Mechanism of Long-Range Chromosome Motion Triggered by Gene Activation

Abstract

Movement of chromosome sites within interphase cells is critical for numerous pathways including RNA transcription and genome organization. Yet, a mechanism for reorganizing chromatin in response to these events had not been reported. Here, we delineate a molecular chaperone-dependent pathway for relocating activated gene loci in yeast. Our presented data support a model in which a two-authentication system mobilizes a gene promoter through a dynamic network of polymeric nuclear actin. Transcription factor-dependent nucleation of a myosin motor propels the gene locus through the actin matrix, and fidelity of the actin association was ensured by ARP-containing chromatin remodelers. Motor activity of nuclear myosin was dependent on the Hsp90 chaperone. Hsp90 further contributed by biasing the remodeler-actin interaction toward nucleosomes with the non-canonical histone H2A.Z, thereby focusing the pathway on select sites such as transcriptionally active genes. Together, the system provides a rapid and effective means to broadly yet selectively mobilize chromatin sites.

Keywords: Hsp90; chromatin motion; chromatin remodeler; genome organization; molecular chaperone; nuclear actin; nucleoskeleton.

Figure 1.. Long-range motion of the INO1 locus to the nuclear periphery is p23 and Hsp90 dependent.

(A) Representative images of the GFP-marked INO1 locus, which is visualized using an integrated array of lac binding sites near INO1 that are bound by GFP-LacI, in the presence of inositol (+) and absence (−). The outer and nuclear membranes are marked by ER05-mCherry (Randise-Hinchliff et al., 2016). (B) The reliance on p23 for inositol-dependent movement of INO1 was checked using p23 null (p23Δ) and parental yeast cells. (C) The impact of Hsp90 on INO1 motion was tested in the absence (DMSO), presence of an Hsp90 inhibitor (Radicicol), or after removal of the drug (Withdrawal). All INO1 motion data represent averages of 3 independent experiments with at least 50 counted cells in each trial and the error bars represent the SEM.

Figure 2.. Motion of INO1 to the nuclear periphery is dependent upon SWR-C and INO80-C but not the ISW1 remodeler.

(A) INO1 position in parental, swr1Δ, or isw1Δ yeast was checked in the presence (+) and absence (−) of inositol (n = 3). (B) INO80-C was translocated to the cytosol utilizing the anchor away system (Rapamycin) or left unperturbed (EtOH) and the nuclear locale of INO1 was checked, as marked (n = 3).

Figure 3.. Hsp90 and p23 modulate the DNA interactions of INO80-C and SWR-C in vivo and in vitro.

(A) ChIP was used to measure the inositol-dependent association of SWR-C (Swr1-TAP) or INO80-C (Ino80-TAP) at the INO1 promoter after 1 h of inositol withdrawal at 30°C in the presence (parent al) or absence of p23 (p23Δ). The data represent the fold enrichment of the indicated remodeler triggered by inositol withdrawal (n = 3). (B) INO80-C and SWR-C DNA binding activities were monitored by EMSA using purified complexes and radiolabeled INO1 promoter DNA, as indicated. The influence of p23 (1 μM) or Hsp90 (1 μM) on DNA binding was determined, as marked (n = 3). The nuclear concentration of Hsp90 is 1 μM and p23 is 4 μM (Echtenkamp et al., 2016).

Figure 4.. INO1 motion relies on remodeler ARP subunits and on actin polymerization.

