Epigenetic control of neuronal activity dependent gene transcription as a basis for long term memory formation

The present work attempts to study and characterize the events that mediate long-term memory consolidation from an epigenetics standpoint, specifically in chromatin modification or “epig

Copyright

Nicodemus Edrick Oey

2014

The brain is a highly plastic structure, capable of changing its responses to the external stimuli it receives This evolutionarily conserved concept of neuroplasticity underlies all forms of memory, from sea slugs, flies, rodents, all the way to humans While short-term memories that last for minutes are mediated

by transient changes in neural connections, long-term memories that last for years require more persistent changes that involve the production of new proteins Decades of research have shown that the molecular mechanisms responsible for the consolidation of long-term memory are unlikely to be mediated

by changes to the static genomic DNA sequence The last twenty years have seen the emergence of epigenetics as a highly sophisticated mechanism by which a neuron can dictate with remarkable specificity which genes should be expressed at precisely which time in response to activity

Neuronal activity-dependent gene transcription depends on the action of several enzymes that respond to activity to specifically regulate the expression of genes that effectuate downstream functions The ability of epigenetic regulators to tag specific locations in the genome for the de-novo transcription of genes has proven to be essential to learning and memory, as indicated by the disastrous consequences of their absence in various clinical syndromes of mental retardation The present work attempts to study and characterize the events that mediate long-term memory consolidation from an epigenetics standpoint, specifically in chromatin modification or “epigenetic tagging” of specific nucleosomes which seem to be involved in both early and late events of memory consolidation along the temporal axis of neural activity Amongst the many epigenetic regulators important for memory function, the TIP60 protein, in particular, is of significant interest due to its involvement both in early events of neuronal activity-dependent gene induction, and also in late events consisting of epigenetic changes leading to long-lasting memory consolidation

Using a combination of biochemistry, super-resolution microscopy, chromatin immunoprecipitation, and mass spectrometry-based techniques, the first part of this thesis presents findings on the role of the Alzheimer’s Disease-associated epigenetic enzyme TIP60 and an X-linked Mental Retardation (XLMR)-associated protein PHF8 in the rapid neuronal activity-dependent transcription of ARC, a crucial regulator of memory consolidation The second part of this thesis will explore the role of TIP60 in mediating the functions of ARC protein itself in the late epigenetic events that eventually result in memory consolidation The last part of the thesis will be devoted to discussing the importance of epigenetic processes of chromatin modification in general neuronal functions such as development and survival, as well as specific functions such as memory consolidation Finally, looking forward to the future, several of the potentially endless possibilities in neuroepigenetics such as clinical applications of epigenetic modifying therapy and ARC-modulating strategies in neuropsychiatric disorders will be offered

Dedication

As you read the words written on this page, neuronal cells in your brain are firing in highly patterned electrical activities across their synapses in order to encode the information you read, some of which you may remember, being eventually stored in your memory This remarkable ability of the brain which is composed of over 200 billion neurons and more than 100 trillion synapses is thanks to the amazing capabilities found in each unique neuron, which is able to change the genes it expresses at any given time in response to the pattern of activity it receives Such is the dynamics of neuronal cells that networks of them are able to underlie our ability

to see, to hear, to smell, to taste, to move, to feel, and most importantly, to think…

I would like to dedicate these four years’ work to my parents, Hoat and Megan Oey, for bringing me to life, for raising me as a good son and brother, and for allowing me

to travel 15349km to Singapore to start living my dream of being a Clinician-Scientist,

a long, arduous journey which this PhD dissertation is a part of: Mom and Dad, I love you I would like to thank my siblings for all their support: my sister Elrika and new brother-in-law Christopher Prest in Waterloo for allowing me to sing at their wedding,

my younger sister Elvina Oey in New York for the invaluable Occupational Therapist and Psychologist point-of-view, my younger brother Edbert Oey for muscling me into the right path no matter how much I strayed, my younger cousin William Tanaka Oey for being the fun, kind-hearted man that he is and Carissa Oey for taking care of my parents when I should be the one doing it… thank you to all my wonderful siblings: I love you guys One incredibly special mention goes out to the love of my life, Christine Chan, PhD candidate, for correcting my drawings of neurons that look like

“Strepsils™”, for those many hours of rehearsals before presentations, and for helping me clean my incredibly messy room: I love you, Hui Shan

I would like to thank the VanDongen Laboratory: Shaun Teo and Caroline Wee, for holding me by the hand when I was first trying to walk (aka run gels), Rajaram Ezhilarasan for the hard work and dedication, Niamh Higgins, Knvul Sheikh, Annabel Tan, Gokul Banumurthy our amazing RA’s who work day and night and on the weekends too, Ju Han the computer genius who taught me Chinese, Mark Dranias for the discussions on synaptic plasticity, Xiaoyu for investigating long-term memory

in cultured neurons, and especially How Wing Leung, our post-doc, who has taught

me everything I know Finally, I would like to say to my lab mom and dad, Tony and Margon VanDongen, “Aren’t you glad I survived?” – thank you Margon for the best spaghetti I’ve ever tasted, for believing in me when no one else did, and for defending me when odds were against it, thank you Tony for mentoring me… you have taught me how to do science, and that is something I will never forget for the rest of my life I am forever in your debt With that, I hope you don’t mind me saying that I am very much looking forward to the exciting research we have planned together on the horizon!

Contents

Title Page i

Abstract Signature ii

Copyright iii

Abstract iv

Dedication v

Table of Contents vi

List of Figures x

List of Abbreviations xii

Chapter 1

Review of Literature 1

I Introduction to Learning and Memory A A short history 1

B Classifications of memory 3

II Molecular Mechanisms of Long-Term Memory Formation 5

A Overview: plasticity and activity 5

B Epigenetics as a mechanism of activity-dependent gene expression 6

C ARC: a master regulator of synaptic plasticity 11

D TIP60: an effector of early and late neuroepigenetic events 15

E PHF8: a specialized neuronal transcriptional co-activator 16

III The Timeline of Neuronal Activation 18

Chapter 2 Early Epigenetic Events: the Characterization of a Chromatin-modifying Complex Composed of PHF8 and TIP60 that Alter H3K9acS10P to Enable Activity-dependent Transcription of Arc .20

I Abstract 20

II Introduction 21

III Materials and Methods 24

A Plasmid Construction and Cloning 24

B Hippocampal and Cortical Neuronal Cell Culture 25

C Transfections and Neuronal Stimulations 26

D Conventional Immunofluorescence 26

E Proximity Ligation In-Situ Assay 27

F Widefield Microscopy, Calcium imaging, and Data Analysis 28

G Co-immunoprecipitation and Western Blotting 29

H Immunoprecipitation followed by Mass Spectrometry 30

I Chromatin Immunoprecipitation (ChIP) and Triton X-Acetic

Acid-Urea histone gel electrophoresis 30

J 3-dimensional Structured Illumination Microscopy 32

K 3-dimensional Stochastic Optical Reconstruction Microscopy 32

IV Results 33

A Transcriptional Activators PHF8 and TIP60 Colocalize in the Interchromatin Space 33

