BTB Domain Dimerization: Development of a Protein-Protein Interaction Assay

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BTB domain dimerization: Development of a protein-protein interaction assay

By Qingniao (Carol) Wang

A thesis submitted in conformity with the requirement for the degree of

Master of Science

Graduate Department of Biochemistry

University of Toronto

BTB domain dimerization: Development of a protein-protein interaction assay

Qingniao (Carol) Wang Masters of Science 2009 Department of Biochemistry University of Toronto

Abstract

In the human genome, 43 BTB (Bric-à-brac, Tramtrack, and Broad Complex) containing BTB-Zinc Finger proteins have been identified, many of which are transcription factors involved in cancer and development. These BTB domains have been shown to form homodimers and heterodimers which raise DNA binding affinity and specificity for transcription factors.

This project was to develop an efficient assay to systematically identify interactions between BTB domains. It combined a co-expression system, fluorescent protein tagging and Ni-NTA plate retention. It was concluded that fourteen analyzed BTB domains formed homodimers, but only certain BTB pairs formed heterodimers, such as BCL6 with Miz1 and Miz1 with RP58. To further understand the specificity of BTB domain interactions, more structural and sequence information is still needed. In conclusion, this assay provided a comprehensive detection method for BTB domain interaction mapping. The information generated provides candidates for further functional and structural studies.

Acknowledgements

This research project would not have been possible without the support of many people. I wish to express my gratitude to my supervisor, Dr. Gil Privé who offered invaluable guidance, assistance and patience. Special thanks are also due to the members of my supervisory committee, Dr. Avi Chakrabarty and Dr. Mitsu Ikura. Without their knowledge and assistance this study would not have been successful.

Deepest gratitude to all past and present members of the Privé lab and the Rose lab who provided a positive atmosphere for knowledge exchange, I learned so much from all these great scientists. Especially Peter Stogios, Tine Nielsen, Alex Ghetu, John Holyoake, Wesley Errington, Dona Ho and Konstatin Popovic for their helpful discussions; Lu Chen and Neil Pomroy for technical supports; as well as Eden Fussner and Seiji Sugiman-Marangos for their input to this project. I would also like to convey my thanks to all labs at 4th floor of TMDT for instrument usages.

Last but not least, I want to thank my beloved families in China and all my friends in Canada for their understanding and endless love throughout these two years.

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Contents

Abstract ...... ii

Acknowledgements ...... iii

Contents ...... iv

List of tables ...... vii

List of figures ...... viii

List of equations ...... ix

List of abbreviations ...... x

Chapter 1: Introduction ...... 1

1.1 Overview of BTB domains ...... 1 1.2 BTB-Zinc Finger proteins ...... 1 1.2.1 BTB-Zinc Finger proteins as transcriptional regulators...... 1 1.2.2 Proteins of interest ...... 3 1.3 BTB domain-domain interactions ...... 5 1.3.1 Transcription factors interactions ...... 5 1.3.2 BTB domain homodimers: functional and structural highlights ...... 8 1.3.3 BTB domain heterodimers: findings and biological roles ...... 11 1.4 Biological roles of 14 BTB domains ...... 13 PLZF ...... 13 BCL6 ...... 15 MIZ1 ...... 16 RP58 ...... 17 BOZF ...... 18 Y441 ...... 19 HKR3 ...... 19 Kaiso ...... 19

ZID ...... 20 FAZF ...... 21 Bioref ...... 22 KUP ...... 22 ZBTB4 ...... 22 LRF ...... 23 1.5 Project goals ...... 25 1.6 Backgrounds in assay development ...... 25 1.6.1 Overview of protein-protein interaction assays ...... 25 1.6.2 Fluorescent protein tags ...... 28 1.6.3 Kinetics in BTB dimer formation ...... 29 1.6.4 Chemical denaturation and refolding ...... 31 1.6.5 The coexpression system ...... 31

Chapter 2: Materials and Methods ...... 33

2.1 Fluorescent protein labeling and optimization ...... 33 2.1.1 Fluorescent protein background minimizations ...... 33 2.1.2 Placement of fluorescent proteins to BTB domains ...... 35 2.2 Dimer formation by chemical denaturation and refolding ...... 35 2.2.1 Protein expression and purification ...... 35 2.2.2 Kinetic studies of BTB domain interactions ...... 36 2.2.3 Dimer formation by chemical denaturation and renaturation ...... 37 2.3 Dimer formation by co-expression system ...... 39 2.3.1 Cloning: insertion of BTB domains into co-expression vectors ...... 39 2.3.2 BTB pairs co-expression in auto-induction media ...... 40 2.3.3 Detection of dimer formation ...... 42 2.3.4 Data analysis ...... 43

Chapter 3: Results ...... 44

3.1 Fluorescent protein interactions ...... 44 3.2 Results from chemical denaturation and refolding assay ...... 45 3.2.1 FP-BTB proteins expression and purification ...... 45 3.2.2 Kinetic studies of BTB domain interactions ...... 46 3.2.3 BTB dimer formation by chemical denaturation and refolding ...... 48 3.3 Results from co-expression and plate retention assay ...... 54 3.3.1 Plate binding background control for fluorescent proteins ...... 54 3.3.2 Dimerization of PLZF, FAZF, Kaiso, LRF, BCL6 and Miz1 ...... 56 3.3.3 Dimerization of 8 other BTB domains ...... 61

Chapter 4: Discussion ...... 67

4.1 Assay development ...... 67 4.1.1 Why do we need a new assay? ...... 67 4.1.2 Troubleshooting in FRET assay development ...... 68 4.1.3 Further improvements in coexpression assays ...... 71 4.2 BTB dimers detected by FRET based assay ...... 75 4.2.1 FAZF homodimers undergo spontaneous exchange ...... 75 4.2.2 BTB homodimers and heterodimers formed after refolding ...... 76 4.2.3 FRET signal variance caused by structural differences ...... 78 4.3 BTB dimer detection by a coexpression assay ...... 79 4.4 Comparison with literature findings ...... 81 4.5 Does BTB dimerization specificity relate to sequences? ...... 82

Chapter 5: Conclusion and future directions ...... 86

5.1 Conclusion: assay development and BTB dimer detection ...... 86 5.2 More BTB domain interaction mapping using this assay ...... 87 5.3 Functional and structural studies on identified heterodimers ...... 88

Reference ...... 89

Appendix ...... 97

List of tables

Table 1: List of BTB-ZF proteins studied in this project...... 4 Table 2: List of proteins used in chemical denaturation and refolding assay ...... 35 Table 3: BTB pairing matrix in kinetic and chemical denaturation and refolding experiments ... 37 Table 4: List of plasmids cloned for co-expression assay...... 40 Table 5: 14×14 BTB coexpression matrix ...... 41 Table 6: Kd of fluorescent protein self-interactions ...... 45 Table 7: Pairs of BTB domains tested in chemical denaturation and refolding assay...... 48 Table 8: Coexpression matrix of 6 BTB domains ...... 56 Table 9: Interaction mapping of 6×6 BTB domains...... 60 Table 10: Interaction mappings of 14×14 BTB domains ...... 66 Table 11: Pair-wise sequence comparisons of 14 BTB domains...... 83

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List of figures

Figure 1: Summary of BTB-ZF proteins studied in this project...... 3 Figure 2: DNA binding and dimerization domains of major eukaryotic TF families...... 6 Figure 3: Crystal structures of 6 BTB homodimers...... 9 Figure 4: Schematic of the BTB fold from BTB-ZF family ...... 10 Figure 5: Dimerization interfaces of BTB domains from PLZF and FAZF...... 11 Figure 6: CFP and YFP excitation spectrum and emission spectrum...... 29 Figure 7: Proposed kinetic pathway in BTB heterodimer formation...... 30 Figure 8: SDS-PAGE gel image of fractions in C-PLZF protein purification...... 46 Figure 9: Total FRET spectrum in BTB kinetic studies...... 47 Figure 10: CD spectroscopy of V-PLZF+PLZF-C before and after refolding...... 49 Figure 11: Total FRET spectra for BTB homodimers formed by denaturation and refolding. .... 50 Figure 12: Total FRET spectra for Miz1-BTB dimers formed by denaturation and refolding..... 51 Figure 13: LRF dimer formed after refolding in a time-course trypsin digests...... 53 Figure 14: BTB domain interaction mapping by chemical denaturation and refolding assay...... 54 Figure 15: Background control of Cerulean and Venus binding to plates...... 55 Figure 16: Data processing of Cerulean and Venus levels observed before and after binding. ... 58 Figure 17: Level of V-BTB retained by six different h-BTB-C in coexpression assay...... 59 Figure 18: Native PAGE images of coexpression supernatant...... 61 Figure 19: Levels of V-BTB retained by 8 other BTB domains...... 64 Figure 20: Proposed structures of BTB homodimers tagged with Venus and Cerulean...... 70 Figure 21: BTB dimers produced after coexpression...... 72 Figure 22: Rationale of Ni-NTA plate retention assay ...... 73 Figure 23: Comparison of four BTB-BTB interaction assays...... 74 Figure 24: Sequence alignments of 14 BTB domains ...... 85

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List of equations

Equation 1: FRET efficiency Q%...... 34 Equation 2: FRET efficiency and Kd fitting equation ...... 34 Equation 3: FRET Emission ratio...... 38

List of abbreviations

AML: Acute Myelogenous Leukaemia

APL: Promyelocytic Leukemia

BCL6: B-cell lymphoma 6

BCoR: BCL6 co-repressor bHLH: basic-region helix-loop-helix

BTB: Bric-à-brac, Tramtrack, and Broad Complex bZIP: basic region leucine zipper

C: Cerulean

CCS-3: cervical cancer suppressor 3

ChIP: chromatin immunoprecipitation

Co-IP: Coimmunoprecipitation ()

DLBCL: diffuse large B cell lymphoma

ECFP: Enhanced Cyan Fluorescent Protein

ER: emission ratio

ETO: Eight Twenty One

FP: Fluorescent Polarization

FRET: Förster resonance energy transfer

GFP: Green Fluorescent Protein

GST: Glutathione-S-Transferase

h-BTB-C: His tagged BTB-Cerulean proteins

HDACs: histone deacetylases

His: Histidine

HKR3: Human Krüppel-Related 3

Kd: disassociation constant

KUP: KrUppel homologous Protein

LRF: Leukemia/lymphoma related factor

MBPs: methyl-DNA-binding proteins

MCR: multi-cloning region

Miz1: Myc-interacting zinc finger protein

NCoR: nuclear co-repressor

OTG: Octyl β-Thioglucopyranoside

PLZF: Promyelocytic leukemia Zinc finger

RARα: retinoic acid receptor alpha

RFI: the relative fluorescent intensity

RFU: the relative fluorescent unit

RP58: Repressor protein with MW 58kDa

SH2: Src homology 2

SMRT: silencing mediator of retinoic acid transduction

SPR: Surface Plasmon Resonance

STAT: signal transducers and activators of transcription

V: Venus

Y2H: yeast two-hybrid

YFP: Yellow Fluorescent Protein

ZF: Zinc Finger

ZID: Zinc finger protein with Interaction Domain

xii Chapter 1: Introduction

Chapter 1

Introduction

1.1 Overview of BTB domains

The BTB domain (also known as POZ domain) was first discovered in 1993 and named after the Drosophila zinc finger transcription factors Bric-à-brac, Tramtrack, and Broad Complex.

This domain is an evolutionary conserved protein-protein interaction motif found throughout the eukaryotes [1]. BTB domains have high sequence variability, but overall tertiary structures are well-conserved [2].

There are 183 different BTB-containing proteins identified in the human genome [3].

These proteins exhibit a wide range of functional roles, such as protein ubiquitination [4], ion channels assembly [5], cytoskeleton regulation [6] and transcription repression [7] .

1.2 BTB-Zinc Finger proteins

1.2.1 BTB-Zinc Finger proteins as transcriptional regulators

1 Chapter 1: Introduction

Among the 183 BTB domain proteins, 43 are found upstream of multiple zinc finger domains, hence the name of BTB-Zinc Finger (ZF) proteins [3]. These BTB-ZF proteins are characterized as important transcription factors implicated in cancer and development. One of the most studied transcriptional factors is B-cell lymphoma 6 (BCL6), a proto-oncoprotein that belongs to the BTB-ZF protein family. BCL6 binds to specific DNA sequences and suppresses transcription by recruitment of histone deacetylases (HDACs) through the co-repressors silencing mediator of retinoic acid transduction (SMRT), BCL6 co-repressor

(BCoR), and nuclear co-repressor (NCoR) [8, 9]. BCL6 is required for germinal center formation, and down-regulation of BCL6 is necessary for B-cell maturation and differentiation [8]. In non-Hodgkin’s lymphoma, particularly diffuse large B-cell lymphomas,

BCL6 is over-expressed due to chromosomal translocations [8].

BTB-ZF proteins share a common architecture: a BTB domain at the N-terminus followed by an unstructured linker region and multiple copies of Kruppel-type Cys2His2

(C2H2) zinc finger domains at the C-terminus [2]. The Zinc finger domain mediates DNA binding, while the BTB domain promotes protein-protein interactions and co-factor recruitment [1, 10, 11]. Once ZF recognizes and binds to the regulatory region of target genes, the BTB domain recruits co-factors, such as SMRT, N-CoR, and HDACs, which prevent transcription by modifying histone structures [8, 9].

Most of these transcription factors are sequence-specific. Interactions mediated by BTB domains, especially self-interactions of BTB domains seem to play a role in regulating gene expressions [1]. These homodimeric, heterodimeric and higher-order associations via BTB domains can direct transcription factors to DNA binding, which leads to raised binding affinities and expanded functions [1]. A better characterization of the BTB domain

2 Chapter 1: Introduction interaction network would allow further understanding of general mechanism in expression regulations by transcription factors.

1.2.2 Proteins of interest

In order to investigate BTB domain interactions, 14 BTB-ZF proteins (Figure 1, Table 1) were selected from 43 human BTB-ZF proteins based on biological significance and evidence of recombinant expressions in E. coli. This section aims to provide a general profile of these proteins. Detailed functional descriptions of each BTB-ZF protein are elaborated in later sections.

Figure 1: Summary of BTB-ZF proteins studied in this project. The overall architecture of BTB-ZF proteins is highly conserved. N-terminal BTB domains colored pink are followed by multiple copies of zinc finger motifs colored blue. The start and end sites of BTB domains are also marked. The BTB domain in ZBTB4 contains an internal gap.

