Character count: 49105

This project is my own work except where indicated. All text, figures, tables, data or results

which are not my own work are indicated and the sources acknowledged.

Signed: Date:

 

 

MARCIAGROUP,EMBLGRENOBLE

Biochemicalandstructural

characterisationofhumanJARID2YearinIndustryProjectReport

Francesca Chandler

2016/2017 

 

 

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Contents

1. Abstract ......................................................................................................................... 2

2. Introduction ................................................................................................................... 3

2.1 Regulation of gene expression by Polycomb group proteins........................... 3

2.2 JARID2 – a Polycomb group protein ................................................................ 4

2.3 JARID2 – a structural challenge ....................................................................... 8

2.4 Open questions ................................................................................................. 8

2.5 Aim of this work ................................................................................................. 8

3. Methods ...................................................................................................................... 10

3.1 Cloning and plasmid preparation .................................................................... 10

3.2 Protein expression and production in E. coli .................................................. 10

3.3 Protein expression in insect cells (MultibacTM system) .................................. 11

3.4 Nuclear purification ......................................................................................... 11

3.5 Expression screening (E. coli) ........................................................................ 12

3.6 Strep affinity chromatography ......................................................................... 12

3.7 Ni-NTA affinity chromatography ..................................................................... 12

3.8 Ion-exchange and heparin chromatography .................................................. 13

3.9 Size exclusion chromatography ...................................................................... 13

3.10 SDS- PAGE…………………………………………………………………….13

3.11 Western blotting.……………………………………………………………….14

3.12 In-gel tryptic digestion coupled to mass spectrometry…………………….14

4. Results ........................................................................................................................ 15

4.1 Full length JARID2 in E. coli ........................................................................... 15

4.2 Full length JARID2 in insect cells ................................................................... 17

4.3 Designing and generating a library of JARID2 constructs ............................. 21

4.4 Characterisation of ShortRBR ........................................................................ 21

4.5 Characterisation of Cterm ............................................................................... 25

4.6 Characterisation of LongRBR ......................................................................... 28

5. Discussion and outlooks ............................................................................................. 37

6. References .................................................................................................................. 40

7. Acknowledgements ..................................................................................................... 43

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1. Abstract

Polycomb group (PcG) proteins are epigenetic regulators involved in modulating chromatin

structure to maintain gene repression. JARID2 is a cofactor of Polycomb repressive complex 2

(PRC2), a PcG protein, which adds repressive marks to histone tails. JARID2 regulates the

repressive enzymatic activity of PRC2 and is predicted to recruit PRC2 to chromatin via its two

DNA-binding domains. A recently identified RNA-binding region at the N-terminus of JARID2

has revealed a potential role for long non-coding RNAs (lncRNAs) in enhancing PRC2 assembly

on chromatin. It has been proposed that lncRNAs may be acting as a scaffold between JARID2

and PRC2 to stimulate recruitment. However, there is no structural data available for JARID2,

lncRNA and PRC2-mediated gene repression and thus, the complex molecular mechanisms of

such interactions are unknown. By studying JARID2 and gaining insights into its structure, we

will be able to answer important questions underpinning Polycomb-mediated gene silencing.

Therefore, I sought to express and purify JARID2 for structural and biochemical analysis. To

achieve this objective, I generated a library of JARID2 constructs. Initially, I cloned full length

JARID2 for expression in E.coli and in insect cells. Given the size and disorder of JARID2, the

main focus of studying the full length protein was to identify stable and soluble fragments as

potential targets for future expression. I designed and expressed shorter JARID2 constructs,

focussing on constructs encompassing domains of functional relevance. Here I describe the

experimental approaches used to enhance each construct, and the steps taken towards

obtaining a pure form of JARID2 for use in future biophysical studies and biochemical assays

with lncRNAs and PRC2.

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2. Introduction

2.1 Regulation of gene expression by Polycomb group proteins

Eukaryotic gene expression is a complex, multi-layered system which is highly regulated for

orchestrating development and for responding to environmental stimuli. Transcription factors

and variable chromatin structure are crucial for transcriptional regulation, and thus, essential in

controlling gene expression.

The Polycomb group (PcG) proteins, first discovered in Hox genes in Drosophila melanogaster

(Lewis, 1978), control gene expression in developing embryos through to adulthood. PcG

proteins are a collection of chromatin-associated protein factors which form two functional

families: Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2

(PRC2). Both multimeric complexes are responsible for adding repressive post-translational

modifications to histone tails.

The Ezh2 subunit of PRC2 possesses enzymatic activity, and tri-methylates Lys27 on Histone 3

(H3K27me3) (Simon and Kingston, 2009). PRC1 has E3 ligase activity and catalyses the mono-

ubiquitination of Lys119 on Histone H2A (H2AK119u1) (Wang et al., 2004). Such histone

modifications play two roles. Firstly, they are involved in modulating chromatin architecture to

contribute to gene silencing. Secondly, H2AK119u1 and H3K27me3 are signals which facilitate

a functional crosstalk between PRC1 and PRC2. One isoform of PRC1, canonical PRC1,

specifically binds the H3K27 tri-methylation and in this way, PRC2 is thought to stimulate PRC1

recruitment to chromatin. In addition, more recent studies show the H2AK119u1 mark added by

variant PRC1 complexes can also play a role in PRC2 and H3K27me3 occupancy (Blackledge

et al., 2014).

In Drosophila, PcG proteins are targeted to Polycomb response elements (PRE), which are cis-

regulatory sequences. PREs are proposed to act as an assembly platform to which PcG

complexes bind to maintain the repressed chromatin state (Müller and Kassis, 2006). In

mammals, the story is much more complex, and there is no defined DNA element found to be

responsible for PcG targeting. Instead, CpG islands and long non-coding RNAs (lncRNAs) are

proposed to have an important role in the recruitment of PcG complexes to chromatin (Simon

and Kingston, 2009).

Alongside CpG islands and lncRNAs, accessory proteins JARID2 and SCML2A are also

predicted to enhance PRC1 and PRC2 recruitment (Figure 1). JARID2 and SCML2A both

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contain an RNA-binding region (RBR). These regions have confirmed RNA binding function in

vitro and in vivo. It is proposed that lncRNAs interact with the RBRs of JARID2 and SCML2A to

recruit chromatin remodelling complexes PRC1 and PRC2 to chromatin. (Bonasio et al., 2014;

Kaneko et al., 2014) However, there is no structural information for these interactions, and the

molecular mechanisms by which gene expression is regulated by Polycomb group proteins are

unknown.

Figure 1: Regulation of gene expression. Schematic representation of Polycomb group proteins

(including JARID2) interactions with lncRNAs, initiating histone modifications and downstream gene

repression (Figure adapted (Christophersen and Helin, 2010))

2.2 JARID2 – a Polycomb group protein

JARID2, Jumonji, AT-rich interactive domain 2, is the founding member of the Jumonji family of

histone demethylases. The Jumonji gene (Jmj) was first identified in mouse, where studies of

Jmj homozygous mutants revealed a critical role of Jmj in neural tube formation and cardiac

development (Takeuchi et al., 1995; Lee et al., 2000). Jmj is a transcriptional repressor with

DNA-binding capacity (Kim et al., 2003), but it lacks the key residues in its JmjC domain

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required for demethylase activity (Klose, Kallin and Zhang, 2006). JARID2 is thought to be a cell

cycle regulator due to its role of repressing cyclin D1 expression (Toyoda et al., 2003).

At the molecular level, human JARID2 protein (1246 residues) has eight proposed domains

which give insight to its function (Figure 2A). From the N-terminus, these domains are the

ubiquitin interaction motif (UIM), the PRC2 interaction domain, the RNA-binding region (RBR)

and the nucleosome interaction domain (NID). Towards the C-terminus are the two DNA-binding

domains, ARID and a zinc-finger domain, and the JmjN and JmjC domains which frequently co-

occur in the jumonji family of transcription factors.

The N-terminal ubiquitin interaction motif (UIM) is a conserved 17 amino acid region which

facilitates interaction with H2AK119u1 modified nucleosomes in vivo and in vitro (Cooper et al.,

2016). This repressive modification is the hallmark of PRC1 activity, and via binding this,

JARID2 acts as a mediator between PRC1 and PRC2.