B The Histone Demethylase PHF8 Physically Associates the Histone Acetyltransferase TIP60 37

C PHF8 and TIP60 Form a Dual-Function Complex that Increases Histone Acetylation on H3K4me3-bearing Chromatin 39

D PHF8 Removes Transcriptionally Suppressive H3K9me2 and Associates with Transcriptionally Active H3K9ac 42

E PHF8 and TIP60 are Activity-dependent and Co-regulate H3K9acS10P in Response to Neuronal Activity 43

F The PHF8-TIP60 Complex Modulates activity-induced H3K9acS10P 46

G The PHF8-TIP60 Interactome is Rich in Proteins Involved in Transcription and Includes the Neuronal Splicing Factor PSF 50

H Super-resolution Microscopy Situates Endogenous PHF8, TIP60, and PSF Within 30nm of Each Other in the Activated Neuronal Nucleus 53

V Discussion 56

Chapter 3 Late Epigenetic Events: the Interaction Between TIP60 and ARC Functions to Regulate H4K12ac, a Learning-induced Chromatin Modification Involved in Ageing-associated Memory Impairment 64

I Abstract 64

II Introduction 66

III Materials and Methods 68

A Constructs and Cloning 68

B Cell Culture 69

C Transfections and Stimulations 70

D Immunofluorescence 70

E Imaging and Data Analysis 71

F 3-dimensional Structured Illumination Microscopy 72

G Photo-activated Localization Microscopy (PALM) and Direct Stochastic Optical Reconstruction Microscopy (dSTORM) 72

H Immunoprecipitation and Western Blotting 73

I Induction of Arc gene expression by stimulation of neural network activity 74

IV Results 75

A ARC Protein Interacts with betaSpIVSigma5, PHF8, PML and TIP60 and Components of the TIP60 Chromatin Remodeling Complex 75

B PML, TIP60, and ARC Form a Tight Complex in the Nucleus of Activated Neurons 77

C TIP60 and ARC Overexpression Increases H4K12 Acetylation but not H3K9, H3K14, H2AK5, or H2BK5 Acetylation 78

D ARC, PML, and PHF8 Modulate TIP60’s Acetyltransferase Activity 80

E Endogenous ARC Interacts with TIP60 in a Variety of Dynamic Nuclear Structures as Seen on Localization Microscopy 81

F Endogenous ARC is Correlated with High TIP60 Nuclear Levels in Activated Neurons 84

G ARC Recruits TIP60 to PML Bodies 85

H Activity-induced ARC Increases H4K12 Acetylation at a Timepoint that Correlates with Memory Consolidation in Neurons 86

I The Enzymatically Inactive Mutant of TIP60 Fails to Induce H4K12 Acetylation in Hippocampal Neurons 88

J ARC Associates at Single-Molecule Level with the

Learning-Induced Histone Mark H4K12ac 89

II Preliminary Results and Discussion

A In-vitro Neural Network Activity Leads to Specific Site-Directed Changes in Chromatin Modification 101

B In-vivo Novel Environment Enrichment Leads to Specific Patterns

of Chromatin Modification Partly Mediated by PHF8 and TIP60102

C PHF8 and TIP60 are Activity-Dependent Chromatin-modifying Enzymes With Different Promoter Occupancy Profiles 103

D The Transcriptional Activator PHF8 is Found Within Nanometres

of PTB-associated Splicing Factor and Nascent RNA 105

E The Recruitment of PHF8 to Active Transcriptional Start Sites

Precedes RNA Polymerase II Binding at the Arc and c-Fos

Genomic Loci Following Neuronal Activation 106

F Specific Regulation of Arc Gene Expression by ERK and p38

MAPK Signaling Pathways 107

G The Interactome of PHF8, TIP60, and ARC Give Novel Clues to the Processes that Lead Ultimately to Memory Consolidation 110

Chapter 5

Conclusions and Future Directions: Towards Epigenetically Informed Translational and Clinical Trials 117 Bibliography 125

Appendix A

Publications accepted or under review 138

Figure 3: PHF8 removes the repressive histone mark H3K9me2 and associates with the

activating histone mark H3K9ac 42

Figure 4: Neuronal activity reorganizes PHF8 and TIP60 in the nucleus and effectuate

histone methylation and acetylation changes 45

Figure 5: PHF8 and TIP60 modulate neuronal activity-induced histone acetylation at

H3K9acS10P and activation of the Arc gene 48

Figure 6: Knockdown of PHF8 impairs activity-dependent induction of H3K9acS10P and Arc

and c-Fos expression 49

Figure 7: PHF8, TIP60, and H3K9acS10P are specifically enriched in the transcriptional start

site of the Arc gene 50

Figure 8: Common interacting partners between PHF8 and TIP60 function primarily in

transcription and mRNA processing 52

Figure 9: Endogenous TIP60 is located within 30nm of PHF8 in the activated hippocampal

neuronal nucleus 54

Figure 10: PHF8 and TIP60 form a tripartite complex with the splicing factor PSF/SFPQ 55 Figure 11: Four-color immunofluorescence of a quaternary complex formed between ARC,

PML, bSpectrin, and TIP60 77

Figure 12: ARC protein interacts with two members of the TIP60 chromatin remodeling

complex: the transcriptional coactivator BRG1 and AMIDA 77

Figure 13: Endogenous ARC is able to localize TIP60 to PML bodies 79 Figure 14: ARC+TIP60 overexpression had a mild effect on global H4K12 acetylation 80 Figure 15: ARC has a positive modulatory effect on TIP60-mediated H4K12 acetylation 81 Figure 16: 3D Stimulated Emission Depletion Microscopy shows association of endogenous

Arc and Tip60 83

Figure 17: Dual-color super-resolution microcopy of Arc-mEOS2 and endogenous Tip60 in

the activated neuronal nucleus 84

Figure 18: ARC protein levels correlate with that of TIP60 at the 4-hour mark of sustained

neural activity 85

Figure 19: ARC recruits Tip60 to PML bodies 86 Figure 20: ARC expression increases H4K12ac levels 89 Figure 21: A Tip60 mutant lacking acetyltransferase activity decreases H4K12 acetylation.

