UASrpg can function as a heterochromatin boundary element in yeast

Abstract

The HM loci in Saccharomyces cerevisiae constitute region-specific but gene-nonspecific repression domains, as a number of heterologous genes transcribed by RNA polymerase II or III are silenced when placed at these loci. The promoters of the Ashbya gossypii TEF gene and the S. cerevisiae TEF1 and TEF2 genes, however, are resistant to transcriptional silencing by the HM silencers in yeast. Moreover, when interposed between the HML alpha genes and the E silencer, certain segments of these promoters block the repression effect of the silencer on the alpha genes. All of these fragments contain UASrpg (upstream activation sequence of ribosome protein genes) composed of multiple binding sites for Rap1. In fact, a 149-bp segment consisting essentially of only three tandem Rap1-binding sites from the UASrpg of yeast TEF2 exhibits silencer-blocking activity. This element also exhibits insulating activity and orientation dependence characteristic of known chromatin boundary elements. Finally, the element blocks the physical spread of heterochromatin initiated at a silencer. This segment provides the first example of chromatin domain boundary or insulator elements in yeast.

Figure 1

The kanMX module is refractory to SIR-mediated transcriptional silencing. (A) Modified HM loci in strain YXB15. The kanMX module (P_AgTEF–kan^R–T_AgTEF) and ADE2 gene were inserted at HML and HMR, respectively. (Open bars) HML silencers. (Shaded bars) HMR silencers. (Arrows in open bars) HMLα genes. (Arrows in shaded bar) HMRa genes. (Solid arrows) FRT sites. See Materials and Methods for details. (B) Growth phenotypes of YXB15 (HMLα:: KanMX HMRa::ADE2 ade2-1 SIR⁺), YXB15s (HMLα::KanMX HMRa::ADE2 ade2-1 sir3), Y2047b (ΔE–HMLα–SUP4–o–ΔI-HMRa ade2-1 SIR⁺) and YXB1 (HMLα HMRa ade2-1 SIR⁺). Cells were grown to late log phase and serial dilutions (10-fold) were spotted on test plates and allowed to grow for 3 days. Geneticin (G418) was used at 250 μg/ml. (C) Northern blot analysis of kanMX expression. Cells of each strain were grown to late log phase before being harvested and their total RNA extracted as described (Kaiser et al. 1994). Ten micrograms of total RNA was loaded in each lane. The gel was stained with ethidium bromide to reveal the 25S and 18S rRNAs as loading controls. The kan^R mRNA was detected by Northern blotting and hybridization with a radioactive probe made from the ORF sequence of kan^R. Note that strain YXB13-I was the parental strain of YXB15 (Materials and Methods; Fig. 2).

Figure 2

The A. gossypii and yeast TEF promoters confer resistance to transcriptional silencing. (Left) Modified HML loci in the strains tested. YXB19 contains unmodified ADE2 gene (open block arrows) at HML. YXB13 has the kanMX module (solid block arrows) at HML. YXB21 and YXB22 have chimeric genes P_AgTEF–ADE2–T_AgTEF (solid regions of the block arrows represent AgTEF sequences and open regions represent the ADE2 coding region) and P_TEF2–ADE2 (shaded region of the block arrows represents the TEF2 promoter and open regions represent the ADE2-coding region) at HML, respectively. (Middle) Growth phenotypes examined as described in legend to Fig. 1. (Right) Mating efficiency as measured by quantitative mating (Materials and Methods). The mating efficiency of YXB1 was taken as one.

Figure 3

Insertion of a silencing-resistant gene at HML does not abolish the SIR-dependent silent chromatin structure. Cells of the indicated strains were grown in YPR medium to log phase before galactose was added and the cultures were incubated for 2.5 hr. DNA isolated from cells was fractionated by agarose gel electrophoresis in the presence of 30 μg/ml chloroquine. Under this condition, the more negatively supercoiled circles migrate more slowly in the gel. HML and HMR circles were revealed by Southern blotting. The Gaussian center of each distribution of topoisomers is indicated (●). (HML′ and HMR′) The modified HML and HMR loci in strain YXB15, respectively (Fig. 1A).