In embryonic stem cells, JARID2 is associated to PRC2 (Peng et al., 2009), an interaction which

has been isolated to residues 111- 121. JARID2 and PRC2 share >90% overlapping binding

profiles of target genes and inhibition of JARID2 causes a decrease in PRC2 binding to such

target genes (Pasini et al., 2010), revealing a role for JARID2 in PRC2 recruitment. The

enzymatic activity of PRC2 involves an allosteric feedback loop. The methyltransferase activity

of the Ezh2 subunit is activated by recognition and binding of H3K27me3 repressive marks to

an aromatic domain formed by the Eed subunit (Margueron et al., 2009). This feedback

mechanism enables the propagation of H3K27me3 marks along chromatin, maintaining the

repressive chromatin state. Furthermore, JARID2 is also tri-methylated by PRC2 at K116, and

K116me3 can mimic the Eed subunit binding by H3K27me3 and promote PRC2 activity (Sanulli

et al., 2015). This molecular interaction has been captured in the crystal structure (PDB: 5HYN)

of PRC2 with a short, methylated JARID2 peptide (Justin et al., 2016).

In addition to the PRC2-JARID2 duality, lncRNAs are also implicated in this epigenetic pathway.

LncRNAs are a class of non-protein coding transcripts which are longer than 200 nucleotides in

length (Cao, 2014). As an emerging field, the role of lncRNAs in gene regulation is largely

unknown. A 27 amino acid RNA-binding region (RBR) has been identified in JARID2 (residues

332-358). LncRNA MEG3, a 1.5 kB lncRNA involved in the p53 tumour suppressor pathway,

binds the RBR domain of JARID2, and Ezh2, the catalytic subunit of PRC2. The presence of

MEG3, and other lncRNAs, increases the JARID2 mediated recruitment of PRC2 to chromatin

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in vivo (Kaneko et al., 2014). The predicted mechanism of these interactions is purely

speculative.

The most studied mechanism of non-coding RNAs (ncRNAs) modulating gene repression is

during X-chromosome inactivation (XCI) in female mammals. The XCI process, which is vital for

dosage compensation between males and females, is underpinned by the coating of one X-

chromosome in Xist RNA, resulting in gene silencing and heterochromatinisation. JARID2 has

now been implicated in this important process as crucial for targeting PRC2 to the Xist-coated

X-chromosome (Teixeira da Rocha et al., 2014). JARID2 and PRC2 are thought to maintain the

inactive state by propagating H3K27me3 repressive marks and modulating chromatin

architecture (Escamilla-Del-Arenal et al., 2011). The N-terminal region of JARID2 (residues 1-

583) is the minimal region required to act as an intermediate, tethering PRC2 to Xist RNA. This

role in XCI is independent of the RBR domain of JARID2 (Teixeira da Rocha et al., 2014), which

suggests other parts of the N-terminus of JARID2 are interacting with ncRNAs. This model of

PRC2, JARID2 and ncRNA interaction supports the proposed mechanism of JARID2 and

lncRNAs recruiting PRC2 to chromatin to mediate gene repression.

Furthermore, another domain which could enhance the binding of JARID2 to chromatin is the

nucleosome interaction domain (NID). The NID is composed of a 100 amino acid region,

partially overlapping with the RBR at the N-terminus, and has been found to enhance PRC2

binding to nucleosomes in vitro (Son et al., 2013).

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The two DNA-binding domains of JARID2, independent of the NID, are also thought to be

critical for its function. The largest of these is the ARID (AT-rich interaction domain) motif. This

~100 amino acid binding module is conserved in eukaryotes and commonly found in

transcription factors, notably the Dead ringer protein of Drosophila (Gregory et al., 1996). A

number of ARID structures have been studied by NMR, revealing a non-canonical helix-turn-

helix (HTH) motif, contacting DNA via the major and minor grooves (Patsialou, Wilsker and

Moran, 2005). At the C-terminus of JARID2 is a predicted C5HC2 zinc finger domain. Zinc finger

domains were first discovered in transcription factor TFIIA (Miller, Mclachlan and Klug, 1985)

and have well-reported DNA and RNA-binding potential. This could be a domain involved in

RNA interactions outside of the defined RBR domain. These DNA-binding regions support the

hypothesis that JARID2 is binding DNA in chromatin to recruit PRC2. However, studies have

shown the HTH motif of the ARID domain binds DNA with low affinity (Nayak, Xu and Min,

2011). If such a low affinity interaction is also seen in the ARID domain of JARID2, this provides

more evidence for the need of other factors, such as lncRNAs, to recruit PRC2 and reduce gene

expression.

Figure 2. JARID2 – an intrinsically disordered protein. (A) Domain organisation of human JARID2. (B) Statistical

prediction of the degree of disorder in JARID2 – based on primary amino acid sequence. (Zsuzsanna Dosztányi et al.

(2005)). Values above 0.5 are residues which are predicted to be intrinsically disordered. The N-terminus of JARID2

is largely disordered, and the C-terminus is predicted to have more defined tertiary structure.

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2.3 JARID2 – a structural challenge

In terms of structural analysis, JARID2 remains uncharacterised. Despite the identification of

important functional regions, the only structure available for JARID2 encompasses an 8-amino-

acid peptide bound to the Eed subunit of PRC2 (Justin et al., 2016). The lack of success in the

structural characterisation of JARID2 is likely due to its unusually large size (139 kDa), and to its

predicted flexible tertiary structure. The median protein length in H. sapiens is 375 amino acids

(45 kDa) (Brocchieri and Karlin, 2005), and JARID2 is more than three times this size, which

can be a limiting factor for structural analysis. Regarding the flexibility of JARID2, statistical

analysis reveals a highly disordered N-terminal region (Figure 2) (Dosztányi et al., 2005). This

region is rich in interaction domains important for its function in transcriptional repression. It

could be the flexible structure of JARID2 that makes it suitable for binding such a variety of

complexes; protein and RNA alike. The size and lack of defined tertiary structure make JARID2

an unlikely target for crystallisation. However, the structural characterisation of intrinsically

disordered proteins (IDPs) is an emerging field, notably with NMR spectroscopy. For example, a

well-studied transcriptional activator, Ets-1, contains a flexible DNA-binding segment which

becomes ordered on DNA-binding (Pufall et al., 2005). Successful structural characterisation of

JARID2 could reveal similar mechanisms, giving important insights for future studies of

disordered proteins.

2.4 Open questions

In spite of its limitations as a structural target, JARID2 is an accessory protein at the interface of

PcG-mediated transcriptional repression. There have been many studies investigating the role

of Polycomb group proteins in gene silencing, but there are important questions which remain

unanswered: What are the important structural features of JARID2 which enable it to bind

lncRNAs? What is the molecular mechanism by which lncRNAs interact with Polycomb group

proteins to be recruited to chromatin? Is JARID2 interacting with PRC1 via ubiquitin modified

histones? How is JARID2 binding DNA in chromatin? Structural and functional studies of

JARID2 could provide important answers in this complex puzzle of gene regulation.

2.5 Aim of this work

The goal of my project was to obtain a pure form of JARID2 or of a shorter construct

encompassing the RBR domain. On achieving this goal, JARID2 could then be characterised

biochemically and structurally to further our understanding of its role in transcriptional regulation.

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To achieve such goals, I had to;

- Order the synthetic clone for human JARID2

- Clone this gene into suitable vectors for expression in bacterial and eukaryotic systems

- Design a library of expression constructs, encompassing the RBR domain to maximise

the chances of obtaining a pure and homogeneous target

- Screen expression of these constructs in E. coli and insect cells

- Purify and optimise the purification strategy for each construct

- Assess purity, stability and homogeneity by biochemical and biophysical methods

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3. Methods

3.1 Cloning and plasmid preparation

A plasmid containing the cDNA clone of human JARID2 (IMAGE: 4520786) was obtained by

gene synthesis (Source BioScience). From this synthetic vector, the full length sequence of

JARID2 was amplified by PCR. In parallel, the pET26G expression vector (Surade et al., 2006)

containing sequences for His- and Strep-tags was amplified by PCR, and the JARID2 sequence

inserted by sequence- and ligation-independent cloning (SLIC) into the pET26G vector. The

resulting vector was named pET26G-JARID2. Plasmids pETG-J2LongRBR, pETG-J2ShortRBR

and pETG-J2Cterm were obtained from the SLIC cloning of shorter constructs using the

pET26G-J2 plasmid as a template. For compatibility with the MultiBacTM baculovirus expression

system, full length JARID2 (PCR amplified from pET26G-J2) was cloned by SLIC cloning into

pACEbac1 acceptor plasmid. The resulting construct was named pACEbac1-J2. The presence

of the correct insert in the plasmid library was confirmed by enzyme digestion and agarose gel

electrophoresis. DNA sequencing (Eurofins) validated the sequence of the plasmids. All cloning

and sequencing primers were designed using Gentle, verified using OligoEvaluatorTM (Sigma

Aldrich) and produced by Eurofins. Competent E. coli MachI (ThemoFisher) cells were

transformed with each plasmid for plasmid amplification. The plasmid DNA was extracted and

purified with a Miniprep kit (Qiagen).