H3K9acS10P in a subset of neurons in the brain 104

Figure 26: NEE induces specific H3K9acS10P within a subset of neurons that have TIP60 and

ARC in the hippocampus 107

Figure 28: Single-molecule localization microscopy situates PHF8 and its binding partner

PSF in areas of active RNA transcription 109

Figure 29: PHF8 is recruited to TSS of neuronal activity-dependent genes before RNA

Polymerase II 110

Figure 30: Control of specific activity-induced gene expression by modulation of upstream

signaling kinase pathways 112

Figure 31: The interactome of PHF8, TIP60, and ARC reveals a major uniting mechanism of

mRNA metabolism, transcriptional regulation, and mRNA splicing 115

Figure 32: A graphical abstract of activity-dependent DNA, histone, RNA, and protein

changes in a single brain cell 116

Figure 1 – Graphical Summary of Present Dissertation 122

List of Abbreviations

Arc = Activity-Regulated Cytoskeletal-associated gene (NCBI gene ID 11838 in Mus musculus)

CFP = Cyan Fluorescent Protein

DAPI = 4',6-diamidino-2-phenylindole; marker of cellular DNA

Fos = FBJ osteosarcoma oncogene (NCBI gene ID 14281 in Mus musculus )

GFP = Green Fluorescent Protein

H3 = Histone 3

H3K9 = Histone 3 Lysine 9

H3K9ac = Histone 3 Lysine 9 acetylated

H3S10p = Histone 3 Serine 10 phosphorylated

H3K9acS10P = Histone 3 Lysine 9 acetylated Serine 10 phosphorylated

HEK293 = Human Embryonic Kidney cells 293

IEG = Immediate-Early Gene

MYST = MOZ, YBF2, SAS2, TIP60 family of acetyltransferases

nm = nanometer (1×10−9 m)

NEE = Novel Environmental Enrichment

PHF8 = PHD-Finger protein 8 (NCBI gene ID 320595 in Mus musculus)

Pol II = RNA Polymerase II

PSF = Polypyrimidine Tract Binding protein-associated Splicing Factor, also known as SFPQ (Splicing Factor Proline/Glutamine rich, NCBI gene ID 71514 in Mus Musculus)

SIM = Structured Illumination Microscopy

STORM = Stochastic Optical Reconstruction Microscopy

TIP60 = Tat-Interacting Protein 60kDa (also known as Kat5, K(lysine) acetyltransferase 5) YFP = Yellow Fluorescent Protein

Chapter 1

Review of Literature

1.I Introduction to Learning and Memory

1.I.A A short history

“You have to begin to lose your memory, if only in bits and pieces, to realize that memory is what makes our lives Life without memory is no life at all Our memory is our coherence, our reason, our feeling, even our action Without it we are nothing.”

― Luis Buñuel

A major goal of neurobiology is to establish how complex behaviors such as learning and memory are encoded in the cells of the brain itself In the 28,470 days that constitutes the average human lifespan1, a person may come in contact with over 100,000 other individuals and a multitude of things, places, tastes, sounds, smells, and other sensations which form the bulk of his experiences in life, all of which will largely influence the way he thinks and behaves We refer to this ability as learning, which is defined as a measurable, adaptable change in a person’s behavior due to experience Memory, on the other hand, is the storage and recall of that experience which

is required for a person to use what was learned Although there has been much discord between psychologists using “top-down” approaches and physiologists who employ “bottom-up” approaches in formulating theories about learning and memory, studies of direct removal of certain areas of the brain such as in the case of H.M2 definitively show that the underlying mechanisms of memory are found in the nervous system Experiments conducted by Karl Lashley in the 1930s were amongst the first to dissect out the existence of a memory trace in the brain: using surgical lesions to disrupt

an already-formed spatial memory in animals, it was concluded that memories are not localized in any one structure but are rather distributed diffusely in equipotent areas throughout the cortex3

“This series of experiments has yielded a good bit of information about what and where the memory trace is not It has discovered nothing directly of the real nature of the engram […] I believe that even the reservation of individual synapses for special associative reactions is impossible The alternative is, perhaps, that the dendrites and cell body may be locally modified in such a manner that the cell responds differentially, at least in the timing of its firing, according to the pattern of combination

of axon feet through which excitation is received.” [Lashley K In search of the engram SympSocExp Biol 1950;4:454–82.]

Remarkably even at this early stage of neurobiological research, Lashley had already held the view that memory formation involved the activity of thousands to millions of neurons connected in circuits wherein each individual neuron and its synaptic connections “may be locally modified” This view was corroborated by seminal work by Donald Hebb and Jerzy Konorski who suggested that synaptic connections may be made more effective by neuronal activity which produce both a short increase in excitability, thought to underlie short-term memory, and a long-lasting structural change in the cell body and synapse itself, thought to underlie long-term memory4 This theory, which continues to undergo rigorous validation through studies done at the anatomical, biochemical, electrophysiological and now molecular level, is based on the principle that for learning and memory to occur, changes in the environment must be translated into measurable, adaptable changes in the physiology of the neuron The changes exerted by differing patterns of neuronal activity that result in behavioral modification constitute a mysterious phenomenon commonly referred to as neuroplasticity

It is on this contextual framework of neuroplasticity that I now address the main issue of this thesis which is how does the environment affect individual neuronal behavior to learn and encode meaningful memories? I will begin by first exploring what types of memory are working at the broad, psychological level I will then delve deeper into the proposed mechanisms accounting for memory at the neural network and cellular level, which are all the result of many decades of research Finally, I will focus on the molecular mechanisms

of memory formation, including an overview of the nascent field of neuroepigenetics on which this thesis is built

1.I.B Classifications of Memory

To highlight the gaps in our knowledge about memory formation, it is necessary to briefly review the currently dominant theory of how memory works At the moment of this writing, the generally accepted model, attributed

to Atkinson and Shiffrin in 1968, is that there exist at least three distinct systems that flow naturally from one to another, all of which function together

to form a lasting memory5 Information in the form of stimuli detected by our senses (vision, hearing, touch) can be either ignored and disappear or perceived and enter into our sensory register: a “buffer” or temporary storage which works subconsciously on a time scale of less than one second6,7 When attended to with the proper amount of attention, it is thought that some information is transferred to short-term memory, which works on a time scale

of less than one minute8 Finally, information from short-term memory, through

a mechanism called consolidation, can then enter long-term memory, which may last for days, months, or even in some cases a lifetime9

