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Control of chromatin remodeling activity and and higher order genome organisation by non-coding RNA

Principal Supervisor: Paul Badenhorst - Institute of Cancer and Genomic Sciences

Co-supervisor: So Yeon Kwon PhD

Project title: Control of chromatin remodeling activity and and higher order genome organisation by non-coding RNA

University of Registration: Birmingham

Project outline:

The developmental complexity of higher eukaryotes demands intricate tissue-specific and temporally-regulated control of gene expression. This is achieved through complex interplay between transcription control elements that can be scattered over many thousands of base pairs of the genomic landscape. Models for enhancer activity indicate that functional association and cooperation of dispersed regulatory elements is mediated by spatial organisation of the nucleus through the establishment of higher-order chromatin structures. A major challenge is to identify the mechanisms controlling genome organisation and how specificity between dispersed regulatory elements is regulated. Key to this is understanding the function of discrete genome elements called insulators that were first identified and characterized in Drosophila. Data from our laboratory demonstrate that chromatin dynamics, regulated by the conserved ATP-dependent chromatin remodeling enzyme NURF, is critical for insulator function and genome organisation1.

ATP-dependent chromatin remodeling enzymes are multi-subunit protein complexes that utilize the energy of ATP hydrolysis to alter nucleosome dynamics and are divided into broad families based on the core catalytic subunit and effects on nucleosomes - eviction, sliding or variant histone replacement2. To define nucleosome targets of the NURF remodeler we generated comprehensive genome-wide nucleosome maps for both normal and NURF deficient cells. This research showed that, NURF functionally interacts with and co-localizes with insulator proteins to establish nucleosome-depleted regions1.

This insulator function of NURF was most apparent at subclasses of insulators that mark the boundaries of chromatin domains, so-called topologically-associated domains (TADs). TADs have been identified in both vertebrates3,4 and Drosophila5 using sequencing-based chromosome conformation capture (HiC) techniques and reflect regions of the genome with a high propensity for local interactions but which interact infrequently with sequences in adjacent TADs. TADs present an elegant solution to control enhancer-promoter specificity as they limit enhancer interactions to promoters in the same domain. However, recent data suggest that rapid wholesale reorganisation of TADs can occur6. We speculate that control of remodeler activity and thus insulator function at TAD boundaries provides a means by which dramatic alteration of transcription programmes can be achieved. As such, understanding the mechanisms of recruitment and action of remodelers at insulators and their impact on higher order chromatin organisation are a high priority.

Data from our laboratory suggests that regulatory RNAs control remodeler activity at insulators. Interactions between NURF and insulator components are RNase-sensitive1 and largest subunit of NURF contains an eAT motif, a domain recently shown to bind RNA7, providing a mechanism for RNA interaction. A growing body evidence indicates non-coding RNAs exert important roles in insulator function, and potentially in the establishment of higher order nuclear structures8. Modulation of chromatin remodeling enzyme targeting and/or activity is an attractive mechanism through which these effects could be mediated.

A key priority is to identify the RNA components associated with NURF and insulator complexes to discriminate joint function in control of higher order genome organisation. In this project we will use targeted chromosome conformation capture sequencing to generate both high-resolution maps of NURF and insulator mediated higher-order genome interactions. We will use both RNA immunoprecipitation sequencing (RIP-seq) and formaldehyde RNA immunoprecipitation sequencing (fRIP-Seq) using NURF and insulator antibodies to identify associated RNAs. RNAs so characterized will be ablated by CRISPR genome editing, and function in higher order genome organisation and NURF-insulator dynamics determined.

References:

  1. Kwon, S.Y, et al. 2016. PLOS Genetics, 12, e1005969.
  2. Tessarz P, Kouzarides T. 2014. Nat. Rev. Mol. Cell Biol. 15, 703.
  3. Lieberman-Aiden, E. et al. 2009. Science. 326, 289
  4. Zhang, Y. 2012. Cell. 148, 908
  5. Sexton, T. et al. Cell. 148, 458
  6. Li L, et al. 2015. Mol Cell. 58, 216.
  7. Filarsky M et al. 2015. RNA Biol. 12, 864.
  8. Schubert T,et al. 2012 Mol Cell. 48, 434.

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

  • Chromosome conformation capture sequencing (ChiA-PET)
  • RNA immunoprecipitation sequencing (RIP-seq)
  • Formaldehyde RNA immunoprecipitation sequencing (fRIP-Seq)
  • Single molecule imaging of remodeler complexes in live cells
  • CRISPR-mediated gene deletion

Contact: Dr Paul Badenhorst, University of Birmingham