Cbf1p modulates chromatin structure, transcription and repair at the Saccharomyces cerevisiae MET16 locus

Abstract

The presence of damage in the transcribed strand (TS) of active genes and its position in relation to nucleosomes influence nucleotide excision repair (NER) efficiency. We examined chromatin structure, transcription and repair at the MET16 gene of wild-type and cbf1Delta Saccharomyces cerevisiae cells under repressing or derepressing conditions. Cbf1p is a sequence-specific DNA binding protein required for MET16 chromatin remodelling. Irrespective of the level of transcription, repair at the MspI restriction fragment of MET16 exhibits periodicity in line with nucleosome positions in both strands of the regulatory region and the non-transcribed strand of the coding region. However, repair in the coding region of the TS is always faster, but exhibits periodicity only when MET16 is repressed. In general, absence of Cbf1p decreased repair in the sequences examined, although the effects were more dramatic in the Cbf1p remodelled area, with repair being reduced to the lowest levels within the nucleosome cores of this region. Our results indicate that repair at the promoter and coding regions of this lowly transcribed gene are dependent on both chromatin structure and the level of transcription. The data are discussed in light of current models relating NER and chromatin structure.

Figure 1

(A) The MET16 gene structure. The three regulatory elements, CDE1, AP-1 and TATA-box, and relevant restriction enzymes sites are shown. Positions are indicated in relation to the first codon of the protein. (B) Representative results of MET16 transcription in CBF1 (lanes 1–5) and cbf1Δ (lanes 6–10) strains. Hybridization with ACT1 probe (top) and with MET16 (bottom) is shown. Lanes 3 and 6 correspond to samples grown in YPD medium. Lanes 1, 2 and 7, 8 correspond to samples grown for 1 and 2 h in repressing conditions, and lanes 4, 5 and 9, 10 to samples grown in derepressing conditions.

Figure 2

Nucleosome mapping at the MET16 locus of naked (N lanes) and chromatin DNA isolated from both strains and growth conditions. Indicated are specific bands/regions as mentioned in the text. (A) Low resolution MNase digestion pattern of the ∼9 kb EcoRI restriction fragment including MET16. Relevant locus components and restriction sites are shown on the left. The black line corresponds to the probe used for the Southern blot. A, B, C and D band(s) indicate the regions more sensitive to MNase within the MspI restriction fragment of MET16. (B) MNase digestion pattern at nucleotide resolution of the MspI restriction fragment of the MET16 TS. (C) MNase digestion pattern at nucleotide resolution of the MspI restriction fragment of the MET16 NTS. Some nucleotide positions are shown for reference. Proposed nucleosomes in the wild-type (white ovals) and mutant strain (black ovals) are shown on the right at their approximate nucleotide positions.

Figure 3

Representative autoradiographs at nucleotide resolution showing the repair of CPDs in the TS and NTS of the MspI fragment of MET16 in wild-type and cbf1Δ cells grown in 10 µM (derepressing) and 1 mM methionine conditions (repressing). Sanger A+G and T+C sequencing ladders are included to determine the position of the CPDs induced. U, untreated cells; 0, cells treated with UV light no repair; (1–4), UV-treated cells allowed to repair the damage for 1–4 h. The intense top band corresponds to the undamaged MspI fragment of MET16, the bands below represent DNA fragments with a CPD lesion that was cut by the CPD-specific endonuclease. Proposed nucleosomes in CBF1 (white ovals) and cbf1Δ cells (black ovals) are shown at their approximate nucleotide positions.

Figure 4

Repair of individual CPDs at MET16 versus the nucleotide position in (A) derepressing and (B) repressing conditions. The rate of repair in CBF1 (diamonds) and cbf1Δ cells (squares) is expressed as the T_50% for CPDs in the TS (top), and NTS (bottom). Position 1 corresponds to the start of the coding region. Cbf1p binding site (black box), Gcn4p binding site (striped box), TATA-box (white box) and coding region (grey box). Approximate nucleosome positions according to the high resolution mapping of the MET16 TS are shown for the CBF1 (white ovals) and cbf1Δ strain (black ovals).

Figure 5

Relative ratios of T_50% values for CPD repair between CBF1 and cbf1Δ cells in the MET16 TS (squares) and NTS (triangles) in (A) derepressing and (B) repressing conditions. Values above 1 indicate faster repair in the CBF1 strain and those below 1 faster repair in the cbf1Δ mutant. Position 1 corresponds to the start of the coding region. Cbf1p binding site (black box), Gcn4p binding site (striped box), TATA-box (white box) and coding region (grey box). Approximate nucleosome positions according to the high resolution mapping of the MET16 TS are shown for the CBF1 (white ovals) and cbf1Δ strain (black ovals).

Figure 6

Relative ratios of T_50% values for CPD repair between MET16 derepressing and repressing conditions in the TS (diamonds) and NTS (circles) of (A) CBF1 cells and (B) cbf1Δ cells. Values above 1 indicate faster repair when transcription is derepressed, and values below 1, faster repair with transcription repressed. Position 1 corresponds to the start of the coding region. Cbf1p binding site (black box), Gcn4p binding site (striped box), TATA-box (white box) and coding region (grey box). Approximate nucleosome positions according to the high resolution mapping of the MET16 TS are shown for the CBF1 (white ovals) and cbf1Δ strain (black ovals).