Regardless of what framework one chooses to study memory, one of the most crucial questions in neuroscience remains how does information become represented by the millions of cells in the brain? Between top-down approaches that look at human behavior, and studies that attempt to look at this question from the “bottom up” or from the molecules that constitute the brain, is the “black box” which is the neural network The brain is made up of hundreds of thousands of networks each of which is built from thousands of individual neurons connected together Due to the elusiveness of the engram, the idea that memories are stored in a distributed manner in the brain has become increasingly tangible In support of this idea, researchers are now able to simulate biologically plausible networks in silico10, which have the capacity to recognize faces and learn to differentiate different types of music, thanks to the emergent properties of the network: that is, capabilities that are not explained by the physiology of each individual neuronal cell These computational experiments have since been validated in living neuronal networks11 In this respect, memory has been attributed to the ability of an interconnected network of neurons to transiently store information through modification of the strength of the synapses that link them Although this may serve as a plausible explanation for working memory that lasts several seconds at maximum, the fact that long-term memory as we know it may persist for months and even years point toward the existence of another, more long-lasting mechanism

1.II Molecular Mechanisms of Long-Term Memory

Formation

1.II.A Overview

In contrast to short-term memory which relies on reverberating activity between neurons and therefore lasts only a few seconds, long-term memory can last for decades and therefore require more permanent changes at the structural level which persist in the absence of transient action potentials Research into the cellular and molecular mechanisms underpinning complex cognitive functions began with the groundbreaking discovery that neurons can adapt in response to the external environment not only during development but also in adulthood, where neuroplasticity governs diverse phenomena ranging from vision, somatosensory maps, to learning and memory Early studies, performed in many different species, involving intracerebral injections

of mRNA and protein synthesis inhibitors showed that inhibiting protein synthesis circumjacent to a training period does not affect short-term memory but abolishes the ability of animals to form long-term memory, indicating that there is a window of time during which gene transcription and protein synthesis is crucial for the formation of long-term memory12-14 It has therefore been known for several decades that there is a fundamental mechanistic difference in the way short- and long-term memory are formed15

Unlike short-term memory, the formation of long-term memory is entirely dependent on gene transcription and subsequent protein synthesis, which

was first demonstrated by elegant studies in the model organism Aplysia 16,17

Since then, extensive work in Drosophila and rodents has validated the notion

that activity-dependent neuronal plasticity underlies long-term memory18-21 Central to all of these studies is the basic principle that external stimuli, through alterations of neurotransmitter release, ion channel opening, Ca2+influx, or electrical activity at neuronal synapses, are able to induce changes

in gene expression that occur inside the neuronal nucleus22-24 The basic premise that gene regulation in the nucleus can be directly altered by neural activity at the synapse lies at the core of the concept of neuronal plasticity that underlies many complex aspects of cognition and behavior But how does a neuron dynamically change its gene expression programs in response to synaptic activity?

1.II.B Epigenetics as a mechanism of activity-dependent gene expression

In the process of differentiation, pluripotent embryonic stem cells divide into highly specialized cell types such as hepatocytes, melanocytes, or neurons themselves which, despite having the same genetic material as the original stem cell, continue to “remember” they are liver, pigment, or brain cells even

as they divide further and replicate their entire genome de novo Upon

exposure to various antigens, T lymphocyte precursors of the mammalian immune system commit themselves to an almost limitless array of differentiated states each of which expresses different genes, forming an immunological memory in response to transient signals from foreign invaders

In much the same way, the remarkable ability of neurons to adapt to the environment can be traced to a fundamental property of all cells, which is that

of cellular memory: this is the concept of epigenetics (epi – from the Greek επί

meaning over or outside of), which is the study of all physiological processes

that are not caused by changes in DNA sequence but can nevertheless highly

influence gene expression25 Previously thought to be hard-wired only into developing cells, epigenetic mechanisms are now also thought to function in the dynamic regulation of gene expression in the post-mitotic neurons of the adult nervous system26 The four cardinal mechanisms of epigenetics include: 1) DNA methylation: the addition of methyl groups to DNA bases, 2) non-coding RNAs: RNA editing and DNA recoding, 3) nucleosome remodeling: the higher-order 3-dimensional arrangement of chromatin, and 4) histone code: post-translational modifications of histone molecules that package DNA itself27

The present work focuses on the role of chromatin modification as an important phenomenon in regulating gene expression programs which mediate activity-dependent plasticity in the nervous system In neurons, genetic material does not exist naked in the nucleus but is compacted in a highly ordered structure consisting of histones and DNA, collectively called chromatin28 Due to the immense levels of compaction, wherein a 3-meter long stretch of chromosomal DNA is able to fit into a nucleus that is 3-microns

in length, a large majority of the genome is virtually inaccessible to gene transcriptional machinery Hence, in a system that requires dynamic gene expression changes, there is a need for a mechanism to attach molecular labels to parts of the genome that needs to be activated at the right time

The basic organization of chromatin is a stretch of 146bp of DNA wrapped around an octameric core histone complex, which altogether is termed the nucleosome Each individual histone, including H2A, H2B, H3, and H4, is an

evolutionarily conserved, highly basic protein which has a protruding terminal tail that can be modified post-translationally through processes not limited to acetylation, phosphorylation, and methylation of specific lysine residues, all of which can affect gene expression through direct modification

N-of chromatin structure or by serving as platforms for the binding N-of transcription factors While histone acetylation has been traditionally thought

to be transcriptionally activating, the role of phosphorylation and methylation can be either activating or repressing, making the dynamic, combinatorial regulation of these modifications an extremely complicated process

Neurons in the brain continually change the genes they express in response

to synaptic activity, which is highly specific to the stimuli the organism receives The physical mechanism of “tagging” nucleosomes inside the neuronal nucleus with post-translational histone modifications can be likened

to ascribing dynamic postal codes or addresses which can potentially serve to dictate exactly “when” and “where” specific genes must be expressed: a highly desirable ability in neurons given the static nature of genomic DNA Importantly, these “tags” or modifications are reversible and continually changing such that at any given moment in time, each nucleosome may be bearing different sets of modifications, with varying effects on gene transcription The epigenetic modification of histone molecules therefore represents a rich, highly modifiable way to regulate gene transcription, which

is very attractive as a potential biochemical basis of information storage in the nervous system

The workhorses that are responsible for reading, writing, or erasing these modifications may be collectively termed epigenetic or chromatin regulators

