Conservation of histone binding and transcriptional repressor functions in a Schizosaccharomyces pombe Tup1p homolog

Abstract

The Ssn6p-Tup1p corepressor complex is important to the regulation of several diverse genes in Saccharomyces cerevisiae and serves as a model for corepressor functions. To investigate the evolutionary conservation of these functions, sequences homologous to the S. cerevisiae TUP1 gene were cloned from Kluyveromyces lactis (TUP1) and Schizosaccharomyces pombe (tup11(+)). Interestingly, while the K. lactis TUP1 gene complemented an S. cerevisiae tup1 null mutation, the S. pombe tup11(+) gene did not, even when expressed under the control of the S. cerevisiae TUP1 promoter. However, an S. pombe Tup11p-LexA fusion protein repressed transcription of a corresponding reporter gene, indicating that this Tup1p homolog has intrinsic repressor activity. Moreover, a chimeric protein containing the amino-terminal Ssn6p-binding domain of S. cerevisiae Tup1p and 544 amino acids from the C-terminal region of S. pombe Tup11p complemented the S. cerevisiae tup1 mutation. The failure of native S. pombe Tup11p to complement loss of Tup1p functions in S. cerevisiae corresponds to an inability to bind to S. cerevisiae Ssn6p in vitro. Disruption of tup11(+) in combination with a disruption of tup12(+), another TUP1 homolog gene in S. pombe, causes a defect in glucose repression of fbp1(+), suggesting that S. pombe Tup1p homologs function as repressors in S. pombe. Furthermore, Tup11p binds specifically to histones H3 and H4 in vitro, indicating that both the repression and histone binding functions of Tup1p-related proteins are conserved across species.

FIG. 1

Comparison of primary structure of Tup1p homologs in yeast. (A) Alignment of Tup1p homologs. Amino acids identical among three or four yeast species are in gray or black, respectively. The predicted Ssn6p-binding region and seven WD repeats are indicated. Triangles represent the positions where introns were inserted in S. pombe tup11⁺. ScTup1, S. cerevisiae Tup1p; KlTup1, K. lactis Tup1p; CaTup1, C. albicans Tup1p (2); SpTup11, S. pombe Tup11p. (B) Comparison of functional domains within Tup1p homologs. Values indicate the percent identity with S. cerevisiae Tup1p. Dotted and closed boxes represent the Ssn6p-binding and WD repeat domains, respectively. The bars indicate the functional domains of S. cerevisiae Tup1p.

FIG. 2

Regulation of Tup1p-repressed genes in S. cerevisiae tup1 mutants expressing Tup1p homologs. Total RNA samples were prepared from cells of tup1-disrupted strain YMH427 having the plasmid carrying S. cerevisiae TUP1 (lane 1), K. lactis TUP1 (lane 2), or S. pombe tup11⁺ (lane 3) or the vector plasmid YCp50 alone (lane 4). Each RNA sample (2 μg per lane) was separated on an agarose gel in the presence of formaldehyde, blotted onto a nylon membrane, and hybridized with ³²P-labeled a-specific STE2, the glucose-repressed SUC2, or the oxygen-repressed ANB1 probe. The same membranes were rehybridized with ³²P-labeled ACT1 as an internal marker. The ANB1 DNA fragments were also hybridized with the Tr transcript which is not regulated by TUP1.

FIG. 3

Identification of functional domains in S. pombe Tup11p by creation of chimeric proteins. The tup1 strain YMH427 was transformed with plasmids harboring the indicated S. cerevisiae-S. pombe Tup1p hybrids. The resulting transformants were assayed for mating ability, flocculation, and STE6-PHO5 expression (APase activity [in milliunits], the average of three measurements with a margin of error of <20%). The open boxes indicate regions derived from S. cerevisiae Tup1p, and the closed boxes indicate regions derived from S. pombe Tup11p. The amino acid positions of the junctions are indicated. Flo, flocculation; −, nonflocculent; +, flocculent; Non, nonmating ability.

FIG. 4

Interaction of S. pombe Tup11p with S. cerevisiae α2p and Ssn6p in vitro. (A) Binding of S. pombe Tup11p to α2p. In vitro ³⁵S-labeled S. pombe Tup11p was incubated with beads bound to GST-α2p (lane 1) or to GST alone (lane 2). After the beads were washed, proteins bound to the beads were analyzed by SDS-PAGE. Shown are autoradiograms detecting the labeled proteins. Input (lane 3) represents 10% of the labeled Tup11p used in the binding reaction. (B) Binding of S. pombe Tup11p with S. cerevisiae Ssn6p. In vitro ³⁵S-labeled S. cerevisiae Ssn6p was incubated with beads bound to GST-S. cerevisiae Tup1p (7-253) (lane 1), GST-S. pombe Tup11p (1-298) (lane 2), or GST alone (lane 3). Input (lane 4) represents 10% of the labeled Ssn6p used in the binding reaction.

FIG. 5

Interaction of S. pombe Tup11p with histones. (A) GST pull-down analysis. Semipurified histones were incubated with beads bound to GST-S. cerevisiae Tup1p (amino acids 7 to 253) (lanes 2 and 3), GST-S. pombe Tup11p (amino acids 1 to 298) (lanes 4 and 5), or GST alone (lanes 6 and 7). After washing, bound (lanes 2, 4, and 6) and unbound (lanes 3, 5, and 7) fractions were analyzed by SDS-PAGE and visualized by staining with Coomassie brilliant blue R-250. Input (lane 1) represents the total amount of histones used in each binding reaction. (B) Far-Western blot analysis. Samples of yeast histone proteins were separated by SDS-PAGE, electroblotted onto a nylon membrane, and probed with an ³⁵S-labeled S. cerevisiae Tup1p (ScTup1) or S. pombe Tup11p (SpTup11) probe. A parallel lane was stained with Coomassie brilliant blue R-250. (Coomassie).

FIG. 6

Transcription of the glucose-repressed fbp1⁺ gene in tup11 and tup12 mutants. Total RNA samples were prepared from cells of JY741 (tup11⁺ tup12⁺) (lanes 1 and 2), JY741-Δtup11U (tup11::ura4⁺) (lanes 3 and 4), JY741-Δtup12L (tup12::LEU2) (lanes 5 and 6), JY741-Δtup11U, or Δtup12L (tup11::ura4⁺ tup12::LEU2) (lanes 7 and 8) grown in repressing (R; 8% glucose; lanes 1, 3, 5, and 7) or derepressing (D; 0.1% glucose and 3% glycerol; lanes 2, 4, 6, and 8) conditions. Each RNA sample (10 μg per lane) was separated on an agarose gel in the presence of formaldehyde, blotted onto a nylon membrane, and hybridized with a ³²P-labeled PCR product harboring the coding region of fbp1⁺. The same membrane was rehybridized with a ³²P-labeled leu1⁺ PCR product as an internal control. Normalized levels of fbp1⁺ RNA relative to leu1⁺ (values averaged from three independent experiments) are shown below the lane numbers. The level of fbp1⁺ in the wild-type strain (WT) under derepressing conditions was set to 1.0 for comparison purposes. Standard deviations were within 10% for all samples except lanes 6 and 8, for which standard deviations were 21 and 27%, respectively.