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Ubiquitin

Ubiquitin The ubiquitin relay Multitasking Ubiquitin Ligases in the DNA Lys

Damage Response Cys E4 E1 Promega tour, Karolinska Institutet, March 8, 2013 Cys E2 Nico Dantuma E3 Lys Department of Cell and Molecular Biology Karolinska Institutet, Stockholm

substrate

Nico Dantuma March 8, 2013

The ubiquitin-proteasome system Different forms of ubiquitin chains ubiquitin

1 KK KKK K K 76 Ubiquitylation Degradation 611 27 29 33 48 63 poly-ubiquitination

mono-ubiquitination

proteasome E3 peptides

monoubiquitination membrane trafficking, DNA repair, transcriptional regulation substrate Lys 6 polyubiquitination signalling (rare?) Lys 29 polyubiquitination proteasomal degradation (rare?)

Lys 48 polyubiquitination proteasomal degradation

Lys 63 polyubiquitination DNA repair, signalling

Nico Dantuma March 8, 2013 Nico Dantuma March 8, 2013

GFP-tagged ubiquitin Dynamics of GFP-tagged ubiquitin

GFP-Ub GFP-UbK0G76V

Ub bleach Cyt Cyt bleachCyt Ub pre 0 min 10 min pre 0 min 10 min GFP Ub GFP Ub GFP ubiquitylation Ub Ub GFP Ub K Nuc bleach Nuc

pre0 min 10 min bleach Nuc pre 0 min 10 min

5 K0,G76V 3 GFP-Ub Nuc/Cyt ratio GFP-Ub Nuc/Cyt ratio Cytosolic bleach 4 Cyt bleach 3 2 Cytosolic bleach 2 Cyt bleach Nuc/Cyt ratio Nuc/Cyt Nuc/Cyt ratio Nuc/Cyt 1 1 NuclearNuc bleach bleach NuclearNuc bleach bleach 0 0 02468101214 02 46 8101214

Dantuma et al J. Cell Biol. 2006 Time (min) Time (min) Nico Dantuma March 8, 2013 Nico Dantuma March 8, 2013

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Response of GFP-Ub in nucleus Accumulation of GFP-tagged ubiquitin over long time

Nico Dantuma March 8, 2013 Nico Dantuma March 8, 2013

GFP-ubiquitin accumulation DNA damage-induced ubiquitylation of in bleached spot histone H2A

Bergink et al Dev. 2006

Nico Dantuma March 8, 2013 Nico Dantuma October 8, 2010

H2A is also ubiquitylated in response to Recruitment BRCA1 and 53BP1 double strand breaks

Cell 2007 Cell 2007 ATM

MRN

MDC1 PNAS 2007 H2 P Science 2007 AX RNF8 P

X RNF168

BRCA1 remodelling- assisted ubiquitylation RAP (CHD4) Cell 2009 Cell 2009 80 H2 A

Ubiquitin-dependent extraction from 53BP1 Me H4 chromatin (VCP/p97)

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DNA damage response is activated Tethered RNF8 gives local ubiquitylation of H2A by tethering of ubiquitin ligases RNF8

mCherry-LacR-RNF8α-ubiquitin Merge RNF8 RNF8 WT P LacR MDC1 X LacO

RING*

DNA repair DNA repair

Nico Dantuma March 8, 2013 Nico Dantuma March 8, 2013

Tethered RNF8 recruits RNF168 Tethered RNF8 and RNF168 induces H2A ubiquitylation RNF8 RNF8 mCherry-LacR-RNF.. Ubiquitylated H2A Merge

X X

MIU MIU RNF RNF 168 168

H2A

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Tethered RNF8 and RNF168 gives Lys63- RNF8 and RNF168 recruit BRCA1 linked ubiquitin chains RNF8 RNF8 Lys63 ubiquitin LacR-RNF8 BRCA1 mCherry-LacR-RNF.. chain Merge

X X

MIU MIU RNF RNF 168 LacR-RNF168 BRCA1 168

H2A H2A Y Y

BRCA1

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Tethering of RNF8 causes chromatin The chromatin remodeller CHD4 decondensation is recruited to RNF8

Note: Array in NIH2/4 cells is 10 kb Array in AO3 cells is 90 Mb

Nico Dantuma

Nico Dantuma March 8, 2013 March 8, 2013

CHD4 is part of NuRD complex CHD4 tethering causes decondensation which is dependent on ATPase activity