As with the rules of physics, the rules of chromatin regulation follow the general principle that for every action there is an equal but opposite reaction: while Histone Acetyltransferases (HATs) tag histones with acetylation marks, Histone Deacetylases (HDACs) remove acetyl groups Likewise, Histone Methyltransferases (HMTs) write methylation marks whereas Histone Demethylases (HDMs) erase methyl groups from histones Hundreds of other proteins are able to “read” these nucleosomal tags and latch on to potentially very specific areas of the genome, by virtue of PHD (Plant Homeo-Domain),

or bromo-domains which bind to methylated and acetylated histones respectively

Perhaps the most striking manifestation of the importance of chromatin regulation is the clinical observation that defects in the enzymes responsible for specifying histone modifications lead to specific and disastrous consequences in cognitive function29 Notwithstanding evidence implicating epigenetic enzymes in numerous neurological and neuropsychiatric disorders29,30, an increasingly large number chromatin-modifying proteins is now recognized to be crucial in learning and memory31 For instance, defects

in the enzyme CREB Binding Protein (CBP), a histone acetyltransferase and transcriptional co-activator, cause Rubinstein-Taybi syndrome which is characterized by severe cognitive disability32 On the other hand, mutations in RSK2, a histone kinase that phosphorylates serine-10 of H3, are implicated in Coffin-Lowry Syndrome, which is also marked by prominent mental retardation Finally, inactivating mutations in PHD Finger Protein 8 (PHF8), a histone demethylase, also cause mental retardation33 These seemingly different mechanisms target different histone marks, yet all of them exert a

highly specific neurological phenotype of intellectual disability, prompting the search for the exact roles of histone acetylation, phosphorylation, and methylation not only in neuronal development but also in adult neuroplasticity and memory formation

While it is important to identify the epigenetic enzymes which contribute to cognitive function, the next step will be to understand how these enzymatic machineries are regulated, what chromatin modifications are effectuated, and ultimately, what gene targets they act upon to produce synaptic, network, and behavioral changes The study of the epigenetics of memory is further complicated by the fact that each individual epigenetic enzyme appears to be responsible for controlling distinct gene sets with varying temporal specificities Given that there exist hundreds of epigenetic regulators affecting

a potentially unlimited number of chromatin and downstream gene targets, the study of the molecular mechanism of memory formation at least from the chromatin viewpoint seems like an amazingly daunting task It can be conceptualized, however, that for a single neuron, one can systematically organize the events that eventually lead to memory consolidation in a temporally defined manner Much like how the cellular correlate of memory, Long Term Potentiation, has an early and a late phase, which parallels the timeline of short-term memory acquisition and long-term memory consolidation, epigenetic events can also be divided into early events that occur within minutes of neuronal activation, and late events that take a longer time to develop In this model, early epigenetic events may underlie early-phase LTP and short-term plasticity while late events may be responsible for late-phase LTP and memory consolidation

1.II.C Arc – a regulator of synaptic memory

The leading mechanisms to explain memory at the level of neuronal synapses are Long Term Potentiation (LTP) and Long Term Depression (LTD), which are defined by the measurable, persistent increase (for LTP) or decrease (for LTD) in the strength of the post-synaptic neuronal response in response to varying stimuli34,35 These stimuli-dependent changes in synaptic weights are thought to operate in every single one of the hundred trillion synapses connecting the neurons in the brain and mediate all forms of activity-dependent plasticity36 While LTP can be thought of as being a positive regulator of a memory trace by strengthening response to a stimulus, the seemingly counterintuitive LTD turns out to be crucial for memory as well, apparently by producing a diminished response to a stimulus37 As such, both early and late LTP and LTD are directly paralleled by many behavioral correlates of learning and memory And just like in short vs long-term memory, only the late phase of LTP and LTD requires novel induction of gene and protein synthesis38,39 Although many proteins have so far been implicated in synaptic plasticity, one particular gene product, ARC (Activity-Regulated Cytoskeletal protein), is situated at the nexus of LTP, LTD, and protein synthesis-dependent memory

Arc is a single copy gene that is speculated to have appeared late in evolution

as no reasonable homolog exists in invertebrate species though it is highly conserved in vertebrates, suggesting that this unique gene may have highly specific functions in higher organisms40,41 In excitatory pyramidal neurons of

the cortex and hippocampus where it is enriched, Arc expression has proven

to be one of the most tightly coupled events to neuronal activity and a plethora

of behavioral paradigms ranging from simple exposures to sound42, emotional and fear memory43, spatial exploration44, taste memory45, visual stimuli46, and novel environments47 Remarkably, total deletion of the Arc gene in the mouse

completely abrogated the ability to form long-term memories without disrupting short-term memory formation48 Subsequently, it was found that Arc

expression was related to virtually all cases of long-term memory formation, whether it be in birds49, rodents50, and even humans51 Arc may be the only

gene currently known to be induced by neuronal activity and required for all

forms of LTP and LTD: early Arc synthesis is required for early LTP expression, whereas sustained Arc synthesis is needed for late-phase LTP

maintenance52,53; on the other hand, Arc is also required for LTD54,55 This

bidirectional involvement suggests that Arc may be a master regulator of

activity-dependent changes in synaptic weights, regardless of the direction of the change56

In view of its tight coupling with neural activity and its undisputed role in consolidating long-term memories, much research has focused on

determining the exact molecular functions of Arc and the pathways that govern Arc expression and regulation Consistent with its highly crucial role in memory consolidation, Arc gene expression is tightly controlled at many

levels, including transcription, mRNA degradation, translation, and protein degradation57 Although we now know that diverse mechanisms including NMDA receptors58, the ERK/MAPK pathway59, metabotropic Glutamate receptors60, and Voltage-Gated Calcium Channels61 may all play a role in

regulating Arc expression, the precise signaling cascades that connect

synaptic activity with the actual transcription of Arc gene are complex and not

well understood

Given the complexity in its regulation, there is a high likelihood that the

dynamic, experience-dependent increase in Arc expression may be mediated

by epigenetic mechanisms Although one study has shown that DNA

methylation may be responsible for shutting down Arc expression at the

24-hour time point after electroconvulsive seizures62, the effects of chromatin

modification on Arc transcription have not yet been elucidated Interestingly, recent findings indicate that the rapid induction of Arc transcription that occurs

within minutes of neuronal activity is controlled by RNA Polymerase II which is

“paused” at the promoter region of the Arc gene63 In Chapter 2 of this thesis,

I propose a mechanism by which early Arc transcription is regulated by an

epigenetic mechanism involving a dual-function chromatin-modifying complex and neuronal activity-dependent post-translational histone modification

While it is important to study how such an important gene is regulated at the level of transcription, it is the translated ARC protein is ultimately responsible

for its function Arc translation is also very complex and is known to happen at

multiple sites including dendrites64 The fact that emotional memories are highly Arc-dependent is reflected by the complex regulatory mechanisms involved in ARC protein translation, being modulated by dopamine and beta-adrenergic receptor signaling65 Indeed, infusions of beta-adrenergic agonists into the amygdala increases ARC protein levels in the hippocampus and enhanced the performance of mice trained under an inhibitory avoidance paradigm, an emotionally relevant memory66

