Inheritance of CENP-A Nucleosomes during DNA Replication Requires HJURP

Zasadzińska, Ewelina; Huang, J.S.T.; Bailey, Aaron O.; Guo, Lucie Y.; Lee, Nancy S.; Srivastava, Shashank; Wong, Kelvin; French, Bradley T.; Black, Ben E.; Foltz, Daniel R.

doi:10.1016/j.devcel.2018.09.003

Cited by 91 publications

(97 citation statements)

References 89 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…RNAi knockdown of cul-1 resulted in a modest but statistically significant increase in the 394 abundance of AID::GFP in the AC compared to control(RNAi) treatment (+19%, n = 31 395 and 33, respectively, P < 0.0001) (Figure 4C). This result suggests that there is TIR-dependent, auxin-independent depletion of AID::GFP, similar to reports in other systems 397 (Morawska and Ulrich 2013;Nishimura and Fukagawa 2017;Zasadzińska et al 2018). 398…”

supporting

confidence: 87%

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

Martinez

Kinney

Medwig-Kinney

et al. 2019

Preprint

View full text Add to dashboard Cite

As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system, allows for spatial and temporal control of protein degradation, functioning through the activity of a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID method, utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 minutes of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 interacts with C. elegans SKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the Ftz-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work provides a conceptual improvement to the AID system for dissecting gene function at the single-cell level during C. elegans development.

show abstract

supporting

confidence: 87%

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

Martinez

Kinney

Medwig-Kinney

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…During DNA replication the MCM2-7 replicative helicase along with other histone chaperones like HJURP, are instrumental for the stable transmission of parental CENP-A during S-phase (Huang et al 2015;Petryk et al 2018;Zasadzińska et al 2018).…”

Section: Discussionmentioning

confidence: 99%

“…Recent work identified the MCM2-7 replicative helicase to recycle previously deposited H3/H4, H3.3/H4 and CENP-A/H4 tetramers together with other chaperones (Alabert & Groth 2012;Burgess & Zhang 2013;Foltman et al 2013;Gérard et al 2006;Huang et al 2015;Petryk et al 2018). This ensures the transfer of parental nucleosomes to freshly replicated DNA and in human cells also includes the CENP-A specific chaperon HJURP previously only assigned to CENP-A loading (Zasadzińska et al 2018).…”

Section: Introductionmentioning

confidence: 99%

Spt6 is a maintenance factor for centromeric CENP-A

Bobkov

Huang

Berg

et al. 2019

Preprint

View full text Add to dashboard Cite

Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This constitutes both an opportunity 2 to remodel the underlying chromatin as well as the potential danger of losing epigenetic information. Centromeric transcription has been shown to be required for stable incorporation of the centromere-specific histone dCENP-A in M/G1-phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes.Here we demonstrate that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and shows enhanced association with non-phosphorylatable dCENP-A mutants compared to histone H3, while phosphomimetic residues alleviate association with Spt6. We conclude that Spt6 acts as a conserved CENP-A maintenance factor, which is required during transcription-mediated chromatin remodelling at the centromere to ensure long-term stability of epigenetic centromere identity.

show abstract

“…This auxin-inducible degron system was engineered to function outside plant cells (1). Although the AID system has been widely adopted to degrade tagged proteins of interest, recent studies report auxin-independent depletion of endogenously tagged proteins (12)(13)(14). For instance, tagging the centromeric histone chaperone, HJURP, results in depletion of more than 90% of protein in the absence of auxin in human cell lines (14).…”

Section: Introductionmentioning

confidence: 99%

“…Although the AID system has been widely adopted to degrade tagged proteins of interest, recent studies report auxin-independent depletion of endogenously tagged proteins (12)(13)(14). For instance, tagging the centromeric histone chaperone, HJURP, results in depletion of more than 90% of protein in the absence of auxin in human cell lines (14). Chronic depletion has also been reported in chicken cells (13) and in yeast cells (12), suggesting that auxinindependent depletion may be a systemic problem when tagging endogenous genes.…”

Section: Introductionmentioning

confidence: 99%

An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion

Sathyan

McKenna

Anderson

et al. 2019

Preprint

View full text Add to dashboard Cite

Rapid perturbation of protein function permits the ability to define primary molecular responses while avoiding downstream cumulative effects of protein dysregulation. The auxininducible degron (AID) system was developed as a tool to achieve rapid and inducible protein degradation in non-plant systems. However, tagging proteins at their endogenous loci results in chronic, auxin-independent degradation by the proteasome. To correct this deficiency, we expressed the Auxin Response Transcription Factor (ARF) in an improved inducible degron system. ARF is absent from previously engineered AID systems, but ARF is a critical component of native auxin signaling. In plants, ARF directly interacts with AID in the absence of auxin and we found that expression of the ARF Phox and Bem1 (PB1) domain suppresses constitutive degradation of AID-tagged proteins. Moreover, the rate of auxin-induced AID degradation is substantially faster in the ARF-AID system. To test the ARF-AID system in a quantitative and sensitive manner, we measured genome-wide changes in nascent transcription after rapidly depleting the ZNF143 transcription factor. Transciptional profiling indicates that ZNF143 activates transcription in cis and ZNF143 regulates promoterproximal paused RNA Polymerase density. Rapidly inducible degradation systems that preserve the target protein's native expression levels and patterns will revolutionize the study of biological systems by enabling specific and temporally defined protein dysregulation. Auxin Inducible Degron | Auxin Response Factor | ZNF143 | Transcription Factors | RNA Polymerase Pausing

show abstract

Inheritance of CENP-A Nucleosomes during DNA Replication Requires HJURP

Cited by 91 publications

References 89 publications

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

Spt6 is a maintenance factor for centromeric CENP-A

An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion

Contact Info

Product

Resources

About