3 Chapter 1: Introduction

BTB ID Name Aliases Expression 1 PLZF Promyelocytic leukemia Zinc finger, ZBTB16, ZNP145 Expressed in hematopoietic system, ovary, intestine, spleen, muscle and heart tissues [12] 2 BCL6 B cell lymphoma 6, Zinc finger protein 51, LAZ-3 protein, ZBTB27 Expressed in germinal center T and B cells, primary in immature dendritic cells [12, 13] 5 MIZ1 Myc interacting zinc finger 1, ZBTB17, ZNP151 Widely expressed, high levels in leukemia chronic myelogeneous cell lines [14]

8 RP58 Repressor protein with MW 58kDa, ZNF238, ZBTB18,TAZ1 Expressed in lymphoid tissues, testis, heart, brain, skeletal muscle, and pancreas [14] 25 BOZF BTB/POZ and zinc-finger domains factor on chromosome 1, ZBTB8 Widely expressed, with high levels in fetal brain, fetal lung and pancreas [14]

28 Y441 ZBTB24, KIAA0441, ZNF450 Widely expressed, with high levels in T-cells and B-cells [14]

29 HKR3 Krüeppel-related zinc finger protein 3, ZBTB48 Ubiquitous expressed, with high levels in human fetal and adult nervous tissues [14] 4 31 Kaiso ZBTB33, ZNF348 Ubiquitously expressed with relatively lower level in brain and testis [14] 34 ZID Zinc finger protein with interaction domain, ZBTB6, ZNP 482 Widely expressed with highest levels in the brain [14] 38 FAZF Fanconi anemia zinc finger, TZFP, ZBTB32 Predominantly expressed in testis, some isoforms are ubiquitously expressed [15] 39 Bioref ZBTB26, ZNF481 Ubiquitous expressed [14] 40 KUP ZBTB25, ZNF46 Expressed mainly in hematopoietic cells and testis [14]

42 ZBTB4 Zinc finger and BTB domain containing 4 Expressed in brain, lung, kidney, muscle heart,placenta, liver, spleen and thymus [15] 46 LRF Leukemia/lymphoma related factor, Pokemon, FBI-1, ZBTB7 Widely expressed in adult tissues and cell lines, up-regulated in various cancer [16]

Table 1: List of BTB-ZF proteins studied in this project. BTB ID, aliases and expression profiles listed here are adapted from BTB database [3], and GNFSymAtlas [14].

Chapter 1: Introduction

1.3 BTB domain-domain interactions

The roles BTB-ZF proteins play in transcription regulation are largely dependent on the versatile interactions mediated by BTB domains. BTB domain-domain interactions are the major focus of this project. From this point on, BTB domains mentioned here all belong to

BTB-ZF family proteins unless stated otherwise.

1.3.1 Transcription factors interactions

In eukaryotic cells, protein expression is tightly regulated at the transcriptional level and post-transcriptional level to achieve phase-specific and location-specific manners [17, 18].

The transcriptional regulation is the first step in the process, where transcription factors (TFs) directly bind to their genomic DNA targets, resulting in an activation or repression of target gene expression. Up to 10% of coding region of the human genome is to TFs [17], and each

TF likely regulates many targets in different gene regulatory contexts. Transcriptional regulation is extremely intricate because it is controlled by post-translational modifications of TFs, interactions with cofactors, thermodynamics of TF-DNA interactions, and TF-TF interactions [18]. The main discussion here is on TF-TF interactions.

Many eukaryotic TFs bind to their target genes as functional dimers, where a physical interaction with an identical molecule or with another molecule within the same family is required [17, 19]. The best studied TF families that form dimers include basic region leucine zipper (bZIP), basic-region helix-loop-helix (bHLH) and signal transducers and activators of transcription (STAT). In humans, these TFs create more than 500 dimers with distinct biological roles including cell cycle, reproduction, development, homeostasis, metabolism,

5 Chapter 1: Introduction immunity, and programmed cell death [19]. In these TF families, DNA binding and dimerization are mediated through highly conserved domains (Figure 2).

Figure 2: DNA binding and dimerization domains of major eukaryotic TF families. BTB-ZF proteins are compared with other well-studied TF families in the literature. All of these TFs have dimerization domains (colored red) and DNA-binding domains (colored blue).

TF dimerization has important functional implications. Homodimer formation is common, sometimes functionally required for TFs. For example, STAT1, STAT 3, STAT4,

STAT5 and STAT6 all have to be phosphorylated to form active homodimers via Src homology 2 (SH2) domains before entering the nucleus and activating transcription [20].

During transcription regulations, formation of homodimers gives TFs higher DNA binding affinity and specificity [19]. On the other hand, TF heterodimers combine TFs with distinct

6 Chapter 1: Introduction

DNA binding specificity, thereby facilitating spatial and indirect control of different DNA elements, and offering a limited number of TFs an expanded functional range. For instance,

Jun and ATF2 bZIP proteins interact through C-terminal leucine zippers, resulting in heterodimers with distinct binding activities from their parental homodimers [21]. The decision of which dimer to form is determined by factors like protein concentrations, posttranslational modifications, and binding affinities [19-21].

TF dimerization makes important contributions to the flexibility and complexity of gene regulation. By forming either homodimers or heterodimers, one TF can have multiple binding partners through its dimerization domain. In theory, N genes of a given TF family could potentially generate N homodimers and N(N-1)/2 unique pairs of heterodimers [19]. In actuality, the structural and physio-chemical specificities required for dimer formation limit binding options. For example, for the 51 bZIPs identified, there are potentially 1326 unique dimers in theory, while only ~350 unique bZIP dimers are estimated by experimental methods [19]. Thanks to recent advances in high-throughput genomic and biophysical methods, more and more dimers have been identified [20-22]. Structural and comparative analysis based on these findings reveals key residues involved in diverse dimerization patterns, which help to refine structural rules that regulate dimerization specificity.

Elucidation of rules of dimerization specificity is crucial not only for understanding biological complexity but also for developing novel therapeutic strategies [19]. In the future, it will be important to comprehensively map all dimerization interactions between TFs and to incorporate this information into construction of transcription regulation network models

[17, 18].

7 Chapter 1: Introduction

1.3.2 BTB domain homodimers: functional and structural highlights

In BTB-ZF proteins, dimerization is mediated by BTB domains, as confirmed by many solution studies and several crystallographic structures of BTB domains.

Comparing numerous in vitro and in vivo studies, BTB-ZF proteins have similar behavior in solution despite their sequence differences [1, 2, 11, 23-30]. More importantly, it is found that BTB-ZF proteins form exclusively homodimers in solution via BTB domains. BTB domain dimerization is functionally required. These homdimers appear to be strong and stable, as monomers of BTB domains are never detected. PLZF is a good representative of the BTB-ZF protein family. Gel filtration, dynamic light scattering, and equilibrium sedimentation experiments show that BTB domain in PLZF forms a homodimer with a Kd below 200 nM [31]. Differential scanning calorimetry and equilibrium denaturation experiments consistently show that PLZF-BTB homodimer undergoes a two-state folding/unfolding transition: the native dimer state and the denatured monomer state with no stable intermediate in between [31, 32].

Crystal structures of BTB domains confirm BTB homodimers in solution, and provide valuable structural information. Figure 3 shows crystal structures of several of the BTB domains mentioned above. These BTB domains contain an N-terminal extension of α1 and

β1 followed by the conserved core elements of three β-strands and five α-helices (Figure 4)

[2].

8 Chapter 1: Introduction

Figure 3: Crystal structures of 6 BTB homodimers. BTB homodimer structures of (A) BCL6 [33], (B) LRF [29], (C) PLZF [34], (D) Kaiso [35], (E) Miz1 [35] and (F) FAZF [35] are shown here. BTB domains all exist as homodimers in the crystal. Moreover, structures of these 6 BTB domains demonstrate highly conserved overall folds.

9 Chapter 1: Introduction

Figure 4: Schematic of the BTB fold from BTB-ZF family This figure is adapted from [2]. BTB domains contain α1 and β1 at the N-terminus followed by series of β strands and α helices in the core. Typical BTB homodimers involve α1 interactions with α2 and α3 from the other chain; as well as the packing of β1 and β5 from the other chain.

The dimer interface of PLZF-BTB demonstrates two important features of BTB homodimers: the packing of α1 from one monomer with α2 and α3 from the other monomer; and an intermolecular association of β1 from one monomer and β5 of the other monomer, which is often referred as strand-exchange (Figure 5A) [2]. Many crystal structures of BTB domains share these two features, such as BCL6 and Miz1. However, the only exception is the BTB domain from FAZF, where dimers are solely hold by interactions between α helices without any strand-exchange (Figure 5B).

Based on information gathered from the dimer interface, many highly conserved residues have been identified. Sequence studies of 43 BTB domains in human BTB-ZF family confirm these residues in predicted dimer interface, thereby proposing that most BTB domains can dimerize via interactions by α1, β1 and β5, similar to the exchanged dimer structure. However, a smaller interface that involves only α1 residues is also possible

10 Chapter 1: Introduction between non-exchanged BTB homodimers, as seen in the FAZF homodimer structure [2].

Figure 5: Dimerization interfaces of BTB domains from PLZF and FAZF. Figure 5A shows the dimer structure of BTB domains in PLZF, where residues involved in dimerization are highlighted in blue. It demonstrates a typical strand-exchanged structure found in many BTB homodimers. The exception is FAZF homodimers, as shown in figure 5B. FAZF BTB domains do not exist as domain-exchanged dimers where residues involved in the interface are colored orange. There is only α1 interaction with α2 and α3 from the other chain, but not the inter-domain β packing found in other BTB domains.

1.3.3 BTB domain heterodimers: findings and biological roles

Heterodimers are readily observed for transcriptional factors in the same family, especially close homologues. As the dimerization module for BTB-ZF proteins, BTB domains are proposed to mediate heterodimeric interactions in the same way as homodimers. BTB domains are highly conserved in their dimer interfaces, which make these domains potentially capable of forming heterodimers. Studies have applied various techniques to detect BTB-ZF heterodimers.

FAZF was first identified as an analogue of PLZF. They share 35% sequence identity in their BTB domains and bind to the same DNA target sequence [36]. One study showed that

11 Chapter 1: Introduction

FAZF formed heterodimers with PLZF based on yeast-two-hybrid and coimmunoprecipitation assays [36]. However, it also showed that this heterodimer was not mediated by BTB domains. Instead, the interaction might be through an unidentified binding intermediate [36].

LRF is another close homologue of PLZF. It was found that LRF did not interact with

PLZF despite their sequence similarities [16]. Instead, LRF was shown to associate with

BCL6, where elevated LRF level was often observed with high levels of BCL6 in many human cancers [16]. In COS-1 cell line, LRF was colocalized with BCL6 in the nucleus [16].

It was still undetermined whether BTB domains or ZF domains were responsible for the interaction based on yeast-two hybrid assay performed [16].

A similar story has been developed for BCL6 and PLZF interactions. Because of their similar functional roles, BCL6 and PLZF was coexpressed and found to colocalize in nuclear dots in many cell lines [37]. This study also pointed out that the colocalization between two proteins did not rely on BTB domains, but two zinc finger regions [37].

Another study proposed that BCL6 heterodimerize with Miz1 in germinal center B cells

[38]. BCL6 and Miz1 were coimmunoprecipitated in B cell lines nuclear extracts with endogenous BCL6 and Miz1. More importantly, chromatin immunoprecipitation (ChIP) indicated that BCL6 was recruited to the CDKN1A promoter region by Miz1. It explained why even without its binding site in the promoter, BCL6 could still repress CDKN1A transcription, which played an essential role in DNA damage-induced p53-independent growth arrest and apoptosis pathways in GC B cells. Results also implied that BTB domains in BCL6 and Miz1 were mediating this heterodimer formation [38].

12 Chapter 1: Introduction

A very recent study performed similar experiments on Miz1 and ZBTB4. Miz1 and

ZBTB4 were coimmunoprecipitated from colon carcinoma cells [39]. In addition, ChIP experiments showed that both Miz1 and ZBTB4 bound to the core promoter of P21CIP1 genes [39]. It was proposed that ZBTB4 heterodimerizes with Miz1 forming a DNA-binding complex, which repressed the p53-mediated P21CIP expressions in multiple human tumors

[39]. Domains involved in Miz1-ZBTB4 heterodimers were not further investigated in this study.

These BTB-ZF heterodimers are identified by different techniques with a wide spectrum of proposed functional roles. Different from homodimers, formation of BTB heterodimers is not solely associated with sequence similarities. This is consistent with some well-studied TF families, where the dimerization specificity is determined by structural and physico–chemical properties of two interacting proteins [19].

As described in Section 1.4, BTB domain mediated interactions are fundamental to their roles in development and many human cancers. Determining how BTB domains control their dimerization specificity is important to understand the BTB-ZF interaction network.

1.4 Biological roles of 14 BTB domains

PLZF

PLZF (Promyelocytic leukemia Zinc finger) protein is encoded by ZBTB16. It is a 673 amino acid transcriptional repressor found in hematopoietic system, intestine, spleen, muscle and heart tissues [40]. Similar to other BTB-ZF proteins, PLZF has an N-terminal BTB domain, which recruits SMRT, NCoR, and HDACs, and nine copies of zinc fingers at the

13 Chapter 1: Introduction

C-terminal. PLZF is implicated in cancer, development and stem cell biology.

The proto-oncogene PLZF was first identified in t(11;17)(q23;q21) chromosomal translocation associated Acute Promyelocytic Leukemia (APL), where PLZF was fused to retinoic acid receptor alpha (RARα) to form the PLZF-RARα product [41]. BTB domains in this fusion protein recruit corepressors and HDACs to RARα target genes, thus inhibiting expression of key genes required for normal myeloid differentiation [42]. A very recent study shows PLZF controls the development of invariant natural killer T cell effector functions

[43]. In addition, PLZF has been implicated in the pathogenesis of Acute Myelogenous

Leukaemia (AML)-associated translocation t(8;21), where DNA binding of PLZF is impaired due to interactions with ETO (Eight Twenty One) protein [44]. A few references have also indicated PLZF roles on solid tumors, such as melanoma tumourigenesis [45] and prostate cancer [46]. A recent study suggests that BTB domain of PLZF interacts with a novel cervical cancer suppressor 3 (CCS-3), which inhibits the cell growth by inducing apoptosis [47].

PLZF acts as a transcriptional repressor through spatial and temporal binding to Hox gene, which is essential for cell fate decision, patterning and embryogenesis [7]. Knock-out mice studies suggest that PLZF inactivation results in altered expression of Hox genes and patterning defects affecting both axial and appendicular skeleton [48]. Interestingly, Hox genes have also been implicated in the control of proliferation and differentiation of primitive haemopoietic cells [7], which may further exacerbate the roles PLZF play in myeloid haemopoietic cells.

PLZF is also proposed to be involved in stem cell biology because it dynamically

14 Chapter 1: Introduction expresses during embryogenesis and it reduces cell proliferation by induction of G0/G1 cell cycle arrest [7]. PLZF is required for self-renewal and maintenance of the stem cell pool by chromatin remodeling and transcriptional regulation during those processes [49].

BCL6

The BCL6 (B-cell lymphoma 6) protein is encoded by ZBTB27. It is a 706 amino acid transcription repressor composed of an N-terminal BTB domain and six zinc finger domains at the C-terminus. This sequence-specific transcription repressor inhibits gene expression by recruitments of N-CoR, SMRT, HDACs and B-CoR forming a large co-repressor complex

[9]. Since it was cloned in 1993 [30], considerable studies have characterized roles of BCL6 in germinal center development and lymphomagenesis [50].