What could be the function of such an exquisitely regulated gene with respect

to neural function? Despite decades of research, the only currently known

function of Arc is the endocytosis of GluR2-specific AMPA receptors at

neuronal synapses67, which may mediate its effects on LTD, but does not explain its role in maintenance of late LTP Although some ARC protein exists

in the dendrites where they may control LTD, the majority of ARC protein is located in the nucleus where it binds to a neuronal spectrin betaSpIVSigma5 and associates with Promyelocytic Leukemia (PML) bodies, which are major sites of transcriptional regulation68 Subsequent to this, a recent study has shown that ARC protein has both an export signal that allows it to exit into the cytoplasm as well as a retention domain and localization signal that targets it

to the nucleus where it may play a role in the PML-dependent transcriptional downregulation of the glutamate receptor gene GluA169 The function ascribed

to ARC seems to be still confined to a homeostatic regulation of synaptic strength, without much of a well-defined role in long-term memory consolidation In light of its pervasive role in memory consolidation in many different species including humans, discovering the function of this highly important yet enigmatic molecule will be key in advancing the science of neurobiology In the subsequent chapters of this thesis, I will first present

results confirming the rapid activity-dependent expression of Arc and the

mechanisms involved in its early RNA Polymerase II-dependent transcriptional elongation (Chapter 2) In Chapter 3 of this thesis, I will be

exploring recent findings from our laboratory implicating Arc protein in the

regulation of H4K12ac, a learning-induced histone acetylation mark that is

affected in ageing-associated memory impairment, through its interaction with the epigenetic enzyme Tip6070

1.II.D TIP60 – a crucial effector of early and late epigenetic events

With regard to synaptic plasticity and memory formation, histone acetylation is

by far the most well-studied epigenetic modification71, and has been implicated in neurodegenerative diseases and aging-related memory impairment72 Studies have shown that highly specific histone lysines are acetylated in response to different paradigms of learning and memory73,74, yet the identity of the enzymes responsible for effectuating these specific changes has largely been understudied Conversely, in cases where the enzyme identity is known, as in the role of p300/CBP in Rubinstein-Taybi syndromic mental retardation, the exact histone lysines affected and the roles they play

in mediating memory formation defects are not yet known75 One particular epigenetic regulator, the HAT enzyme TIP60 (HIV Tat interactive Protein, 60 kDa, also known as KAT5), has emerged as an important effector of neuronal plasticity and memory formation76

Initially discovered as an HIV Tat-interacting protein77, TIP60 is a member of the MYST (MOZ, YBF2, SAS2, TIP60) family of nuclear histone acetyltransferases, and is evolutionarily conserved from yeast to humans The importance of TIP60 in gene transcription is highlighted by the finding that it is recruited to virtually all active protein-coding genes in the yeast

Saccharomyces cereviseae 78 Furthermore, Tip60 is one of six “hub” genes

that interacts with more than one quarter of genes in the genome, as

discovered in a recent genetic interaction screen in the worm C elegans 79 Remarkably, depletion of TIP60’s HAT activity resulted in the dysregulation of predominantly nervous system-specific genes80, suggesting that this ubiquitously expressed and highly important chromatin regulator may have neuron-specific roles81 Indeed, numerous studies have shown that TIP60 is transcriptionally active in neurons, promotes neuronal survival, and encourages axonal growth in neurodegenerative conditions82-85 As with ARC, TIP60 protein also localizes to PML nuclear bodies, implicating genomic and transcriptional roles86 A recent genome-wide ChIP-Seq analysis in Drosophila

has clearly demonstrated the recruitment of TIP60 to the promoters of genes involved in memory formation76

In summary, despite several recent advances, it is still not clear how TIP60 induces the transcription of highly specific genes, which histone lysines it acts

on and what mechanisms may be activating it to do so Importantly, how does

a ubiquitously expressed protein become highly targeted to specific locations

of non-neuronal genes are required for normal functioning of the brain The

fact that histone methylation is important in this story of transcriptional regulation in neurons is underscored by the observation that the enzymes responsible for specific patterns of methylation at lysine 4 and lysine 9 of histone H3 are crucial for neuronal development and cognitive function from mollusks to vertebrates87,88 The writer enzyme, or histone methyltransferase,

as well as the eraser, or histone demethylase, that specifically regulate H3K4me3, for instance, both have an essential role in long-term memory formation73,76,89-91 Similarly at the H3K9me2 locus, mutations of the writer enzyme, G9a/GLP, lead to aberrant neuronal expression of non-neuronal genes The eraser enzyme that is specific to H3K9me2 is PHD finger protein

8 (PHF8), which is an alphaketoglutarate-dependent histone lysine demethylase enzyme that, when mutated, causes a severe cognitive disability33 PHF8 is interesting because it has a PHD domain that binds specifically to H3K4me3, thereby allowing it to perform demethylation of H3K9me2 in a highly context-specific manner92 In the nucleus, PHF8 is thought to function by increasing mRNA transcription by directly modulating RNA Polymerase II93 In regulating neuronal function, PHF8 also has affinity for specific DNA sequences, through its association with zinc-finger proteins94, and through the regulation of cytoskeleton-associated genes95 Finally, like ARC and TIP60, PHF8 is also implicated in PML nuclear body function, albeit

in a more well-defined and specific manner of transcriptional upregulation96

Despite this apparent specificity in its binding to various locations spread across the genome, the gene targets that PHF8 transcriptionally regulates in neurons are not known The signaling cascades that activate it, the cross-talk

of the chromatin modification it causes and how it causes mental retardation

ultimately are still the subject of intense research The present work places PHF8 at the centre of an elaborate macromolecular mechanism of enzymatic histone demethylation, chromatin modification cross-talk, and transcriptional

elongation that subserves the extremely rapid upregulation of Arc

transcription In so doing, PHF8 thereby may be playing the role of an epigenetic “gateway” that modulates the consolidation of long-term memory

1.III The Timeline of Neuronal Activation

The organization of this thesis will attempt to follow the temporal sequence of epigenetic events and discuss the roles of PHF8, TIP60, and ARC proteins in the neuron as it receives electrical stimuli Changes that occur at the level of chromatin structure, gene transcription, and protein translation will be described, along with the methods used to study them Both in-vitro and in-vivo models are used: 1) in-vitro, increased neural network activity is achieved

by combining downregulation of inhibitory GABA currents by bicuculline with the potassium channel blocker 4-aminopyridine97, 2) in-vivo, the widely used paradigm of environmental enrichment is used to model experience-dependent plasticity in living animal brains98 In the present work, the timeline

of neuronal activation has been greatly simplified to early events, which occur

as early as 5 minutes within the start of increased synaptic activity, and late events which are traditionally protein synthesis-dependent and take place post 2 hours of neuronal activation

