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. 2012 Apr 3;31(10):2391–2402. doi: 10.1038/emboj.2012.82

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Copyright © 2012, European Molecular Biology Organization

This is an open-access article distributed under the terms of the Creative Commons Attribution Noncommercial Share Alike 3.0 Unported License, which allows readers to alter, transform, or build upon the article and then distribute the resulting work under the same or similar license to this one. The work must be attributed back to the original author and commercial use is not permitted without specific permission.

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HAT tethering on tetO-HAC induced expansion of newly synthesized CENP-A assembly through HJURP. (A) A HAC cell line (HeLa-HAC-R5) was transfected with a set of tetR-EYFP-fusion expressing vectors. (B) Timetable for the experiment. HA-tagged CENP-A expression vector (pCDNA5-HA-CENP-A) was co-transfected with tetR-EYFP-fusion expressing vector. (C) Representative images of newly synthesized CENP-A assembly. Cells were stained with DAPI, anti-GFP (recognize EYFP; green) and anti-HA (red). Arrowheads indicate tetO-HAC position. Scale bar, 5 μm. (D) Schematic timetable for gene depletion and new CENP-A assembly assay. HeLa-HAC-R5 cells were firstly transfected with siRNA. After 24 h incubation, HA-CENP-A and a set of tetR-EYFP-fusion expression vectors were co-transfected. Cells were stained with DAPI, anti-GFP and anti-HA. (E) hMis18α or HJURP depletion using siRNA. siRNAs for hMis18α (sihMis18α) and for HJURP (siHJURP) as well as for a negative control (siControl: siNegative, ambion) were used for transfection. Total RNA was purified two days after transfection and quantified by real-time PCR. hMis18α or HJURP mRNA levels were normalized by HPRT transcripts. Horizontal axis indicates relative hMis18α or HJURP mRNA level against a negative control (siControl). Error bar, s.d. (n=3). (F) HA-CENP-A assembly frequency on endogenous centromere was counted in each sample (n?100). Error bar, s.d. (n=3). (G) A frequency of expanded HA-CENP-A assembly induced by HAT tethering (example is shown in panel C bottom) was counted in each sample (n?100). Error bar, s.d. (n=3). Column colors indicate subpopulations of cells, which had CENP-A assembly at endogenous centromere (red) and had no assembly (orange). *P-values of t-test are 0.001 (red column) and 0.017 (orange column). **P-values of t-test are 0.006 (red column) and 0.033 (orange column).