BCL6 is highly expressed in B cells undergoing affinity maturation within germinal centers, and its expression is downregulated upon selection for apoptosis or differentiation

[51]. Mice with Bcl6 disrupted display a phenotype characterized by a defect in germinal center formation and massive inflammatory response in many organs, especially the heart and lung, corresponding to a typical Th2 hyper-immune response [52]. Further studies suggest that BCL6 regulates the expression of multiple Th2 cytokine, which is essential for

GC memory B-cell development and antigen-specific T cell generation [50]. In addition,

BCL6 is implicated in anti-apoptotic activities in GCB-cells by forming a regulation loop with p53 [38]. In the physiological context of DNA breaks related to somatic mutations (SM) and immunoglobulin class switch recombination, p53 is activated and induces BCL6 expression, which down-regulates p53 and spares the cell from apoptosis [38]. This

15 Chapter 1: Introduction down-regulation of p53, in turn, attenuates BCL6 expression and allows B-cell maturation to complete [53].

In addition to its normal roles, BCL6 is associated with lymphomas due to genetic alternations, such as chromosomal translocations, deletions and somatic mutations [54-56], which deregulate BCL6 expression and shift B-cells towards lymphomagenic pathway. A

3q27 translocation affecting the BCL6 gene is observed in 20%-40% of diffuse large B cell lymphomas (DLBCL), the most common form of non-Hodgkin’s lymphoma (NHL), in 15% of follicular lymphomas (FL), and also in a wide spectrum of B-cell lymphomas [50].

Notably, studies have demonstrated that BCL6 gene was negatively regulated by its gene product, which constitute an autoregulatory circuit [57]. In NHL, high levels of BCL6 gene expression seem to have favorable prognostic value [58].

BCL6 has been considered a new therapeutic target in B-cell lymphomas by regulating its availability and activity [50, 59]. In a recent study, BCL6 inactivation is achieved by cell-penetrating peptide targeting BTB domains of BCL6, which inhibits co-repressor recruitments and thus transcriptional repression [59, 60].

MIZ1

The Miz1 (Myc-interacting zinc finger protein) is encoded by ZBTB17. It is an

803-amino-acid long transcription factor with 13 zinc fingers and a BTB domain at

N-terminus. Miz1 activates genes involved in cell cycle arrest, differentiation and

DNA-damage response. Some studies show that upon UV radiation induced DNA damage,

Miz1 is activated and then used to trans-activate the expression of cell cycle regulator

16 Chapter 1: Introduction

P21CIP1 [61]. Miz1 is also required in embryonic development during gastrulation [62]. It is shown that Miz1−/− embryos succumb to massive apoptosis thus fail to undergo proper gastrulation [62].

On the other hand, upon binding to Myc, a transcription factor coded by proto-oncogene c-myc, transcriptional activation by Miz1 is abolished, and instead the Myc-Miz1 complex acts as a transcriptional repressor [62]. Recent accumulated evidence suggest that genes encoding cell-cycle inhibitory proteins p15Ink4, p21Cip1 and the Myc-antagonist Mad4 are repressed by Myc through interaction with Miz1 [63-65]. Myc-Miz1 repressor complex has also been shown to interfere with tumor-suppressor and DNA-damage-dependent signal pathways [61].

Although the detailed mechanism of Myc-Miz1 transcription regulation is still unknown, several models have been proposed [61]. These models all agreed that Miz1 binding to core promoter [63, 64, 66], protein-protein interactions between Myc and Miz1 [61, 64-66] and the integrity of BTB domains in Miz1 [65] are required for transcription repression activity of Myc-Miz1 complex.

RP58

RP58 (Repressor protein with MW 58kDa), encoded by ZBTB18, is a protein with 522 amino acids and a molecular weight of 58 kDa [67]. Besides the BTB domain at the

N-terminus, RP58 has four sets of Krüppel-type zinc finger motifs [67]. It was first cloned in

1998 and was shown to repress transcription, hence the name RP58 (Repressor Protein with a predicted molecular mass of 58kDa) [24].

17 Chapter 1: Introduction

Immunogold electron microscopic study reveals that almost all RP58 is localized in condensed chromatin regions, suggesting a role for the sequence-specific transcriptional repression in the heterochromatin structure [24, 67]. Northern blot has found RP58 mRNA expresses in lymphoid tissues, testis, heart, brain, skeletal muscle, and pancreas but much lower level in other tissues [24]. Expression of RP58 protein is several hundredfold higher in brain than in any other tissues [24], indicating post-transcriptional controls. A recent study proposes that RP58 plays a critical role during cerebral cortical layer formation due to high expression in the mouse embryonic cerebral cortex [68].

In transient cotransfection experiment, RP58 is shown to repress transcription from binding sequence contains the E box sequence [67]. E box binding activity is also found for members of the helix-loop-helix class of proteins, including c-Myc [67]. Interestingly, another BTB-ZF protein Miz1 interacts with c-Myc [63-65] as stated above. A more recent study detects RP58 and its analogue simiRP58 co-localize in nucleus when co-expressed in

COS-7 cells [69], which leads to a possibility that RP58 and simiRP58 can synergistically repress transcriptions.

BOZF

BOZF is also known as Zinc Finger and BTB domain-containing protein 8 (ZBTB8).

Information about this protein is limited to its basic architecture. It contains one BTB domain of 120 amino acids, in addition to two zinc finger motifs at the C-terminus. It is also shown that BOZF has a natural variant (E191K), which was detected in a colorectal cancer sample [70].

18 Chapter 1: Introduction

Y441

Human Y441 is encoded by ZBTB24 genes. It has 697 amino acids with a 123-amino-acid

BTB domain, one AT rich DNA-binding domain and eight zinc finger domains [14]. No functional or structural studies have been addressed to Y441.

HKR3

HKR3 (Human Krüppel-Related 3) is encoded by ZBTB48. It has 688 amino acids with a

115-amino-acid BTB domain and 11 zinc finger motifs. HKR3 was first identified as a zinc finger gene mapped within chromosome subbands 1p36.2-.3 [71]. This region is commonly rearranged or deleted in advanced neuroblastomas. Studies have suggested that HKR3 is a candidate for the 1p36 neuroblastoma tumor suppressor gene [72]. HKR3 is ubiquitously expressed in human tissues, but especially high levels were present in human fetal and adult nervous tissues [14]. Detailed functional and structural information are not available at this point.

Kaiso

Kaiso, encoded by ZBTB33, was first identified from yeast two-hybrid screening as binding partner of armadillo-domain protein p120 catenin and later shown to function as a transcriptional repressor [73]. Kaiso has 672 amino acids with a 120 amino-acid long BTB domain and a unique three-zinc-finger motif.

19 Chapter 1: Introduction

Kaiso exhibits dual-specificity for DNA binding: it can bind to sequence specific DNA consensus, TCCTGCnA, as well as methylated CpGs [74]. It is also found that p120 catenin inhibits Kaiso-DNA binding because the p120-Kaiso binding site encompasses Kaiso’s zinc finger domain [75]. Kaiso acts as transcription repressors by recruitments of macromolecular

HDAC corepressors and insulator protein CTCF via BTB domain [76]. Interestingly, Kaiso can activate transcription of the neuromuscular gene rapsyn mainly through the acidic regions between BTB and ZF domains [75]. Kaiso mRNA is ubiquitously expressed with relatively lower level in brain and testis [73]. In human tissues, in contrast to cultured cells,

Kaiso is often localized to the cytosol rather than the nuclei [25]. It is suggested that the subcelllular localization of Kaiso is dynamic rather than static [25].

Many studies have demonstrated that Kaiso play a role in tumorigenesis. First piece of evidences comes from the fact that Kaiso target genes, MTA2, MMP7, siamois, cyclinD1, are linked to cell proliferation or tumor metastasis [75]. Secondly, studies in xenopus model system find Kaiso a negative regulator of canonical Wnt signaling, which plays a key role in embryonic growth and development [76]. Suprisingly, Kaiso-deficient mice are still viable with no detectable developmental defects or tumors [77]. It is very likely that Kaiso-related proteins, such as ZBTB4, may compensate for Kaiso loss in knockout mice [75, 76].

Moreover, the finding that Kaiso binds methylated CpGs, an extensively studied hallmark of tumour-suppressor silencing, further indicates a potential role for Kaiso in tumorigenesis

[76].

ZID

ZID (Zinc finger protein with Interaction Domain), encoded by ZBTB6, is a 424 amino acid

20 Chapter 1: Introduction transcriptional factor first identified in 1994. It contains a 120-amino-acid BTB domain and

4 copies of zinc finger motifs. Compared with other BTB domains, ZID is much less characterized in the literature. ZID is found to express in many cell lines and tissues with highest level in the brain [14]. In limited functional studies, ZID is thought to inhibit the interaction of its associated finger regions with DNA [26]. In addition, ZID is proved to interact with itself, but not with another BTB domain Ttk from Drosophila [26].

FAZF

FAZF protein is encoded by Zbtb32 gene, with 487 amino acids and three C-terminal zinc finger motifs [3]. It was first identified as a binding partner for the Fanconi Anemia (FA) group C protein (FANCC) and another BTB-ZF protein PLZF [36]. FAZF is also known as

PLZF-like zinc finger proteins (PLZP), testis zinc finger proteins (TZFP) or repressor of

GATA (RoG) in the literature [7].

FAZF is proposed to play a role in the pathogenesis of the autosomal recessive bone marrow failure, congenital developmental defects and cancer-predisposing syndrome in FA and in APL [36]. Thanks to the 68% identity in zinc finger domains with PLZF [36], FAZF binds to similar DNA sequences as PLZF, which is associated with cell proliferation and

APL [7]. FAZF also interacts with other TFs implicated in hemopoiesis, such as GATA 3, where FAZF represses GATA3-induced cytokine gene expression required for the development of the T cell lineage [78]. It is predicted that FAZF can directly recruit HDAC to the cytokine promoter, thereby repressing interleukin-4 expression in CD8+ T cells.

These findings are consistent with results in mouse knockout studies. FAZF-deficient

21 Chapter 1: Introduction mice exhibit increased T lymphocyte cell proliferation and increased cytokine production in

CD8+ and CD4+ T cells. In addition, these animals have increased number of HSCs in the G1 phase of the cell cycle, which supports the role for FAZF in cell proliferation. Despite the high expression of FAZF in the testis, FAZF-deficient mice show no gross defects, suggesting that some other proteins may compensate the loss of FAZF.

Bioref

Bioref is also known as Zinc finger and BTB domain-containing protein 26. It has 441 amino acids, of which, there are one 122-amino-acid BTB domain, and 4 zinc finger motifs.

The expression of Bioref found ubiquitous in all cell types [14]. However, there has been no study of the biological roles of Bioref.

KUP

KUP (KrUppel homologous Protein) genes were first identified in region q23-q24 of the long arm of human chromosome 14. KUP has 435 amino acids with a 107-amino-acid BTB domain and two distantly spaced zinc finger motifs. There is limited functional information about KUP in the literature. One study verifies its DNA binding activity, although the precise function and DNA target sequence remain unknown [28].

ZBTB4

ZBTB4 is a transcription factor of 1013 amino acids. It was first identified as one of methyl-DNA-binding proteins (MBPs) by BLAST search on the human genome against

22 Chapter 1: Introduction

Kaiso [74]. ZBTB4 has six copies of zinc finger domains near the C-terminus, with one unique triple-zinc-finger domain resembles to Kaiso [74]. Compared with other BTB-ZF proteins, 175-amino-acid BTB domain in ZBTB4 is interrupted by a stretch of 60 residues.

This stretch is located between α-helix 3 and β-sheet 4, which is not in the dimerization interface [74].

Gel retardation assay demonstrates that ZBTB4 has a similar bimodal specificity as

Kaiso, where it can bind to both methylated sequence CGCG and the consensus sequence

KBS (TCCTGCNA) [74]. Interestingly, ZBTB4 is able to bind a single methylated CpG, while Kaiso requires at least two consecutive CpGs for binding [74]. Luciferase assay in mammalian cells proves that ZBTB4 is a potent repressor of the methylated reporter [74].

Northern blotting on adults mouse tissues reveals that ZBTB4 transcripts are detected at high levels in the brain, lung, kidney, muscle and heart, intermediate levels in the placenta, liver, spleen and thymus, and low levels in the testis [74]. ZBTB4 has a broad distribution but seems to be particularly expressed in the brain [74]. In situ hybridization experiments suggests that ZBTB4 controls gene expression in different types of neurons, which can be involved in olfactory, motor and hippocampal functions [74].

LRF

LRF (Leukemia/lymphoma related factor), encoded by ZBTB7, is a 584 amino acid BTB-ZF transcription factor with four zinc finger motifs. It is also known as Pokemon, ZBTB7, and

FBI-1 in the literature. LRF is encoded by Zbtb7 genes, which locates to syntenic chromosomal regions, and are widely expressed in adult tissues and cell lines [16].

23 Chapter 1: Introduction

Up-regulation of LRF was also found in a subset of lymphomas, as well as in a subset of breast, lung, colon, prostate and bladder carcinomas [79].

LRF was first identified as the binding partner of HIV-1 promoter IST [29]. As a transcription repressor, LRF recognizes consensus binding sequences that are often repeated in different orientations and spacing [29]. One of the examples is the transcription repression of human cartilage oligomeric matrix protein (COMP) gene by LRF [80]. It is found that

LRF represses COMP expression by directly binding to the COMP gene promoter in its negative regulatory elements, followed by recruitments of HDAC1 [80].

LRF is known as a PLZF homologue able to interact with BCL6, both of which are involved in many human cancers [16]. Besides that, its oncogenic properties have been shown by many studies. It is found that inactivation of zbtb7 results embryonic lethality due to severe anaemia and impaired cellular differentiation in multiple tissues [7]. In immature T and B lymphoid lineage cells with overexpressed ZBTB7 develop aggressive tumours [81].

Recently, LRF is characterized as a crucial regulator of the ARF tumor suppressor gene

(p19Arf in the mouse, and p14Arf in human) [79]. Through multiple binding sites in the promoter, LRF represses p19Arf transcription thus degradations of nuclear p53 and oncogenic transformation [29]. On the other hand, depletion of LRF inhibited the transformation of various oncogenes, which leads to senescence in mouse embryonic fibroblasts [79]. Due to its critical roles in oncogenesis, LRF has become an attractive target for therapeutic intervention.

24 Chapter 1: Introduction 1.5 Project goals

BTB domains in BTB-ZF proteins illustrate how a family of fairly simple protein domains can adapt a variety of interactions and a wide range of functions. Understanding BTB domain interactions and their networks is essential for understanding the mechanisms of related biological processes on a molecular level. Previous studies have identified many

BTB homodimers but only a few pairs of BTB heterodimers. In order to complete the overall picture of how BTB domains interact, a more robust and systematic interaction mapping is needed.

The aim of this project is to develop a medium-to-high throughput protein-protein interaction assay that can systematically analyze BTB domains interaction networks. By mapping interactions and analyzing structural/sequence information of 14 BTB domains, we attempt to identify key residues involved in BTB interactions interface, and to understand rules governing BTB domain interactions, especially BTB domain heterodimers.

Furthermore, this screening also serves to provide candidate BTB domain heterodimers for

X-ray crystallographic studies to obtain the first BTB heterodimer structure.

1.6 Backgrounds in assay development

1.6.1 Overview of protein-protein interaction assays

Protein interactions are vital to most cellular processes, which makes the analysis of protein-protein interactions important in understanding the mechanism of biological processes on a molecular level. Many comprehensive interaction-detection systems have

25 Chapter 1: Introduction been developed to facilitate the analysis. These systems were designed either in vitro or in vivo with different characterization approaches and inherent limitations.