3.2 Protein expression and production in E. coli

The expression vectors were transformed in chemically competent E. coli BL21(DE3),

BL21(DE3) Rosetta and BL21(DE3)pLysS cells (ThermoFisher) (cell type varying with

construct). Selected plasmids were grown in 50 mL cultures overnight at 37°C. 20 mL of the

overnight culture was used to inoculate 1 L of L.B broth with kanamycin. 1 L cultures were

grown to an optical density of 0.6-0.7 at 600 nm wavelength. Expression was induced with 0.2

mM, 0.4 mM or 1 mM IPTG and incubated at 37°C for 2, 4 or 6 hours or at 18°C overnight. Cells

were harvested by centrifugation (4500g, 15 minutes), followed by resuspension and second

centrifugation step (4000g, 10 minutes). Cell pellets were stored at -20°C.

Cells were lysed by thawing and re-suspension in 20 mL lysis buffer (e.g. 50 mM Tris pH 7.5,

100 mM KCl, 0.1% Nonidet P-40 (Sigma Aldrich) with cOmpleteTM EDTA-free protease inhibitor

cocktail (Roche). Resuspension was sonicated (50% amplitude, 5 min, 10s ON, 20s OFF) and

cells clarified by centrifugation (20000g, 30 min, 4°C). The supernatant obtained was used for

proceeding purifications.

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3.3 Protein expression in insect cells (MultiBacTM system)

pACEbac1-J2 plasmid was transformed into DH10EMBacY E. coli cells (ThermoFisher) to

integrate gene of interest into the baculoviral genome (Bieniossek et al., 2011). Bacmid DNA

was isolated and Spodoptera frugiperda (Sf21) insect cells transfected with the bacmid. The first

generation of virus (V0) was harvested 48 hours after transfection and used to infect 25 mL

flasks of Sf21 cells (at a density of 0.5 x 106 cells/mL). Cell proliferation was monitored each day

and cells diluted with Sf-900 III SFM (ThermoFisher) to maintain a density of 0.5 – 1.0 x 106

cells/mL. Expression of YFP-coding gene in baculovirus genome enables qualitative analysis of

protein expression from the same promoter (polh). One million cell aliquots were taken to

monitor YFP fluorescence using a microplate spectrophotometer (Tecan) and to confirm protein

expression by SDS-PAGE and Western Blotting. V1 virus was harvested 24 hours after the ‘Day

after Proliferation Arrest (DPA)’ – ‘DPA+24’. DPA was confirmed by cell counting and a plateau

in YFP fluorescence (absorbance at 529 nm). 50 mL cell cultures were centrifuged at 100g, 10

minutes, and the V1 virus harvested as the supernatant. V1 virus stored at 4°C.

A small-scale expression test was carried out to determine the strength of the J2 V1 virus. 50 mL

cell cultures were infected at 0.5 x 106 cells/mL density with different volumes of J2 V1 virus (25,

50, 100 and 125 µL). Protein expression was monitored by YFP and analysed by SDS-PAGE

and Western blot (anti-His and anti-Strep).

JARID2 was expressed in large-scale by infecting 500 mL cells (0.5 x 106 cells/mL density) with

2 mL V1. Cells were harvested on ‘DPA+24’ by centrifugation (1000g, 15 minutes). Pellets were

stored at -20°C.

3.4 Nuclear purification

Cell pellets from 50 mL infected insect cells (~50 million cells) were used. Cells were thawed on

ice and re-suspended in 5 mL lysis buffer (50 mM HEPES pH 8.0, 100 mM KCl, 0.1% NP40).

Re-suspension centrifuged at 4000g/5 min/4°C, supernatant collected. Pellet re-suspended in

another 5 mL lysis buffer and centrifugation repeated. This step repeated three times more until

five cytosolic fractions were collected. The pellet transferred to an Eppendorf tube and

centrifuged 4000g/5 min/4°C. ~1 mL supernatant ‘nuclei supernatant’ was collected. The cell

pellet was once more re-suspended in 200 µL lysis buffer and sonicated 30s ON, 30s OFF, 5

minutes with Bioruptor® (Diagenode). Bioruptor® was used instead of a normal sonicator to

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efficiently shear chromatin. After chromatin shearing, the sample was centrifuged 15000g/10

minutes/4°C, and resulting supernatant collected.

3.5 Expression screening (E. coli)

Cell cultures were prepared and expression induced as previously described (3.2). Protein

expression was analysed by harvesting and centrifuging (13000 rpm/5 min/RT) 1 mL cell

aliquots from 50 mL cell cultures. Resulting 1 mL cell pellets were dissolved by resuspension in

20% SDS. Resuspensions were shaken at 800 rpm/15min/RT, followed by centrifugation

(13000 rpm/20 min/RT). 5X sample loading buffer was added to the supernatants, before boiling

samples at 95°C/5 min for analysis by SDS-PAGE and Western blotting.

3.6 Strep affinity chromatography

Soluble protein fraction obtained by centrifugation (3.2) added directly to Strep column. For a 1

L cell culture, 5 mL Strep-Tactin Superflow beads (Qiagen) were used with a batch purification

protocol. Before the addition of supernatant, Strep beads (stored in 50 mM Tris pH 8, 150 mM

NaCl) were washed and equilibrated with lysis buffer. Where the Strep column was used

following a Ni-NTA column, the sample was first re-buffered using a PD-10 Desalting Column

(GE Healthcare) following the gravity protocol. Flow-through collected for SDS-PAGE analysis.

Column washed with 3CV wash buffer (50 mM Tris pH 8, 150 mM NaCl). Protein eluted with

3CV elution buffer (50 mM Tris pH 8, 150 mM NaCl, 2.5 mM desthiobiotin). All buffers filtered

(0.22µM) (Merck). Strep-Tactin Superflow beads were re-generated using 1X Strep-Tactin

regeneration buffer (Qiagen) and washed extensively (>10CV) in wash buffer.

3.7 Ni-NTA affinity chromatography

Ni-NTA affinity columns were used at different stages depending on the purification strategy

applied. Where Ni-NTA was used as the first purification stage, the supernatant was added

directly to Ni-NTA agarose beads (Qiagen). When a Ni-NTA column was used after a Strep

column, the elution from the Strep column was added to the Ni-NTA beads. The mixture was

incubated for 2 hours/4°C on a rotor. After incubation, the flow-through was collected. The

column was washed with 3CV high salt buffer (50 mM Tris pH 7.5, 1 M KCl, 20 mM imidazole),

followed by 3CV Ni-NTA wash buffer (50 mM Tris pH 7.5, 100 mM KCl, 20 mM imidazole).

Proteins eluted with increasing concentrations of imidazole (100 mM, 200 mM and 400 mM)

with 2CV buffer.

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3.8 Ion-exchange and heparin chromatography

HiTrap 1 mL columns (SP FF, CM FF, Q FF, DEAE FF) (GE Healthcare) were used for cation-

and anion-exchange chromatography. HiTrap Heparin 1 mL (GE Healthcare) columns were

used for heparin chromatography. HiTrap columns were used following a Ni-NTA column and

PD-10 Desalting column. 50 mM HEPES pH 7.5, 75 mM NaCl and 5 mM βME was used as the

starting buffer for ion-exchange. Samples were injected using a 10 mL Superloop (GE

Healthcare). Proteins eluted over a 30CV gradient up to 1 M NaCl. For heparin affinity

chromatography, samples were bound in 10 mM sodium phosphate pH 7.4 and samples eluted

over a 20CV gradient up to 2 M NaCl. Following ion exchange and heparin exchange, samples

were re-buffered in 50 mM HEPES pH 7.5, 75 mM NaCl and 5 mM βME with a PD-10 column.

3.9 Size exclusion chromatography

Samples eluted from purification columns were concentrated using Amicon Ultra centrifugal

filters (Merck) prior to injection on Superdex75 10/300 (ShortRBR), Superdex200 10/300

(LongRBR, Cterm) or Superdex200 5/150 Increase (LongRBR)(GE Healthcare). For LongRBR

and ShortRBR, 0.5 mL, 4 mL and 15 mL 10 kDa filters were used. For Cterm, 0.5 mL and 4 mL

30 kDa filters were used. With Superdex S75 and S200 10/300, samples were generally

concentrated to 500 µL and 1 mL for injections between 0.1 mg/mL and 9 mg/mL. Samples

were separated in 50 mM Tris pH 7.5, 150 mM NaCl and 5 mM βME at a flow rate of 0.5 mL/min

using the ÄKTApurifier system. Using the ÄKTAmicro system with Superdex S200 5/150

Increase, 50 µL of sample was injected at concentrations between 0.1 mg/mL and 2 mg/mL.

