Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Jun 2;23(11):2246-57.
doi: 10.1038/sj.emboj.7600227. Epub 2004 Apr 29.

Ssn6-Tup1 requires the ISW2 complex to position nucleosomes in Saccharomyces cerevisiae

Zhengjian Zhang  1 , Joseph C Reese
Affiliations

Ssn6-Tup1 requires the ISW2 complex to position nucleosomes in Saccharomyces cerevisiae

Zhengjian Zhang et al. EMBO J. .

Abstract

The Imitation SWItch (ISWI) chromatin remodeling factors have been implicated in nucleosome positioning. In vitro, they can mobilize nucleosomes bi-directionally, making it difficult to envision how they can establish precise translational positioning of nucleosomes in vivo. It has been proposed that they require other cellular factors to do so, but none has been identified thus far. Here, we demonstrate that both ISW2 and TUP1 are required to position nucleosomes across the entire coding sequence of the DNA damage-inducible gene RNR3. The chromatin structure downstream of the URS is indistinguishable in Deltaisw2 and Deltatup1 mutants, and the crosslinking of Tup1 and Isw2 to RNR3 is independent of each other, indicating that both complexes are required to maintain repressive chromatin structure. Furthermore, Tup1 repressed RNR3 and blocked preinitiation complex formation in the Deltaisw2 mutant, even though nucleosome positioning was completely disrupted over the promoter and ORF. Our study has revealed a novel collaboration between two nucleosome-positioning activities in vivo, and suggests that disruption of nucleosome positioning is insufficient to cause a high level of transcription.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Nucleosome positioning at the RNR3 locus. Nuclei isolated from wild type (WT) with or without MMS (0.03%, 2 h) treatment, Δtup1, and Δcrt1 strains were subjected to micrococcal nuclease (MNase) digestion and detected by indirect end labeling. On the top of each panel, M is a genomic DNA molecular marker digested with the appropriate combinations of restriction enzymes; ND is MNase-digested naked DNA; and 0 represents the undigested sample. Within each panel, the filled triangles represent the internucleosomal hypersensitive sites in the WT chromatin samples; the open triangle indicates the hypersensitive site over the DREs in the WT chromatin; and the open circles indicate the chromatin change in the upstream to the DREs associated with chromatin remodeling. (D) A schematic summary of the chromatin structure shown in (A), (B) and (C), with the position and orientation of RNR3 and the neighboring ORFs indicated by arrows.
Figure 2
Figure 2
ISW2 is required for nucleosome positioning over RNR3. (A) Chromatin structure around the RNR3 promoter was analyzed in wild type, Δisw1, Δisw2, Δchd1 and sth1-ΔC strains. (B–D) The chromatin structure was analyzed across RNR3 in Δisw2 cells, in parallel with wild-type (−/+ MMS) and Δtup1 strains. See Figure 1 legend for more details.
Figure 3
Figure 3
Histone H4 crosslinking to RNR3. Wild-type (WCS484) and Δisw2 (YJR792) strains containing an Myc-tagged version of histone H4 were used in the ChIP assay. (A) Schematic of the PCR probes amplified at the RNR3 locus. The center (in base pairs) of each PCR fragment is indicated in parentheses. (B) Summary of ChIP experiments using anti-Myc monoclonal ascites fluid. For each PCR fragment, the IP signal was normalized to the input signal and expressed as ‘percentage IP'. With the RNR3 translation start site set as +1, the PCR products are: A (−1000–792), B (−788–540), C, URS (−448–236), D, promoter (−179–+8), E (+56–320), F (+333–563), I (+2014–2292), J (+2304–2542) and L (+3055–3263).
Figure 4
Figure 4
Transcription and PIC formation in repressed Δisw2 cells. (A) Northern blot analysis of RNR3 expression in wild-type and Δisw2 strain. The small cellular RNA (scR1) was used as a loading control. (B) Chromatin IP analysis of TBP and RNA polymerase recruitment to the RNR3 promoter. Primers amplifying RNR3 promoter (−179 to +8) were used for the PCR analysis. The signals from the untreated wild-type strain were arbitrarily set as 1.
Figure 5
Figure 5
Tup1 is recruited to RNR3 independent of ISW2. (A) Schematic of the PCR probes amplified at the RNR3 locus. (B) Summary of results using anti-serum raised to full-length Tup1. The preimmune serum was used as an IP background control. A pair of primers directed to the coding region of POL1 was used as a control for the specificity of Tup1 localization. The location of PCR products over RNR3 is indicated in the legend of Figure 3, with the addition of regions G (+1465–1703), H (+1742–1968) and K (+2811–3045).
Figure 6
Figure 6
Isw2 crosslinking to the RNR3 locus is widespread and independent of Ssn6–Tup1. The crosslinking of Isw2 containing nine Myc epitopes at the C-terminus (ISW2-MYC9) was examined by the ChIP assay. IPs from an untagged strain were used as the negative control. (A) Isw2–Myc crosslinking was detected across the RNR3 locus. Primers for SUC2 (−298 to +19) and INO1 (−112 to +102) promoters were used as positive controls. The RNR3 primers are described in the legends of Figures 3 and 5. (B) Crosslinking of Isw2–Myc in Δssn6 and Δtup1 strains using the URS PCR primer pair C.
Figure 7
Figure 7
Collaboration of Ssn6–Tup1 and ISW2 at the ENA1 locus. (A) MNase mapping of the ENA1 promoter detected by indirect end labeling. Filled triangles represent the regularly spaced hypersensitive sites in the wild-type chromatin, which are interpreted as internucleosomal sites. The open circles extending beyond nucleosome 6 (left of panel) indicate the possibility that nucleosome positioning continues, but could not be resolved by this gel. (B) ChIP assay for Tup1 and Isw2-Myc crosslinking to the ENA1 URS. Tup1 and Myc antibody were used, with the preimmune or IPs from an untagged strain used as negative controls, respectively. The PCR fragment corresponding to −632 to −316 relative to the translation start site flanks the Sko1 and Mig1/2 binding sites.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alen C, Kent NA, Jones HS, O'Sullivan J, Aranda A, Proudfoot NJ (2002) A role for chromatin remodeling in transcriptional termination by RNA polymerase II. Mol Cell 10: 1441–1452 - PubMed
    1. Basrai MA, Velculescu VE, Kinzler KW, Hieter P (1999) NORF5/HUG1 is a component of the MEC1-mediated checkpoint response to DNA damage and replication arrest in Saccharomyces cerevisiae. Mol Cell Biol 19: 7041–7049 - PMC - PubMed
    1. Becker PB, Horz W (2002) ATP-dependent nucleosome remodeling. Annu Rev Biochem 71: 247–273 - PubMed
    1. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14: 115–132 - PubMed
    1. Clapier CR, Langst G, Corona DF, Becker PB, Nightingale KP (2001) Critical role for the histone H4 N terminus in nucleosome remodeling by ISWI. Mol Cell Biol 21: 875–883 - PMC - PubMed

Publication types

MeSH terms

-