Coimmunoprecipitation (Co-IP) is a popular technique for protein interaction discovery in vivo [82, 83]. Co-IP works by selecting an antibody specific to the protein of interest to add to a cell lysate. The antibody-protein complex is then precipitated usually using protein-G or protein-A sepharose which binds most antibodies. If there are any protein/molecules that bind to the first protein, they will also be precipitated. Co-precipitated protein can then be identified by Western blot analysis or by sequencing a purified protein band. Problem with these assays is the possibility that an observed interaction is mediated not by direct contact between proteins, but instead by indirect protein interactions or nucleic acid mediated interactions, which causes a false positive result in this type of protein-protein interaction assay [82, 83].

With the increasing number of proteins that have been discovered, the development of protein interaction assays is moving towards high-throughput techniques to handle large amount of data.

The yeast two-hybrid (Y2H) method is based on the fact that many eukaryotic transcription activators have at least two distinct domains: a DNA binding domain (DBD) and a transcription activation domain (TAD) [84]. In this system, protein of interest (bait) is fused with the DBD, and prey protein is fused with the TAD. When protein-protein interaction occurs, two domains are brought to proximity and the transcription is activated

[84]. The Y2H method has been applied to screen entire genomes, where each bait clone is mated with an array of prey clones or a prey library [85]. Although the Y2H method has

26 Chapter 1: Introduction considerably accelerated the screening of protein-protein interactions in vivo, the experimental data generated has low consistency [85]. Y2H methods tend to bias towards nonspecific interactions and fusing transcription activator to target proteins imposes difficulties in folding [83-85]. It is suggested that different in vitro techniques should be used to validate Y2H protein interactions in vivo [83-85].

Förster resonance energy transfer (FRET) is a powerful technique to detect direct protein-protein interactions that can be used in vitro and in vivo [84]. FRET is an energy transition from donor fluorophores to acceptor fluorophores, which occurs when two molecules are positioned within 10nm of each other [86]. In a FRET-based assay, bait and prey proteins are fused with donor and acceptor fluorophores, and protein-protein interaction is characterized by the detected FRET signal [84]. This technique can also be used for high-throughput screening when combined with flow cytometry [86]. FRET-based assays can suffer the signal contamination caused by overlap between donor and acceptor fluorophores [86-88]. In addition, because the efficiency of energy transfer varies inversely with the sixth power of the distance between fluorophores [86], FRET signal is heavily influenced by the distance between fluorophores. FRET signal variation can be misleading in some cases as the distance between fluorophores in the interacting protein complex is structurally dependent.

Previous work in our lab has examined BTB domains interaction in vitro using a size exclusion chromatography assay [11]. In this assay, “short” versions of BTB domains without thioredoxin tags and “long” versions of BTB domains with thioredoxin tags were mixed and analyzed by size exclusion chromatography. In parallel, samples were denatured and refolded prior to chromatography in order to over come kinetic barrier to exchange [11].

27 Chapter 1: Introduction

This assay offered useful information in BTB domain interactions but it was complicated in operation and not amenable to studying many interactions in a high-throughput manner. In order to expand the interaction mapping to up to 43 human BTB domains, a more efficient assay is needed.

1.6.2 Fluorescent protein tags

Since it was isolated from Jellyfish more than 30 years ago [89], Green Fluorescent Protein

(GFP) and many genetically modified variants have been used extensively in molecular biology. These proteins enable dynamic detection and quantification using fluorescence microscopy. More importantly, fluorescent proteins that exhibit Förster resonance energy transfer (FRET) have served as versatile biosensors to detect protein-protein interactions

[86].

Advances in fluorescent protein engineering have developed new GFP variants with enhanced brightness, quantum yield and signal-to-noise level [87, 88, 90]. Cyan Fluorescent

Protein (CFP) and Yellow Fluorescent Protein (YFP) are the most common partners for intermolecular FRET [87, 88]. However, it was shown that fluorescent protein might interact with each other at high concentrations [90], which would result in false-positive interactions.

It was suggested that dimerization could be greatly reduced by mutating hydrophobic residues at the dimer interface [90, 91]. The resulting fluorescent proteins have been optimized and developed in recent studies [87, 88, 91].

In this study, a modified Enhanced Cyan Fluorescent Protein (ECFP) Cerulean and a modified Yellow Fluorescent Protein (YFP) Venus were used to facilitate detection of

28 Chapter 1: Introduction protein-protein interactions (Figure 6) [86]. CFP Cerulean has maximum excitation at 433 nm and maximum emission at 475 nm [86]. YFP Venus has maximum excitation at 514 nm and maximum emission at 527 nm [86]. When two proteins are in proximity (<10nm), excitation near 433 nm will result emission at 527 nm due to energy transfer from Cerulean to Venus [86].

Figure 6: CFP and YFP excitation spectrum and emission spectrum. CFP emission spectrum (blue dash line) and YFP excitation spectrum (yellow solid line) are overlapped, which results in FRET when two fluorophores are in proximity [86]. In this case, when samples are excited around 400-420nm, emission at 530-550nm can be detected. Y-axis is in the relative fluorescent unit (RFU). In this graph, all spectra were normalized to a maximum excitation or emission of 1.0.

1.6.3 Kinetics in BTB dimer formation

The kinetics of dimer formation is usually complicated. Figure 7 illustrates the kinetic pathway in BTB heterodimer formation from BTB homodimers. The first step involves the disassociation of BTB homodimers to BTB monomers, which re-associate to form new heterodimers in the second step.

29 Chapter 1: Introduction

In the solution, BTB domains are found as strongly associated homodimers and BTB monomers have not been detected [2, 11]. The low rate of disassociation is presumably due to a high kinetic barrier in step 1. As a consequence, BTB heterodimers can not form from mixtures of homodimers under non-denaturing conditions. It is proposed that once the kinetic barrier to disassociate is overcome, BTB domains can reassociate to form homodimers or heterodimers. Therefore, in order to detect BTB dimer formation, additional strategies have to be considered.

Homodimers A A B B

1 1

Monomers A A B B

2 2

Heterodimer 2 A B

Figure 7: Proposed kinetic pathway in BTB heterodimer formation. Two steps are proposed to form BTB heterodimers from BTB homodimers. The first step is the disassociation of BTB homodimers. The second step is the re-association of BTB monomers to a heterodimer.

30 Chapter 1: Introduction

1.6.4 Chemical denaturation and refolding

Protein denaturation and refolding are commonly used in folding pathway studies [92]. In these studies, proteins are chemically denatured. With gradual removal of denaturants, proteins are restored to their native states. The entire process can be monitored by biophysical methods until proteins are properly refolded [92].

BTB domains exhibit strand-exchanged homodimer structures and monomers have not been detected [2, 11]. Most BTB dimers are predicted two-state dimers, which only have native and unfolded state but no stable intermediate in the folding/unfolding process [32]. It is hypothesized that BTB domains can interact from the unfolded state thereby forming homodimers or heterodimers. This was tested in previous studies, where BTB dimers were detected by size exclusion chromatography after chemical denaturation and refolding [11].

This project began with an optimization of an assay using FRET based on dimer formation by chemical denaturation and refolding.

1.6.5 The coexpression system

As an improved alternative to the above method, we hypothesized that coexpression is an experimentally simpler method to circumvent kinetic barriers for BTB dimer formation.

Aiming for greater efficiencies in detecting BTB domain interactions, this assay is based on the coexpression of BTB domains tagged with fluorescent proteins. Importantly, formation of BTB heterodimers by coexpression system provides a starting point for structural characterizations of BTB heterodimers.

31 Chapter 1: Introduction

The coexpression of multiple genes in E. coli has been established as one of novel approaches to study protein complexes [93], such as heterogeneous proteins [94]. Two strategies can be used: 1) target genes are cloned in tandem on the same plasmid; 2) target genes are cloned on two plasmids with compatible replicons and antibiotic resistance [95].

This system has also been applied to identification and characterization of protein-protein interactions [95, 96], and structural analysis of protein complexes [97]. It is believed that coexpression allows proteins to interact during folding, which mimics the formation of two-state dimer in vivo [32].

32 Chapter 2: Material and methods

Chapter 2

Materials and Methods

2.1 Fluorescent protein labeling and optimization

2.1.1 Fluorescent protein background minimizations

In order to minimize the background caused by CFP and YFP self-interactions, mutation

A206K was introduced to fluorescent proteins by Quick Change. This mutation was shown to disrupt fluorescent protein dimer formation in previous studies [88].

Mutated Cerulean (mC) and mutated Venus (mV), together with wild-type Cerulean (wtC) and wild-type Venus (wtV), were cloned into pGEX 6P1CH vector (GE Healthcare) with

GST purification tag followed by a 3C digestion site. GST-3C-Venus and GST-3C-Cerulean were expressed in E. coli using BL21DE3 codon(+) cells after IPTG induction at 37°C for three hours. After that, proteins were purified using a glutathione affinity column. The GST tag was subsequently cleaved by 3C protease, and final purified proteins were stored in PBS buffer pH 7.4.

33 Chapter 2: Material and methods

To determine their interactions, these proteins were assigned into 4 pairs: wtC+wtV, wtC+mV, mC+wtV, and mC+mV. Venus proteins at high concentrations were gradually titrated into 10μM Cerulean solution. Samples were excited at 420 nm and emission was monitored between 430 and 600 nm with 2nm increments using a Shimadzu RF5301 spectrofluorometer.

Fluorescence intensities at 475 nm were used to calculate the FRET efficiency Q% using equation 1. FRET efficiency and protein concentrations were fitted into equation 2 using the program Labfit to generate disassociation constants (Kd).

F Q% =1− F0

Equation 1: FRET efficiency Q%.

F0 is the fluorescence intensity without any ligand protein present; F is the fluorescence intensities in the presence of ligand protein.

([C]+[V ]+ Kd − ([C]+[V ]+ Kd)2 − 4[C][V ]) Q = Q + (Q − Q )× min max min 2[C]

Equation 2: FRET efficiency and Kd fitting equation Qmin is the initial FRET efficiency, Qmax is the final FRET efficiency, [C] is the total Cerulean concentration, [V] is the total Venus concentration, and Kd is the dissociation constant.

It was found that wild-type Cerulean and mutated Venus was the pair with the weakest interaction based on Kd (Chapter 3). Therefore, they were cloned into the plasmids as fluorescent tags. From this point on, Cerulean (C) stands for wild-type Cerulean and Venus

(V) stands for mutated Venus unless otherwise stated.

34 Chapter 2: Material and methods

2.1.2 Placement of fluorescent proteins to BTB domains

In order to obtain highest signal to noise ratio (discussed in later section), Venus was placed at the N-terminus of the BTB domain while Cerulean was place at the C-terminus of the

BTB domain. Coding region for Venus was inserted into vector plasmids between the Afl II and Bam HI restriction sites, followed by different BTB domains between the Bam HI and

Hind III restriction sites. Cerulean genes were placed after BTB domains between the Hind

III and Not I restriction sites.

2.2 Dimer formation by chemical denaturation and refolding

2.2.1 Protein expression and purification

In this assay, genes encoding for 6 BTB domains were placed into the Cerulean version and the Venus version of pGEX 6P1CH vectors (Table 2). All plasmids were verified by sequencing.

Protein inserted Cerulean version Venus version - C V PLZF PLZF-C V-PLZF FAZF FAZF-C V-FAZF Kaiso Kaiso-C V-Kaiso LRF LRF-C V-LRF BCL6 BCL6-C V-BCL6 MIZ1 MIZ1-C V-MIZ1 Table 2: List of proteins used in chemical denaturation and refolding assay Two sets of plasmids were created for each BTB protein: one tagged with Cerulean (C), the other one tagged with Venus (V).

35 Chapter 2: Material and methods

These 12 fusion proteinswere then individually expressed and purified in parallel as described below. Plasmids were transformed into BL21 DE3 codon (+) cells; and proteins were expressed overnight at room temperature after 1mM IPTG induction. Harvest cells were spun down at 6000rpm for 30 minutes. Cell paste was resuspended in lysis buffer

(50mM TRIS pH 8.0, 150mM NaCl and 1mM DTT) and lysed by passing through an

Emulsiflux 4 times. Cell lysate was then centrifuged at 15,000 rpm at 4°C for 25 minutes.

Collected supernatants were loaded onto 10 ml pre-equilibrated

Glutathione-S-Transferase (GST)-agarose column (Sigma) at 1mL/min. The column was subsequently washed using washing buffer (50mM TRIS pH 8.0, 150mM NaCl). Proteins were eluted with elution buffer (50mM TRIS pH 8.0, 150mM NaCl, 15mM reduced

Glutathione and 1mM DTT). After buffer exchange to cleavage buffer (50mM TRIS pH 8.0,

150mM NaCl, 1mM EDTA and 1mM DTT), proteins were incubated with 3C protease at

4°C overnight. Cleaved GST tags were then removed by a second pass through a GST column. All fractions were collected and analyzed by SDS-PAGE.

Final purified protein solutions were stored in PBS buffer pH 7.4 and the concentrations were determined spectroscopically by the absorbance of the fused fluorescent protein tags.

Concentrations were calculated with molar extinction coefficients of Cerulean of 41537

M-1cm-1 at 433nm and Venus of 118698 M-1cm-1 at 515nm. Samples were stored at -80°C for use at later times.

2.2.2 Kinetic studies of BTB domain interactions

To examine whether BTB domains undergo spontaneous exchange, kinetic studies were

36 Chapter 2: Material and methods performed. In this experiment, 1μM Cerulean tagged BTB protein was added to 1μM Venus tagged BTB protein. Mixed solutions were excited at 414nm, and emission was scanned from 440nm to 600nm with 2nm increments. Spectra were measured at different time points from 0 to 20 minutes.

2.2.3 Dimer formation by chemical denaturation and renaturation

In denaturation-refolding experiments, Cerulean tagged BTB protein A and Venus tagged

BTB protein B were mixed according to Table 3. To detect homodimer formation, BTB-C proteins were mated with homo-species V-BTB, such as V-PLZF and PLZF-C, which were shown in the diagonal of the table. On the other hand, heterodimer formation used BTB-C to mate with hetero-species V-BTB, such as V-PLZF and FAZF-C, which were shown in the off diagonal region of the table.

A B V-PLZF V-FAZF V-Kaiso V-LRF V-BCL6 V-Miz1

PLZF -C FAZF -C Kaiso-C LRF-C BCL6-C

Miz1-C

Table 3: BTB pairing matrix in kinetic and chemical denaturation and refolding experiments Pairs of BTB proteins were selected based on this 6×6 matrix. When A and B were homo-species BTB proteins with different fluorescent tags, such as V-PLZF+PLZF-C, formation of homodimer was tested. These pairs were found in the diagonal of this table. On the other hand, hetero-species BTB proteins were mixed, such as V-PLZF+FAZF-C, for testing heterodimer formation, as shown in cells off-diagonal.