Samples were separated in 50 mM Tris pH 7.5, 150 mM NaCl and 5 mM βME at a flow rate of

0.3 mL/min.

After ion exchange chromatography, a Superdex200 5/150 Increase (GE Healthcare) column

was used with the ÄKTAmicro system. Samples were separated in 50 mM HEPES pH 7.5, 75

mM NaCl and 5 mM βME at a flow rate of 0.3 mL/min.

Protein concentration was determined with a Nanodrop spectrophotometer (ThermoFisher).

3.10 SDS-PAGE

Self-cast Tris-Glycine gels (10%, 12% and 15%) were used for SDS-PAGE. Prior to SDS-

PAGE, protein samples were mixed with sample buffer at a 1:5 ratio and boiled at 95°C for 5

minutes. Gels were run in Tris-Glycine-SDS running buffer at 220V for 1 hour. After

14  

electrophoresis, gels were stained with InstantBlueTM (Expedeon) and imaged with GelDoc

system (BioRad). For silver staining, the SilverQuestTM Silver Staining Kit (Invitrogen) protocol

was followed.

3.11 Western blotting

Samples were run on SDS-PAGE before transfer onto a nitrocellulose membrane (GE

Healthcare) (100V, 1h). Membranes were blocked in blocking solution (2% BSA, in Tris-buffered

saline (TBS) with 0.05% Tween20) for 1 hour at room temperature or at 4°C overnight.

Membranes were washed with Avidin-TBST buffer (2 mg/mL Avidin (Sigma)) and TBST buffer.

Membranes were incubated with anti-Strep (Precision Protein StrepTactin-AP Conjugate

(BioRad)) or anti-His (Anti-poly-histidine antibody Alkaline Phosphatase conjugate (Sigma)) for

2 hours at room temperature. Blots were washed with TBST buffer and alkaline phosphatase

(AP) buffer before colour developed with NBT/BCIP (both SigmaAldrich) in AP buffer.

3.12 In-gel tryptic digestion coupled to mass spectrometry

Gels were prepared as described in 3.9. After electrophoresis, gels were stained with

Coomassie blue. Samples were sent for analysis at the Proteomics Core Facility at EMBL

Heidelberg, following a standard protocol (Shevchenko et al., 2007). Trypsin cleaves proteins by

hydrolysing peptide bonds after lysine and arginine residues, generating a number of peptides.

These peptides are then identified by mass spectrometry. The data we receive outlines the

peptides and subsequent proteins identified from the band in the Coomassie gel, as well as the

sequence coverage for each protein and number of peptides identified.

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4. Results

4.1 Full length JARID2 in E. coli – expression and identification of a unique, stable

50 kDa fragment

I cloned (see Methods 3.1) and expressed full length JARID2 with His- and Strep- tags in E. coli

(Fig. 3A) in BL21(DE3) and BL21(DE3) Rosetta cells. I optimised expression with induction of

0.4 mM IPTG and growth at 18°C overnight. The 143 kDa protein was not soluble (Fig. 3A).

However, there was a soluble 50 kDa protein which was detected by Western blotting with an

anti-Strep antibody. I observed this 50 kDa Strep signal consistently throughout all expression

trials and solubility optimisation. To determine if this protein was a fragment of JARID2 with

potential for soluble expression, I sent a gel for analysis by in-gel trypsin digestion coupled to

mass spectrometry (Shevchenko et al., 2007). The protein was not detected by Western blotting

with anti-His antibody, so I predicted that the fragment contained the Strep-tag with the more

structurally ordered C-terminal fragment of JARID2. I isolated the pure 50 kDa fragment by

Strep affinity purification (Fig. 3B), where it eluted from the Strep column with 2.5 mM

desthiobiotin. The analysis of this stable, soluble sample (Fig. 5C) revealed it contained

peptides from the N-terminus (residues 35 – 418) and the C-terminus (1081- 1278). The

peptides produced from the trypsin digest represented 13% (166 residues) coverage of the full

length and tagged JARID2 sequence. This analysis revealed a potentially unique interaction

between the N-terminus and C-terminus of JARID2, which could be implicated in its structure.

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Figure 3. Expression of full length JARID2 in E. coli. (A) Anti-Strep western blot depicting expression of full length

JARID2 (142 kDa). Protein retained in insoluble fraction. (B) Coomassie stained gel (left) of sample eluted from Strep

column, containing 50 kDa fragment and anti-Strep western blot (right) showing antibody signal for 50 kDa fragment.

Sample analysed by in-gel tryptic digest and mass spectrometry (PCF, EMBL Heidelberg) (C) Analysis identified N- and

C-terminal peptides from JARID2 form a soluble 50 kDa fragment.

17  

4.2 Full length JARID2 in insect cells – investigating solubility

I expressed full length JARID2 using the MultiBacTM baculovirus expression system due to

numerous advantages over E. coli expression. The insect cell system possesses eukaryotic

protein processing capabilities (Jarvis, 2009), including post-translational modifications which

could enable JARID2 to adopt its native structure.

Small-scale expression screening after infecting with V1 J2 virus showed some soluble protein

after sonication and centrifugation. This protein was detected by anti-Strep Western blotting

(Fig. 4A). Expression of soluble protein was low compared to expression in total cell lysate,

confirmed by antibody staining.

Under larger scale expression conditions, JARID2 was insoluble after sonication and

centrifugation (Fig. 4B). I hypothesised that this could be due to cell lysis conditions. The

predicted pI (isoelectric point) of JARID2 is 9.44 (Gasteiger E. Gattiker A., Duvaud S., Wilkins

M.R., Appel R.D., Bairoch A., 2005). To investigate the effect of lysis buffer pH on the solubility

of the sample, I screened five different lysis buffers below the theoretical pI (Fig. 4C). From this

data, it can be concluded that it is not the pH of the lysis buffer which is limiting the solubility of

the sample. I then considered the physiological nature of JARID2 and how this could be

affecting its solubility in vitro.

JARID2 is a nuclear protein (Lee et al., 2000) and has five predicted nuclear localization

sequences (Kosugi et al., 2009). It is possible JARID2 was retained in the insoluble fraction due

to its strong tendency to bind to nuclear chromatin. Therefore, I carried out a nuclear purification

with the theory that less intense lysis could release some protein in the soluble fraction. Figure

5A outlines the cell lysis process. JARID2 is expressed in the total cell lysate, but not expressed

in the cytosolic fractions, which include five steps of centrifugation and resuspension. After the

fifth lysis step, the remaining cell pellet is centrifuged. On a small scale, JARID2 could be

isolated in the supernatant after the sixth centrifugation step (Fig. 5A, 5B). After isolating some

soluble full length JARID2 by this protocol, I used a Ni-NTA pull-down assay to see if the soluble

protein could be obtained in a purer form (Fig. 5C). The initial solubility of JARID2 on a small

scale was not reproducible, and the protein is clearly retained in the cell pellet fraction.

Following this, I identified an over-expressed, soluble protein of around 36 kDa. This protein

appeared to have some Ni-NTA binding capability, as not all protein was found in the flow-

through from the Ni-NTA column. This protein was isolated in the high-salt wash from the Ni-

18  

NTA column, where around half of the protein was eluted. I confirmed it was not a Sf21 protein

by extracting a sample of non-infected Sf21 cells and analysing the sample on SDS-PAGE (Fig.

5C). Therefore, I hypothesised that this protein could be a soluble fragment of JARID2. I sent

this protein for analysis by in-gel tryptic digestion coupled to mass spectrometry. The results

confirmed it was not a Sf21 cell protein, but also that it was not a fragment of JARID2. The band

corresponding to a soluble, 31 kDa Autographa californica nucleopolyhedrovirus (AcMNPV)

nuclear matrix-associated phosphoprotein. The AcMNPV is the baculovirus from which the

MultiBacTM expression system is based, which explains the presence of a protein which is not a

fragment JARID2, nor a native Sf21 protein.

19  

Figure 4. Expression of full length JARID2 in insect cells. (A) Anti-Strep western blot of a small scale expression

of JARID2. Antibody detected in total cell lysate and some soluble protein in the supernatant. (B) Anti-Strep western

blot depicting JARID2 expression and insolubility after lysis (Lysis buffer: 50 mM Tris pH 7.5, 100 mM KCl, 0.1%

NP40), sonication and centrifugation. (C) Solubility screening - varying pH of lysis buffer (pH 5.5 – pH 9.5)

(Coomassie gel)

20  

Figure 5. Investigating solubility of full length JARID2 in insect cells. (A) Coomassie gel of J2 nuclear

purification with extensive lysis steps. (B) Anti-Strep Western blot of supernatant sample after nuclear

purification. (C) Nuclear purification with Ni-NTA pull down. An over-expressed and soluble 36 kDa protein can

be isolated in the high salt wash of Ni-NTA column.

