Volume 4, Issue 1, Pages 75-83 (July 1999) A Role for Transcriptional Repressors in Targeting the Yeast Swi/Snf Complex  Dessislava Dimova, Zeena Nackerdien, Seth Furgeson, Sayaka Eguchi, Mary Ann Osley  Molecular Cell  Volume 4, Issue 1, Pages 75-83 (July 1999) DOI: 10.1016/S1097-2765(00)80189-6

Figure 1 Role of Negative Regulation in the Swi/Snf Dependence of the Histone HTA1–HTB1 Locus (A) Strains W303–1A (SNF) and JR2–19A (snf5Δ) were transformed with plasmids carrying an HTA1–lacZ reporter gene with (+) or without (−) the HTA1–HTB1 negative site. (B and C) Strains W303–1A (SNF), W303Δ1 (hir1Δ), PKY534 (hir2Δ), JR2–19A (snf5Δ), JR4–20D (snf5Δhir1Δ), and DY19–25B (snf5Δhir2Δ) (B) and strains W303–1A (SNF), W303Δ1 (hir1Δ), PKY534 (hir2Δ), DY21–33A (snf2Δ), DY21–36B (snf2Δhir1Δ), and DY22–21A (snf2Δhir2Δ) (C) were transformed with a plasmid carrying an HTA1–lacZ reporter gene with the HTA1–HTB1 negative site. (D) Untransformed strains W303–1A (SNF), JR2–19A (snf5Δ), DY21–33A (snf2Δ), W303Δ1 (hir1Δ), W303Δ2 (hir2Δ), JR4–20D (snf5Δhir1Δ), and DY19–25B (snf5Δhir2). (A–C) Levels of lacZ and RP51A mRNAs as measured by an S1 nuclease protection assay. (D) Levels of HTB1 and ACT1 mRNAs as measured by Northern blot analysis. Molecular Cell 1999 4, 75-83DOI: (10.1016/S1097-2765(00)80189-6)

Figure 2 Hir Protein Recruitment Creates a Requirement for Snf5p Plasmids pBTM116 (LexA), pBTM-HIR1 (LexA–Hir1), pBTM-HIR2 (LexA–Hir2), and pBTM-HIR1(C-term) (LexA–Hir1p[C-term]) were cotransformed into strains W303–1A (SNF5) and JR2–19A (snf5Δ) with HTA1–lacZ reporter gene plasmids in which the HTA1–HTB1 negative site was absent (−OP) or in which lexA operator sequences (+OP) were substituted for the HTA1–HTB1 negative site. The levels of lacZ and RP51A mRNAs were measured by an S1 nuclease protection assay. Molecular Cell 1999 4, 75-83DOI: (10.1016/S1097-2765(00)80189-6)

Figure 3 Role of Promoter Strength versus Negative Regulation in the Snf5p Dependence of the HTA1–HTB1 Locus HTA1–HTB1 promoter elements were substituted for the two CYC1 UAS elements in a CYC1–lacZ reporter gene carried on plasmid pLGΔ178. After transformation into strains W303–1A (SNF5) and JR2–19A (snf5Δ), β-galactosidase assays were performed in duplicate on more than five independent transformants. The results represent mean Miller units with standard deviation. (A), pURS; (B), pURSΔneg; (C), pUAS2/3; (D), pUAS1 with 16 bp UAS consensus; and (E), pUAS2/3 + neg. HTA1–HTB1 UAS elements: +, open boxes; HTA1–HTB1 negative site: −, closed box. Molecular Cell 1999 4, 75-83DOI: (10.1016/S1097-2765(00)80189-6)

Figure 4 Coimmunoprecipitation of Swi/Snf Components with Hir1p and Hir2p Strains BJ5465 or W303–1A were transformed with plasmids carrying HIR1–HA or HIR2–HA genes. The HA epitope–tagged Hir proteins were precipitated (IP) from whole-cell lysates (CL) by incubation with monoclonal antibody against HA (αHA) or with a nonspecific rabbit anti-mouse antibody (NS). Western blot analysis was performed with αHA monoclonal antibody and with (A) αSnf5p, (B) αSnf2p, or (C) αSwi3p polyclonal antibodies to detect coimmunoprecipitation of native Swi/Snf proteins. (A and B) 1/200 or (C) 1/1000 of input cell lysates and αHA unbound fractions (αHA Sup) were also analyzed. Molecular Cell 1999 4, 75-83DOI: (10.1016/S1097-2765(00)80189-6)

Figure 5 Snf5p Is Associated with HTA1–HTB1 Promoter Sequences In Vivo Chromatin solutions were prepared from strains: (A), W303 (SNF5) and JR2–19A (snf5Δ); (B), W303 (SNF5), W303Δ1Δ2 (hir1Δhir2Δ), PKY581 (HTA1Δneg), DY21–33A (snf2Δ), and BLY14 (swi3Δ). Immunoprecipitations were performed with αSnf5p polyclonal antibody or mock treated (−Ab), DNA was purified from immunoprecipitated (IP) and input (Total) chromatin, and 5 μl of each DNA was amplified by PCR with primers specific for HTA1–HTB1 promoter sequences (HTA1) and ACT1 sequences. The PCR products were separated on a 10% acrylamide gel. (A) Lane 1, no DNA template; lanes 2 and 3, mock IP of snf5Δ and SNF5 chromatin (1:2 dilution); lanes 4 and 5, αSnf5 IP of snf5Δ and SNF5 chromatin (1:2 dilution); lanes 6–9, total chromatin solutions from snf5Δ (undiluted, 1:5 dilution) and SNF5 (1:2, 1:5 dilution) strains. (B) Lane 1, no DNA template; lanes 2–4, mock IP of fixed chromatin (undiluted) from snf2Δ, swi3Δ, and wild-type cells; lanes 5–7, αSnf5 IP of snf2Δ, swi3Δ, and wild-type chromatin (undiluted); lanes 8–13, total chromatin solutions (1:10, 1:20). (C) Lane 1, no DNA template; lanes 2–4, mock IP of fixed chromatin (undiluted) from hir1Δhir2Δ, HTA1Δneg, and SNF5 strains; lanes 5–7, αSnf5p IP of fixed chromatin (undiluted) from hir1Δhir2ΔHTA1Δneg, and SNF5 strains; lanes 8–10, total chromatin solutions (1:20). The HTA1Δneg PCR product is smaller by 54 bp from the HTA1 PCR product. (D) Western blot analysis of Snf5p levels in αSnf5p immunoprecipitates from wild-type and mutant strains. Lanes 1–4, IP from 3 mg of chromatin lysates; lanes 5–6, IP from 1.5 mg of chromatin lysates. Molecular Cell 1999 4, 75-83DOI: (10.1016/S1097-2765(00)80189-6)