1μM Cerulean tagged protein A was mixed with 1 μM Venus tagged proteins B in 120μL

37 Chapter 2: Material and methods storage buffer (PBS buffer pH 7.4 and 1mM DTT). Solid Guanidine HCl was added to a final concentration of 6M to denature mixed proteins. After a 30 minutes incubation at room temperature, the solution was diluted 10 fold using refolding buffer (PBS buffer pH 7.4

+10mM DTT), and incubated for another 30 minutes. Guanidine was gradually removed by concentrating and diluting twice using Eppendorf Spin Column with 10K cutoff to final volume of 120μL. A 1μM Venus and 1μM Cerulean mixture was used as negative control.

Recovered solution was excited at 414nm, and emission was scanned from 440nm to 600nm with 2nm increments using a Shimadzu fluorescence spectrophotometer. Spectra were taken at native state before denaturation, and after refolding. The FRET emission ratio (ER) was introduced and calculated as fluorescent intensities of acceptor over donor (Equation 3). ER value is inversely proportional to the distance between fluorophores, which can be used to evaluate the strength of interactions detected. In addition, CD spectra were taken before and after refolding to ensure proteins were properly folded.

Equation 3: FRET Emission ratio. The emission of acceptors is the relative fluorescent intensity (RFI) of Venus measured at 527nm. The emission of donors is the RFI of Cerulean at 475nm.

A trypsin digest was performed on selected pairs after refolding. 17μg/mL trypsin was added into the refolded protein solution. Total FRET spectra (excitation at 414 nm, emission at 440 nm to 600 nm), Venus excitation spectra (excitation at 400 nm to 560 nm, emission at

580 nm), and Cerulean excitation spectra (excitation at 360 nm to 480 nm, emission at 500

38 Chapter 2: Material and methods nm) were taken at different time points from 0 to 25 minutes.

2.3 Dimer formation by co-expression system

In order to achieve a higher throughput in this protein-protein interaction assay and remove compilations from variable FRET signals (see Chapter 3 and 4), dimer formation by coexpression and coprecipitation was introduced to this study. In this method, a His-tagged

BTB-Cerulean fusion protein was coexpressed with a non-tagged Venus-BTB protein.

Following Ni-NTA purification, the proteins were quantified by measuring the fluorescent proteins’ excitation spectra.

2.3.1 Cloning: insertion of BTB domains into co-expression vectors

BTB domains tagged with fluorescent proteins were inserted into co-expression vectors from

Novagen. Vectors used are pETDuet™-1 and pACYCDuet™-1 from Novagen. Genes encoding for BTB-C proteins were placed in multi-cloning region (MCR) 1 of pACYCDuet-1 vector, which is Chlormphenicol-resistant. These clones were used as bait clones encoding for His-BTB-Cerulean fusion proteins (h-BTB-C). On the other hand,

V-BTB proteins were placed in MCR2 of ampicillin-resistant pETDuet-1, which were used as prey cones encoding for Venus-BTB proteins (V-BTB) with no purification tag.

Both pACYCDuet-1 and pETDuet-1 were modified for more convenient cloning. Genes coding for Cerulean and Venus were first placed into corresponding vectors. These plasmids were later used as background controls as they contained no BTB domains. Based on these

39 Chapter 2: Material and methods two plasmids, further modifications unified restriction sites to Bam H1 and Hind III, so that

BTB domains could be inserted in parallel. In the end, BTB domains were cloned into 30 corresponding plasmids including Cerulean and Venus controls. These 30 plasmids (Table 4) were all verified by sequencing.

Protein inserted Bait clones Prey clones pACYCDuet pETDuet Blank h-C V FAZF h-FAZF-C V-FAZF PLZF h-PLZF-C V-PLZF LRF h-LRF-C V-LRF Kaiso h-Kaiso-C V-Kaiso BCL6 h-BCL6-C V-BCL6 MIZ1 h-MIZ1-C V-MIZ1 RP58 h-RP58-C V-RP58 BOZF h-BOZF-C V-BOZF Y441 h-Y441-C V-Y441 HKR3 h-HKR3-C V-HKR3 ZID h-ZID-C V-ZID Bioref h-Bioref-C V-Bioref KUP h-KUP-C V-KUP ZBTB4 h-ZBTB4-C V-ZBTB4

Table 4: List of plasmids cloned for co-expression assay. In coexpression based assay, two sets of clones were created: bait clones, encoding for h-BTB-C proteins, and prey clones, encoding for V-BTB proteins. Including the controls, there were total 30 clones created.

2.3.2 BTB pairs co-expression in auto-induction media

According to the matrix below (Table 5), one bait clone from pACYC series and one prey clone from pET series were mated as one co-expression pair. When homo-species BTB clones were mixed, BTB homodimer formation was tested. On the other hand, when

40 Chapter 2: Material and methods hetero-species BTB clones were mixed, BTB heterodimer formation was tested. Two plasmids were incubated with 50μL XL1 Blue competent cells for 10 minutes on ice. Cells were then heat shocked at 42°C for 45 seconds and quickly recovered on ice for 2 minutes.

Cells were allowed for one hour growth in SOC media at 37°C before plating with

Chlormphenicol and Ampicillin selection.

Bait Clones: His-BTB-C PLZF FazF Kaiso LRF BCL6 Miz1 RP58 Bioref KUP BOZF Y441 HKR3 ZID ZBTB4 PLZF FazF Kaiso LRF BCL6 Miz1 RP58 Bioref KUP BOZF Y441

Prey Clones: V-BTB Clones: Prey HKR3 ZID ZBTB4

Table 5: 14×14 BTB coexpression matrix Coexpressions of prey and bait clones were based on this 14×14 matrix. His-BTB-Cerulean proteins were expressed by bait clones (colored in green). On the other hand, Venus-BTB proteins were expressed by prey clones thereby was colored in yellow. Similar with previous denaturation and refolding assay, diagonal cells indicate homodimer detections, while off-diagonal cells indicate heterodimer detections.

The next day, colonies were picked for outgrowth in auto-induction media. For every co-expression pair, four different colonies were picked and transferred into 2mL of auto-induction media (Novagen Overnight Express Instant TB) with 34μg/mL chlormphenicol and 100μg/mL ampicillin, preloaded onto 24 deep-well plates (Qiagen).

Culture plates were then shaken at 220rpm at 37°C overnight. Each well on the plate had a maximum volume of 10mL, and were sealed by airpore seal (Qiagen) ensuring sufficient air flow for cell growth and minimum medium evaporation. 1mL cell culture from each sample

41 Chapter 2: Material and methods was transferred into a 96-deep-well plate and cells were harvested by centrifugation at 4000 rpm for 25 minutes. From this point on, cells were all handled in 96-well plate scale.

2.3.3 Detection of dimer formation

100μL lysis mix (23μg/mL DNase I, 200μg/mL lysozyme) were added to each sample on

96-well plates. After 30 minute incubation on nutator at room temperature, detergent OTG

(Octyl β-Thioglucopyranoside) was added into the mixture to a final concentration of 1%.

Plates were continued to shake at room temperature until cell debris started to precipitate. In order to separate out the supernatant, plates were centrifuged at 4000 rpm for 25 minutes at

4°C. 100μL supernatant were then transferred onto a 96-well filter plate preloaded with

50μL Ni-NTA beads allowing binding at room temperature for 20 minutes.

This loaded plate was placed into SpectraMax M5 Microplate Reader from Molecular

Device to determine Venus level and Cerulean level in each sample. Venus excitation spectrum was taken at excitation scan from 400 nm to 560 nm with 5 nm increments and emission at 580 nm. Cerulean excitation spectrum was taken at excitation scan from 360 nm to 480 nm with 5 nm increments and emission at 500 nm. Because Venus tagged proteins do not have His-tag, only Cerulean tagged proteins can bind to Ni-NTA beads. Any unbound proteins were centrifuged down after centrifugation at 600 rpm for 5 minutes. Ni-NTA beads on the filter plate were washed with 150μL washing buffer (lysis buffer, 40mM imidazole) before 100μL lysis buffer was added to recover the initial volume. Microplate fluorescent reader measured Venus and Cerulean levels retained on the plate after binding and washing.

In addition, 20μl supernatant of selected coexpression pairs were loaded onto the 10%

42 Chapter 2: Material and methods non-denaturing PAGE gel at pH 7.5. The PAGE gel was analyzed by Typhoon 8600 Variable

Mode Imager (Amersham Biosciences) to capture images for Venus. The image probing

Venus was measured at emission wavelength greater than 526nm when excited at 532nm.

2.3.4 Data analysis

Excitation spectrum was used to determine Venus and Cerulean level before and after binding. In Venus excitation spectrum, the fluorescent intensities at 515 nm were calculated as an average from 510nm to 520nm. Venus levels detected in coexpression samples were corrected by negative controls. Similarly, for Cerulean spectrum, the fluorescent intensities at 435 nm were calculated as average from 430 nm to 440 nm.

For every co-expression pair, 4 independent colonies were grown and analyzed.

Statistical analysis and plot construction were completed using Microsoft Excel.

43 Chapter 3: Results

Chapter 3

Results

3.1 Fluorescent protein interactions

To test and select a pair of fluorescent proteins for FRET assay, disassociation constants of 4 pairs of fluorescent proteins were measured and compared (Table 6). Although mutated

Cerulean and mutated Venus did show weaker interaction compared with wild type Cerulean and wild type Venus, they still exhibit a disassociation constant (Kd) of 7.6μM, much lower than 74mM stated in the literature [91]. Comparing the disassociation constants (Table 6), it was found that wild type Cerulean and mutated Venus had the weakest interaction among all the other pairs of fluorescent proteins tested. In addition, this pair showed a weaker interaction when protein concentration was less than 1μM. Therefore, wild type Cerulean and mutated Venus were selected as fluorescent protein tags, and the optimal protein concentration used in this study was 1μM or less.

44 Chapter 3: Results

Reaction Kd (uM) wtC+wtV 3.7 wtC+mV 12.11 mC+mV 7.6 mC+wtV 10.98

Table 6: Kd of fluorescent protein self-interactions Disassociation constants (Kd) for different pairs of Venus and Cerulean are compared in this table. It was found that wild-type Cerulean and mutated Venus had the highest Kd, thus the weakest interaction.

3.2 Results from chemical denaturation and refolding assay

Previous studies have shown that: 1) BTB domains are homodimers in solution; 2) chemical denaturation and refolding is required for BTB-BTB exchange even in homodimers [11].

The one exception was FAZF, which exchanged without denaturation [11]. Here, a similar chemical denaturation and refolding assay using fluorescent protein-BTB (FP-BTB) proteins confirmed previous findings and provided more efficient detections of BTB dimer formation.

3.2.1 FP-BTB proteins expression and purification

All plasmid constructs listed in Table 2 were successfully made. 13 of the 14 encoded proteins were expressed except for GST-BCL6-C. A typical purification and digestion was showed in Figure 8. The final 13 proteins were stored as stock solutions of concentration of

40-50μM.

45 Chapter 3: Results

Figure 8: SDS-PAGE gel image of fractions in C-PLZF protein purification. This gel represents a typical purification process of BTB domain-fluorescent protein fusion proteins. Over expressed fusion protein GST-C-PLZF in the supernatant were eluted from GST column. After that, GST tags were cleaved by 3C digest, leaving the C-PLZF proteins of molecular weight around 40kDa, indicated by the red arrow in the gel.

3.2.2 Kinetic studies of BTB domain interactions

To monitor how BTB domains interact with each other in solution, kinetic studies were performed by mixing the proteins under non-denaturing conditions. Different pairs of BTB proteins were mixed according to Table 3, and total FRET spectra were measured from 0 to

20 minutes.

It was found that for most pairs, there was no change in fluorescent intensity as shown in

Figure 9A, where all spectra were overlaid. Similar spectra were observed for the Cerulean and Venus pair, which acted as a background control. This indicated that there was no interaction between most of these BTB homodimers under non-denaturing conditions.

The only exception was V-FAZF + FAZF-C, which had a decreasing Cerulean

46 Chapter 3: Results fluorescent intensity at 475nm, and an increasing FRET fluorescent intensity at 527nm from

0 to 20 minutes (Figure 8A). Although very subtle, changes in fluorescent intensities reached saturation by the end of 20 minutes. The decreasing Cerulean signal, the increasing FRET signal and the final saturation were indications of protein-protein interactions. Therefore, it was concluded that V-FAZF and FAZF-C could exchange subunits in solution. Combined with previous studies [11], it was believed that this interaction was spontaneous FAZF homodimer self-exchange, which will be discussed in later sections.

0 Min 1 Min 2 Min 5 Min 10 Min RFU 25 Min

V‐PLZF and PLZF‐C

440 460 480 500 520 540 560 580 600

0 Min 1 Min 2 Min 5 Min 10 Min RFU 25 Min

V‐FAZF and FAZF‐C

440 460 480 500 520 540 560 580 600

Figure 9: Total FRET spectrum in BTB kinetic studies. Figure 8A shows overlaid spectra measured after V-PLZF mixed with PLZF-C. It is found that there was no change in FRET signal at 527nm from 0-25 minutes, indicating that there was no interaction between V-PLZF and PLZF-C. Figure 8B shows overlaid spectra of V-FAZF mixed with FAZF-C in 25 minutes time course. It is found that there was a gradual decrease in Cerulean signal at 475nm with a gradual increase in FRET signal at 527nm. These changes reach static state after 10 minutes. It indicated that V-FAZF was interacting with FAZF-C in solution, and this interaction reached equilibrium in 10 minutes.

47 Chapter 3: Results

3.2.3 BTB dimer formation by chemical denaturation and refolding

BTB domains were matched according to Table 7. Pairs in the diagonal represent BTB homodimers, such as V-PLZF+PLZF-C. Pairs off the diagonal represent BTB heterodimers, such as V-PLZF+FAZF-C. Shaded pairs found in table 6 were chemically denaturated and refolded for dimer detection. CD spectra of proteins at native state, unfolded state, and refolded state were compared to ensure proper refolding (Figure 10). Total FRET spectra measured before and after refolding (Figure 11 and 12) were normalized for comparison for

Cerulean emission due to varied recoveries in different pairs.

A B V-PLZF V-FAZF V-Kaiso V-LRF V-BCL6 V-Miz1 PLZF -C FAZF -C Kaiso-C LRF-C BCL6-C ND ND ND ND ND ND Miz1-C

Table 7: Pairs of BTB domains tested in chemical denaturation and refolding assay. Shaded pairs were tested in chemical denaturation and refolding assay. BCL6-C did not express in E.coli cells. Reciprocal pairs using V-BCL6 and BTB-C covered all BCL6 heterodimer detection. However, BCL6 homodimer interaction could not be tested (ND).

48 Chapter 3: Results

Figure 10: CD spectroscopy of V-PLZF+PLZF-C before and after refolding. CD spectra measured for V-PLZF+PLZF-C protein pair at their native (blue), unfolded (red) and refolded (green) states were overlaid. The native state spectrum shows characteristics of α helices from the BTB domain. The unfolded state spectrum does not showα helices or β sheets characteristics, indicating that protein was unfolded. More importantly, the CD spectrum taken after refolding confirmed restoral of secondary structures.