(A) The position of INO1 in parental or cells deficient in an SWR-C ARP subunit (arp6Δ) or INO80-C ARP subunits (arp8Δ and ies6Δ as it results in loss of the Arp5-module) were monitored in the presence (+) and absence (−) of inositol (n = 3). (B) Parental yeast were treated with the carrier (DMSO) or Latrunculin-A (Lat-A) for 30 min then shifted to inositol free media. INO1 nuclear position was determined before (+) or after 30 min or 3 h of inositol starvation (−) (n = 3). (C) The INO1 GFP-marked yeast were transformed with an expression vector for an actin chromobody and examined using a DeltaVision OMX high-resolution microscope. The outer and nuclear membrane is visualized with ER05-mCherry and the bright green spot in the nucleus is the GFP-marked INO1 locus. (D) The nuclear actin signal was observed using an Airyscan confocal microscope. A still image captured from a high-speed Video (Video S7) is shown. (E) To assess the dynamic properties of the nuclear actin the images from 4 consecutive frames of an Airyscan video were averaged. A still image from Video S8 is shown. (F) The influence of expressing wild type actin fused to an NLS or the actin polymerization mutant R62D was checked in parental INO1-marked yeast in the presence (+) and absence (−) of inositol, as indicated (n = 3).

Figure 5.. Select actin binding proteins (ABPs) control INO1 movement including the Hsp90-dependent Myo3 myosin motor.

(A) The effect of the Arp2/3 inhibitor CK666 on INO1 motion was determined. Parental yeast were grown in the presence (+) or absence (−) of inositol along with carrier (Ethanol) or CK666 (50 μM) (n = 3). (B) Actin binding proteins (ABPs) influence the inositol-dependent transition of INO1 to the nuclear periphery. The genes encoding ABPs with established genetic interactions with either Hsp90 or p23 were knocked out and the nuclear location of INO1 was determined in the presence (+) and absence (−) of inositol (n = 3). The statistical significance of movement in each genetic background relative to WT was determined using non-paired t-tests: vrp1Δ (p=0.05), sla1Δ (p=0.87), myo3Δ (p=0.01), bnr1Δ (p=0.01), lsb1Δ (p=0.01), and tpm1Δ (p=0.003). (C) The inositol-dependent mobility of INO1 was checked in parental (WT) and she4Δ yeast (n = 3).

Figure 6.. DNA zip code-bound transcription factors nucleate type I myosins at the INO1 locus.

(A) ChIP was used to measure the inositol-dependent association of Myo1, Myo2, Myo3, and Myo5 myosin motor proteins, as marked, at either GRS I or GRS II after inositol withdrawal (n = 3). (B) The nucleation of Myo3-TAP at either GRS I or GRS II after inositol withdrawal was determined in either parental, cbf1Δ, or put3Δ yeast, as indicated (n = 3). All ChIP data represent the fold enrichment of the indicated myosin protein following inositol withdrawal. (C) The influence of purified Myo2 or Myo3 on Put3 bound to either a UAS or GRS I oligonucleotide was determined by EMSA (n = 3). The position of free probe is marked. (D) The sufficiency of either full-length (FL) Myo3 or truncation proteins having the indicated combination of the amino-terminal motor (1), central TH (2), or carboxyl-terminal SH3 (3) domains to support inositol-dependent INO1 motion was determined, as indicated (n = 3).

Figure 7.. The ARP-containing nucleosome remodelers interact directly with actin in a chaperone-dependent manner.

(A) Purified INO80-C, SWR-C, Myo2, and Myo3 bind directly to purified f-actin based on an actin spin-down assay. Dependence on actin to pellet each protein was checked using reactions without (−) or with (+) actin, as marked. The propensity of each protein to pellet with f-actin was quantified and the average ratio of the pellet to supernatant signal from 3 independent experiments is shown. The presence of Ino80-TAP, Swr1-TAP, Put3-TAP, Myo2-TAP, or Myo3-TAP in either the supernatant (S) or pellet (P) fractions was detected by immunoblot analysis using an anti-TAP antibody. (B) Hsp90 regulates the INO80-C/f-actin interaction in a nucleosome-dependent manner while INO80-C bound f-actin independent of nucleosome presence. The influence of Hsp90 (1 μM) and/or p23 (1 μM) on the INO80-C association with f-actin was determined in the absence or presence of canonical or H2A.Z-containing non-canonical nucleosomes, as marked. The presence of Ino80-TAP in either the supernatant (S) or pellet (P) fractions was detected by immunoblot analysis using an anti-TAP antibody (n = 3).