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