49 Chapter 3: Results

Native State Native State After Refolding After Refolding RFU RFU

Background V+C V‐PLZF+PLZF‐C

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Native State Native State After Refolding After Refolding RFU RFU

V‐FAZF+FAZF‐C V‐Kaiso+Kaiso‐C

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Native State Native State After Refolding After Refolding RFU RFU

V‐LRF+LRF‐C V‐Miz1+Miz1‐C

440 460 480 500 520 540 560 580 600 440 460 480 500 520 540 560 580 600

Figure 11: Total FRET spectra for BTB homodimers formed by denaturation and refolding. Spectra were taken for different pairs of homo-species BTB domains at their native state and after refolding from chemical denaturation. Spectra measured after refolding were normalized for easier comparison. Pure Venus was mixed with pure Cerulean as a negative control (A).

50 Chapter 3: Results

Native State After Refolding RFU

Background V+C

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Native State Native State After Refolding After Refolding RFU RFU

V‐PLZF+Miz1‐C V‐FAZF+Miz1‐C

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Native State Native State After Refolding After Refolding RFU RFU

V‐Kaiso+Miz1‐C V‐LRF+Miz1‐C

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Native State Native State After Refolding RFU RFU

V‐BCL6+Miz1‐C V‐Miz1+Miz1‐C

440 460 480 500 520 540 560 580 600 440 460 480 500 520 540 560 580 600

Figure 12: Total FRET spectra for Miz1-BTB dimers formed by denaturation and refolding. Spectra were taken for different pairs of hetero-species BTB domains at their native state and after refolding from chemical denaturation. Spectra measured after refolding were normalized for easier comparison. Pure Venus was mixed with pure Cerulean as a negative control (A). Miz1 homo-species pair was used as a positive control (G).

51 Chapter 3: Results

It was found that for most homo-species BTB pairs, there were much stronger FRET signals at 527nm after refolding, indicating homodimeric interaction were established. On the other hand, there was only slight difference between spectra measured before and after refolding for V-FAZF and FAZF-C mixture. The spectrum of V-FAZF and FAZF-C at the native state was taken within 1 minute after mixing, when the equilibrium was not established (Figure 11C). The refolded spectrum was similar to the equilibrated spectra shown in Figure 9B. Despite the small FRET signal, it was still believed that FAZF formed homodimers after refolding, which will be discussed in later sections. For heterodimers formed with Miz1, it was found that FAZF+Miz1 did not exhibit a FRET signal after refolding, while PLZF+Miz1, Kasio+Miz1, LRF+Miz1, and BCL6+Miz1 produced weak

FRET signals. Many factors might cause the FRET signal variance here, as discussed in later sections.

In order to confirm that the emission signal at 527nm was caused by BTB-BTB interactions, a trypsin digest was performed in the LRF homodimers formed after refolding

(Figure 13A). Both BTB and fluorescent proteins are resistant to trypsin digest, while the short linker between the two parts of the fusion proteins is susceptible. Upon the addition of trypin from 0 minute to 25 minutes, it was found that the emission at 475nm was gradually increasing, while the emission at 527nm was gradually decreasing, which indicates the loss of established interactions. At the same time, Cerulean levels changed by less than 5%

(Figure 13B) and Venus level remained unchanged, as measured by excitation spectra

(Figure 13C).

52 Chapter 3: Results

0 minute 3 minutes RFU 5 minutes 10 minutes 360 380 400 420 440 460 480 15 minutes RFU 20 minutes 25 minutes 30 minutes RFU

440 460 480 500 520 540 560 580 600

400 420 440 460 480 500 520 540 560

Figure 13: LRF dimer formed after refolding in a time-course trypsin digests. LRF dimers formed after denaturation and refolding were monitored in trypsin digest from 0-30 minutes with total FRET spectra (Figure 13A), Cerulean excitation spectra (Figure 13B) and Venus excitation spectra (Figure 13C). It was found that Cerulean signal at 475nm gradually increased and FRET signal at 527nm was gradually decreased. Changes reached static in 30 minutes. At the same time, Venus level was constant, while Cerulean levels changed by 5%.

Total FRET spectra showed that some BTB pairs had very strong FRET signal (Figure

12G), while some has weak ones (Figure 12B) or none (Figure 12A). Variations in FRET signals made them hard to compare. However, based on ER values, BTB pairs were categorized into three classes: no interaction (ER between 0.3-0.5), weak interaction (ER between 0.5-0.7), and strong interaction (ER greater than 0.7). An overall BTB interaction mapped was summarized based on this system (Figure 14).

It was found that all BTB domains form homodimers with variations in FRET signal and

ER value. It was found that many BTB pairs did not form heterodimers after refolding. Some

BTB pairs were found to have ER values indicating BTB-BTB interactions, such as Miz1 and BCL6.

53 Chapter 3: Results A B V-PLZF V-FAZF V-Kaiso V-LRF V-BCL6 V-Miz1 PLZF -C ++ − − + + − FAZF -C − + − − − − Kaiso-C − − ++ − + + LRF-C − − − ++ − + BCL6-C ND ND ND ND ND ND Miz1-C + − + + + ++ RFU RFU RFU

V‐FAZF+Miz1‐C V‐PLZF+Miz1‐C V‐Kaiso+Kaiso‐C

440 460 480 500 520 540 560 580 600 440 460 480 500 520 540 560 580 600 440 460 480 500 520 540 560 580 600

Figure 14: BTB domain interaction mapping by chemical denaturation and refolding assay. Interaction mapping of 6 BTB domains were summarized in the matrix. These BTB pairs were categorized based on emission ratios observed. Pairs with ER 0.3-0.5 were considered no interaction (colored blue); ones with ER 0.5-0.6 were considered weak interactions (colored pink); and last ones with ER greater than 0.6 were strong interaction pairs (colored red). BCL6-C did not express, so related pairs were not detectable (ND).

3.3 Results from co-expression and plate retention assay

To further improve the throughput of this detection assay, an alternative co-expression and

Ni-NTA plate retention assay was developed. Initially, 6 BTB domains: PLZF, FAZF, Kaiso,

LRF, BCL6 and Miz1 were used to validate and optimize the assay. Finalized assay was then applied to detect interactions of 8 other human BTB domains.

3.3.1 Plate binding background control for fluorescent proteins

Background controls were tested using different His tagged BTB-Cerulean proteins

(h-BTB-C) as the bait and Venus as the prey in coexpressions. Figure 15 shows plots

54 Chapter 3: Results comparing Cerulean and Venus level before and after binding as determined by excitation spectra. After binding and washing, h-BTB-C proteins were retained on the plate through binding to Ni-NTA (Figure 15A). On the other hand, Venus proteins were washed off the plate (Figure 15B). Therefore, it was concluded that h-BTB-C bound to Ni-NTA beads successfully and that Venus could not bind to Ni-NTA. Most importantly, there was no interaction between Venus and Cerulean.

Cerulean level before and after binding 35000 Before Binding After Binding 28000

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10000 Before Binding After Binding 8000

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Figure 15: Background control of Cerulean and Venus binding to plates. Prey clone encoding for Venus only was coexpressed with bait clones encoding for series of His-BTB-Cerulean proteins (h-BTB-C). Cerulean and Venus levels observed before and after binding were used to quantify bait and prey proteins retained on the Ni-NTA plates. It was found that majority of bait proteins containing Cerulean were retained on the plate with reasonable variance. On the other hand, prey proteins containing only Venus were not retained on the plate.

55 Chapter 3: Results

3.3.2 Dimerization of PLZF, FAZF, Kaiso, LRF, BCL6 and Miz1

Bait and prey clones were matched and co-transformed based on the BTB interaction matrix

(Table 8). Coexpression pairs listed in the table all achieved reasonable expression levels for detection.

His-BTB-C PLZF FAZF Kaiso LRF BCL6 Miz1 PLZF FAZF Kaiso LRF

V-BTB BCL6 Miz1

Table 8: Coexpression matrix of 6 BTB domains Bait clones His-BTB-C were coexpressed with prey clones V-BTB according to this table. All shaded coexpression pairs achieved optimal yield for the detection.

To easily compare the data, coexpressions of one prey V-BTB with 6 different bait h-BTB-C proteins were grouped together. For example, Venus excitation spectra detected before and after binding were measured for V-BCL6 and h-BTB-C coexpression pairs in

Figure 16A and 16B. Fluorescent intensities at 435nm indicate Cerulean levels and ones at

515nm indicate Venus levels. It was found that almost all h-BTB-C bound to the plate after binding and washing with variations in a reasonable range. The V-BCL6 retained on the plate is shown Figure 16C.

Compared with the negative control (V-BCL6+h-C), there was significant amount of

V-BCL6 retained by His-tagged BCL6-C (p<0.05). In the other word, BCL6-BCL6 homodimer formed in this coexpression pair. Since BTB homodimers have been widely

56 Chapter 3: Results observed, they served as an internal positive control in each of the group. In addition, significant amount of V-BCL6 was also retained by h-Miz1-C, indicating interactions between Miz1 and BCL6. Levels of Venus retained on the plate for the other groups were plotted and compared in a similar manner to V-BCL6 (Figure 17).

57 Chapter 3: Results

Venus excitation spectra before binding 16000

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10000 V‐BCL6+h‐PLZF‐C V‐BCL6+h‐FazF‐C 8000 V‐BCL6+h‐Kaiso‐C 6000 V‐BCL6+h‐LRF‐C 4000 V‐BCL6+h‐BCL6‐C 2000 V‐BCL6+h‐Miz1‐C

0 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560

Venus excitation spectra after binding 16000

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10000 V‐BCL6+h‐PLZF‐C V‐BCL6+h‐FazF‐C 8000 V‐BCL6+h‐Kaiso‐C 6000 V‐BCL6+h‐LRF‐C 4000 V‐BCL6+h‐BCL6‐C 2000 V‐BCL6+h‐Miz1‐C

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Figure 16: Data processing of Cerulean and Venus levels observed before and after binding. Figure 16A and Figure 16B are Venus excitation spectra measured before and after binding for BCL6 group. Figure 16C plotted levels of V-BCL6 retained on the Ni-NTA plate by h-BTB-C. V-PLZF coexpressed with Cerulean was served as the negative control (p<0.05). Venus excitation scans were taken from 400 to 560nm with emission at 500nm. Both Venus and Cerulean could be excited at this given range, and their emission could be measured. For more rigorous Cerulean quantitation, Cerulean excitation spectra were measured as described in the methods section.

58 Chapter 3: Results

V‐PLZF retained by BTB‐C V‐FAZF retained by BTB‐C 3500 3500

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V‐Kaiso retained by BTB‐C V‐LRF retained by BTB‐C 3500 3500

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V‐BCL6 retained by BTB‐C V‐Miz1 retained by BTB‐C 3500 3500

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Figure 17: Level of V-BTB retained by six different h-BTB-C in coexpression assay. Data for V-BTB retained by different h-BTB-C were plotted in groups. Figure 17A compared V-PLZF retained by different bait proteins. Similarly, figure 17B represented for V-FAZF group; figure 17C represented for V-Kaiso group; figure 17D represented for V-LRF group; figure 17E represented for V-BCL6 group; and figure 17F represented for V-Miz1 group. In every group, V-BTB+h-C coexpression was used as the negative control. Compared with the negative control, all BTB domains formed homodimers, which retained significantly higher amount of prey V-BTB proteins on the plate (p<0.05). In addition, V-BCL6 was retained by h-Miz1-C, while V-Miz1 was retained by h-BCL6-C, which indicated heterodimeric interactions between Miz1 and BCL6.

59 Chapter 3: Results

His-BTB-C C PLZF FAZF Kaiso LRF BCL6 Miz1 PLZF √ FAZF √ Kaiso √ LRF √ V-BTB BCL6 √√ Miz1 √√

Table 9: Interaction mapping of 6×6 BTB domains. This table summarizes results generated from coexpression of 6 BTB domains in addition to the negative control with Cerulean. It was found that coexpressions formed 6 BTB homodimers, as shown in orange boxes. In addition, V-BCL6+Miz1-C and V-Miz1+BCL6-C formed heterodimers in coexpression, which were shown in red boxes.

Table 9 summarized results generated from the coexpression assay. It was found that all

6 BTB domains formed homodimers, as shown in the diagonal of the table. This proved our hypothesis that BTB domain could form dimers in the coexpression. Besides homodimers, the BTB heterodimers V-BCL6+h-Miz1-C and V-Miz1+h-BCL6-C were also detected. To assess the relevance of detected interactions, supernatants of these two coexpression pairs were analyzed by native PAGE followed by fluorescence imaging. Captured images probing

Venus verified the formation of Miz1-BCL6 heterodimer in both cases (Figure 18).

Representative images from the rest of native PAGE images are shown in the appendix.

Compared with the previous assay, Miz1-BCL6 heterodimers were also listed among dimers found in chemical denaturation and refolding. However, other pairs shown to dimerize after in vitro refolding did not form dimers in coexpressions.

60 Chapter 3: Results

Figure 18: Native PAGE images of coexpression supernatant. Fluorescent images probing Venus were used to analyze different coexpression supernatants. One band for V-BCL6 is detected in V-BCL6 and Cerulean coexpression (lane 1); while two bands are detected in V-BCL6 and Miz1-C coexpression (lane 2). The upper band represents V-BCL6, as it is found in lane 1. The lower extra band represents V-BCL6 in V-BCL6-Miz1-C heterodimers. Similarly in V-Miz1+BCL6-C coexpression (lane 4), V-Miz1 are detected to have two bands, where the lower one is V-Miz1 as determined in lane 3; and the other one is V-Miz1-BCL6-C heterodimers.

3.3.3 Dimerization of 8 other BTB domains

After validations and optimizations, this assay was extended to detect interactions of additional 8 BTB domains. These BTB domains were selected based on their expression levels in E. coli. It was found that most of these BTB domains expressed successfully under the coexpression condition. However, some proteins did not achieve the desired expression level, thus could not be tested for dimer detection. Table 10 showed coexpression pairs completed and their results were shown in Figure 19 as plots comparing V-BTB levels retained.

61 Chapter 3: Results

V‐HKR3 retained by BTB‐C 3500

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62 Chapter 3: Results

V‐BOZF retained by BTB‐C 5600 4900 4200 3500 2800 2100 1400

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63 Chapter 3: Results

Figure 19: Levels of V-BTB retained by 8 other BTB domains. Levels of V-RP58 (figure 19A), V-KUP (figure 19B), V-HKR3 (figure 19C), V-Bioref (figure 19D), V-BOZF (figure 19E), V-ZID (figure 19F), V-Y441 (figure 19G) and V-ZBTB4 (figure 19H) retained by different BTB-C bait proteins are plotted and compared (p<0.05).

64 Chapter 3: Results 3.4 Summary: 14 BTB domain interaction map

In summary, an interaction assay was developed for BTB domains interactions.

Incorporation of fluorescent proteins and autoinductive coexpression greatly improved the throughput for the assay. It not only was verified using 6 well-studied BTB domains, but also was also applicable to other 8 BTB domains in the family. Most BTB coexpression pairs achieved minimum yield for detection, while some pairs did not. Summarizing all results generated, table 10 systematically mapped interactions of 14×14 BTB domains studied in this project.

