DNA-barcodednucleosome library
Literature seminar #2
2020.5.25
M1 Tamiko Nozaki

1

Contents

1. Introduc,on
2. DNA-barcoded nucleosome library
3. Regula,on of chroma,n remodeling ac,vity by PTMs
4. Func,onal crosstalk between histone PTMs
5. Summary

2

Contents

1. Introduc,on
2. DNA-barcoded nucleosome library
3. Regula,on of chroma,n remodeling ac,vity by PTMs
4. Func,onal crosstalk between histone PTMs
5. Summary3

1.Introduc+on

High-throughput sequencing-based methods and DNA-encoded molecules

Liszczak, G., & Muir, T. W. (2019). ACIE, 58(13), 4144-4162.

4

1.Introduc+on

Hits to therapeu-c targets iden-ﬁed using DEC methods

Kunig, V. et al., (2018), Biological chemistry, 399(7), 691-710.

…
Reports of high-aﬃnity hits for biological targets by usind DNA encoded library are now common.
Goodnow Jr, R. A. et al., (2017). Nature Reviews Drug Discovery, 16(2), 131.

5

1.Introduc+on

Barcode technology in yeast

microarray

DNA seq

Pierce, S. E. et al.,(2006). Nature methods, 3(8), 601-603.

The simultaneous measurement by using barcode
array provides a powerful system for identifying thegenes required for growth in any condition of interest

Delneri, D. (2010). FEMS yeast research, 10(8), 1083-1089.

6

1.Introduc+on

Chemical Methods for Appending DNA to Natural and Unnatural Func:onal Groups
e.g. DNA-antibody conjugations

Ullal, A. V. et al. (2014). Science transla-onal medicine, 6(219), 219ra9-219ra9.

Promising strategy for targe0ng a single site in proteins
Liszczak, G., & Muir, T. W. (2019). ACIE, 58(13), 4144-4162.

7

1.Introduc+on

An#body–DNA conjugate applica#ons

An$bodies represent one of the most common classes of proteins to which amino acid side chain-DNA
conjuga$on methods have been applied.
Liszczak, G., & Muir, T. W. (2019). ACIE, 58(13), 4144-4162.

8

Contents

1. Introduc,on
2. DNA-barcoded nucleosome library
3. Regula,on of chroma,n remodelling ac,vity by PTMs
4. Func,onal crosstalk between histone PTMs
5. Summary9

2. DNA-barcoded nucleosome library

Organiza(on and analysis of chroma(nPTMs are dynamically inscribed into chroma<n through covalent modiﬁca<ons of DNA and histones
by 'writer' and 'eraser' enzymes; 'readers' further convert this chroma<n landscape into deﬁned
transcrip<onal outputs.
Nucleosomes are indispensable substrates in biochemical studies that require the three-dimensional
nucleosome architecture.

Fierz, B., & Muir, T. W. (2012). Nature chemical biology, 8(5), 417.

10

2. DNA-barcoded nucleosome libraryDNA-barcoded nucleosome libraryDNA-barcoded nucleosome library a versa6le pla8orm with high throughput and sensi6vity→ greatly accelerates biochemical inves6ga6ons into chroma6n recogni6on and signaling
Liszczak, G., & Muir, T. W. (2019). ACIE, 58(13), 4144-4162.

11

2. DNA-barcoded nucleosome library

Synthesis of modiﬁed mononucleosomes via a variety of protein chemistry approaches
Mono-Ubiqui?nated nucleosome
Lys120EPL

thiazolidine deprotec?on

H2B118-125

H2B118-125

H2B118-125
H2B1-116EPL

2 : Ub1-75-thioester

H2B118-125
Ala117

Desulfuriza?on

H2B118-125), ) EPL

McGinty, R. K.et al,(2008).
Nature, 453(7196), 812-816.

Fierz, B. et al.,(2012). JACS. 134, 19548–19551 (2012). modified

Dawson, P. E. et al.,(1994). Science, 266(5186),776-779.

12

2. DNA-barcoded nucleosome library

Synthesis of modiﬁed mononucleosomes via a variety of protein chemistry approaches
N-acylurea

hydrazide

Pep$de with PTM

C-terminal fragment(N-terminal A→C)
N-terminal fragment

Pep$de with PTM
N-terminal fragment C-terminal fragment(N-terminal C→A)A

Dawson, P. E. et al.,(1994). Science, 266(5186),776-779.

