Epigenetic regulation of ribosomal gene
transcription during malignant transformation
Jeannine Diesch
Oncogenic Signalling and Growth Control Program
Peter MacCallum Cancer Centre
Melbourne, Australia
The process of ribosome biogenesis
Hein et al., Trends Mol Med 2013
CX-5461
Bywater et al., Cancer Cell 2012
Organisation of ribosomal genes
• 200 ribosomal DNA (rDNA) repeats are organized as clustered repeats in nucleolar organizer
regions (NORs) on acrocentric chromosomes
• rDNA transcription by is the most rate limiting step in ribosomal biogenesis and accounts for 80% of
the global transcription
Scheer et al, J Chrom 1997
Organisation of ribosomal genes
• rDNA exists in two distinct epigenetic states
– Active repeats: euchromatic,
enriched in H3K4me3, H3 and H4 actelylation
unmethylated
– Inactive repeats: heterochromatic
enriched in H3K9me3, H3K30me3, H3K27me3
methylated
• Majority of rDNA repeats are epigenetically repressed or silenced
The nucleolus
• Most prominent substructure in the nucleus
• Nucleoli form around actively transcribed rDNA repeats
• Functions besides ribosome biogenesis:
– Growth and cell cycle regulation
– Biogenesis of nonribosomal RNAs and ribonucleoproteins
– DNA replication and repair
– Stress signaling
– Aging
• Enlarged nucleoli due to increased rate of rDNA transcription are characteristic for cell
transformation and cancer
Drygin et al., Ann Rev Pharm&Tox,2010
Carcinoma Normal Tissue
Ribosomal RNA FISH
NIH3T3
Elaine Sanij
• Model of spontaneous Myc-driven B-cell lymphoma
• Myc oncogene under control of immunoglobulin heavy chain enhancer (Ig)
• Eμ-Myc mice have perturbed B cell development
Eμ-Myc transgenic mouse model
Megan Bywater
c-MYC Eμ disease
latency
B-cell lymphoma
Dysregulation of rRNA synthesis in Eμ-Myc mice
• Increased rRNA production in premalignant and malignant Eμ-Myc compared to wild type
littermates.
• Increased UBF occupancy of rDNA in malignant Eμ-Myc.
• Increased active rDNA in malignant Eμ-Myc without changes in rDNA methylation.
Megan Bywater
RNA-FISH
wt prem mal
0
1×107
2×107
3×107
4×107
Co
rrecte
d n
ucle
ola
r IT
S
Hypothesis: MYC-driven malignancies require
rDNA transcription to survive
Aims:
1. To investigate chromatin changes at the rDNA repeats during malignant
transformation
What induces activation of rDNA from premalignant to malignant Eμ-Myc?
Why are MYC-driven cancer cells more sensitive to Pol I inhibition?
2. To examine the role of the nucleolus in the three-dimensional organisation
and expression of the genome during malignancy
– perinucleolar / nucleolar-associated domains (NAD)
• consist of DNA, other than rDNA, that is associated with nucleolus
• significantly enriched in transcriptionally repressed genes
• E.g. inactive X chromosome
Do changes in rDNA structure have a genome-wide impact?
Aim 1: Chromatin changes at rDNA during malignancy
1. ChIP assay:
– histone marks for active and inactive repeats
– rDNA-binding proteins:
1. Capture rDNA using sureselect target enrichment
2. Next generation sequencing
• Comparison between wt/prem/mal Eμ-Myc
- H3K4me3 - H3K27me3
- H3K27ac - H4K20me3
- H3K9ac - H3K9me3
- UBF - Pol I - Tip60 - CSB
- Nucleolin - H1 - Tip5 - RUNX2
- CTCF - H2Az - Treacle - HDAC1
- SIRT1 - SIRT7 - PRMT5 - TTF1
Circularized chromosome conformation capture (4C)
• Approach to examine spatial organisation of the genome by detecting interactions between two
genomic loci (intra- and inter-chromosomal)
• Cross-linking of a known region (viewpoint) with interacting regions followed by restriction digest
and proximity based ligation
• Assay design in collaboration with Justin O’Sullivan (Liggins Institute, University of Auckland)
Digestion of rDNA with DpnII (cuts rDNA 93x)
15 viewpoints across rDNA:
– 6 in 18s rDNA
– 1 in 5.8s rDNA
– 6 in 28s rDNA
– 1 in IGS
– 1 in upstream terminator (T0)
• Comparison between wt/prem/mal Eμ-Myc
• 4C with Eμ-Myc wt and Eμ-Myc prem:
Pool PCR products
Illumina HiSeq: 100bp, PE
1 Ladder
2 3 4 9 5 6 7 8
wt
1 Ladder
2 3 4 9 5 6 7 8
prem
Chromatin interactions of rDNA repeats
Chromatin interactions of rDNA repeats
• Average of 3 x 106 mapped reads per viewpoint
• Enriched gene families:
– Zinc-finger (ZNF) genes
– Olfactory receptor (OR) genes
– RNA genes (SnoRNAs, spliceosomal RNAs, 5s RNA)
– Ribosomal protein pseudogenes
– LINE1 transposable elements
Eμ-Myc wt
18s IGS 28s
4C-seq reads identified in premalignant EμMyc
Data range: 0-1000
3
7
8
9
10
14
13
Chr
Refseq
genes
12
1 2 3 4 5 6 7 8 9 10 11 13 14 15 12 16 18 19 X 17
Integrative Genomics Viewer (IGV)
3
7
8 – 13
14
18s rDNA
5.8s rDNA
28s rDNA
IGS
Summary
• Ribosome biogenesis is dysregulated in Eμ-Myc lymphoma cells.
• Eμ-Myc cells display reactivation of silenced rDNA repeats from premalignancy to
malignancy and robust changes in chromatin structure.
• Eμ-Myc lymphoma cells are highly sensitive to Pol I inhibition in vivo
inhibition of rDNA transcription can be used to selectively target MYC-driven
malignancies.
• Next steps:
– 4C-seq analysis
– Epigenomic profiling of rDNA repeats
highlights the importance of rDNA transcription in malignant transformation
Acknowledgements
Growth control lab
Ross Hannan
Gretchen Poortinga
Elaine Sanij
Armadeep Dhillon
Amee George
Nadine Hein
Analia Lesmana
Don Cameron
Jaclyn Quin
Omer Gilan
Megan Bywater
Oncogenic signalling and
growth control program
Grant McArthur
Rick Pearson
Kate Hannan
PMCC FACS core
Viki Milovac
Sophie Kotsakidis
PMCC Molecular genomics
core
Gisela Mir
Tim Holloway
Tim Semple
Richard Totthill
PMCC Animal house
Justin O’Sullivan
Liggins Institute, University of
Auckland
Austen Ganley
Massey University, Auckland