Spt4p co-localizes with kinetochores and associates with chromosomal loci. (A) Epitope-tagged SPT4 is functional. Growth of five-fold dilutions of wild-type (YPH499) and spt4-138 (YMB54) strains was compared to SPT4-GFP (YMB1859) and HA-SPT4 (GHY262) strains on YPD plates incubated at 30°C and 37°C for 3–4 days. The spt4-138 strain exhibits a slower growth compared to the wild-type strain even at the permissive temperature of 30°C. (B) Subset of Spt4-GFP foci co-localizes with kinetochore. Chromosome spreads from strain (YMB1859) expressing Ndc10p-HA, and Spt4-GFP probed with anti-GFP or anti-HA antibodies. The arrows in the merged panel indicate co-localization of Ndc10p-HA (red), and Spt4-GFP (green) is shown in yellow. (C) HA-Spt4p associates with chromosomal loci. ChIP experiments were carried out using wild-type strain (GHY262) expressing chromosomally tagged HASPT4 grown to logarithmic phase at 30°C. Different dilutions of chromatin samples from total (T) (undiluted, 1/100, 1/500), immunoprecipitated with anti-HA (HA), and mock (M) were analyzed using primers for core CEN1, core CEN3, core CEN16, HMRa, TELVIR, and ACT1 or intergenic regions (IR₁ and IR₂) devoid of ORFs (chromosome IV, co-ordinates 1 157 000–1 157 200 and 1 523 000–1 523 180, respectively). Values for the quantitation of the data are shown above each of the lanes, with the value for undiluted total set to 100 for each row (lane 1). These data show that the PCR yield is proportional to the amount of starting material.

Ndc10p is required for the association of HA-Spt4p to the core CEN loci. ChIP experiments were carried out using wild-type strain (GHY262) or ndc10-1 strain expressing HASPT4 (YMB1659) grown at 30 or 25°C and shifted to 37°C for 4 h. (A, B) Chromatin samples from T, HA, and M were analyzed using primers for core CEN16, TELVIR, or ACT1. (C) Chromatin samples from T (row 1), HA (rows 2 and 3) and M (row 3) were analyzed using primers for core CEN16 (lane 4) and those that flank on either side (lanes 1–3 and 5–6).

Centromeric chromatin structure is altered in the spt4-138 strain. (A) Representation of three DraI sites within CDEII of the CEN3, the resulting DraI–EcoRI or EcoRI–EcoRI fragments, and the probe used for Southern blot analysis. (B) CEN3 chromatin structure of wild-type and spt4-138 mutant. Nuclei prepared from logarithmic growing cultures of wild-type (PKY090) and spt4-138 (YMB2192) strains grown at 37°C for 6 h were incubated with 0, 5, 15, 50, and 150 U/ml of DraI at 37°C for 30 min. Southern blot analysis of the EcoRI digested DNA was performed and probed with a ³²P-dCTP-labeled 0.9 kb HindIII–BamHI fragment as shown in (A). The fraction of CEN3 accessible to DraI (% cut) was determined by quantification of the cut band (2.9 kb) divided by the sum of the uncut (5.1 kb) and cut bands (2.9 kb) using a Fuji Phosphorimager. The results were reproducible in multiple experiments, with values not differing by more than 0.01%.

Spt4p is required for restricting the association of Cse4p-HA with kinetochores. (A, B) Cse4p-HA, but not Mtw1-GFP, shows an altered localization pattern in the spt4-138 strain. Chromosome spreads from wild-type (YPH98) and spt4-138 (YMB54) strains expressing CSE4-HA (YMB2142 and YMB2140, respectively) or MTW1 (YMB2231 and YMB2230, respectively) grown at 30°C were probed with anti-HA and anti-GFP antibodies. (C) Graphic representation of the percentage of wild-type and spt4-138 cells showing Cse4p-HA and Mtw1-GFP foci corresponding to data in (A) and (B), respectively. At least 100 nuclei were counted in two independent experiments. Similar results were obtained with the spt4Δ strain (data not shown). (D) Chromosomal association of Cse4p-HA is altered in the spt4-138 strain. ChIP experiments were carried out using wild-type (YMB2142) and spt4-138 (YMB2140) strains expressing Cse4p-HA grown at 30°C. Chromatin samples from T, HA, and M were analyzed using primers to core CEN16 and flanking DNA on either side at −3.0 or +5.0 kb, HMRa, TELVIR, or ACT1. (E) Expression of Cse4p-HA is not affected in the spt4-138 strain. Western blot analysis was performed using the following strains: wild-type (lane 1), wild-type, and spt4-138 strains expressing CSE4-HA (lanes 2 and 3). Blots were probed with anti-HA or anti-PGK (loading control) antibodies.