Desulfuriza$on
Desulfurization

Blanco-Canosa, J. B. et al.(2008),ACIE, 47(36), 6851-6855.

Zheng, J. S. et al., (2013). Nature protocols, 8(12), 2483.

Fang, G. M., Li et al., (2011). ACIE, 50(33), 7645-7649.

13

2. DNA-barcoded nucleosome library

Barcoded nucleosome assembly

Octamer formation

NUC formation

BarcodeDis&nct combina&ons of histones were combined with 5ʹ-barcoded versions of the strong nucleosome
posi&oning sequence 601.

Nguyen, U. T et al,(2014). Nature methods, 11(8), 834-840.

14

2. DNA-barcoded nucleosome library

Prepara&on of a DNL and its use in in vitro ChIP-seq experiments

DNL are treated with purified effectors or the combined chromatin recognizing andmodifying activities of the nuclear proteome
The desired products are isolated by chromatin immunoprecipitation, followed bymultiplexed DNA-barcode sequencing
Nguyen, U. T et al,(2014). Nature methods, 11(8), 834-840.

15

2. DNA-barcoded nucleosome library

Prepara&on of a DNL and its use in in vitro ChIP-seq experiments

https://www.abcam.com/epigenetics/studying-epigenetics-using-chip16

2. DNA-barcoded nucleosome libraryProﬁling the substrate preference of histone-mark “readers”
BPTF the BD–PHD ﬁnger transcrip,on factor
Previous pep,de and nucleosome binding studies
Nucleosomal binding increases with H3K4me3-H4K16ac
Kac-binding pocket of BPTF's BD can accommodate only one Kac

Ruthenburg, A. J., et al.
(2011). Cell, 145(5), 692-706.

Only the BPTF-PHD-BD fusion associated bivalently to substrates carrying an
H3K4me3 mark in conjunc,on with a monoacetylated lysine residue on H4
A strong enhancement was found with H4Kac5-containing nucleosomes
Nguyen, U. T et al,(2014). Nature methods, 11(8), 834-840.

17

2. DNA-barcoded nucleosome libraryProﬁling the substrate preference of histone-mark “readers”
P300transcrip.onal coac.vator, acetyltransferase
Previous pep.de study
Pep.des that interacted with BD-PHD contained at least one acetylated residue, and pep.des that
displayed the strongest aﬃnity contained combina.ons of mul.ple acetylated histone H4 tail.

A BD-mediated robust associa.on to substrates with hyperacetylated H4 tails was observed.

Nguyen, U. T et al,(2014). Nature methods, 11(8), 834-840.

18

2. DNA-barcoded nucleosome libraryProﬁling the regula/on of histone-mark “writers”
P300acetyltransferase

The p300 positive feedback loop is mediated mainly by nucleosomal
recruitment through the BD as opposed to an allosteric process
Nguyen, U. T et al,(2014). Nature methods, 11(8), 834-840.

h?ps://www.cellsignal.com/contents/science-cst-pathwaysepigeneEcs/epigeneEc-writers-and-erasers-of-histone-h3/pathways-epi-h3

19

2. DNA-barcoded nucleosome libraryDNA-barcoded mononucleosomes as internal standards to calibrate ChIP-seq data
Internal Standard Calibrated ChIP (ICeChIP) → unbiased trans-experimental comparisons‘‘barcode’’ that encodes each member’s concentraFon
The greatest source of experimental error of
ChIP is the frequently poor affinity, specificity,and reproducibility of the antibodies
employed to capture desired epitopes.

Grzybowski, A. T. et al. (2015). Molecular cell, 58(5), 886-899.

20

Contents

1. Introduc,on
2. DNA-barcoded nucleosome library
3. Regula,on of chroma,n remodeling ac,vity by PTMs
4. Func,onal crosstalk between histone PTMs
5. Summary21

3. Functional crosstalk between histone PTMs

Chroma'n remodelers
ISWI family

The ISWI chroma<n remodelling ATPase cons<tutes
an important subfamily within the SNF2 superfamily ofATPase.
A basic patch at the base of the H4 tail is a major
determinant of ISWI recogni<on.
ISWI remodelers are s<mulated by histone variant
H2A.Z.
ISWI remodellers
ACF

WHICH

RSF

Toto, M. et al, (2014). Chromosoma, 123(1-2), 91-102.

