Genome-wide Estrogen Receptor-α activation is sustained, not cyclical

  1. Andrew N Holding  Is a corresponding author
  2. Amy E Cullen
  3. Florian Markowetz
  1. University of Cambridge, United Kingdom

Abstract

Estrogen Receptor-alpha (ER) drives 75% of breast cancers. Stimulation of the ER by estra-2-diol forms a transcriptionally-active chromatin-bound complex. Previous studies reported that ER binding follows a cyclical pattern. However, most studies have been limited to individual ER target genes and without replicates. Thus, the robustness and generality of ER cycling are not well understood. We present a comprehensive genome-wide analysis of the ER after activation, based on 6 replicates at 10 time-points, using our method for precise quantification of binding, Parallel-Factor ChIP-seq. In contrast to previous studies, we identified a sustained increase in affinity, alongside a class of estra-2-diol independent binding sites. Our results are corroborated by quantitative re-analysis of multiple independent studies. Our new model reconciles the conflicting studies into the ER at the TFF1 promoter and provides a detailed understanding in the context of the ER's role as both the driver and therapeutic target of breast cancer.

Data availability

Sequencing data have been deposited in GEO under accession code GSE119057.

The following data sets were generated

Article and author information

Author details

  1. Andrew N Holding

    Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom
    For correspondence
    andrew.holding@cruk.cam.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8459-7048
  2. Amy E Cullen

    Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Florian Markowetz

    Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

Funding

Cancer Research UK (C14303/A17197)

  • Florian Markowetz

Breast Cancer Now (2012NovPR042)

  • Florian Markowetz

Cancer Research UK (C60571/A24631)

  • Andrew N Holding

Cancer Research UK (A19274)

  • Florian Markowetz

Alan Turing Institute (EPSRC grant EP/N510129/129/1)

  • Andrew N Holding

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

Copyright

© 2018, Holding 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

  • 3,249
    views
  • 376
    downloads
  • 15
    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. Andrew N Holding
  2. Amy E Cullen
  3. Florian Markowetz
(2018)
Genome-wide Estrogen Receptor-α activation is sustained, not cyclical
eLife 7:e40854.
https://doi.org/10.7554/eLife.40854

Share this article

https://doi.org/10.7554/eLife.40854

Further reading

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Hans Tobias Gustafsson, Lucas Ferguson ... Oliver J Rando
    Research Article

    Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA structure and nucleotide modifications can each interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We show that OTTR successfully captures both full-length tRNAs and tRNA fragments in budding yeast and in mouse reproductive tissues without any prior enzymatic treatment, and that tRNA cloning efficiency can be further enhanced via AlkB-mediated demethylation of modified nucleotides. As with other recent tRNA cloning protocols, we find that a subset of nucleotide modifications leave misincorporation signatures in OTTR datasets, enabling their detection without any additional protocol steps. Focusing on tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to "ground truth" Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRNA fragments - including both 5′ and 3′ tRNA halves derived from the majority of tRNAs – than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Omid Gholamalamdari, Tom van Schaik ... Andrew S Belmont
    Research Article

    Models of nuclear genome organization often propose a binary division into active versus inactive compartments yet typically overlook nuclear bodies. Here, we integrated analysis of sequencing and image-based data to compare genome organization in four human cell types relative to three different nuclear locales: the nuclear lamina, nuclear speckles, and nucleoli. Although gene expression correlates mostly with nuclear speckle proximity, DNA replication timing correlates with proximity to multiple nuclear locales. Speckle attachment regions emerge as DNA replication initiation zones whose replication timing and gene composition vary with their attachment frequency. Most facultative LADs retain a partially repressed state as iLADs, despite their positioning in the nuclear interior. Knock out of two lamina proteins, Lamin A and LBR, causes a shift of H3K9me3-enriched LADs from lamina to nucleolus, and a reciprocal relocation of H3K27me3-enriched partially repressed iLADs from nucleolus to lamina. Thus, these partially repressed iLADs appear to compete with LADs for nuclear lamina attachment with consequences for replication timing. The nuclear organization in adherent cells is polarized with nuclear bodies and genomic regions segregating both radially and relative to the equatorial plane. Together, our results underscore the importance of considering genome organization relative to nuclear locales for a more complete understanding of the spatial and functional organization of the human genome.