Heat shock factor 1 suppression induces spindle abnormalities and sensitizes cells to antimitotic drugs

Background Heat shock factor 1 (HSF1) is the master regulator of the heat shock response and supports malignant cell transformation. Recent work has shown that HSF1 can access the promoters of heat shock proteins (HSPs) and allow HSP expression during mitosis. It also acts as a mitotic regulator, controlling chromosome segregation. In this study, we investigated whether the transactivation activity of HSF1 is required for the assembly of mitotic spindles. Results Our results showed that phosphorylation of HSF1 at serine 326 (S326) and its transactivation activity were increased during mitosis. Inhibition of the transactivation activity of HSF1 by KRIBB11 or CCT251263 during mitosis significantly increased the proportion of mitotic cells with abnormal spindles. It also hampered the reassembly of spindle microtubules after nocodazole treatment and washout by impeding the formation of chromosomal microtubule asters. Depletion of HSF1 led to defects in mitotic spindle assembly, subsequently attenuating cell proliferation and anchorage-independent cell growth (AIG). These HSF1 depletion-induced effects could be rescued by ectopically expressing wild-type HSF1 or a constitutively active mutant (∆202-316, caHSF1) but not the S326A or dominant negative (∆361-529, dnHSF1) mutants. In addition, overexpression of HSP70 partially reduced HSF1 depletion-induced spindle abnormalities. These results indicate that HSF1 may support cell proliferation and AIG by maintaining spindle integrity through its transactivation activity. Furthermore, inhibition of HSF1 transactivation activity by KRIBB11 or CCT251236 can enhance diverse anti-mitosis drug-induced spindle defects and cell death. Conclusions The increased transactivation activity of HSF1 during mitosis appears to be required for accurate assembly of mitotic spindles, thereby supporting cell viability and probably AIG. In addition, inhibition of the transactivation activity of HSF1 may enhance the mitotic errors and cell death induced by anti-mitosis drugs.

Overexpression of BBX18 Promotes Thermomorphogenesis Through the PRR5-PIF4 Pathway

Thermomorphogenesis is the morphological response of plants to an elevation in the ambient temperature, which is mediated by the bHLH transcription factor PIF4. The evening-expressed clock component, PRR5, directly represses the expression of PIF4 mRNA. Additionally, PRR5 interacts with PIF4 protein and represses its transactivation activity, which in turn suppresses the thermoresponsive growth in the evening. Here, we found that the B-box zinc finger protein, BBX18, interacts with PRR5 through the B-Box2 domain. Deletion of the B-Box2 domain abolished the functions of BBX18, including the stimulation of PIF4 mRNA expression and hypocotyl growth. Overexpression of BBX18, and not of B-Box2-deleted BBX18, restored the expression of thermoresponsive genes in the evening. We further show that BBX18 prevents PRR5 from inhibiting PIF4-mediated high temperature responses. Taken together, our results suggest that BBX18 regulates thermoresponsive growth through the PRR5-PIF4 pathway.

Direct photoresponsive inhibition of a p53-like transcription activation domain in PIF3 by Arabidopsis phytochrome B

Photoactivated phytochrome B (PHYB) binds to antagonistically acting PHYTOCHROME-INTERACTING transcription FACTORs (PIFs) to regulate hundreds of light responsive genes in Arabidopsis by promoting PIF degradation. However, whether PHYB directly controls the transactivation activity of PIFs remains ambiguous. Here we show that the prototypic PIF, PIF3, possesses a p53-like transcription activation domain (AD) consisting of a hydrophobic activator motif flanked by acidic residues. A PIF3mAD mutant, in which the activator motif is replaced with alanines, fails to activate PIF3 target genes in Arabidopsis, validating the functions of the PIF3 AD in vivo. Intriguingly, the N-terminal photosensory module of PHYB binds immediately adjacent to the PIF3 AD to repress PIF3’s transactivation activity, demonstrating a novel PHYB signaling mechanism through direct interference of the transactivation activity of PIF3. Our findings indicate that PHYB, likely also PHYA, controls the stability and activity of PIFs via structurally separable dual signaling mechanisms.