In Chapter 2 – early events (<30 minutes), I describe a mechanism by which

TIP60 may be involved in the rapid upregulation of the neuronal gene Arc, a

master regulator of synaptic plasticity I propose that TIP60 is able to acetylate a very specific histone lysine, namely H3K9acS10P, upon forming a dual-function complex with the histone demethylase PHF8 The specific

induction of H3K9acS10P at the Transcriptional Start Site of Arc contributes to the early, rapid transcription of the Arc gene through a mechanism that likely

involves RNA Polymerase II unpausing

In Chapter 3 – late events (>2hours), I will explore the possible mechanisms

by which TIP60 may be involved in the late epigenetic events leading to memory consolidation, specifically through its role as an H4K12ac modulator

I will discuss the role that ARC, beta-Spectrin, and PML proteins may play in mediating changes that occur several hours after neuronal activation

In Chapter 4, I will attempt to integrate these findings of early and late epigenetic events that eventually result in memory consolidation I will describe efforts taken to elucidate the identity of the gene targets of TIP60, PHF8, and ARC in the context of neuronal activation in-vitro and in-vivo Finally, in order to consolidate these current findings and discuss the possibilities afforded for future studies, the thesis will end with Chapter 5 wherein several clinical trials based on these findings will be described

induced Arc gene expression, whereas interfering with the function of this

complex abrogates it A global proteomics screen revealed that the majority of common interactors of PHF8 and TIP60 were involved in mRNA processing, including Polypyrimidine Tract Binding protein-associated Splicing Factor

(PSF), an important molecule involved in neuronal gene regulation Finally, using super-resolution microscopy, I show that PHF8 and TIP60 interact at the single molecule level with PSF, thereby situating this chromatin modifying complex at the crossroads of transcriptional activation These findings point toward a mechanism by which an epigenetic pathway can regulate neuronal activity-dependent gene transcription, which has implications in the development of novel therapeutics for disorders of learning and memory

2.II Introduction

Activity-dependent gene transcription, a pre-requisite for memory formation, is

a highly complex process99,100 Several epigenetic mechanisms have been put forth to explain this remarkable ability of neurons to dynamically regulate gene expression, including chromatin modification, which is capable of altering gene expression programs and even induce alternative splicing101 The importance of chromatin modification in learning and memory is demonstrated

by the fact that dysfunction of chromatin-modifying enzymes causes severe memory impairment, which ranges from Alzheimer’s disease to intellectual disability102-104 Recent evidence has shown that the rapid induction of

immediate-early genes (IEGs) such as Arc in response to neuronal activity is

mediated by a mechanism involving the escape of promoter-proximal RNA Polymerase II into transcriptional elongation63,105 The idea that stimulus-dependent rapid gene induction is controlled at the level of transcriptional elongation and mRNA processing is conserved across many cell types and is likely to be mediated by modification to chromatin structure106 Nevertheless, although it is known that enzymes are likely responsible for the chromatin

modifications that contribute to neuronal gene activation, the nature of these epigenetic regulators is still obscure

In this chapter, I report that PHF8 cooperates with TIP60 in an dependent manner to enable the rapid induction of the immediate-early gene

activity-Arc by specifically regulating H3K9acS10P, a dual-chromatin mark that is

required for transcriptional activation As no direct interaction between a demethylase and an acetyltransferase has yet been reported, I focused on precisely characterizing the localization of PHF8 and TIP60 using multi-colour super-resolution microscopy and investigated their physical interaction using co-immunoprecipitation and proximity in-situ ligation Within minutes of neural network activation, I found that the complex containing PHF8 and TIP60 specifically upregulated the transcriptional-elongation associated mark H3K9acS10P, which is required for rapid gene induction through a mechanism that likely involves transcriptional elongation Upon verifying that this complex is able to synergistically regulate the methylation and acetylation

of transcriptionally active H3K4me3-positive histones, I examined the TIP60 interactome through immunoprecipitation followed by mass spectrometry, which revealed that the majority of PHF8 and TIP60 interacting partners are indeed involved in transcription and RNA processing Overexpression of PHF8, but not the inactive mutant PHF8-F279S107,

PHF8-increased neuronal H3K9acS10P and Arc expression, whereas

RNAi-mediated knockdown of PHF8 inhibited both activity-induced H3K9acS10P

and Arc Furthermore, both PHF8 and TIP60 were found to be recruited to the

Arc promoter within minutes of neuronal activation Finally, using

single-molecule imaging techniques, I demonstrate that the two chromatin-modifying

enzymes have a well-defined 3-dimentional spatial relationship with each

other, with each molecule occupying long-stranded structures which are

closely associated with their common binding partner, PTB-associated

Splicing Factor (PSF), at the nucleosomal scale The direct interaction

between the chromatin modifier PHF8 with PSF, a long term

memory-associated splicing factor 108,109 lends further evidence to the role of chromatin

modification in transcriptional activation and co-transcriptional splicing in

neuronal activity-dependent gene regulation

Chapter 2: Summary – The rapid activity-regulated transcription of Arc is mediated by

the epigenetic enzyme TIP60, through its association with PHF8

2.III Materials and Methods

Full-length PHF8 was cloned through reverse transcriptase reaction of human brain cDNA (Marathon), and confirmed via Sanger sequencing against a construct of FLAG-PHF8 which was a generous gift from Petra de Graaf 93 Fusion fluorescent constructs PHF8-mTurquoise2, PHF8-YFP, PHF8-tdTomato, were cloned by inserting the full-length PHF8 PCR product flanked

by SalI and AgeI sites in-frame into the multiple cloning sites of the respective vectors To generate PHF8-FLAG and TIP60-FLAG, the YFP sequence was excised with NotI and an oligonucleotide encoding the FLAG peptide sequence was annealed and ligated to the C-terminus of PHF8 and TIP60 Mutagenesis of PHF8 into PHF8-F279S was performed using the megaprimer method68 by first performing a PCR with a reverse primer containing the single nucleotide mutation (c.836C>T) to generate a forward primer for a

second PCR reaction amplifying the full-length gene

Hippocampi and cortices from E18 Sprague-Dawley rats of either sex were dissected aseptically, digested using a papain dissociation system (Worthington Biochemical Corporation), and cultured in media supplemented with B27 (Brewer and Price, 1996) The appropriate density of neurons were plated on poly-D-lysine-coated glass-bottom culture dishes (MatTek, Ashland, MA), 8-well Lab-Tek II chambered cover-glass (Nunc, Denmark), or 96-well glass-bottom plates (Nunc, Denmark) that had been double-coated with poly-D-lysine overnight Neurons were fed weekly by replacing half of the medium HEK293, HeLa, and U2OS cells were obtained from the author’s University

Cell Culture Facility, and were cultured in high glucose DMEM (Gibco) with 10% fetal bovine serum (Invitrogen Corporation, Carlsbad, CA) and 1% Penicillin-Streptomycin Cells were plated on the poly-D-lysine coated glass-bottom dishes for imaging or the Lab-Tek II chambered cover-glasses for super-resolution imaging

