOA.
(B) Increase in cell growth is observed in the MED14 overexpression strain compared with the empty vector (EV) control on 5FOA (B) MED14 overexpression does not affect protein levels of cohesin or Scc2. Cohesin and Scc2 Myc tagged strains containing the MED14 galactose overexpression plasmid (MED14 OE +) or empty vector (MED14 OE −) were grown in Gal-LEU and levels of cohesin subunits and Scc2 were analyzed by immunoblot. GAPDH and H2A levels are included as loading controls.
(C) Overexpressing MED14 does not increase bulk Scc2 or cohesin on chromatin. Chromatin fractionation was performed on yeast cells and levels of Myc tagged Scc2 or Scc1 in the whole-cell extract (WCE), supernatant (SUP), and chromatin pellet (CP) were visualized by immunoblotting. GAPDH and H2A served as loading controls and positive controls for the SUP and CP fractions, respectively.
See also Figure S3.