Sum1 and Ndt80 proteins compete for binding to middle sporulation element sequences that control meiotic gene expression

Abstract

A key transition in meiosis is the exit from prophase and entry into the nuclear divisions, which in the yeast Saccharomyces cerevisiae depends upon induction of the middle sporulation genes. Ndt80 is the primary transcriptional activator of the middle sporulation genes and binds to a DNA sequence element termed the middle sporulation element (MSE). Sum1 is a transcriptional repressor that binds to MSEs and represses middle sporulation genes during mitosis and early sporulation. We demonstrate that Sum1 and Ndt80 have overlapping yet distinct sequence requirements for binding to and acting at variant MSEs. Whole-genome expression analysis identified a subset of middle sporulation genes that was derepressed in a sum1 mutant. A comparison of the MSEs in the Sum1-repressible promoters and MSEs from other middle sporulation genes revealed that there are distinct classes of MSEs. We show that Sum1 and Ndt80 compete for binding to MSEs and that small changes in the sequence of an MSE can yield large differences in which protein is bound. Our results provide a mechanism for differentially regulating the expression of middle sporulation genes through the competition between the Sum1 repressor and the Ndt80 activator.

FIG. 1.

Mutations in SMK1 MSE alter Sum1 and Ndt80 binding affinity. EMSA was performed with wild-type and mutant SMK1 MSEs using fivefold serial dilutions of purified His6-tagged Ndt80_1-409 (A) and Sum1_523-1062 (B). Results from four representative mutants are shown. The SMK1 wild-type site is in lanes 1 to 3; C8A mutant, lanes 4 to 6; A12G, lanes 7 to 9; A12T, lanes 10 to 12; and T5G, lanes 13 to 15. The percent binding of each mutant site was determined in comparison to the wild type and is shown in Table 1.

FIG. 2.

Ndt80 competes with Sum1 for binding to the SMK1 MSE in vitro. (A) EMSAs of Ndt80 binding are shown in the absence of Sum1 (top) or in the presence (bottom) of a constant amount of purified Sum1_523-1062. Ndt80_1-409 protein was titrated into the reactions in twofold increments ranging from 0.011 to 0.35 μM final concentrations per reaction (lanes 2 to 7, 9 to 14, and 16 to 21). No Ndt80 was added to lanes 1, 8, and 15 in either panel. The reactions in the bottom panel contain a constant amount of Sum1 at a 0.12 μM final concentration per reaction. Lanes 1 to 7 show competition for binding to the wild-type SMK1 MSE, lanes 8 to 14 show the SMK1 MSE A2C mutant site, and lanes 15 to 21 show the SMK1 MSE A13T mutant site. (B) The percent of probe bound by Ndt80 in each of the competition experiments was quantitated using a phosphorimager. Data are presented for the three highest amounts of Ndt80 in each titration (lanes 5 to 7, 12 to 14, and 19 to 21). Open symbols indicate levels of Ndt80binding in the absence of Sum1 (data from EMSA in panel A, top). Solid symbols indicate binding in the presence of Sum1 (data from EMSA in panel A, bottom). Squares are data for binding to the wild-type SMK1 MSE; circles, A2C mutant site; and diamonds, A13T site.

FIG. 3.

Sum1 and Ndt80 compete for binding to the SMK1 MSE in vivo. (A) Sum1-dependent repression of lacZ reporter expression in the presence and absence of Ndt80. Reporter plasmids containing wild-type (lanes 1 and 3) or A12T mutant (lanes 2 and 4) SMK1 MSE were cotransformed with a SUM1-myc plasmid into sum1Δ strains. One host strain had an integrated copy of a GAL-NDT80 expression construct (MPY4, lanes 3 and 4), and the other did not (JXY3, lanes 1 and 2). Cells were grown in galactose, and the expression levels of the reporter were measured using β-galactosidase liquid assays. The repression of each reporter construct was calculated by comparison with a reporter lacking an MSE in the same strain background. (B) Western blot of Sum1-myc from lysates containing wild-type (lanes 1 to 3) and A12T mutant (lanes 4 to 6) MSE reporters. Lysates in lanes 2, 3, 5, and 6 were from strains that contained the SUM1-myc-tagged gene expressed from the SUM1 promoter. Lanes 1 and 4 are lysates from strains containing an empty vector, pRS415. Lanes 2 and 5 were lysates from strain JXY3, lacking the GAL-NDT80 construct, and lanes 3 and 6 are lysates from strain MPY4, which contains the GAL-NDT80 construct.

FIG. 4.

Models for Sum1 and Ndt80 regulation of the MSE. (A) Sum1 and Ndt80 bind to overlapping sequences within the MSE. The sequences are based on the mutational analysis and alignment of sites from the microarray analysis. Sequence letters include N (A, G, C, or T), Y (C or T), W (A or T), and D (A, G, or T). (B) Regulatory activity of different MSEs during the transition from mitotic growth to the middle stages of meiosis. MSEs that function as only a Sum1 repressor site (open boxes), only an Ndt80 activator site (shaded boxes), or as a site of both Sum1 repression and Ndt80 activation (hatched boxes) are shown. Around the time of meiotic prophase, Sum1 is degraded and Ndt80 is expressed. During this transition, Sum1 and Ndt80 may compete for binding to a subset of the MSEs.