Role of Cassava CC-type Glutaredoxin MeGRXC3 in Regulating Sensitivity to Mannitol-Induced Osmotic Stress Dependent on its Nuclear Activity

BackgroundWe previously identified six drought-inducible CC-type glutaredoxins in cassava cultivars, however, less is known about their potential role in the molecular mechanism by which cassava adapted to abiotic stress. ResultsHerein, we investigate one of cassava drought-responsive CC-type glutaredoxins, namely MeGRXC3, that involved in regulation of mannitol-induced inhibition on seed germination and seedling growth in transgenic Arabidopsis. MeGRXC3 overexpression up-regulates several stress-related transcription factor genes, such as PDF1.2, ERF6, WRKY40, and WRKY53 in Arabidopsis. Protein interaction assays show that MeGRXC3 interacts with Arabidopsis TGA2 and TGA5 in the nucleus. Eliminated nuclear localization of MeGRXC3 failed to result mannitol-induced inhibition of seed germination and seedling growth in transgenic Arabidopsis. Mutation analysis of MeGRXC3 indicates the importance of conserved motifs for its transactivation activity in yeast. Additionally, these motifs are also indispensable for its functionality in regulating mannitol-induced inhibition of seed germination and enhancement of the stress-related transcription factors in transgenic Arabidopsis.ConclusionsMeGRXC3 overexpression confers mannitol sensitivity in transgenic Arabidopsis possibly through interaction with TGA2/5 in the nucleus, and nuclear activity of MeGRXC3 is required for its function.

Non-covalent Interaction With SUMO Enhances the Activity of Human Cytomegalovirus Protein IE1

Viruses interact with the host cellular pathways to optimize cellular conditions for replication. The Human Cytomegalovirus (HCMV) Immediate-Early protein 1 (IE1) is the first viral protein to express during infection. It is a multifunctional and conditionally essential protein for HCMV infection. SUMO signaling regulates several cellular pathways that are also targets of IE1. Consequently, IE1 exploits SUMO signaling to regulate these pathways. The covalent interaction of IE1 and SUMO (IE1-SUMOylation) is well studied. However, the non-covalent interactions between SUMO and IE1 are unknown. We report two SUMO-Interacting Motifs (SIMs) in IE1, one at the end of the core domain and another in the C-terminal domain. NMR titrations showed that IE1-SIMs bind to SUMO1 but not SUMO2. Two critical functions of IE1 are inhibition of SUMOylation of Promyelocytic leukemia protein (PML) and transactivation of viral promoters. Although the non-covalent interaction of IE1 and SUMO is not involved in the inhibition of PML SUMOylation, it contributes to the transactivation activity. The transactivation activity of IE1 was previously correlated to its ability to inhibit PML SUMOylation. Our results suggest that transactivation and inhibition of PML SUMOylation are independent activities of IE1.

Direct photoresponsive inhibition of a p53-like transcription activation domain in PIF3 by Arabidopsis phytochrome B

Phytochrome B (PHYB) triggers diverse light responses in Arabidopsis by binding to a group of antagonistically acting PHYTOCHROME-INTERACTING transcription FACTORs (PIFs) to promote PIF degradation, consequently downregulating PIF target genes. However, whether PHYB directly controls the transactivation activity of PIFs remains ambiguous. Here we show that the prototypic PIF, PIF3, possesses a p53-like transcription activation domain (TAD) consisting of a sequence-specific, hydrophobic activator motif surrounded by acidic residues. A PIF3mTAD mutant in which the activator motif is replaced with alanines fails to activate PIF3 target genes in Arabidopsis in dark, light, and shade conditions, validating the in vivo functions of the PIF3 TAD. Intriguingly, binding of the N-terminal photosensory module of PHYB to the PHYB-binding site adjacent to the TAD inhibits its transactivation activity. These results unveil a photoresponsive transcriptional switching mechanism in which photoactivated PHYB directly masks the transactivation activity of PIF3. Our study also suggests the unexpected conservation of sequence-specific TADs between the animal and plant kingdoms.