21  

4.3 Designing and generating a library of JARID2 constructs

Given the lack of solubility in the full length form, I designed and cloned shorter constructs of

JARID2. I based the design of these constructs on previous literature (Kaneko et al., 2014)

where such shorter forms were used to define the RNA-binding region of JARID2. I focussed on

three shorter constructs, each of which contains important functional domains (Fig. 6). The

LongRBR and ShortRBR both contain the RNA-binding region and PRC2 interaction domain.

However, LongRBR will reportedly bind nucleosomes via its nucleosome interaction domain

(NID) (Son et al., 2013), and ShortRBR will not. The Cterm construct encompasses the

predicted ordered C-terminal half of JARID2, which I would expect to be a suitable target for

structural studies. These constructs were cloned from the pET26G-J2 vector by SLIC cloning for

expression in E. coli, and all contain the His- and Strep-tags with a TEV cleavage site at the N-

terminus.

4.4 Characterisation of ShortRBR

The shortest fragment of JARID2 I expressed is from the ShortRBR construct. This 29.3 kDa

protein contains parts of the PRC2 interaction domain and the RBR. ShortRBR was expressed

in BL21(DE3) and BL21(DE3)Rosetta cells (Fig. 7A) with increasing concentrations of IPTG and

cultures grown under four conditions. Optimal induction conditions were used to induce 50 mL

Figure 6. Schematic of JARID2 constructs. All constructs cloned with N-terminal 10xHis tag and TEV cleavage site, and

C-terminal Strep-tag. Full length human JARID2 cloned in E. coli expression vector pET26G and pACEbac1 acceptor

vector for insect cell expression.

22  

cell cultures. Coomassie gel and anti-Strep Western blotting confirmed induction with 0.4 mM

IPTG followed by overnight incubation at 18°C produced the most soluble ShortRBR (Fig. 7B).

I trialled different purification strategies for ShortRBR, focussing on Strep and Ni-NTA affinity

columns and size exclusion chromatography. Figure 8A outlines the elution profile from a Ni-

NTA and Strep column purification from 1 L of cell culture. The ShortRBR present in the 200

mM and 400 mM imidazole elutions from Ni-NTA column was not eluted in the flow through, the

washing step or eluted during the Strep purification. The next strategy was to use only the Ni-

NTA column for initial purification. This yielded less pure samples, but ShortRBR was enriched

in the 200 mM and 400 mM imidazole elutions (Fig. 8B). I applied a size exclusion step to

separate contaminants and to analyse any aggregation in the sample. The resulting

chromatogram (Fig. 8D) shows significant aggregation. The fractions eluted from the S75

column were analysed by anti-Strep Western blotting (Fig. 8C, right) to confirm where ShortRBR

is eluted. It can be concluded that ShortRBR is aggregating as it is eluted close to the column

void volume.

23  

Figure 7. Characterisation of ShortRBR – induction and solubility optimisation. (A) Expression of ShortRBR (29.3

kDa) in BL21(DE3)Rosetta cells. Expression induced with increasing IPTG concentration (0.2 mM, 0.4 mM, 1 mM) and

cells incubated at 37°C and 18°C for varying time periods as indicated (anti-Strep Western blot). (B) Solubility screening

with 50 mL cell cultures in BL21(DE3) and BL21(DE3)Rosetta cells. Total cell lysate and supernatant (after sonication

and centrifugation) samples used to analyse amount of soluble ShortRBR (anti-Strep Western blot).

24  

Figure 8. Characterisation of ShortRBR –

purification. (A) Ni-NTA and strep column

purification. ShortRBR (29.3 kDa) eluted mainly in

200 mM and 400 mM imidazole elutions from Ni-NTA

column, and put on strep column after desalting with

PD10 column. Protein not detected in flow through,

wash or elution of strep column (anti-strep Western

blot). (B) Ni-NTA affinity purification. (200 mL cell

culture) Protein enriched in 200 mM and 400 mM

imidazole elutions (anti-strep Western blot). (C) (D)

Larger scale Ni-NTA affinity purification (1 L cell

culture) followed by size exclusion chromatography

(Superdex S75 10/300). (C) Elution profile of Ni-NTA

column (left) and elution profile from size exclusion

chromatography. ShortRBR in fractions 9, 10, 11

(anti-strep Western blot) (D) Gel filtration

chromatogram with concentrated 400 mM Ni-NTA

elution (0.259 mg/mL). ShortRBR present in

aggregation peak confirming ShortRBR aggregation.

25  

4.5 Characterisation of Cterm

The largest in the library of shorter JARID2 constructs is Cterm. It has a theoretical molecular

weight of 83.2 kDa and contains the region of JARID2 which is proposed to be more structurally

ordered (Fig. 2A). This makes it an optimal target for biophysical and structural studies. It does

not contain the RBR, so it is an ideal candidate for use as a negative control in RNA-binding

studies. Cterm was expressed in BL21(DE3) and BL21(DE3)Rosetta cells (Fig. 10A). Higher

expression was seen with growth at 37°C. I investigated four induction conditions to analyse the

solubility of Cterm by comparing the amount of protein present in the supernatant and cell pellet

after sonication and centrifugation. The presence of Cterm was confirmed by anti-Strep Western

blotting (Fig. 9B). The total cell lysate contained a <50 kDa protein fragment detected only by

anti-Strep antibody and not anti-His, suggesting it is a degraded fragment incorporating the C-

terminus of Cterm. However, this degraded protein or potential contaminant was not present in

the soluble fraction of Cterm. Cterm was soluble with 0.2 mM IPTG induction and incubation at

37°C for two hours.  

These induction conditions were used to prepare all samples used for purification trials. The first

purification strategy I applied was a Strep affinity column. Cterm was not binding to the Strep

column, as protein could be seen in the flow through and washing elutions (Fig. 10A). The next

strategy was to use a Ni-NTA affinity column. The protein bound to the Ni-NTA column, with

only a small amount of protein lost in the flow through (Fig. 10B). Cterm could be isolated in the

200 mM and 400 mM imidazole elutions (Fig. 10B). These fractions did contain some

degradation (~36 kDa) detected by anti-Strep Western blotting. These fractions were

concentrated before injection onto a size exclusion column. The concentration step eliminated

the smaller contaminating fragments (Fig. 10C). The size exclusion profile (Fig. 10D) reveals the

heterogeneity of the sample. There is also some aggregation. I analysed the size exclusion

fractions by Coomassie stained gels and anti-Strep Western blotting, but the position of Cterm

in the elution profile could not be elucidated. This is likely due to low protein concentrations after

gel filtration. From this data, it is not clear whether Cterm is aggregating or not. Future

experiments would include the addition of an ion-exchange or heparin chromatography step, to

improve the homogeneity of the sample prior to gel filtration. In addition, purification would need

to be carried out at a larger scale to enable sufficient analysis of the behaviour and potential

aggregation of Cterm throughout the purification.

26  

Figure 9. Characterisation of Cterm - induction and solubility optimisation. (A) Expression of Cterm

(83.2 kDa) in BL21(DE3) cells. Expression induced with increasing IPTG concentration (0.2 mM, 0.4 mM, 1

mM) and cells incubated at 37°C and 18°C for varying time periods as indicated (anti-Strep Western blot). (B)

Solubility screening (200 mL cell cultures) with BL21(DE3) and BL21(DE3)Rosetta cells. Cterm most soluble

with 0.2 mM IPTG induction and incubation at 37°C/2h (anti-Strep Western blot).  

27  

Figure 10. Characterisation of Cterm – purification. (A) Strep affinity purification. Cterm (83.2 kDa)

not bound to Strep column, eluted in flow-through and in column washing. Identification of Cterm

identified by anti-Strep (left) and anti-His (right) Western blotting. (B) Ni-NTA affinity purification. Cterm

eluted in 200 mM and 400 mM imidazole elutions from Ni-NTA matrix (anti-Strep Western blot). (C)

Concentrated 200 mM and 400 mM imidazole Ni-NTA elutions injected on Superdex S200 (anti-Strep

Western blot). (D) Size exclusion chromatogram showing aggregation and a heterogeneous sample.

