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
. 2003 Feb 15;31(4):1224-33.
doi: 10.1093/nar/gkg200.

Rap1p and other transcriptional regulators can function in defining distinct domains of gene expression

Qun Yu  1 , Runxiang Qiu, Travis B Foland, Dan Griesen, Carl S Galloway, Ya-Hui Chiu, Joseph Sandmeier, James R Broach, Xin Bi
Affiliations

Rap1p and other transcriptional regulators can function in defining distinct domains of gene expression

Qun Yu et al. Nucleic Acids Res. .

Abstract

Barrier elements that are able to block the propagation of transcriptional silencing in yeast are functionally similar to chromatin boundary/insulator elements in metazoans that delimit functional chromosomal domains. We show that the upstream activating sequences of many highly expressed ribosome protein genes and glycolytic genes exhibit barrier activity. Analyses of these barriers indicate that binding sites for transcriptional regulators Rap1p, Abf1p, Reb1p, Adr1p and Gcn4p may participate in barrier function. We also present evidence suggesting that Rap1p is directly involved in barrier activity, and its barrier function correlates with local changes in chromatin structure. We further demonstrate that tethering the transcriptional activation domain of Rap1p to DNA is sufficient to recapitulate barrier activity. Moreover, targeting the activation domain of Adr1p or Gcn4p also establishes a barrier to silencing. These results support the notion that transcriptional regulators could also participate in delimiting functional domains in the genome.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Identification of new barrier elements that can block the spread of transcriptional silencing. In strain 1, the HML-I silencer was inverted and the URA3 gene was inserted to its right. Each of the rest of the strains listed on the left was derived from strain 1 by inserting the UAS of a gene between HML-I and URA3 (see Materials and Methods). The coordinates indicate the distance (bp) from the translation start codon (ATG) of a gene. Binding sites for Rap1p, Abf1p and Rep1p were shown. Ten-fold serial dilutions of a late log-phase culture of each strain were spotted on synthetic complete (SC) medium and SC supplemented with 1 mg/ml FOA (FOA) and grown for 3 days.
Figure 2
Figure 2
Dissection of barrier elements. Strains 30–32 were derived from strain 1 (Fig. 1) by inserting fragments of RPS10A-UAS between HML-I and URA3. Strains 33–36 had fragments of TPI1-UAS inserted between HML-I and URA3. Strains 37 and 38 had fragments of RPS28A-UAS inserted between HML-I and URA3. Growth phenotypes of the strains on SC and SC+FOA media were shown.
Figure 3
Figure 3
Barrier activity of TEF2-UAS was abolished by mutations in its Rap1p-binding sites. (A) Mutations introduced into the Rap1p-binding sites of TEF2-UAS. Three DNA fragments containing the Rap1p-binding sites (R1, R2 and R3) of TEF2-UAS were used to make radioactive probes for EMSA. One or two C→A mutations were introduced into each sequence resulting in m1, m2 or m3. The mutations were highlighted. The 13 bp consensus sequence for Rap1p binding was shown on the top. (B) EMSA for Rap1p binding. EMSA of radio-labeled R1, R2, R3, m1, m2 or m3 was carried out with (+) or without (–) recombinant Rap1p, respectively. The position of DNA–Rap1p complex was indicated by an arrowhead. Note that a faster-migrating band in the EMSA assay was most likely the result of a degradation product of Rap1p existing in the protein extract. See Wahlin and Cohn (53) for a similar EMSA for Rap1p binding. (C) Effect of mutations in Rap1p sites on the barrier activity of TEF2-UAS. Strain 39 was deleted for the HML-I silencer. Strain 40 was derived from strain 39 by inserting TEF2-UAS between HML-E silencer and the HMLα genes. Strain 41 was identical to strain 40 except that the three Rap1p-binding sites between HML-E and HMLα were mutated as described in (A). The mating efficiency of each strain was shown.
Figure 4
Figure 4
Barrier elements alter local DNA topology. (A) Strain for analysis of topology of HMLΔI DNA. FRT sites were shown as arrowheads. A sequence to test (X) was inserted between HML-E and the HMLα. (B) The X-sequences in strains tested. Rap1p sites (arrows) and the TATA box (open square) were shown. (C) Mating efficiency of strains described in (B). (D) Topology of DNA circles excised from sir3– derivatives of strains described in (B). Cells were grown in YPR (yeast extract + bacto peptone + 2% raffinose) to early log phase. DNA was fractionated on agarose gels with 30 µg/ml chloroquine. Under this condition, more negatively supercoiled circles migrate slower. The center of each distribution of topoisomers is indicated by a circle. An open circle indicates that the position of a distribution center deviates from that predicted based on the size of the circle.
Figure 5
Figure 5
Targeting the activation domain of Rap1p, Adr1p or Gcn4p recapitulates barrier activity. (A) Strain 50 has two tandem LexA-binding sites (black bars) inserted between inverted HML-I and URA3. Plasmids L1 through L6 carry fusion genes encoding various parts of Rap1p linked to LexA. Vector is pRS425. All plasmids carry LEU2. Growth phenotypes of strain 50 bearing each plasmid on leucine minus (–Leu) and –Leu + FOA media were shown. (B) Strain 51 has a single LexA-binding site (black bar) inserted between HML-I and URA3. Plamids L7 and L8 carry genes encoding fusion proteins consisting of LexA linked to the activation domains (AD) of Adr1p (415–467) and Gcn4p (102–149), respectively. Growth phenotypes of strains 50 and 51 bearing each plasmid were shown. (C) Western blotting analyses of LexA-fusion proteins. LexA-fusion proteins from strain 50 bearing each of the L1 through L8 plasmids were detected by immunoblotting with an anti-LexA polyclonal antibody (Invitrogen). Equal amounts of cell extract were loaded. Note that lanes 6–10 were exposed for slightly longer time than lanes 1–5. The asterisk indicates a major peptide that cross-reacted with the α-LexA antibody. Note that some samples contain degradation products of the fusion proteins (e.g. lanes 3, 4 and 8). The positions of LexA (L1) and LexA-fusion proteins (L2–L8) are indicated by arrowheads.
Figure 6
Figure 6
TEF2-UAS blocks ADE2 silencing when it is placed 2 kb downstream of the ADE2 gene. Strains 52 and 53 were grown on YPD medium. In these strains, the ADE2 gene at the HML was the sole copy of it. Cells in which ADE2 was silenced would have a red color (strain 52).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Grunstein M. (1998) Yeast heterochromatin: regulation of its assembly and inheritance by histones. Cell, 93, 325–328. - PubMed
    1. Braunstein M., Rose,A., Holmes,S.G., Allis,C.D. and Broach,J.R. (1993) Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev., 7, 592–604. - PubMed
    1. Imai S., Armstrong,C.M., Kaeberlein,M. and Guarente,L. (2000) Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature, 403, 795–800. - PubMed
    1. Moazed D. (2001) Common themes in mechanisms of gene silencing. Mol. Cell, 8, 489–498. - PubMed
    1. Carmen A.A., Milne,L. and Grunstein,M. (2001) Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3. J. Biol. Chem., 277, 4778–4781. - PubMed

Publication types

MeSH terms