As expected, most BTB domains interacted with themselves forming homodimers. In addition, interestingly, two pairs of BTB heterodimers were also found. They were

BCL6-Miz1 and Miz1-RP5. Only the BCL6-Miz1 heterodimer was previously identified

[38]. The other BTB heterodimer pairs will require in vivo studies to further elucidate their biological relevance and possible functional roles. Overall, the majority of BTB pairs did not interact, which was different to some results shown in the literature [16, 36, 39]. For example, Miz1 was shown to interact with ZBTB4 by recently published research [39].

However, this assay concluded that the interaction was not due to BTB domains of Miz1 and

ZBTB4.

65 Chapter 3: Results

His-BTB-C C PLZF FazF Kaiso LRF BCL6 Miz1 RP58 Bioref KUP BOZF Y441 HKR3 ZBTB4 ZID PLZF √ FAZF √ LRF √ Kaiso √ BCL6 √√ Miz1 √√√ RP58 √√ ND ND Bioref √ ND

V-BTB KUP ND ND ND BOZF √ ND Y441 ND ND 66 HKR3 ND ND ND √ ND ZBTB4 √ ND ZID ND ND ND ND ND ND

Table 10: Interaction mappings of 14×14 BTB domains Results from coexpressions of 14×14 BTB domains are summarized in this table. Shaded boxes indicate coexpression pairs that are successfully expressed and detected.. Pairs that did not achieve sufficient protein expression were not detectable (ND). It is found that BTB homodimers are readily detected, as shown in orange boxes. Two pairs of heterodimers, BCL6-Miz1 and Miz1-RP58, are also detected, as shown in red boxes.

Chapter 4: Discussion

Chapter 4

Discussion

4.1 Assay development

4.1.1 Why do we need a new assay?

In the literature, many protein-protein interaction assays have been developed to serve different purposes. Previous studies characterizing BTB domain proteins interactions have been conducted based on these methods. However, as assays may have been compromised for specificities or higher throughput, each of these studies has their own limitations. For example, heterodimeric interaction of FAZF and PLZF was first identified by coimmunoprecipitation in one study [36]. Although it provided evidence of protein interactions in vivo, it did not imply a direct interaction between two BTB domains. In fact, false positive results are considered one significant shortcoming in Co-IP assays [83]. As a result, another assay is required to provide unambiguous answers to BTB domain interactions.

Many methods developed in vitro detect physical interactions of purified proteins

Chapter 4: Discussion without any interference. One good example is X-ray crystallography, which not only identifies protein-protein interaction, but also provides structural and biological characterizations. As seen previously, many BTB domains have been crystallized under various conditions, and all exhibit homodimeric interactions. This tool is not a high-throughput method to be used in protein interaction screening, instead often used in characterizations at later stages.

Other protein interactions assays with higher throughputs, such as FRET, Fluorescent

Polarization (FP) and Surface Plasmon Resonance (SPR), detect interactions in solution.

These methods are used to detect dimers formed dynamically in the solution. However, these conventional methods are not applicable for BTB dimers. Most BTB domains are strongly associated homodimers, which do not exchange spontaneously. Our lab has not observed

BTB monomers in any of our biochemical studies. Formation of BTB heterodimers requires two steps as illustrated in Figure 7. A high kinetic barrier for BTB homodimers to disassociate restricts the subsequent heterodimer formation.

To address this problem, a previous study [11] took the approach of denaturing BTB homodimers to unfold monomers followed by refolding. Analytical gel filtration detected

BTB dimers after these treatments. This assay successfully produced detectable BTB dimers in vitro, but was laborious and difficult to scale up to measure a large number of interactions.

4.1.2 Troubleshooting in FRET assay development

This project started with modifications in the detection process. As fluorescent protein-based FRET showed great potential in high-throughput protein interaction assays

Chapter 4: Discussion

[86-88], the first modification was to place fluorescent proteins into the system to accelerate the detection. The rationale was to create two versions of BTB domains tagged with different fluorescent proteins. After denaturation and refolding, BTB dimer formation brings fluorescent proteins in proximity, thus creating FRET signals.

Fluorescent proteins used in this study were Cerulean, a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) Venus. These two proteins are commonly used in

FRET due to their overlapping emission and excitation spectra [86]. Cerulean and Venus differ from their parent CFP and YFP by mutations that enhance their brightness and quantum yields [88]. However, these fluorescent proteins tend to dimerize at high concentrations, which often leads to false-positive results. In order to minimize the background interactions caused by Cerulean and Venus interaction, the A206K mutation was introduced to both Venus and Cerulean as suggested by a recent paper [87]. However, there were still significant dimeric associations between mutated fluorescent proteins (Table 6). To find a pair of fluorescent proteins with weakest interactions, mutated and wild-type fluorescent proteins were paired up. Comparing Kd values, wild-type Cerulean (C) and mutated Venus (V) were found to have the highest Kd values, thereby were selected to be used in this project. In addition, the optimum protein concentration used in the assay was determined to be no more than 1µM. This was determined by assessing V+C FRET spectra at different concentrations. V+C spectrum was found to have minimum FRET signal due to self interactions when the concentration was lower than 1µM.

After fluorescent protein tags were selected, placements of these proteins were also taken in consideration. Based on structural information available for BTB domains, it was observed that most BTB homodimers were strand-exchanged (Figure 3). In FRET, signal 69

Chapter 4: Discussion strength is inversely proportional to the distance between fluorophores. Therefore, Cerulean was placed at the C-terminus, and Venus was placed at the N-terminus of BTB domains in order to enhance the FRET signal. The only exception was FAZF BTB domains, as shown in

Figure 20B. Comparing the distances between fluorophores in two types of structures, it was found that the distance in FAZF was about 3 times greater than in other structures. Therefore, it was expected that the FRET signal related to non-domain-swapped BTB domains will be

81 times weaker than domain-swapped BTB dimers.

Figure 20: Proposed structures of BTB homodimers tagged with Venus and Cerulean. PLZF BTB domain (figure 20A) demonstrates a domain-swapped structure found in most BTB domains. FAZF BTB domain (figure 20B) is an exception, where two domains are held by α helices contact in the middle without swapped N termini. Distances between Cerulean and Venus attached to BTB domains are estimated to 21 Å in PLZF, and 58 Å in FAZF.

Chapter 4: Discussion

In this FRET based assay, purified BTB domains with fluorescent protein tags were denatured and refolded. The purpose was to form BTB dimers from BTB monomers during the refolding. Similar to previous studies [11], 6M guanidine was chosen as the denaturant.

The refolding solution was optimized by varying the salt concentration and compositions. In addition, gradual removal of denaturant was achieved by several rounds of concentration and dilution with refolding buffer.

Detection of FRET signals was fairly straightforward. Compared with high throughput fluorescent plate reader, fluorospectrometer was more sensitive, thereby was mainly used in this study. There were several major drawbacks of FRET based assay. The first one was the inefficient refolding process. BTB domains with either Cerulean or Venus had to be expressed and purified individually, and chemical denaturation and refolding was not high throughput. Moreover, FRET signals were largely dependent on the distance between fluorophores, which varied significantly in different structures. The known structures helped guide the interpretation of the FRET data, but this would not be possible with BTB domains of unknown structure, leading to results that would be difficult to interpret.

4.1.3 Further improvements in coexpression assays

Coexpression was the alternative approach to form BTB dimers as stated previously. The hypothesis was that coexpression of BTB domains in E. coli would allow during folding.

This assay is not dependent on the FRET signal. Instead, the amount of copurified protein is quantified by the Venus excitation spectrum of the untagged V-BTB protein. Preliminary studies coexpressed a matrix of 6 BTB domains verified this hypothesis. Coexpression of

Chapter 4: Discussion two plasmids carrying the same BTB domain with different fluorescent protein tags demonstrated a mixed population of BTB homodimers, as seen in the native gel (Figure 18).

Based on the color composition observed, Figure 21A illustrated BTB homodimers formed after the coexpression. It showed that although each plasmid has its independent transcription and translation, BTB domains can still readily interact when they are folding. In fact, mixed Venus and Cerulean tagged homodimers of 6 BTB domains were all detected in coexpression system, which further validated our hypothesis.

A V C C V-BTB1 Homo-species BTB Coexpression BTB1 BTB1 C BTB1 BTB1 BTB1-C BTB1 BTB1

V C C

B Hetero-species V-BTB1 BTB1 BTB1 BTB2 BTB2 BTB Coexpression

BTB2-C C

BTB1 BTB2

Figure 21: BTB dimers produced after coexpression. Coexpression of homo-species (A) and hetero-species (B) BTB domains creates two sets of BTB dimers. In homo-species BTB coexpressions, 3 types of BTB homodimers are predicted to form. In hetero-species BTB coexpression, if two BTB domains do interact, 2 types of BTB homodimers and one heterodimer will form.

Several modifications were introduced to improve the signal to noise level. Proteins were placed into His-tag based plasmids instead of original GST plasmids. In this case, background interactions caused by dimerization of GST was eliminated while purification

Chapter 4: Discussion tags were still preserved. In addition, BTB domains were coexpressed with fluorescent proteins to serve as negative controls and baselines in signal detection. It should be noted that fluorescent proteins were used as fluorophores for protein quantification here, in order to avoid problems associated with FRET, such as self interactions and signal variances due to geometrical differences between fluorophores.

Based on these modifications, two versions of BTB domains were cloned as shown in

Table 7. Bait clones encoding for His-BTB-C were coexpressed with prey clones encoding for V-BTB. This matrix approach was also often used in other high throughput assays, such as yeast-two-hybrid. It was appreciated for its clear organization in systematic screenings.

During coexpressions, the usage of small-scale autoinduction system expedited the assay significantly [98]. In autoinduction system, protein expression from the plasmids was repressed until cells reached saturation and started to induce without the addition of IPTG.

Further testing showed that the culture could be grown directly from plates. Small scale protein expression culture of 2 mL was sufficient for the detection because of high yields using autoinduction system. Therefore, protein expression, lysis and purification were all handled by multi-well plates, similar to other high-throughput assays.

Ni-NTA beads Co-expression supernatant Venus Cerulean

Binding BTB2 BTB1 Washing

HHHHHH

Figure 22: Rationale of Ni-NTA plate retention assay Steps involved in Ni-NTA plate retention assay are illustrated in this figure. Ni-NTA beads were firstly loaded onto filter plate. After washing and equilibration, coexpression supernatant was added for binding. His tagged BTB1-Cerulean serve as bait proteins to bind onto plates. If BTB1 and BTB2 interact, Venus-BTB prey proteins will be retained on the plate, and thus be detected and quantified. Unbound Venus-BTB proteins are washed out to the underdrain plate.

Chapter 4: Discussion

Detection of BTB dimers was achieved by a Ni-NTA retention assay (Figure 22).

Retained BTB prey proteins were quantified by Venus fluorescent intensities using microplate reader. Instead of using Ni-NTA coated 96-well plates, filter plates preloaded with Ni-NTA beads were selected. This was because protein binding and washing on filter plates involved less manual handling, and gave higher signal to noise ratios. Moreover,

Ni-NTA beads could be regenerated, which was more economical.

Native PAGE was also used to detect BTB dimers formed after the coexpression. The

Venus tag facilitated the detection of BTB domains using the fluorescent imager. It provided direct evidence of the presence of BTB heterodimers after coexpression. However, generating a good native PAGE was sometimes difficult. For example, a unclear separation of two proteins was one of the issues often encountered. Therefore, native PAGE was not used as the primary technique for BTB dimer formation. Instead, it was used as a secondary checking for dimers identified from the coexpression assay.

Figure 23: Comparison of four BTB-BTB interaction assays. Summary of three stages in the assay development is listed above. Major improvements from FRET based assay to coexpression based assay are also highlighted.

Chapter 4: Discussion 4.2 BTB dimers detected by FRET based assay

4.2.1 FAZF homodimers undergo spontaneous exchange

In FRET based assays, initial kinetic assay tested whether BTB homodimers underwent spontaneous dimer-dimer exchange. A time-course of FRET spectra indicated that most BTB homodimers did not exchange in solution except for FAZF. Spectra of V-FAZF and FAZF-C mixed solution showed a gradual increase in FRET signal at 527nm accompanied by a gradual decrease at Cerulean emission at 475nm. Although changes were small, they were consistently and reproducibly observed.

Why was spontaneous self-exchange only found with FAZF? Structural comparison of

BTB domains provided a possible explanation. As stated previously, most BTB domain homodimers demonstrated strand-exchanged structures. There were two major contributions in the dimer interface: the tight packing of α1, α2 and α3 between two chains and the interchain β1-β5 interaction. Disassociation of strand-exchanged BTB homodimers required separation of α packing and unwinding of N terminal β strands. The later motion is expected to be difficult because it involves large conformational changes along two chains. Therefore, self-exchange of strand-exchanged BTB homodimers would be extremely unfavorable. In contrast, FAZF was the only one that did not have swapped domains. In FAZF, those two chains were held by the contact between α helices in the core (Figure 20B). Presumably, the lack of interchain β1-β5 interactions significantly lowered the kinetic energy barrier for dimer disassociation. Therefore, among all BTB domains, FAZF was the only possible one that could have spontaneous self-exchange.

Chapter 4: Discussion

4.2.2 BTB homodimers and heterodimers formed after refolding

Because most BTB dimers did not form under non-denaturing conditions, it was then hypothesized that after chemical denaturation, collapsed BTB monomers could co-fold in vitro, reforming BTB dimers. Under this hypothesis, homo-species BTB proteins would allow homodimers to refold, while hetero-species BTB proteins would allow heterodimers to fold.

BTB homodimer was used to test the hypothesis based on the finding that all BTB domains examined form homodimers, which was consistent from both in vivo and crystal structure studies. It was predicted that homodimeric interactions would be observed for all other BTB domains in this family. In the process of assay development, BTB homodimers were particularly useful, as they were used as built-in positive controls, and thus effective measures of the accuracy and sensitivity of the assay.

It was found that strong FRET signals were detected for most homo-species BTB proteins after denaturation and refolding, indicating BTB homodimer formation. For further confirmation, CD spectra measured before and after refolding were compared (Figure 10).

All BTB pairs were shown to regain their conformation after the removal of denaturants.

Trypsin digests also confirmed that the FRET signal was due to BTB-BTB interactions.

Because both BTB domains and fluorescent proteins, but not the linker between them, were resistant to trypsin digest, it was expected that FRET signal would gradually decrease due to release of the BTB domains from the fluorescent proteins. Experimental findings were consistent with the prediction (Figure 13), proving that dimers formed after refolding were not via interactions of fluorescent proteins. Moreover, despite their different fluorescent

Chapter 4: Discussion protein tags, BTB homodimers formed should have the same free energy as BTB homodimers with the same fluorescent tags, indicating that there was a mixed population of

BTB homodimers in the solution (Figure 21). Formation of BTB homodimers after denaturation and refolding validated the hypothesis, which was also applicable in heterodimer detections later on.

After the rationale was validated, FRET signals after denaturation and refolding were detected for hetero-species BTB proteins. Although the refolding was successful, FRET signals were suffered from ambiguities due to varied signal strength, which will be discussed in the next section. FRET emission ratio was used as an effective measure in comparing generated data with positive and negative controls. Based on emission ratios, it was found that most BTB domains did not form heterodimers, which had emission ratios comparable to negative controls. Six pairs of hetero-species BTB proteins were measured with weaker

FRET signals. Weak FRET signal could result from low population of BTB-BTB heterodimers. In addition, increased distance between fluorophores could weaken FRET intensities as well, so that the magnitutde of the FRET sinal could not be taken as a direct measure of the population of the heterodimers. Therefore, it was hard to determine whether

BTB heterodimers formed in these 6 pairs.