28  

4.6 Characterisation of LongRBR

LongRBR (53.2 kDa) contains the full RNA-binding region and the nucleosome interaction

domain. Of the shorter constructs, characterisation of LongRBR will give the most insight into

JARID2 function. I expressed LongRBR in E. coli, and it was well expressed in BL21(DE3) cells

(Fig. 11A). A soluble sample of LongRBR could be isolated after overnight growth at 18°C (Fig.

11B), although around 50% of the expressed protein is retained in the insoluble fraction.

29  

Figure 11. Characterisation of LongRBR – expression and solubility optimisation. (A) Expression of

LongRBR (53.2 kDa) in BL21(DE3) cells. Expression induced with 1 mM IPTG with increasing incubation times. At

37ºC, cells were incubated for 2h, 4h and 6h. At 18ºC expression was analysed after overnight incubation. Anti-

Strep Western blot (left) and anti-His Western blot (right) confirm presence of both protein tags. Anti-His Western

blot shows less degradation of LongRBR when grown at 18°C. (B) Solubility screening with BL21(DE3) and

BL21(DE3)Rosetta cells, both induced with 1 mM IPTG and grown at 18°C overnight. LongRBR is partially soluble

in both cell types, some protein is retained in insoluble fraction after centrifugation. LongRBR has higher levels of

expression in BL21(DE3) cells.

30  

For initial purification screening, I used Strep and Ni-NTA affinity columns. LongRBR was

purified after Strep and Ni-NTA affinity chromatography, but the sample eluted in 200 mM and

400 mM imidazole was degraded from the C-terminus (Fig. 12A). Western blotting also showed

insufficient binding to the Strep column, LongRBR could be identified in the flow-through and

washing steps with the Strep column. To eliminate this loss of protein at an early stage in the

purification, I focussed on Ni-NTA affinity. Here, LongRBR was eluted in all imidazole elutions,

but still degrading. To eliminate the degraded, shorter fragments, I added a size exclusion step.

The sample was not aggregating, and after size exclusion chromatography, LongRBR could be

identified by Western blotting (Fig. 12C). To analyse the stability of the sample, I re-injected it on

a size exclusion column after one week (Fig. 12D). The sample gave a homogeneous peak,

suggesting it is stable after one week, which is important for future biochemical assays.

However, Coomassie gel analysis showed the impurity of the sample and Western blotting

confirmed the degraded proteins had not been removed by size exclusion chromatography.

A purer form of LongRBR was required for it be used in further biochemical and biophysical

studies. Therefore, an ion-exchange step was added to eliminate the contaminants which

remained after Ni-NTA chromatography. The pI of LongRBR is predicted to be 9.96, 2 pH units

above the working pH of 7.5. Therefore, a cation exchange step was introduced. LongRBR was

eluted from the column at 250 mM NaCl (Fig. 13A, 13C). The protein yield in this sample was

very low, and after concentration and injection on size exclusion column LongRBR could no

longer be detected. I then screened a range of ion exchange conditions, including anion

exchange and varying the strength of the ion exchanger. Heparin affinity chromatography was

also used following the rationale that a DNA-binding protein such as LongRBR should have an

affinity for the heparin matrix. In the running conditions I trialled, it did not bind the heparin

column. Strong anion exchange gave the highest yield of LongRBR. It was eluted with 300 mM

NaCl (Fig. 14A), confirmed by Western blotting (Fig. 14C). I analysed this sample by size

exclusion chromatography (Fig. 14B), where LongRBR shows no aggregation. The sample is

not homogeneous, and degradation is seen throughout the purification by a double band.

Given the low yield obtained after ion-exchange and size exclusion chromatography, a Strep

affinity step was once again added to the purification. Previously, in purifications from 1 L of cell

culture, the sample was pure on a Coomassie but the concentration was too low to give a 280

nm signal when analysed by Superdex 200 10/300 size exclusion. Therefore, the sample was

analysed with a Superdex 200 5/150 Increase column, which requires a lower volume of the

sample so a more concentrated sample could be injected. Anti-Strep antibody staining shows a

31  

large proportion of soluble protein not binding to the Strep column, but there is still some soluble

protein which can be enriched after Strep and Ni-NTA affinity purification (Fig. 15A, Fig. 15B).

The 400 mM elution from the Ni-NTA column was concentrated and injected onto the Superdex

200 5/150. The sample was injected at concentrations between 0.255 mg/mL and 1.66 mg/mL

(from purifications using 1L, 3L and 6L of cell culture), the sample was not aggregating and

could be isolated in the first peak in the size exclusion chromatogram (Figure 15C). The

resulting size exclusion fractions were combined and concentrated and showed anti-Strep

staining (Fig. 15B). Figure 15D shows the level of purity which could be achieved after a three

step, Strep, Ni-NTA, and size exclusion purification. To increase the scale of the purification, I

used 6 L of cell culture to purify LongRBR for analysis by Superdex 200 10/300. The sample

was concentrated to 2.7 mg/mL and injected at this concentration. The resulting chromatogram

showed aggregation, but LongRBR could still be isolated in fractions corresponding to peaks

after the initial aggregation peak. Further analysis is required to determine the aggregation

behaviour of the LongRBR at higher concentrations.

In summary, the purity of LongRBR is enriched after a three-step purification protocol. However,

after the soluble fraction is added to the Strep column, a double band (which is stained after

incubation with an anti-Strep antibody) is observed to be the most highly expressed throughout

the purification. Therefore, this purification strategy is producing two proteins, which differ in size

by less than 5 kDa. I predicted the higher of the two bands to correspond to LongRBR, as it has

the same migration pattern as the protein present in the total cell lysate and supernatant

fractions (confirmed by Western blotting). I predicted the slightly (~3 kDa) smaller protein to be a

degraded fragment of LongRBR, degraded from the N-terminus as it still contains the C-terminal

Strep-tag (given its detection by anti-Strep Western blotting). In-gel trypsin digestion/mass

spectrometry analysis confirmed that the higher band (Fig. 15D) contains LongRBR. Mass

spectrometry confirmed the presence of 42 unique peptides corresponding to 65% of the

LongRBR protein. In addition, the lower band also contains LongRBR, with 38 unique peptides

corresponding to 59.4% of the sequence. The peptides identified for each band span the same

range of 468 amino acids, which includes 95.7% of the LongRBR construct. This analysis

confirmed the presence of LongRBR in both bands.

From this data, no proteolysis can be confirmed. There are no lysine or arginine residues until

K34 in the sequence, therefore any degradation from the N-terminus which corresponds to only

3 kDa cannot be identified by trypsin digestion. Alternative enzymes could be used for further

proteomics analysis, for example, GluC is a serine protease which will cleave at the carboxyl

32  

side of aspartic acid and glutamic acid residues (pH depending) (Giansanti et al., 2016). This

would be useful in the case of LongRBR because its N- and C-termini contain aspartic and

glutamic residues out of the range covered by trypsin digestion. This further analysis would

allow the proteolysis of LongRBR to be confirmed. Future constructs will then be designed,

avoiding regions of LongRBR which are susceptible to degradation.

33  

Figure 12. Characterisation of LongRBR – optimising purification. (A) Anti-Strep Western blot outlining

purification process with Strep and Ni-NTA affinity columns. LongRBR eluted in E200 and E400 imidazole elutions.

(B) Anti-Strep Western blot outlining Ni-NTA column only purification steps. Protein eluted in all imidazole elutions.

(C) Size exclusion chromatography (S200 10/300) after Ni-NTA elution. LongRBR not aggregating and detected on

anti-Strep Western blot (right), mainly in Fraction 12. (D) Re-injection of LongRBR Fraction 12 from SEC after one

week. LongRBR not aggregating and present in low, impure fractions after second size exclusion step (anti-Strep

Western blot (right)).

34  

Figure 13. Characterisation of LongRBR – cation exchange purification. (A) Strong cation exchange (HiTrap

SP FF 1 mL) chromatogram. LongRBR eluted with ~250 mM NaCl, confirmed by anti-strep Western blot (C). This

sample injected onto size exclusion column, (B) is resulting gel filtration chromatogram. (C) Anti-Strep Western

blotting of LongRBR eluted from strong cation exchange, concentrated to 0.249 mg/mL. 50 µL used for analytical

size exclusion chromatography (S200 5/150), fractions corresponding to 280 nm peak (B) concentrated. Low

anti-Strep antibody signal after gel filtration.

35  

Figure 14. Characterisation of LongRBR – anion exchange purification. (A) Strong anion exchange (HiTrap

Q FF 1 mL) chromatogram. LongRBR eluted with ~300 mM NaCl. (B) Size exclusion chromatogram (S200

5/150), 50 µL LongRBR isolated from strong anion exchange concentrated to 0.273 mg/mL. Elution

corresponding to the peaks between 1.25 and 2 mL VE concentrated to 0.075 mg/mL. (C) Anti-Strep Western

blotting of LongRBR eluted from strong anion exchange and size exclusion injection and elutions. LongRBR is

not detected after gel filtration.

