Article Figures & Data
Figures
- Figure 1
Tf2 mobilization assay. (A) Schematic of the Tf2-12natAI assay. The endogenous Tf2-12 element was marked with a nat antibiotic resistance cassette interrupted with an artificial intron (AI). Mobilization of this element results in the generation of a functional cassette and the acquisition of resistance to Nat. (B) Nat resistance (NatR) arises as a result of Tf2-12natAI mobilization. Genomic DNA, isolated from a WT (untagged) strain, starting Tf2-natAI strains, and from NatR colonies was analyzed by Southern blotting with a probe specific to the natAI cassette. (C) Mobilization is impaired by inactivation of homologous recombination. The frequency of Tf2-12natAI mobilization was determined for the WT and rad51∆ strains by fluctuation analysis using the method of the median. Values were scaled relative to the WT. Error bars represent ± SEM. * P < 0.05 (t-test).
- Figure 2
Constitutive activation of the SREBP homolog Sre1 results in high levels of Tf2 mobilization. (A) Cells were patched onto YE5S plates and incubated at 30° for 2 days either under normal oxygen conditions or in an anaerobic jar. Cells were then resuspended in H2O and ∼1 × 108 cells spread onto YE5S plates supplemented with Nat. Plates were incubated at 30° under normal oxygen conditions until colonies appeared. (B) The indicated strains were grown to midlog growth phase at 30° in YE5S. Cells were harvested and processed for β-galactosidase assays. Results are the mean of at least three independent assays and are scaled relative to the WT value. Error bars indicate ± SEM. (C) The mobilization frequency of Tf2-12natAI was determined by fluctuation analysis as described in Materials and Methods. Values were scaled relative to the WT. Error bars indicate ± SEM. *** P < 0.001 (t-test).
- Figure 3
Loss of Set1- and Abp1-mediated silencing induces Tf2 mobilization. (A) The indicated strains were grown to midlog growth phase at 30° in YES. Cells were harvested and processed for β-galactosidase assays. Results are the mean of at least three independent assays and are scaled relative to the WT value. Error bars indicate ± SEM. (B) The mobilization frequency of Tf2-12natAI in the indicated strain backgrounds was determined by fluctuation analysis as described in Materials and Methods. Values were scaled relative to the WT. Error bars indicate ± SEM. (C) As for A. (D) Tf2 mRNA levels in the indicated strains was determined by RT-qPCR and normalized to act1+ mRNA. Values are the mean of at least three biological repeats and error bars indicate ± SEM. *** P < 0.001, ** P < 0.01, and * P < 0.05 (t-test).
- Figure 4
Loss of HIRA-mediated silencing does not result in uncontrolled Tf2 element mobilization. (A) Midlog phase cells of the indicated strains were subjected to quantitative β-galactosidase assays. Mean values were determined from at least three independent assays and are scaled relative to WT. Error bars indicate ± SEM. (B) Deletion of hip1+ results in only modest increase in Tf2 mobilization. The frequency of Tf2-12natAI mobilization was determined for the indicated strains by fluctuation analysis using the method of the median. Values were scaled relative to the WT. Error bars represent ± SEM. Data for sre1-N from Figure 2C are included for comparison. (C) Deletion of other HIRA complex genes does not stimulate Tf2 mobilization. Mobilization frequency was determined as described for B. (D) HIRA suppresses expression of the marked Tf2-12natAI element. RNA was prepared from the indicated strains and Tf2-12natAI RNA was determined by strand-specific RT-PCR. (E) Comparison of Tf2 mRNA levels in sre1-N and hip1∆ backgrounds. RNA was prepared from the indicated strains and Tf2 mRNA levels were assayed by RT-qPCR and normalized to act1+ mRNA. Values are the mean of at least three biological repeats and error bars indicate ± SEM. ** P < 0.01, * P < 0.05, and ns (not significant) P > 0.05 (t-test). (F) Comparison of Tf2-lacZ expression with Tf2-12natAI mobilization frequency relative to WT levels in the indicated genetic backgrounds. (G) Increased levels of Tf2 IN in hip1∆ cells. Tf2 IN in WT and two hip1∆ strains were detected by immunoblotting. α-Tubulin (loading control) was detected with anti-tubulin Ab (tat-1).
- Figure 5
Loss of HIRA does not disrupt Tf bodies. (A) FISH analysis was performed using a FISH probe corresponding to the ∼3.6-kb coding region of Tf2 elements. Representative FISH images from the indicated strains (top). Quantitative FISH analysis of observed Tf2 foci/cell in the indicated strains (bar graph; bottom). Number of cells analyzed per strain (n). (B) As for A. Declustering of Tf2s assessed by χ2-test was significant in sre1-N and set1Δ (P < 0.001) but not hip1Δ (P > 0.05).
- Figure 6
The transcriptional repression and clustering functions of Set1 suppress Tf2 mobilization. (A, top) Schematic of the domain structure of Set1 and (bottom) a summary of the properties of the set1 mutants (Mikheyeva et al. 2014). (B) Analysis of Tf2-12natAI mobilization frequency was determined in the indicated set1 mutant backgrounds by fluctuation analysis using the method of the median. Values were scaled relative to the WT. Error bars represent ± SEM. *** P < 0.001 and ns denotes P > 0.05 (t-test).
- Figure 7
Model for the control of mobilization via transcriptional silencing and clustering into Tf bodies. Silencing factors such as Set1, Abp1, HIRA, and HDACs inhibit Tf2 transcription and thereby limit cDNA accumulation. The clustering of dispersed Tf2 elements into Tf bodies limits mobilization by restricting cDNA integration by homologous recombination.
