Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae - pnas.201612422SI

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Conserved forkhead dimerization motif controls DNA replication timing and

spatial organization of chromosomes in S. cerevisiae

Ostrow, A.Z.; Kalhor, R.; Gan, Y.; Villwock, S.K.; Linke, C.; Barberis, M.; Chen, L.; Aparicio,

O.M.

DOI

10.1073/pnas.1612422114

Publication date

2017

Document Version

Other version

Published in

Proceedings of the National Academy of Sciences of the United States of America

Link to publication

Citation for published version (APA):

Ostrow, A. Z., Kalhor, R., Gan, Y., Villwock, S. K., Linke, C., Barberis, M., Chen, L., &

Aparicio, O. M. (2017). Conserved forkhead dimerization motif controls DNA replication timing

and spatial organization of chromosomes in S. cerevisiae. Proceedings of the National

Academy of Sciences of the United States of America, 114(12), E2411-E2419.

https://doi.org/10.1073/pnas.1612422114

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Supporting Information

Ostrow et al. 10.1073/pnas.1612422114

Fig. S1. Fkh1 and Fkh2 share key residues and predicted structure with human FoxP family proteins. (A) Multiple sequence alignment of additional human and fission yeast (sp) forkhead domains. Amino acid numbers are based on Fkh1, and specific residues discussed in the text are highlighted. (B) Amino acid identities and similarities in Forkhead proteins across species. (C) Modeled forkhead domain structures of Fkh1 (orange) and Fkh2 (pink) overlaid on the solved structure of FoxP3 domain-swapped dimer (red).

1 M

A

B

+ + − + + +

+ + + − + −

His-Fkh1-FD

IP Myc

Sol WCE

− −

Fkh1-WT/dsm-Myc

His-Fkh1-FD

to Fkh1

C

1.0

0.8

0.6

0.4

0.2

0

1 2 3 4 5 6

D

E

1.0

0.8

0.6

0.4

0.2

0

Fig. S2. Fkh1 dimerizes in vitro. (A) Coomassie-stained SDS/PAGE of E. coli-expressed and purified His-Fkh1-FD (lane 1). (B) Pull-down assay with Fkh1-Myc or Fkh1-dsm-Myc isolated from yeast whole-cell extracts (strains ZOy46 and ZOy50, respectively) and immobilized on beads, incubated with E. coli-expressed and purified His-Fkh1-FD. Bound material was analyzed by immunoblotting with a polyclonal anti-Fkh1 antibody. (C) Quantification of results of three experiments as in B showing the average (±SD) ratio of His-Fkh1-FD pulled down by Fkh1-dsm-Myc versus Fkh1-Myc. (D) Bacterially produced GST, GST-Fkh1, and GST-Fkh1-dsm were immobilized on Glutathione Sepharose beads and incubated with yeast lysate containing Fkh1-Myc (lanes 1–4) or Fkh1-GST-Fkh1-dsm-Myc (lanes 5–8). Pull-downs (Upper) and Input (Lower) were analyzed by immunoblot analysis using anti-Myc antibody. (E) Quantification of data in D showing the ratio of Fkh1-dsm-Myc pulled down by GST-Fkh1-dsm to Fkh1-Myc pulled-down by GST-Fkh1.

antibody to ORC IP Myc 1 2 antibody to Myc antibody to Fkh1 Orc1 Orc2 Orc4/5 Orc6

Fig. S3. Fkh1 interaction with ORC is unaffected by dsm mutation. Immunoprecipitation of Fkh1-Myc and Fkh-dsm-Myc from cell extracts of strains ZOy46 and ZOy50, respectively, was analyzed by immunoblotting with anti-ORC polyclonal antibody.

Fig. S4. ARS305 4C interactions along chromosome XII. Strains ZOy20 (fkh2Δ) and ZOy10 (fkh1-dsm fkh2Δ) were synchronized in G1phase and subjected to 4C

analysis with ARS305 as bait; only the FKH1 genotype is indicated on the images. Spheres below the chromosomal plots denote replication origins, with red indicating Fkh-activated and green indicating Fkh-repressed.