It was noticed that other BTB heterodimers that have been reported in the literature, such as LRF and PLZF [80], were not identified as strong positives by our FRET assay. They were either categorized to no FRET signal or weak FRET signal. BCL6 and Miz1 was previously identified as a heterodimeric pair [38], but one of the BTB pairs with weak FRET signal here. Because BTB heterodimers could be not be unambiguously confirmed by the denaturation and refolding assay, based on the evidence of BTB heterodimers in previous 77

Chapter 4: Discussion studies [11], we turned to the coexpression assay.

4.2.3 FRET signal variance caused by structural differences

As mentioned previously, FRET signal variance caused problems when determining if a true

BTB dimer was formed after refolding. In the BTB homodimer denaturation and refolding assay, FAZF had a weaker FRET signal compared with other BTB homodimers. On the other hand, for BTB heterodimers, several pairs were also found to have weak FRET signals.

In homo-species BTB denaturation and refolding experiments, most pairs exhibited strong FRET signals after refolding with emission ratios all above 0.6. The only exception was FAZF homodimers with emission ratio between 0.5-0.6. FRET signal was inversely proportional to the 6th order of distance between fluorophores. Therefore, the weaker FRET signal detected for FAZF could be explained with its structural information. FAZF had a non-domain-swapped structure (unpublished data), where N and C termini from two chains were about 58Å apart. When fluorescent proteins Venus and Cerulean were attached to FAZF, the distance between fluorophores was predicted even greater depending on their orientations. It also explains why FAZF homodimers had weak FRET signals in kinetic studies.

For BTB heterodimers, no such explanation could be addressed because no BTB heterodimer structure has been solved to date. FRET signal variance became a major drawback of this assay, as this assay was aimed to detect uncharacterized BTB dimers.

Therefore, a new method was developed to avoid any FRET signal variance due to structural differentiation.

Chapter 4: Discussion 4.3 BTB dimer detection by a coexpression assay

This assay has two core developments compared with the previous assay: 1) usage of coexpression to promote BTB dimer formation; and 2) detection of formed BTB dimers was dependent on fluorescent intensities of Venus and Cerulean instead of FRET signals.

Coexpression of BTB domains in bait and prey clones allowed domains to interact during folding. BTB homodimers were again used as the positive control to test this hypothesis. It was found that for all BTB domains successfully expressed, homodimers were readily formed. On the other hand, different from FRET based assay, where equal amount of

BTB proteins were mixed, coexpressions were concerned with the uneven expression levels due to discrepancies of plasmid copy numbers. Better than expected, most coexpression pairs achieved equivalent protein expressions with reasonable variations.

It was also predicted that BTB homodimers formed in coexpression would be a mixture of different types of BTB homodimers, as found in denaturation and refolding assay. This was confirmed by the native PAGE analysis, where different excitation and emission filters were used to separately detect the Venus and Cerulean fusion proteins. In the BTB homodimers tested, typically three bands were observed: one contained Venus only, one contained Cerulean only, and another one contained both Venus and Cerulean. In fact, the color composition of these bands already revealed their identities. Coexpressions of the same

BTB domains tagged with different fluorescent proteins created a mixture of V-BTB homodimer, BTB-C homodimer, and V-BTB-BTB-C dimer, as shown in Figure 21A.

Compared with the FRET based assay, BTB dimer detection based on fluorescent protein plate retention was able to offer more accurate results and more convenient data

Chapter 4: Discussion interpretation. First of all, fluorescent proteins were solely used as a tool to quantify protein concentrations. Venus levels retained on the plate were not dependent on either the dimer structure or FRET signal between Cerulean and Venus. Therefore, the ambiguity associated with FRET signal variance was avoided. This also simplified the process of data interpretation. In this assay, Venus level retained on the plate was normalized and compared with negative controls in each group. It was found that both BTB homodimers and heterodimers formed in coexpression retained significant higher levels of Venus on the plate.

No Venus could be detected for BTB pairs that did not interact.

This assay not only provided quantitative information of Venus and Cerulean expression level, but also relative ratios of dimer formation. From pairs formed BTB dimers, it was found that Venus level retained on plates were approximately half of the total amount of

Venus before binding. This is consistent with the 1:2:1 relative ratio of dimers formed in the coexpression.

All BTB homodimers that achieved minimum expression level were readily detected in the homo-species BTB coexpression supernatants, which was consistent with previous findings. On the other hand, only a few BTB heterodimers were detected in hetero-species

BTB coexpressions: BCL6 and Miz1, as well as Miz1 and RP58. This indicated that BTB domains were all capable of homodimeric interactions, while BTB heterodimeric interactions were more specific.

Miz1 and BCL6 heterodimer was previously reported in the literature [38]. It was shown that BCL6 formed heterodimers with Miz1 and was recruited to CDKN1A promoter region, where there was binding site for Miz1 but not BCL6. This was shown to be an important step

Chapter 4: Discussion for GC B-cell to pass over P53-independent growth arrest and apoptosis pathway upon DNA damages [38]. Results generated here provided in vitro evidence that BTB domains were mediating this heterodimeric interaction. However, information generated both in vivo and in vitro was still not enough. Structural information of Miz1 and BCL6 heterodimers would provide valuable information to understand the dimer interface. More biological studies should be addressed to understand how Miz1 and BCL6 heterodimer formation was regulated in the cell.

Miz1 and RP58 heterodimer has not been reported before. To probe the possible functional roles of this heterodimer, expression profiles for both proteins were analyzed. As stated before, Miz1 was upregulated in many forms of cancers, while RP58 was expressed in lymphoid tissues, lymphoid, testis, heart, brain, skeletal muscle, and pancreas [14]. It was possible that in cells expressing both Miz1 and RP58, heterodimers would exhibit a distinct transcription regulation pathway as Miz1 and RP58. Interestingly, RP58 has been shown to bind to DNA sequence containing E-box, which also binds to c-Myc. One report suggested that RP58 might compete with c-Myc for DNA binding in certain cell types [67]. Miz1 may act as an intermediate between c-Myc and RP58, as Miz1 can physically interact with c-Myc via C-terminal regions of the protein [66].

4.4 Comparison with literature findings

This section compared results generated in this project with findings in the literature. BTB homodimers detected were consistent with previous structural studies [11, 29, 33-35].

Moreover, BTB heterodimers identified in this study clarified some results in the literature.

Chapter 4: Discussion

In the literature, LRF and PLZF, as well as LRF and BCL6 were found to interact by Co-IP techniques [16]. In this assay focusing on BTB domains, it was found that these heterodimeric interactions were not observed, suggesting that the interactions were not mediated by BTB domains. Either linker regions or zinc finger regions may be responsible for the interaction. A recent study indicated that Miz1 and ZBTB4 were coimmunoprecipitated from colon carcinoma cells [39]. This study did not identify domains mediating this heterodimeric interaction. According to results from my assay, BTB domains of Miz1 and ZBTB4 were not involved in the interaction, indicating other unknown factors might be contributing to the heterodimer formation. In addition, BTB heterodimer pairs

Miz1-RP58 was never reported, which provides a candidate for further research in vivo.

My results proved Miz1-BCL6 heterodimeric interaction shown in previous studies [11,

38]. Miz1-BCL6 heterodimers were formed when two proteins interacted during folding in cells. The fact that Miz1-BCL6 heterodimers could be separated in native PAGE indicated heterodimers could be purified in a large amount. Therefore, Miz1-BCL6 heterodimers will be the BTB heterodimer to be coexpressed and purified for structural studies.

LRF-Miz1 heterodimer was detected in previous studies [11], but not by assays developed here. The reason that causes this discrepancy is still not clear. One possible explanation can be different tags linked to BTB domains. Thioredoxin and GFP tags might lead BTB domains to slightly different folding pathways.

4.5 Does BTB dimerization specificity relate to sequences?

It was concluded that BTB heterodimers formation was specific, although BTB homodimers

Chapter 4: Discussion were regularly formed. 14 BTB domains sequences were compared pair-wise to determine whether heterodimer specificity was related to sequence conservation (Table 11). It was found that most BTB pairs shared about 20%- 30% overall sequence identities, which were found common in BTB-ZF family. Several pairs had higher sequence identities (between

30% and 40%). Heterodimers Miz1-BCL6 and Miz1-RP58 were found in this category.

However, the other BTB pairs with similar sequence identities did not form heterodimers.

ZID and Bioref had the highest sequence identity, which was 62%. Due to the low yield of

ZID expressed in E. coli, whether these two BTB proteins heterodimerize was unclear.

Therefore, overall sequence identities did not provide sufficient information to understand

BTB dimer specificity.

PLZF FAZF Kaiso LRF BCL6 Miz1 RP58 Bioref KUP BOZF Y441 HKR3 ZID ZBTB4 PLZF 100% 30% 25% 36% 25% 30% 28% 21% 25% 25% 24% 25% 24% 15% FAZF 100% 20% 22% 20% 20% 21% 20% 21% 20% 21% 23% 20% 13% Kaiso 100% 26% 18% 28% 25% 21% 25% 25% 26% 27% 23% 22% LRF 100% 29% 38% 33% 22% 24% 33% 27% 31% 27% 22% BCL6 100% 30% 29% 20% 21% 32% 25% 30% 25% 15% Miz1 100% 39% 22% 32% 36% 35% 37% 27% 20% RP58 100% 24% 34% 35% 31% 33% 25% 17% Bioref 100% 19% 21% 20% 24% 62% 13% KUP 100% 28% 29% 26% 19% 16% BOZF 100% 25% 30% 22% 19% Y441 100% 31% 23% 18% HKR3 100% 26% 19% ZID 100% 13% ZBTB4 100%

Table 11: Pair-wise sequence comparisons of 14 BTB domains. Sequence identities of 14 BTB domains are summarized in this table. It is found that sequence identities ranged from the lowest 13% (ZBTB4 with FAZF, Bioref, and ZID) to the highest 62% (ZID with Bioref). However, most BTB domains share about 20%-40% sequence identity.

Besides the overall sequence identity, residues located in dimer interfaces were also

Chapter 4: Discussion taken in consideration. Based on structural information of PLZF, Kaiso, LRF, BCL6, and

Miz1 homodimers, it was predicted that BTB dimers would share similar dimer structures.

Therefore, residues found in dimer interfaces of these 5 homodimers were highlighted and compared for all 14 BTB domains, as shown in figure 24. It should be noticed that FAZF was not included in the discussion because of its unique non-domain-swapped dimer structure, which was different from all other BTB homodimers. It was concluded that among

14 BTB domains, there were 5 residues located in the dimer interface were absolutely conserved. In addition, there were 8 residues in the dimer interface were considered highly conserved and the rest of residues were less conserved. After detailed assessments, conservations of these residues did not exhibit any obvious relationship with the experimental dimerization pattern. Lastly, as most dimers interfaces involved α1 from one domain interacting with α2 and α3 from the other chain, residues pairs from two domains were also examined. It was found that these residues were mostly hydrophobic residues buried inside the core. Moreover, residues pairs extracted from random BTB pairs did not cause more clashes than ones from true BTB dimers.

There were several reasons why sequence information failed to explain BTB dimer specificities. First of all, there was limited information that could be extracted from two BTB heterodimer pairs observed. Secondly, heterodimer interface was assumed the same as the homodimer. However, without any real heterodimer structural information, this assumption might be incorrect. In summary, with limited information on hand, it was challenging to understand the rules governing BTB heterodimer specificity.

Chapter 4: Discussion

Figure 24: Sequence alignments of 14 BTB domains Based on structural information of PLZF, FAZF, Kaiso, LRF, BCL6, and Miz1, sequences of 14 BTB domains are aligned based on secondary structures. Predicted residues involved in homodimer interface of are highlighted and compared. Absolutely conserved residues are colored brown; highly conserved ones are colored orange; and less conserved ones are colored yellow.

Chapter 5: Conclusion and future directions

Chapter 5

Conclusion and future directions

5.1 Conclusion: assay development and BTB dimer detection

BTB-ZF transcription factors are implicated in many types of cancers. BTB domains mediate various BTB-ZF interactions, which expands the functional range for these transcriptional factors. How BTB domain chooses its interaction partner becomes essential to understand how these transcriptional factors regulate protein expression. Because BTB dimers do not exchange under native conditions in vitro, many existing detection methods were not applicable. In order to provide a medium to high-throughput detection for BTB dimers, a new assay combining fluorescent protein tags, coexpression and Ni plate retention has been developed in this project. This assay was validated using 6 BTB domains that have been studied previously. Results generated were consistent and reproducible. Later on, this assay was applied to additional 8 BTB domains from the database. Overall, 14 BTB domains interaction network has been successfully mapped. It was concluded that all BTB domains were able to interact with themselves forming homodimers. Two pairs of BTB heterodimers were also detected. A Miz1-BCL6 heterodimer was previously reported in the literature [38]. 86

Chapter 5: Conclusion and future directions

This assay confirmed that BTB domains from two proteins were able to heterodimerize. The other pair was Miz1-RP58, which was first reported by this assay. BTB dimers identified by this assay provided guidance for further functional and structural studies. Importantly, one of the major conclusions from this study is that most BTB domains do not form heterodimers.

This information will be helpful in understanding BTB-ZF transcription factor network.

5.2 More BTB domain interaction mapping using this assay

In the initial design of this assay, significant amount of work had devoted to achieve higher throughputs. This project managed to incorporate 14 different BTB domains. The final medium to high-throughput assay could be scaled to study the interaction network of all 43 human BTB domains. Although most BTB domains were successfully expressed in E. coli, a couple BTB domains did not express to desired levels, which included ZID, ZBTB4 and

KUP. Further modifications could be done to improve expression yields. For example,

ZBTB4 BTB domain contains an internal gap, which could be truncated to obtain higher expression levels. In addition, there are some important BTB domains not included in the study, especially HIC-1 and MAZR, which have been characterized as important transcriptional regulators in various biological mechanisms. Systematic interaction mappings of all BTB domains will help us to understand BTB interaction specificity, which was still unclear due to lack of information.

Chapter 5: Conclusion and future directions 5.3 Functional and structural studies on identified heterodimers

BTB dimers identified from this assay become promising candidates for further functional and structural studies. Functional studies could examine whether newly detected BTB dimer exist in vivo. Questions should be addressed to whether two BTB proteins expressed in the same cells and, if so, are they localized in the same cell compartment. How BTB heterodimers impact cellular activities could be directly assessed by disruption of the dimer.

This assay also provides excellent candidates for structural studies. BTB heterodimers formed after coexpression achieved high levels of expression, which could be directly purified for crystallization. Moreover, these heterodimers were readily detected by both plate retention assay and native PAGE, indicating an optimal stability required in structural studies.

In the long run, structural information of BTB heterodimers would shed lights to understand the nature of BTB dimer specificities.

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Appendix

Representative native PAGE images for LRF, ZBTB4, and HKR3 coexpression pairs