HA-Spt4p fails to associate with CEN DNA in a cac1Δ hir1Δ strain. ChIP experiments were carried out using wild-type (PKY090), cac1Δ (PKY638), hir1Δ (PKY117), or cac1Δ hir1Δ (PKY632) strains expressing HASPT4 (pMB237) grown at 30°C. Chromatin samples from T, HA, and M were analyzed using primers for core CEN3, HMRa, TELVIR, or ACT1.

RNAPII and Sir3p are not required for the association of HA-Spt4p to core CEN and spt4 strains display a defect in silencing of reporter genes at heterochromatic loci. (A) HA-Spt4p associates with CEN DNA, but not with the actively transcribed genes ACT1 and TUP1 in a temperature-sensitive rpb1-1 strain. ChIP experiments were carried out using wild-type (GHY501) and rpb1-1 (GHY560) strains expressing HASPT4 (pMB237) grown at 30°C and shifted to 37°C for 1 h. Different dilutions of chromatin samples from total (T) (undiluted, 1/100, 1/500), immunoprecipitated with anti-HA (HA), and mock (M) were analyzed using primers for core CEN1, ACT1, or TUP1. Values for the quantitation of the data are shown above each of the lanes, with the value for undiluted total set to 100 for each row (lane 1). These data show that the PCR yield is proportional to the amount of starting material. (B) HA-Spt4p associates with core CEN DNA, but not with HMRa and TEL in an sir3Δ strain. ChIP experiments were carried out using wild-type (BUY668) and sir3Δ (BUY671) strains expressing HASPT4 (pMB237) grown at 30°C. Chromatin samples from T, HA, and M were analyzed using primers for core CEN1, HMRa, TELVIR, or ACT1. These results suggest that Spt4p may not be required for the transcription of HMRa. (C) Spt4p is required for silencing at HMRa and TEL. Silencing phenotypes were assayed by expression of URA3 integrated adjacent to either HMRa in wild-type (BUY545) and spt4Δ (YMB1871) or TELVIIL (TEL) in wild-type (BUY668) and spt4Δ (YMB1849) strains. Five-fold serial dilutions of cells were plated on YPD, and SC+5-FOA, and incubated for 3–4 days at 30°C. As a control, we determined that spt4Δ strains auxotrophic for URA3 (ura3) do not exhibit growth defects on media containing 5-FOA.

Human SPT4 complements the silencing phenotypes of an Scspt4 mutant and associates with S. cerevisiae core CEN DNA in an Ndc10p-dependent manner. (A) and (B) Silencing phenotypes were assayed by determining the expression of URA3 integrated adjacent to telomere VIIL (TEL) and HMRa in wild-type (BUY671 and BUY545, respectively) and spt4Δ strains (YMB1849 and YMB1871, respectively) expressing human SPT4 (HsSPT4) (pMB299), ScSPT4 (ScSPT4) (pMB237), or vector (vector). Serial dilutions of cells were plated on YPD, SC-LEU-URA, and SC-LEU+5-FOA and incubated for 3–4 days at 30°C. (C) ChIP experiments were carried out using ndc10-1 expressing pHsSPT4/LEU2 (YMB1827) strain grown at 25°C and shifted to 37°C for 4 h. Chromatin samples from T, HA, and M were analyzed using primers for core CEN16, HMRa, TELVIR, or ACT1.