Principal Supervisor: Dr Paul BadenhorstLink opens in a new window

Co-supervisor: John Schwabe

PhD project title: Decoding nucleosomal asymmetry – characterisation of asymmetric histone post-translational modifications and structural implications for nucleosomal engagement of reader complexes

University of Registration: University of Birmingham

Project outline:

Aims

Post-translational modification of the amino-terminal tails of histone proteins (HPTMs) and the epigenetic regulators that interpret them are emerging disease targets. We have shown that HPTMs provide so-called “histone codes” that programme distinct cell-specific transcriptional outcomes by selectively recruiting or modifying the activity of reader complexes that include the ATP-dependent Nucleosome Remodeling Factor NURF [1,2,3] (Fig. 1). While it is widely assumed that HPTMs decorate both tails of each histone protein in nucleosomes, emerging data suggests some HPTMs can be asymmetrically deposited onto one of the two histone tails.

Here we examine how HPTM asymmetry can control activity of reader complexes on target nucleosomes. We will use a combination of high throughput genomics and structural biology to discriminate prevalence and function of asymmetric HPTMs. Reader complexes like the NURF remodeler are emerging oncotargets in cancers like melanoma. Understanding the structural basis of asymmetric tail recognition by NURF will leverage development of novel cancer therapeutics.

Background

ATP-dependent chromatin remodelling factors are multi-subunit molecular machines that alter nucleosome organisation to regulate availability of chromatin targets to the transcription, replication and repair machineries. NURF slides nucleosomes, regulating access of transcription factors and the transcription machinery to active genes [4]. We identified domains

on the large selectivity subunit of NURF (BPTF/NURF301) that bind to a combination of active chromatin marks on the H3 and H4 tails resulting in recruitment of NURF to nucleosomes in vivo [2,3,4]. However, NURF activity at these nucleosomes also requires interaction of its ISWI and NURF55 subunits with unmodified H3 and H4 tails. The requirement for both modified and unmodified tails on the same nucleosome implies that the normal substrates of remodelling enzymes like NURF are asymmetrically modified nucleosomes.

Hypothesis

HPTMs at key regulatory sites in the genome are asymmetrically deposited, with nucleosomes containing both an unmodified and modified histone H3 and H4 tail. Asymmetry provides a general mechanism to orient remodelers on one surface of target nucleosomes to achieve directional sliding in vivo.

Experimental Methods and Research Plan

We have already generated a range of engineered TN5 transposons, HALO-tagged NURF cell lines, methods for over-expression and purification of NURF reader domains and NURF lentiviral knockdown and rescue systems for melanoma cells. These will enable four types of experiment:

1. In vivo tagmentation using anti-HPTM antibody-coupled and HALO-ligand coupled TN5 transposons to map HPTM and remodeler asymmetry on target nucleosomes in vivo.
2. Structural characterisation of interaction between isolated reader domains and multiply-modified histone tails and cryo-EM analysis of intact NURF complexes
3. Mutation of NURF301/BPTF HPTM recognition followed by MNase-Seq nucleosome mapping to determine consequences on in vivo nucleosome positions.
4. Screening small-molecule inhibitors of histone tail-recognition and validation of effect on NURF nucleosome recognition by tagmentation ChIP, structural characterisation and RNAse-seq of melanoma cell lines.

This project is a collaboration between the Badenhorst and Schwabe groups. Badenhorst investigates how chromatin modifying complexes are targeted to nucleosomal substrates [1-4] Schwabe is a world leader in X-ray crystallographic and negative stain electron microscopy analysis of epigenetic regulators [5].

 

References:

1. Kwon, S.Y., … Badenhorst, P. (2009) Alternative splicing of NURF301 generates distinct NURF chromatin remodeling complexes with altered modified histone binding specificities. PLOS Genetics 5(7):e1000574.
2. Kwon, S.Y, … Badenhorst, P. (2016). Genome-wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators. PLOS Genetics, 12: e1005969.
3. Wysocka, J , …, Badenhorst, P.,Wu C. and Allis, C.D. (2006) A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodeling. Nature 442: 86-90.
4. Kwon, S.Y., …. Badenhorst, P. (2022). Combinatorial histone modifications target activity of the ATP-dependent chromatin remodeling enzyme NURF. Nature communications, in revision.
5. Turnbull, R., … Schwabe, J.W.R. (2020) The MiDAC histone deacetylase complex is essential for embryonic development and has a unique multivalent structure. Nature Communications 11, 3252.

 

BBSRC Strategic Research Priority: Understanding the rules of life – Structural Biology

 

Techniques that will be undertaken during the project:

1. Protein purification
2. Whole genome chromatin profiling
3. Targeted mutagenesis in Drosophila
4. Bioinformatic data analysis
5. Structural biology (Cryo-electron microscopy)

 

Contact: Dr Paul BadenhorstLink opens in a new window