• ATPase domain (SNF2 family) • In NuRD complex: CHD4, histone deacetylates HDAC1 and 2 and histone demethylase LSD1 • Linked to repression and heterochromatin formation

Nico Dantuma March 8, 2013 Nico Dantuma March 8, 2013

RNF8-induced decondensation is CHD4 is required for efficient ubiquitylation dependent of CHD4 Ubiquitin conjugates

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CHD4 stimulates BRCA1 recruitment Chromatin remodelling-assisted ubiquitylation

I: RNF8 non-catalytic II: RNF8 catalytic

Luijsterburg et al (2012) EMBO J.

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CRL4ADDB1/DDB2 ubiquitin ligase facilitates recognition of Tethering of DDB2 also causes photolesions by XPC chromatin unfolding

• DDB2 tethers the ubiquitin ligase complex CRL4DDB1/DDB2 to photolesions

• Stimulates the recognition of photolesions by XPC

• Mutations in DDB2 cause Xeroderma Pigmentosum type E (XP-E)

Independent of ubiquitylation Probably stimulates recognition of lesion by XPC

Luijsterburg et al (2012) J. Cell Biol.

Nico Dantuma March 8, 2013 Nico Dantuma March 8, 2013

Recruitment BRCA1 and 53BP1 Recruitment of 53BP1 to DNA damage is dependent on ubiquitylation

MDC1 H2A K119 Ub (or K13/15 Ub) H2 P AX RNF8 P

ATM

MRN BRCA1 Chromatin remodelling- assisted ubiquitylation 53BP1 RAP (CHD4) 80 H2 H4K20Me2 A

H2 A RNF168

Ubiquitin-dependent extraction from 53BP1 Me H4 chromatin (VCP/p97)

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RNF8 recruits the ubiquitin-selective Valosin-containing protein (VCP)/p97/Cdc48 AAA-ATPase VCP/p97/Cdc48

• Dependent on ubiquitin ligase activity • Specific for RNF8

Huytin et al 2003 J. Struc. Biol.

• AAA-ATPase • Binds ubiquitin • Critical for proteasomal degradation of ER substrates (ERAD) • Unfoldase/segregase activity

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VCP/p97 is required for efficient recruitment of 53BP1 Kinetics of VCP recruitment

Nico Dantuma November 15, 2012 33 Nico Dantuma March 8, 2013

Hypothesis 53BP1 and L3MBTL1 bind to same H4K20me2 VCP’s involvement explain the need for ubiquitin in 53BP1 recruitment.

But how does VCP faciliate binding of 53BP1?

We know that VCP segregates proteins

Hypothesis: VCP facilitates binding of 53BP1 to H4K20me2 by extracting a protein that occupies the From Min et al (2007) Nat. Struc. Mol. Biol. same histone mark.

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L3MBTL1 is extracted from chromatin at L3MBTL1 extraction is dependent on DNA damage VCP/p97

Nico Dantuma March 8, 2013 Nico Dantuma March 8, 2013

Conclusions Ubiquitin-dependent extraction from chromatin (UDEC) • RNF8 recruits CHD4 independent of its ubiquitin ligase activity

• CHD4 decondensates chromatin and stimulates ubiquitylation

• RNF8 recruits VCP/p97 dependent of its ubiquitin ligase activity

• VCP/p97 extracts L3MBTL1 and facilitates 53BP1 recruitment

Acs, Luijsterburg et al (2011) Nat. Struc. Mol. Biol. Dantuma & Hoppe (2012) Trends Cell Biol.

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Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden Azadeh Arabi Laura Bott Daniela Gödderz Florian Salomons Thibaud Richard Annika Pfeiffer

Former members Martijn Luijsterburg (CHD4/VCP) Klàra Ács (VCP)

University of Cologne, Germany Leena Ackermann Thorsten Hoppe

University of Copenhagen, Denmark Niels Mailand

Erasmus Medical Center, Netherlands Steven Bergink Wim Vermeulen Support: Swedish Research Council, Swedish Cancer Society, HighQ Foundation, The Netherlands Cancer Institute, Netherlands Hereditary Disease Foundation, Wallenberg Jacques Neefjes Foundation, Wenner-Gren Foundation, The Netherlands Organization for Scientific Research, Nordic Center of Excellence Neurodegeneration, EU Rubicon, Marie Curie

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