CHRAC

Dann, G. P. et al, (2017). Nature, 548(7669), 607-611.

NoRCNURF
22

3. Func(onal crosstalk between histone PTMs

Nucleosome remodelling assay for ISWI family chroma6n remodellers using MN library

Restric*on enzyme accessibility assay
High remodeling activity→ More sensitive to restriction
enzyme at early time point

MN libraryDann, G. P. et al, (2017). Nature, 548(7669), 607-611.

23

3. Func(onal crosstalk between histone PTMs

Nucleosome remodelling assay for ISWI family chroma6n remodellers using MN library

Restric*on enzyme accessibility assay

Full length

High remodeling ac*vity→ More sensi*ve to restric*on
enzyme at early *me point

DNA sequencing

PCR ampliﬁca3on
Chroma3n remodeling

MN librarydigestedDann, G. P. et al, (2017). Nature, 548(7669), 607-611.

No ampliﬁca3on24

3. Func(onal crosstalk between histone PTMs

Evaluation of ISWI remodeling assay against the nucleosome library
Heat map displaying ISWI remodeling data against the nucleosome library.

A DNA standard present in the library that isunoccupied by a histone octamer was always
faster than nucleosome remodeling.

Nucleosomes containing modiﬁca=ons or muta=ons in
the basic patch of the H4 tail were poor substrates ofISWI remodelers.
Nucleosomes containing the histone variant H2A.Z led
to enhanced remodeling ac=vity.
→MN library results are consistent with previous work.

Dann, G. P. et al, (2017). Nature, 548(7669), 607-611.

25

3. Func(onal crosstalk between histone PTMs

Specializa)on of ISWI remodelers for diverse nucleosome modiﬁca)ons.

Subsets of the nucleosome library drive diﬀerences in remodeler
ac8vity A speciﬁc nucleosome type had a broad range of eﬀects acrossdiﬀerent remodelers

Single-site modiﬁca8ons mapped onto the nucleosome
Nucleosome remodeling ac8vity across all ISWI remodelers
consistently posi8ve
consistently nega8ve
more accessible regions of the nucleosome
variable eﬀects
 residing under the DNA

Dann, G. P. et al, (2017). Nature, 548(7669), 607-611.

26

3. Func(onal crosstalk between histone PTMs

The nucleosome acidic patch is crucial for remodeling and regulatable by histone PTMs(7.1, H2AE56A; 7.2, H2BE113A; 7.3,H2AE61A; 7.4, H2AE64A;
7.5, H2AD90A; 7.6, H2AE92A; 7.7,H2BE105A; 7.8, H2AE91A; 2.1, N94D
(H2A→H2A.Z); 2.2, K95S (H2A→H2A.Z)

Acidic patch disrupted nucleosome was ineﬃciently remodeled by all ISWI family remodelers examined.
PTMs near the acidic patch showed both sAmulaAon and inhibiAon of nucleosome sliding acAvity.
→ mechanism mediated by the ATPase subunit

The acidic patch is subject to dynamic
regulaAon by nucleosome modiﬁcaAons

Dann, G. P. et al, (2017). Nature, 548(7669), 607-611.

27

Contents

1. Introduction
2. DNA-barcoded nucleosome library
3. Regulation of chromatin remodelling activity by PTMs
4. Functional crosstalk between histone PTMs
5. Summary28

4. Func(onal crosstalk between histone PTMs

H2BK120 Ub
H2BK120 Ub func.on
Ac%ve transcrip%onal elonga%on
Binding of the histone chaperone complex, FACTDecompac%on of higher-order structure of chroma%n
DNA damage response (DDR) to double-strand breaks

Machinery for installing H2BK120
H2BK120ub is localized primarily to ac%vely
transcribed genes
E2 ligase UBE2A/B and the hetero-dimeric Johnsen, S. A. (2012). FEBS letters, 586(11), 1592-1601. Moyal, L et al. (2011). Molecular cell, 41(5), 529-542.
RING-type E3 ligase RNF20/40
Eﬃcient ubiquityla%on of H2B is coupled to ongoing transcrip%onWojcik, F. et al.,(2018). Nature communications, 9(1), 1-11.