36  

Figure 15. Obtaining a pure form of LongRBR. (A) Coomassie gel with samples from Strep, Ni-NTA and size

exclusion purification. (B) Anti-Strep Western blot with samples from Strep, Ni-NTA and size exclusion

purification, LongRBR can be isolated in after size exclusion and gives a double band with anti-Strep staining. (C)

A typical size exclusion chromatogram for LongRBR sample eluted from Ni-NTA column with 400 mM imidazole

(1.6 mg/mL). LongRBR is present in the peak eluted between 1.5 and 2 mL, confirmed by Western blot (B). (D)

Coomassie gel showing purity obtained after size exclusion chromatography.

37  

5. Discussion and outlooks

Despite extensive studies into Polycomb-mediated repression, JARID2 remains enigmatic, both

structurally and functionally. Therefore, the goal of this study was to obtain a pure form of

JARID2 which could be used for biochemical and biophysical characterisation to further

understand its role in regulating gene expression with lncRNAs and Polycomb group proteins.

I have shown that full length JARID2 is expressed in E. coli and in Sf21 cells. Under the

experimental conditions I used, full length JARID2 was insoluble in E. coli. However, using the

E. coli expressed form of JARID2 I uncovered a potential interaction between the N-terminus

and C-terminus of JARID2 by trypsin digestion and mass spectrometry. This interaction may be

protecting important domains from protease digestion and encompasses the RNA-binding

region which we are most interested in characterising. This interaction could be analysed

further, and future studies could include the co-expression of the N-terminus and C-terminus to

produce a soluble, stable form of JARID2.

After expression in insect cells, JARID2 was insoluble. I reasoned that perhaps its chromatin

binding function and nuclear localisation were causing JARID2 to be retained in the insoluble

fraction after centrifugation. An alternative lysis method was used and was able to obtain a low

amount of soluble protein. However, this solubility was not reproducible on a larger scale.

Further studies should focus on screening further solubility conditions of this large 142 kDa

protein. A long-term goal in our lab is to reproduce a ribonucleoprotein in vitro. This would

involve the co-expression of JARID2 with lncRNAs and PRC2 for structural and functional

characterisation. The MultiBacTM expression system is used for co-expression of multi-subunit

complexes or binding partners, and this JARID2 construct is compatible with this expression

system for future studies.

JARID2 is predicted to be disordered (Fig. 2), which can explain some of the difficulties in

expressing and purifying JARID2. In the cell, IDPs exist largely in a bound state, which explains

the high number of JARID2 binding partners, particularly in its unstructured N-terminal region.

When over-expressed and purified, JARID2 is produced without such binding partners and so

probably adopts a flexible undefined structure. This open structure is then more susceptible to

proteases (Suskiewicz et al., 2011) and this could explain why degradation is observed

throughout the expression and purifications of my constructs.

38  

Therefore, the project will progress by using the ESPRIT (expression of soluble proteins by

random incremental truncation) platform, developed by EMBL as part of the Partnership for

Structural Biology (PSB) in Grenoble. This is a novel, library-based screening technology which

uses truncations of the target gene to design potential expression constructs. The platform

assesses expression, yield and solubility of 30, 000 clones. This will identify new JARID2

targets which could be more suitable for structural characterisation.

In this work, I have expressed and enhanced the purification of three shorter constructs of

JARID2 which encompass important functional regions. Due to the large number of JARID2

binding partners, each construct can be used to analyse its interaction with a different player in

PcG-mediated gene repression.

Cterm and ShortRBR are the least characterised of my constructs. They are both well

expressed in E. coli, but the majority of the protein is insoluble. For the scope of this study, I

obtained enough soluble protein to work with and purify the proteins, but increasing the amount

of soluble protein gained would enhance future studies. For example, a maltose-binding protein

(MBP) tag could be added. This would be more appropriate for ShortRBR due to the large size

(42.5 kDa) of the MBP tag, and this could reduce the aggregation (Lebendiker and Danieli,

2014) seen in the current construct (Fig. 8D). For Cterm, larger scale purifications need to be

carried out to enable identification after gel filtration. Size exclusion chromatograms show some

aggregation but it is not clear whether Cterm is aggregating or not. In addition, an ion-exchange

or heparin chromatography step (given the presence of two DNA-binding domains) should be

added to increase the purity of the sample prior to gel filtration. These constructs could also be

expressed using the MultiBacTM insect cell expression system to determine if the solubility or

yield can be increased.

For LongRBR, I have optimised the purification protocol after trialling various affinity

chromatography techniques. LongRBR is enriched (Fig. 15D) after purification with Strep, Ni-

NTA and size exclusion chromatography. Further proteomic analysis will be carried out to

determine if LongRBR is undergoing degradation throughout the purification to give rise to two

species of LongRBR. If this is the case, the construct will be modified to remove the N-terminal

residues to eliminate proteolysis from the purification and to produce one pure form of LongRBR

for use in functional assays. Furthermore, the His-tag could be moved to the C-terminus, or a

Cobalt resin could be used. In the current sample, there is no aggregation when injected on a

size exclusion column in concentrations up to 1.6 mg/mL, and preliminary tests of stability have

39  

shown the sample to be stable after five days. From these results, it can be concluded that

LongRBR is a promising target for lncRNA and nucleosome binding studies. Additionally,

biophysical techniques, such as SEC-MALLS, will be used to determine the viability of

LongRBR for structural studies.

The intrinsically disordered nature of JARID2 can begin to explain some of the characteristics I

have observed in the three constructs. Firstly, intrinsically disordered proteins (IDPs) are more

prone to aggregation (Uversky and Dunker, 2012), which could explain the aggregation of

ShortRBR. In size exclusion chromatography, IDPs do not behave like globular proteins, so are

eluted at an elution volume which corresponds to a higher molecular mass. I have seen this

characteristic with LongRBR (53.2 kDa), where it shares an elution volume with proteins of >175

kDa. This anomalous passage through gel filtration columns has made it hard to isolate the

constructs from a heterogeneous sample, particularly in the case of Cterm. In addition, flexible

structure increases the accessibility for proteases, which could explain why the purest form of

LongRBR contains a slightly smaller, degraded fragment. However, despite the difficulties in

purifying these constructs, the disorder of the N-terminal of JARID2 makes it even more

important to study its structure. This will require implementing biophysical techniques such as

NMR and SAXS/SANS to capture the dynamic structure of JARID2.

In our lab, we are studying lncRNA MEG3, which has been identified as JARID2-interacting in

vitro and in vivo. The LongRBR construct, which contains the full RBR, will be used to confirm

previously shown RNA-binding (Kaneko et al., 2014). Such assays will confirm LongRBR is

actively binding lncRNAs, and thus can then be used to identify the specific structural motifs in

MEG3 which are responsible for JARID2 binding.

With a pure form of JARID2, such as the LongRBR, we can begin to answer some of the

important questions which can explain the fundamental mechanisms of Polycomb group-

mediated gene repression.

40  

6. References

Bieniossek, C. et al. (2011) ‘MultiBac: expanding the research toolbox for multiprotein

complexes’, Trends in Biochemical Sciences, 37, pp. 49–57. doi: 10.1016/j.tibs.2011.10.005.

Blackledge, N. P. et al. (2014) ‘Variant PRC1 Complex-Dependent H2A Ubiquitylation Drives

PRC2 Recruitment and Polycomb Domain Formation’, Cell, 157, pp. 1445–1459. doi:

10.1016/j.cell.2014.05.004.

Bonasio, R. et al. (2014) ‘Interactions with RNA direct the Polycomb group protein SCML2 to

chromatin where it represses target genes’, eLife. doi: 10.7554/eLife.02637.

Brocchieri, L. and Karlin, S. (2005) ‘Protein length in eukaryotic and prokaryotic proteomes’,

Nucleic Acids Research, 33(10), pp. 3390–3400. doi: 10.1093/nar/gki615.

Cao, J. (2014) ‘The functional role of long non-coding RNAs and epigenetics’, 16, pp. 1–13. doi:

10.1186/1480-9222-16-11.

Christophersen, N. S. and Helin, K. (2010) ‘Epigenetic control of embryonic stem cell fate’, The

Journal of Experimental Medicine, 207(11), pp. 2287–2295. doi: 10.1084/jem.20101438.

Cooper, S. et al. (2016) ‘Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk

between Polycomb complexes PRC1 and PRC2’, Nature Communications. Nature Publishing

Group, 7, p. 13661. doi: 10.1038/ncomms13661.

