Genome biology of type II topoisomerases

Michael Wilson

SickKids Research Institute andDepartment of Molecular Genetics

University of Toronto

Epigenetics Eh!UWO, London, Ontario

June 25th, 2014

Top2:

•Unwinds, unknots and untangles DNA

•Introduces transient double stranded breaks

•Sharply bends DNA (150 degrees)

Deweese, J. E. et al. Nucl. Acids Res. 2008 Dong and Berger, Nature 2007

Topoisomerases introduce DNA breaks to solve topological problems

-- Expressed in most all tissues; prominent in post-mitotic cells

-- Expressed and essential in proliferating cells

Top2a

Top2b

Vertebrates encode two topoisomerases II isozymes:Top2a and Top2b:

3Thakurela S. et al 2013

• Brain (Lyu 2003)• Retina (Nevin et al 2011, Li et al 2014) • T cells (Daev et al 1994)• Testes (Shaman 2006, Martins 2007, Leduc 2008)• Ovarian follicules (Zhang et al 2013)• …

How does Top2b’s genomic occupancy relates to its gene regulatory function?

Liis Uusküla-ReimandHuayun Hou

Enhancers

Chromatin

structure/bound

ary

Top2b widely overlaps with several key genomic features

Promoter/gene body

(Cohesin and liver TFs from Faure et al 2012)

Top2b is associated with CTCF and the cohesin complex

CTCF/Rad21/Top2b “triple sites” have strong CTCF binding

Most triple sites occur at conserved CTCF binding sites

• 92% of CTCF/RAD21/Top2B are shared in >3 tissues

Top2b and the cohesin complex organize around CTCF

Orient CTCF motif

Summary:

1) Top2b localizes at various regulatory regions.

2) Top2b co-localizes with CTCF and the cohesin complex with a prominent orientation.

3) Top2b’s non-random interaction with the genome makes it an intriguing epigenetic regulator

Questions for Matteo and Sue• Do Rad21-Ctcf-Top2b triple sites overlap liver TADs (or other

HiC liver features)? Does this occur more than rad21-ctcf (no Top2b) sites?

• Do Top2b-transcription factor (non Ctcf) sites, engage in loops/3D interactions more frequently than transcription factor binding events that lack Top2B?

• Does the orientation of Rad21-Ctcf-Top2b have any special relationship with active regulatory domains? This could be in general, or compared to the sites with Top2b-Ctcf-Rad21. – Huayun and Liis are still looking, but we have not found anything

striking yet. A negative answer is valuable as it would indicate this spatial relationship is just how these 3 factors interact and that their orientation does not itself provide any discernable function. Still I feel there might be something important here and the Top2b should help find it. Maybe you can find it with your 3D glasses

Wilson Lab:

Huayun Hou*Liis Uusküla-Reimand* Alejandra Medina-RiveraTed YoungLina AntouniansMinggao LiangXuefei Yuan Azad AlizadaXiaoli Lu

Acknowledgments

Odom lab (U of Cambridge)Dominic SchmidtDuncan Odom

Flicek lab (EBI)Petra SchwallieAndre FaurePaul Flicek

Anne-Claude Gingras lab (U of T)Payman Samavarchi-Tehrani

David Bazett-Jones (U of T)Liron Even-Faitelson

Carroll lab (U of Cambridge)Hisham Mohammed

Hepatocyte specific Top2B KO (Lyu and Wang 2003):

Top2b LoxP strain × albumin-Cre strain

Loss of Top2B in hepatocytes does not overtly alter transcriptional regulation

WB analysis:WT mouse Top2b knockout

mouse

IHC analysis:

Top2B staining is brown; nuclei staining is blue

ChIP-seq (CTCF, Rad21, H3K27ac, HNF6)

-- Minor changes in chromatin

Illumina Gene Expression Microarray Analysis:

-- Minor changes in global liver gene expression