1. Chromosomes and Gene Expression

The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure

  1. Hsiang-Chen Chou
  2. Kuhulika Bhalla
  3. Osama El Demerdesh
  4. Olaf Klingbeil
  5. Kaarina Hanington
  6. Sergey Aganezov
  7. Peter Andrews
  8. Habeeb Alsudani
  9. Kenneth Chang
  10. Christopher R Vakoc
  11. Michael C Schatz
  12. W Richard McCombie
  13. Bruce Stillman  Is a corresponding author
  1. Cold Spring Harbor Laboratory, United States
  2. John Hopkins University, United States
  3. Johns Hopkins University, United States
https://doi.org/10.7554/eLife.61797
Share this article
Cite this article
  1. Hsiang-Chen Chou
  2. Kuhulika Bhalla
  3. Osama El Demerdesh
  4. Olaf Klingbeil
  5. Kaarina Hanington
  6. Sergey Aganezov
  7. Peter Andrews
  8. Habeeb Alsudani
  9. Kenneth Chang
  10. Christopher R Vakoc
  11. Michael C Schatz
  12. W Richard McCombie
  13. Bruce Stillman
(2021)
The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure
eLife 10:e61797.
https://doi.org/10.7554/eLife.61797
  2. Download BibTeX
  3. Download .RIS

Abstract

The Origin Recognition Complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here, using tiling-sgRNA CRISPR screens, we report that each subunit of ORC and CDC6 is essential in human cells. Using an auxin-inducible degradation system, we created stable cell lines capable of ablating ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defects in the absence of ORC2 were cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2 deficient cells were also large, with decompacted heterochromatin. Some ORC2 deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization.

Data availability

The data has been deposited to the Dryad database.

The following data sets were generated

Article and author information

Author details

  1. Hsiang-Chen Chou

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.

  2. Kuhulika Bhalla

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.

  3. Osama El Demerdesh

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.

  4. Olaf Klingbeil

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.

  5. Kaarina Hanington

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.

  6. Sergey Aganezov

    Department of Computer Science, John Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.

  7. Peter Andrews

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.

  8. Habeeb Alsudani

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.

  9. Kenneth Chang

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.

  10. Christopher R Vakoc

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1158-7180

  11. Michael C Schatz

    Department of Computer Science, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.

  12. W Richard McCombie

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    Competing interests
    No competing interests declared.

  13. Bruce Stillman

    Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
    For correspondence
    stillman@cshl.edu
    Competing interests
    Bruce Stillman, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9453-4091

Funding

National Cancer Institute (P01-CA13106)

  • Bruce Stillman

National Cancer Institute (P50-CA045508)

  • Bruce Stillman

National Science Foundation (DBI-1627442)

  • Michael C Schatz

National Cancer Institute (P01-CA13106)

  • Christopher R Vakoc

National Cancer Institute (P01-CA13106)

  • W Richard McCombie

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2021, Chou et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 3,096
    views
  • 416
    downloads
  • 29
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Hsiang-Chen Chou
  2. Kuhulika Bhalla
  3. Osama El Demerdesh
  4. Olaf Klingbeil
  5. Kaarina Hanington
  6. Sergey Aganezov
  7. Peter Andrews
  8. Habeeb Alsudani
  9. Kenneth Chang
  10. Christopher R Vakoc
  11. Michael C Schatz
  12. W Richard McCombie
  13. Bruce Stillman
(2021)
The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure
eLife 10:e61797.
https://doi.org/10.7554/eLife.61797

Share this article

https://doi.org/10.7554/eLife.61797

Categories and tags

Research organism

Further reading

    1. Chromosomes and Gene Expression

    Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy

    Carmina Lichauco, Eric J Foss ... Antonio Bedalov
    Research Article

    The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA (rDNA) arrays. We have previously reported that in the absence of SIR2, a de-repressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

    1. Chromosomes and Gene Expression

    Cryogenic Electron Microscopy: MagIC beads for scarce macromolecules

    Carlos Moreno-Yruela, Beat Fierz
    Insight

    Specialized magnetic beads that bind target proteins to a cryogenic electron microscopy grid make it possible to study the structure of protein complexes from dilute samples.

    1. Chromosomes and Gene Expression
    2. Structural Biology and Molecular Biophysics

    Surprising features of nuclear receptor interaction networks revealed by live-cell single-molecule imaging

    Liza Dahal, Thomas GW Graham ... Xavier Darzacq
    Research Article

    Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.