Dosztányi, Z. et al. (2005) ‘IUPred: web server for the prediction of intrinsically unstructured

regions of proteins based on estimated energy content’, BIOINFORMATICS APPLICATIONS

NOTE, 21(16), pp. 3433–343410. doi: 10.1093/bioinformatics/bti541.

Escamilla-Del-Arenal, M. et al. (2011) ‘Evolutionary diversity and developmental regulation of X-

chromosome inactivation’. doi: 10.1007/s00439-011-1029-2.

Gasteiger E. Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A., H. C. (2005) ‘Protein

Identification and Analysis Tools on the ExPASy Server’, in The Proteomics Protocols

Handbook, Humana Press. Humana Press, pp. 571–607. doi: 10.1385/1-59259-890-0:571.

Giansanti, P. et al. (2016) ‘Six alternative proteases for mass spectrometry–based proteomics

beyond trypsin’, Nature Protocols, 11. doi: 10.1038/nprot.2016.057.

Gregory, S. L. et al. (1996) ‘Characterization of the dead ringer Gene Identifies a Novel, Highly

41  

Conserved Family of Sequence-Specific DNA-Binding Proteins’, MOLECULAR AND

CELLULAR BIOLOGY, 16(3), pp. 792–799. Available at:

http://mcb.asm.org/content/16/3/792.full.pdf (Accessed: 17 May 2017).

Jarvis, D. L. (2009) ‘Chapter 14 Baculovirus-Insect Cell Expression Systems’, Methods in

Enzymology, pp. 191–222. doi: 10.1016/S0076-6879(09)63014-7.

Justin, N. et al. (2016) ‘Structural basis of oncogenic histone H3K27M inhibition of human

polycomb repressive complex 2’, Nature Communications. Nature Publishing Group, 7, p.

11316. doi: 10.1038/ncomms11316.

Kaneko, S. et al. (2014) ‘Interactions between JARID2 and Noncoding RNAs Regulate PRC2

Recruitment to Chromatin’, Molecular Cell, 53(2), pp. 290–300. doi:

10.1016/j.molcel.2013.11.012.

Kim, T.-G. et al. (2003) ‘JUMONJI, a Critical Factor for Cardiac Development, Functions as a

Transcriptional Repressor*’. doi: 10.1074/jbc.M307386200.

Klose, R. J., Kallin, E. M. and Zhang, Y. (2006) ‘JmjC-domain-containing proteins and histone

demethylation’, Nature Reviews Genetics, 7(9), pp. 715–727. doi: 10.1038/nrg1945.

Kosugi, S. et al. (2009) ‘Systematic identification of cell cycle-dependent yeast

nucleocytoplasmic shuttling proteins by prediction of composite motifs.’, Proceedings of the

National Academy of Sciences of the United States of America. National Academy of Sciences,

106(25), pp. 10171–6. doi: 10.1073/pnas.0900604106.

Lebendiker, M. and Danieli, T. (2014) ‘Production of prone-to-aggregate proteins’, FEBS

Letters. doi: 10.1016/j.febslet.2013.10.044.

Lee, L. Y. et al. (2000) ‘a Nuclear Protein That Is Necessary for Normal Heart Development

Jumonji, a Nuclear Protein That Is Necessary for Normal Heart Development’, Circ Res, 86, pp.

932–938. doi: 10.1161/01.RES.86.9.932.

Lewis, E. B. (1978) ‘A gene complex controlling segmentation in Drosophila.’, Nature,

276(5688), pp. 565–70. doi: 10.1128/AAC.03728-14.

Margueron, R. et al. (2009) ‘Role of the polycomb protein EED in the propagation of repressive

histone marks’, Nature. Nature Publishing Group, 461(7265), pp. 762–767. doi:

10.1038/nature08398.

42  

Miller, J., Mclachlan, A. D. and Klug, A. (1985) ‘Repetitive zinc-binding domains in the protein

transcription factor LiA from Xenopus oocytes’, The EMBO Journal, 4(6), pp. 1609–1614.

Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC554390/pdf/emboj00271-0237.pdf

(Accessed: 17 May 2017).

Müller, J. and Kassis, J. A. (2006) ‘Polycomb response elements and targeting of Polycomb

group proteins in Drosophila’, Current Opinion in Genetics and Development, pp. 476–484. doi:

10.1016/j.gde.2006.08.005.

Nayak, V., Xu, C. and Min, J. (2011) ‘Composition, recruitment and regulation of the PRC2

complex’, Nucleus, 2(4), pp. 277–282. doi: 10.4161/nucl.2.4.16266.

Pasini, D. et al. (2010) ‘JARID2 regulates binding of the Polycomb repressive complex 2 to

target genes in ES cells.’, Nature, 464(7286), pp. 306–310. doi: 10.1038/nature08788.

Patsialou, A., Wilsker, D. and Moran, E. (2005) ‘DNA-binding properties of ARID family

proteins’, Nucleic Acids Research. doi: 10.1093/nar/gki145.

Peng, J. C. et al. (2009) ‘Jarid2/Jumonji Coordinates Control of PRC2 Enzymatic Activity and

Target Gene Occupancy in Pluripotent Cells’, Cell, 139(7), pp. 1290–1302. doi:

10.1016/j.cell.2009.12.002.

Pufall, M. A. et al. (2005) ‘Variable Control of Ets-1 DNA Binding by Multiple Phosphates in an

Unstructured Region’, Science, 309(5731). Available at:

http://science.sciencemag.org/content/309/5731/142/tab-pdf (Accessed: 12 June 2017).

Sanulli, S. et al. (2015) ‘Jarid2 Methylation via the PRC2 Complex Regulates H3K27me3

Deposition during Cell Differentiation’, Molecular Cell, 57(5), pp. 769–783. doi:

10.1016/j.molcel.2014.12.020.

Shevchenko, A. et al. (2007) ‘In-gel digestion for mass spectrometric characterization of

proteins and proteomes’. doi: 10.1038/nprot.2006.468.

Simon, J. A. and Kingston, R. E. (2009) ‘Mechanisms of Polycomb gene silencing: knowns and

unknowns’, Nature Reviews Molecular Cell Biology. doi: 10.1038/nrm2763.

Son, J. et al. (2013) ‘Nucleosome-binding activities within JARID2 and EZH1 regulate the

function of PRC2 on chromatin’, Genes and Development, 27(24), pp. 2663–2677. doi:

10.1101/gad.225888.113.

43  

Surade, S. et al. (2006) ‘Comparative analysis and “expression space” coverage of the

production of prokaryotic membrane proteins for structural genomics.’, Protein science : a

publication of the Protein Society, 15(9), pp. 2178–2189. doi: 10.1110/ps.062312706.

Suskiewicz, M. J. et al. (2011) ‘REVIEW Context-dependent resistance to proteolysis of

intrinsically disordered proteins’. doi: 10.1002/pro.657.

Takeuchi, T. et al. (1995) ‘Gene trap capture of a novel mouse gene, jumonji, required for neural

tube formation’, Genes and Development. doi: 10.1101/gad.9.10.1211.

Teixeira da Rocha, S. et al. (2014) ‘Jarid2 Is Implicated in the Initial Xist-Induced Targeting of

PRC2 to the Inactive X Chromosome’, Molecular Cell, 53, pp. 301–316. doi:

10.1016/j.molcel.2014.01.002.

Toyoda, M. et al. (2003) ‘jumonji Downregulates Cardiac Cell Proliferation by Repressing cyclin

D1 Expression During this phase, growth signals are transduced from the extracellular

environment, ultimately reaching the cell cycle machinery operating in the cell nucleus, which’,

Developmental Cell, 5, pp. 85–97. Available at: http://www.cell.com/developmental-

cell/pdf/S1534-5807(03)00189-8.pdf (Accessed: 2 June 2017).

Uversky, V. N. and Dunker, A. K. (eds) (2012) Intrinsically Disordered Protein Analysis. New

York, NY: Springer New York (Methods in Molecular Biology). doi: 10.1007/978-1-4614-3704-8.

Wang, H. et al. (2004) ‘Role of histone H2A ubiquitination in Polycomb silencing’, Nature.

Nature Publishing Group, 431(7010), pp. 873–878. doi: 10.1038/nature02985.

7. Acknowledgements

I would like to thank Marco Marcia for this project, and for his supervision and support

throughout the year. I am grateful to all the members of the Marcia group, for their constant

advice and encouragement, particularly Ombeline Pessey for initial lab supervision. I would also

like to thank Alice Aubert (Eukaryotic Expression Facility) for her help with insect cell

expression, and the Proteomics Core Facility at EMBL Heidelberg for the mass spectrometry

analysis.