RCB initiates Arabidopsis thermomorphogenesis by stabilizing the thermoregulator PIF4 in the daytime

Daytime warm temperature elicits thermomorphogenesis in Arabidopsis by stabilizing the central thermoregulator PHYTOCHROME INTERACTING transcription FACTOR 4 (PIF4), whose degradation is otherwise promoted by the photoreceptor and thermosensor phytochrome B. PIF4 stabilization in the light requires a transcriptional activator, HEMERA (HMR), and is abrogated when HMR’s transactivation activity is impaired in hmr-22. Here, we report the identification of a hmr-22 suppressor mutant, rcb-101, which surprisingly carries an A275V mutation in REGULATOR OF CHLOROPLAST BIOGENESIS (RCB). rcb-101/hmr-22 restores thermoresponsive PIF4 accumulation and reverts the defects of hmr-22 in chloroplast biogenesis and photomorphogenesis. Strikingly, similar to hmr, the null rcb-10 mutant impedes PIF4 accumulation and thereby loses the warm-temperature response. rcb-101 rescues hmr-22 in an allele-specific manner. Consistently, RCB interacts directly with HMR. Together, these results unveil RCB as a novel temperature signaling component that functions collaboratively with HMR to initiate thermomorphogenesis by selectively stabilizing PIF4 in the daytime.

The oncogenicity of tumor-derived mutant p53 is enhanced by the recruitment of PLK3

Abstractp53 mutations with single amino acid changes in cancer often lead to dominant oncogenic changes. Here, we have developed a mouse model of gain-of-function (GOF) p53-driven lung cancer utilizing conditionally active LSL p53-R172H and LSL K-Ras-G12D knock-in alleles that can be activated by Cre in lung club cells. Mutation of the p53 transactivation domain (TAD) (p53-L25Q/W26S/R172H) eliminating significant transactivation activity resulted in loss of tumorigenicity, demonstrating that transactivation mediated by or dependent on TAD is required for oncogenicity by GOF p53. GOF p53 TAD mutations significantly reduce phosphorylation of nearby p53 serine 20 (S20), which is a target for PLK3 phosphorylation. Knocking out PLK3 attenuated S20 phosphorylation along with transactivation and oncogenicity by GOF p53, indicating that GOF p53 exploits PLK3 to trigger its transactivation capability and exert oncogenic functions. Our data show a mechanistic involvement of PLK3 in mutant p53 pathway of oncogenesis.

Kaposi’s sarcoma-associated herpesvirus processivity factor (PF-8) recruits cellular E3 ubiquitin ligase CHFR to promote PARP1 degradation and lytic replication

Kaposi’s sarcoma–associated herpesvirus (KSHV), which belongs to the gammaherpesvirus subfamily, is associated with the pathogenesis of various tumors. Nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) catalyzes the polymerization of ADP-ribose units on target proteins. In KSHV-infected cells, PARP1 inhibits replication and transcription activator (RTA), a molecular switch that initiates lytic replication, through direct interaction. Thus, for efficient replication, KSHV has to overcome the molecular barrier in the form of PARP1. Previously, we have demonstrated that KSHV downregulates the expression of PARP1 through PF-8, a viral processivity factor. PF-8 induces ubiquitin–proteasome system–mediated degradation of PARP1 via direct physical association and enhances RTA transactivation activity. Here, we showed that dimerization domains of PF-8 are crucial not only for PARP1 interaction and degradation but also for enhancement of the RTA transactivation activity. PF-8 recruited CHFR for the PARP1 degradation. A knockdown of CHFR attenuated the PF-8–induced PARP1 degradation and enhancement of the RTA transactivation activity, leading to reduced KSHV lytic replication. These findings reveal a mechanism by which KSHV PF-8 recruits a cellular E3 ligase to curtail the inhibitory effect of PARP1 on KSHV lytic replication.