Primary neurons were transfected between DIV 12 and 21 as previously described, with a few modifications 110 Briefly, Lipofectamine:DNA complexes were formed in a suitable amount of Neuronal Transfection Media (BrainBits, UK) for 15 minutes at room temperature (RT) Neuronal growth medium was aspirated and the complexes were added to the neurons for 15 minutes, after which the neuronal medium was restored HEK293, U2OS, and HeLa cells were transfected similarly, except that DMEM with high glucose media was used and the Lipofectamine 2000/ DNA mixture was added directly to existing media Each well in a six-well plate was transfected with a 2:1 ratio of transfection reagent to plasmid DNA To stimulate synaptic NMDA receptors and network activity 97, a combination of 4-Aminopyridine (4AP), Bicuculline (Bic), and Forskolin at a final concentration of 100μM, 50μM, and 50 μM respectively were added to the medium for the appropriate amount of time In control wells, the same volume of vehicle (DMSO) was added to neurons before lysates were collected

For construct co-expression experiments, transfected neuron/HEK293 cells were fixed with a solution containing 4% paraformaldehyde (PFA), 4% sucrose, and 1× PBS for 15 min at 4 °C The cells were subsequently

incubated with 1μM DAPI for 10 minutes, and preserved in 97% Thiodiethanol (TDE, Sigma) For immunostaining, cells were fixed with 100% MeOH at -20°C for 10 min Neurons/HEK293 cells were blocked with a solution containing 10% goat or donkey serum, 2% bovine serum albumin (BSA), and 1× PBS for 1 hour at room temperature (RT), except when the goat-anti-TIP60 (K-17, Santa Cruz) antibody was used, in which case blocking was done with 10% Horse Serum in PBS-0.1% Triton X The primary antibodies were incubated for 1 hour at RT in a dilution buffer containing 1:1 block solution and

PBS-Triton X solution at the following dilutions: Mouse-anti-Arc (C7) 1:300

(Santa Cruz), Goat-anti-TIP60 (K17) 1:300, Rabbit-anti-TIP60 1:300 (Novus Biologicals), Rabbit-anti-H3K9acS10P 1:300 (Abcam) Dishes were washed 5

x 10 minutes with PBS-Triton X and incubated with Fluor488, Fluor568, or Alexa-Fluor647 conjugated secondary antibodies (Molecular Probes-Invitrogen) 1:1000 in dilution buffer for 1 hour at RT Washing was repeated as per the above for 5 x 10 min and dishes were then stained with 1

Alexa-μM DAPI for 10 minutes to label DNA, followed by mounting in 10%, 25%, 50%, and finally 97% Thiodiethanol (TDE)

Stably transfected HEK293 cells expressing PHF8-YFP were fixed with 4% PFA for 15 min at 4 °C, and blocked and permeabilized using a solution containing 10% donkey serum and 0.5% Tween-20 at 37 °C Cells were incubated overnight with primary antibodies: Mouse-anti-GFP (1:1000, Roche) and Goat-anti-TIP60 (1:300, Santa Cruz) were used to detect PHF8 and TIP60, respectively Cells were washed five times with Buffer A(10 mM Tris,

150 mM NaCl, and 0.05% Tween-20), and then incubated for 2 hours with

secondary antibodies conjugated to PLA probes: Duolink II anti-Mouse plus and Duolink II anti-Goat minus were diluted in antibody diluent to a concentration of 1:5 (OLink Bioscience) at 37 °C After five more washes with buffer A at room temperature, hybridization was performed by incubating at 37

°C with the ligation solution (Duolink II Ligase, 1:40) for exactly 30 minutes Ligation was stopped by a wash step and detection of the amplified probe was done with the Duolink II Detection Reagents Kit (Red) After a final wash step

of 15 minutes x 5 in buffer B (200 mM Tris and 100 mM NaCl), cells were mounted and imaged Negative controls were obtained by transfecting the mutant PHF8 (F279S) and by repeating the procedure with no primary antibodies

Fluorescence images were obtained using a motorized inverted wide-field epifluorescence microscope (Nikon Eclipse Ti-E), using 40x and 60x Plan-Apo oil objectives, with numerical apertures of 1.35 and 1.49 respectively Motorized excitation and emission filter wheels (Ludl electronics, Hawthorne, NY) fitted with a DAPI/CFP/YFP/DsRed quad filter set (#86010, Chroma, Rockingham, VT) were used together with filter cubes for DAPI, CFP, YFP and TxRed (Chroma) to select specific fluorescence signals Z-stacks were obtained spanning the entire nucleus and out-of-focus fluorescence was removed using the AutoQuant deconvolution algorithm (Media Cybernetics) Calcium imaging was done either through a cell-permeable Ca2+-sensitive dye (Fluo-4 AM, Invitrogen) or the transfection of a genetically encoded Ca2+sensor (gCamp6, medium isoform) Images were obtained in time series of

100ms/frame, and quantification was performed through the Time Measurement feature of NIS Elements For all purposes, Images were digitized using a cooled EM-CCD camera (iXon EM+ 885, Andor, Belfast, Northern Ireland) Image acquisition was performed using NIS Elements AR 4.2 software (Nikon) NIS Elements Binary and ROI Analysis tools were used

to segment nuclei based on DAPI signal intensity

Transfected HEK293 cells growing in 6-well plates were allowed to express overnight at 37 °C to yield >90% transfection efficiency Throughout the entire procedure, cultures and subsequent lysates were kept on ice or at 4 °C For co-immunoprecipitation, the cultures were washed once with 1 ml of PBS, and lysed in 500 μl of lysis buffer for 30 min, then scraped into 1.5 ml tubes Lysis buffer consisted of 5 mM HEPES pH 7.2, 0.5% NP40, 250 mM NaCl, 2 mM EDTA, 10% glycerol, 1:100 dilution of protease inhibitor cocktail (Sigma-Aldrich) The lysates were spun down for 20 min at 16,000×g to pellet cell debris 500ul of the supernatant was then incubated on a rotator with 5 μl mouse-anti GFP111 for 90 min, followed by 100 μl of Protein-A/G Plus-Agarose (Santa Cruz Biotechnology) for another 60 min on a rotator The beads were spun down at 1000×g for 5 min and the supernatant was removed IP fractions were then washed and re-suspended in 1 ml lysis buffer for a total of

3 times The beads and input lysates were resuspended and boiled at 95 °C for 5 min in sample buffer, resolved by SDS-PAGE with Tris-glycine gels (Bio-Rad), transferred to 0.2um PVDF membranes (Invitrogen), and imunoblotted The primary antibodies used were anti-GFP (mouse-monoclonal; Roche), anti-FLAG (mouse-monoclonal; Sigma)