29

4. Func(onal crosstalk between histone PTMs

Library-based screen for the eﬀects of PTMs on de novo H2B ubiquityla=on
Do pre-exis-ng chroma-n modiﬁca-ons or within gene coding regions inﬂuence H2BK120ub?
in vitro screen of ubiquityla-on using MN Library

In vitro ubiquitylation with RING-type enzyme and HA-tagged ubiquitin

MN library based screening corresponds to several hundred enzyma-c measurements
Wojcik, F. et al.,(2018). Nature communications, 9(1), 1-11.

30

4. Func(onal crosstalk between histone PTMs

H2BK120ub deposi0on is sensi0ve to some nucleosomal modiﬁca0ons
H2A.Z. and Kac on the N-terminal
tail of H2A inhibit H2BK120ub.

PTMs proximal to the DNA
entry/exit site s9mulate H2BK120ub

H2BS112GlcNAc (reported)UBE2A:RNF20/40 apparatus is extremely sensitive tochanges in the H2A tailThe presence of positive charges on both K13 andK15 is especially important.
Wojcik, F. et al.,(2018). Nature communications, 9(1), 1-11.

31

4. Func(onal crosstalk between histone PTMs

Modiﬁca(ons to the H2A tail aﬀect H2BK120ub in cells
In vitro

Replacement of H2AK15 either alone or in tandem with K13 led to reduc:on of H2BK120ub comparedwith a WT H2A control.
The majority of the isolated cellular MNs will be asymmetric, containing both WT (i.e., endogenous)
and mutant copies of H2A.
ü acetyla:on of the H2A N terminus nega:ve regulates H2B ubiquityla:on.
Wojcik, F. et al.,(2018). Nature communications, 9(1), 1-11.

32

4. Func(onal crosstalk between histone PTMs

Crosstalk between H2BK120ub and modiﬁca:ons in the DNA entry/exit site
In vitro 12merBrehove, M. et al., (2015). , Journal of Biological Chemistry, 290(37), 22612-22621.

H3Y41disrupt DNA-histone contacts leading to increased
breathing of DNA on the nucleosome
H3Y41

H3Y41 is proximal to the DNA entry/exit.
DNA entry/exit site on the nucleosome
a region previously implicated in the binding of E3 ligase
Bre1 (the yeast version of the RNF20/40 )ModiﬁcaQons that modulate the local structure of the DNA
Gallego, L. D. et al., Proc. Natl Acad. Sci. USA 113, 10553–10558 (2016). entry/exit site, such as H3Y41ph, might sQmulate ubiquitylaQonacQvity through an enzyme-binding mechanism.
Wojcik, F. et al.,(2018). Nature communications, 9(1), 1-11.

33

4. Func(onal crosstalk between histone PTMs

Model for the crosstalk between H2BK120ub and the H2A/H2A.Z N-terminal tailPanier, S., & Boulton, S. J. (2014)., Nature reviews Molecular cell biology, 15(1), 7-18.

⊣H2AK15Ac ⊣ H2AK15Ub by the RNF168 →DDR pathway
H2BK120Ub by RNF20/40 →DDR pathway

Jacquet, K. et al., Mol. Cell. 62, 409–421 (2016).

The crosstalk may help control the Tming of H2B K120Ub at DDR (DNA damage response ).

Acetylated H2A tailH2A tail would posiTon ubiquiTn correctly so that the transfer from RNF20/40-UBE2AUb to H2BK120 is ensured.
Wojcik, F. et al.,(2018). Nature communications, 9(1), 1-11.

34

Contents

1. Introduc-on
2. DNA-barcoded nucleosome library
3. Regula-on of chroma-n remodelling ac-vity by PTMs
4. Func-onal crosstalk between histone PTMs
5. Summary

35

Summaryü DNA barcoded nucleosome library allows high throughput and sensi:ve PTM analysis.
ü DNL enables to inves:gate PTM-based recruitment and modula:on of histone-markreaders and writers, and shows how PTM signals, alone or synergis:cally, result incomposite systems-level signal outputs through the combined ac:on of the nuclear
proteome.
ü A systema:c analysis of the eﬀects of nucleosome modiﬁca:ons on chroma:n remodeling
ac:vity, generated a dataset that exists as a resource to drive the design of futurebiochemical and cell-based studies.
ü Crosstalk between PTM was revealed by using DNA barcoded mononucleosome library.

36