Figure 4

Silencing-resistant genes exhibit silencer-blocking activity. (Left) Modified HML loci in the strains tested. The HML I silencer was deleted from each strain. The P_TEF2–ADE2 module was inserted between the E silencer and the HMLα genes in YXB25. (Middle) Growth phenotypes examined as described in legend to Fig. 1. (Right) Mating efficiency as measured by quantitative mating. The mating efficiency of YXB26 was taken as one.

Figure 5

UAS_rpg exhibits silencer-blocking activity. (A) Various fragments from the yeast TEF2 promoter were inserted at the SpeI site between HML E silencer and the α genes at HMLΔI (left); their effect on α gene expression was analyzed by quantitative mating (right). The direction of an insertion is indicated by a half arrow. In the diagram of the TEF2 promoter the following are indicated: (solid bars) Rap1-binding sites; (open bar) TATA box; (♦) CT box (Gcr1-binding site); (shaded bar) T-rich region. (B) Similar analyses of A. gossypii TEF promoter and the yeast ADE2 gene. (○) TGACTC sequence. Other symbols are as in A.

Figure 6

UAS_rpg shows position-dependent silencer-blocking activity and insulator activity. (A) UAS_rpg shows position-dependent silencer-blocking activity. The 149-bp P_TEF2 fragment (open arrow; Fig. 5A) was inserted E-proximal (YXB48-I and YXB48-II) or E-distal (YXB58-I and YXB58-II) to the α genes at HMLΔI, respectively. The effect of the insertion on α gene expression was examined by quantitative mating. The mating efficiency of YXB26 was taken as one. (B) UAS_rpg shows insulator activity. In strains YXB57-A to YXB57-D, HML α genes were bracketed by a pair of the 149-bp element of TEF2 (open arrow), which were in turn flanked by the E and I silencers. The mating efficiency of YXB1 was taken as one. (C) UAS_rpg can insulate the URA3 gene from Sir-mediated repression. In strains YXB61-I and YXB61-II, the HMLα genes were replaced by the URA3 gene as detailed in Materials and Methods. A pair of the 149-bp fragment of TEF2 were inserted at sites bracketing URA3 in strains YXB61-I and YXB61-II, resulting in strains YXB62-I and YXB62-II. The growth phenotypes of the YXB61 and YXB62 strains and their sir⁻ derivatives on medium containing 5-FOA are shown at right.

Figure 7

UAS_rpg blocks the spread of SIR-dependent silent chromatin structure initiated at a silencer. (A) Diagram of the HML locus in strains in which separate segments of the locus can be simultaneously excised as circles for topological examination. (Thick arrows) FRT sites; (shaded box) the 149-bp fragment of P_TEF2 containing UAS_rpg. (B) Topological states of separate parts of HML. DNA circles from the HML locus of each strain diagramed in A were analyzed as described in Materials and Methods. The topoisomers of the two circles in each strain are significantly different in size (∼1 vs. ∼3 kb) so that they can be fractionated without overlap in the same track of the gel. The nicked (from II) circles are indicated. The Gaussian center of each distribution of topoisomers is indicated (●).

Figure 8

A model for the heterochromatin boundary activity of UAS_rpg. Transcriptionally silenced chromatin initiates from a silencer and emanates outward as a result of the spreading of the SIR complex along contiguous nucleosomes, leading to an extended complex of Sir proteins across hypoacetylated nucleosomes (left; see introductory section and Discussion for detailed descriptions). Binding of multiple Rap1 molecules to UAS_rpg excludes formation of several nucleosomes across the region and this nucleosomal ‘hole’ in the chromatin blocks the migration of the SIR complex (right). See Discussion for a detailed description. (B) Abf1; (R) Rap1; (O) ORC; (Sir’s) complex of Sir2, Sir3, and Sir4.