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Figure 1 | Epigenetics & Chromatin

Figure 1

From: Dot1 binding induces chromatin rearrangements by histone methylation-dependent and -independent mechanisms

Figure 1

Dot1 is a derepressor. (A) Targeting of LexA or LexA fusion proteins to LexA operators proximal to a promoterless HIS3 gene or a telomeric URA3 gene. A telomeric URA3 reporter is silenced by the silencing complex (SirCx) that spreads from the telomeric repeats (TEL). LexA-Dot1 was targeted to LexA operators between the telomeric repeats and URA3 in a barrier assay, and to LexA operators distal of the telomeric repeats and URA3 in a desilencing assay. (B) Cells were plated in 10-fold serial dilutions on selective media with or without histidine. Transcriptional activation of HIS3 leads to growth on media lacking histidine (strain L40). LexA alone is indicated with a dash. The transcriptional activator domain of Adr1 was used as a positive control. (C) Barrier and desilencing assays of Dot1 and Rpd3 targeted to telomere VIIL (strains NKI5128 and NKI5376). A strain without LexA operators (NKI5240) and LexA alone were used as controls. Cells were plated in 10-fold serial dilutions on selective media (yeast culture; YC) with or without 5FOA. Cells that silence URA3 can grow on 5FOA media whereas cells that express URA3 cannot. (D) URA3 silencing was not disrupted upon targeting of human Lamin C (pLexA-Lamin), a mutant form of human CyclinE (R130A; pLexA-MCycE) or yeast Ecm5 (pLexA-Ecm5; NKI5128). (E) Barrier and desilencing assay of Dot1 at the HML α mating-type locus with an inverted I-silencer (HMLi; YQY10, YQY09). A strain without LexA operators was used as a negative control (YXB85-n). (F) Chromatin immunoprecipitation (ChIP) using specific antibodies against Sir2 and Sir3 [24] was followed by quantitative PCR to determine binding to telomeric URA3 and HML α upon targeting of Dot1 or Dot1G401R (NKI5128). Average ChIP signals were normalized to input levels and Sir protein binding at URA3 relative to HML α was plotted (n = 2, +/- SE <). Similar results were obtained with an active (ACT1) reference gene (see Additional File 1). (G) Immunoblot analysis of Sir2 and Sir3 protein levels in a strain expressing LexA, LexA-Dot1 or LexA-Dot1G401R (NKI5128). Pgk1 was used as loading control.

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