TFIIIB subunit Bdp1p is required for periodic integration of the Ty1 retrotransposon and targeting of Isw2p to S. cerevisiae tDNAs

Abstract

Retrotransposons are RNA elements that reverse transcribe their RNA genomes and make a cDNA copy that is inserted back into a new genomic location by the element-encoded integrase protein. Ty1 is a long terminal repeat (LTR) retrotransposon in Saccharomyces cerevisiae that inserts into an approximately 700-bp integration window upstream of tRNA genes with a periodicity of approximately 80 bp. ATP-dependent chromatin remodeling by Isw2 upstream of tRNA genes leads to changes in chromatin structure and Ty1 integration site selection. We show that the N terminus of Bdp1p, a component of the RNA polymerase III transcription factor TFIIIB, is required for periodic integration of Ty1 into the integration window. Deletion of the Bdp1p N terminus and mutation of ISW2 result in similar disruption of nucleosome positioning upstream of some tRNA genes, and the N-terminal domain of Bdp1p is required for targeting of Isw2 complex to tRNA genes. This study provides the first example for recruitment of an ATP-dependent chromatin-remodeling factor by a general transcription factor in vivo.

Figure 1.

PCR assay and pattern of Ty1 integration in bdp1 N-terminal deletion mutants. (A) Schematic of the PCR assay used in this work. Galactose-induction of a plasmid-borne transposon pVIT41 containing a unique sequence marker (“SSB,” narrow black rectangle) in the LTR (triangle) results in integration into the yeast genome. PCR using the SSB primer and a primer complementary to the tDNA results in amplification of a population of insertion events upstream of genes within the same tDNA family. Use of a primer complementary to a unique region downstream of a specific tDNA amplifies insertions upstream of a single tDNA target. Insertions in both orientations can be amplified using either the SSB primer or its reverse complement. Because of the asymmetric position of the SSB in the LTR, PCR products generated with the orientation 1 (OR1) primer will be 278 bp larger than those using the reverse complement, orientation 2 (OR2). (B) Removal of the first 240 residues of Bdp1p results in altered patterns of Ty1 insertions upstream of tGly^GCC (16 copies in the yeast genome) and tThr^AGU genes (11 copies). Strains expressing either the wild-type Bdp1p (1-594) or the truncation alleles (241-594 and 241-521) were transformed with pVIT41, and four independent transformants were induced for transposition. Insertions upstream of tGly^GCC (top) and tThr^AGU (bottom) were analyzed by PCR using the orientation 1 primer.

Figure 2.

Pattern of integration in all bdp1 mutants. Residues between 139 and 241 of Bdp1p are responsible for the altered pattern of Ty1 insertion. Strains expressing each bdp1 allele were transformed with pVIT41, and two independent transformants were induced for transposition. Genomic DNA was prepared from the population of induced cells and insertions were analyzed by PCR with all four primer pairs. (Top two panels) tGly^GCC-specific primer with orientation 1 primer (panel 1) and orientation 2 primer (panel 2). (Bottom two panels) tThr^AGU-specific PCR with orientation 1 primer (panel 3) and orientation 2 primer (panel 4). PCR failed in lane 13 of panel 4. Residues present in the Bdp1p forms are noted at the top. (Lanes 7-10) A bracket denotes the lanes of PCR products from the N-terminal bdp1 deletion strains that show defects in periodic integration.

Figure 3.

High-resolution analysis of PCR products from Ty1 integration events in bdp1 mutants. (A) Two transformants per negative control (lanes 1-8) or Bdp1p form (lanes 9-24) were analyzed by PCR. The orientation 2 primer was end-labeled with ³²P, and insertions upstream of tGly^GCC genes were analyzed by PCR and run on a 4% polyacrylamide denaturing gel. For each transformant, 28 cycles of PCR (odd-numbered lanes) and 30 cycles of PCR (even-numbered lanes) are shown. Black brackets indicate regions where insertions are seen in the Bdp1p 241-594 and 241-521 mutants but not the full-length Bdp1p. (Glu) No induction on galactose; (pX3) Ty plasmid with no SSB marker in the LTR; (none) no plasmid; (pVIT41^★) pVIT41 donor plasmid with a Ty1 element expressing a defective integrase protein; (MW) molecular-weight markers for comparison to size of PCR products on gel. Number in parentheses indicates the relative position upstream of the tDNA transcription start site, which was determined by subtracting the length of the tDNA and the LTR amplified by the OR2 primer (123 bp) from the size of the molecular-weight marker. (B) Traces of the polyacrylamide gel. Radioactivity is plotted vs. distance upstream of the tDNA start site. The trace from full-length Bdp1p (dashed line, trace of lane 9) is compared with Bdp1p 1-509 (top panel, solid line, trace of lane 21), Bdp1p 241-594 (middle panel, solid line, trace of lane 19), and Bdp1p 241-521 (bottom panel, solid line, trace of lane 13). The amount of radioactivity represents the abundance of a PCR product of a given length, which can also be interpreted as the frequency of insertion into a certain position. (C) Loading control: PCR of actin gene using the genomic DNA used in the integration PCR.

Figure 4.

Ty1 integration pattern upstream of a single and multiple copies of tGly^GCC in the bdp1-Δ240 and isw2-K215R single mutants and the bdp1-Δ240isw2-K215R double mutant. (Top) The orientation 1 primer was combined with the tGly^GCC primer to amplify insertions upstream of the family of tGly^GCC genes. (Bottom) Insertions upstream of a single tGly^GCC, tG(GCC)G2, were amplified using the orientation 1 primer and a primer complementary to unique sequence downstream of the tRNA gene. The resulting products are therefore larger than those generated in the top panel. Two independent transformants of each strain are shown. Circles denote the four bands characteristic of periodic Ty1 integration, and triangles denote the major bands present in the mutant strains but not in the wild type.

Figure 5.

Nucleosome mapping by MNase digestion in the bdp1 mutants compared with isw2 mutants at two different tRNA genes, tG(GCC)G2 and tT(AGU)D. (N) Naked DNA digestion; (Isw2p-K215R) catalytically inactive mutant of Isw2p; (FL) full-length; (M) molecular weight markers. Circles to the right of a lane denote cut sites absent in the wild-type, full-length Bdp1p, and Bdp1p 1-509 but present in the other mutant strains. Triangles denote sites present in wild-type, full-length Bdp1p and Bdp1p 1-509 but absent in the other mutant strains.

Figure 6.

ChIP of Isw2p-K215R upstream of three tDNAs in the bdp1 mutant strains. ChIPs were performed in each strain using a 3× Flag-tagged Isw2p-K215R. For each sample, ChIP of the tDNA and PPA1 (loading control) is calculated as a percentage of the input.

Figure 7.

A model for Isw2 recruitment by the Bdp1p N terminus and its effect on Ty1 integration pattern. (A) The pol III preinitiation factor TFIIIC is constitutively bound to the internal promoter of tDNAs, and nucleosomes in unstable positions may be present in the surrounding area. (B) TFIIIB binding results in a distortion of the DNA by virtue of the DNA-bending activities of TBP and Bdp1p. Isw2 is recruited to the tDNA upstream region by the Bdp1p N terminus, and acts on the nucleosomes there, sliding them into a regular array. (C) Ty1 integration occurs into the nucleosomal DNA at regular intervals of ∼80 bp, based on the spacing of the nucleosomes. The position of integration on the nucleosome is not known. Insertion events are shown here occurring into DNA at the dyad axis.