Olig1) and Its Post

FUNCTION OF OLIGODENDROCYTE TRANSCRIPTION FACTOR 1 (OLIG1) AND ITS POST-

TRANSLATIONAL MODIFICATION

JINXIANG DAI

M.S., Institute of Neuroscience, Shanghai Jiao Tong University, Shanghai, China, 2009

B.S., Shanghai Jiao Tong University, Shanghai, China, 2006

A thesis submitted to the

Faculty of the Graduate School of the

University of Colorado in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

Cell Biology, Stem Cell and Development Program

2015 This thesis for the Doctor of Philosophy degree by

Jinxiang Dai

has been approved for the

Cell Biology, Stem Cell and Development Program

Linda Barlow, Chair

Wendy B. Macklin, Advisor

Bruce Appel

David Clouthier

Brad Bendiak

Aaron Johnson

Date: 8 /14/2015

Dai, Jinxiang (Ph.D., Cell Biology, Stem Cell and Development)

Function of Oligodendrocyte Transcription Factor 1 (Olig1) and its Post-Translational Modification

Thesis directed by Professor Wendy B. Macklin

ABSTRACT

Oligodendrocytes and myelin are essential for maintaining the structural integrity and function of the axons in the central nervous system (CNS). The progressive decline of remyelination in multiple sclerosis is partially due to the failure of oligodendrocyte differentiation within chronic lesions.

Oligodendrocyte specific bHLH transcription factors, Olig1 and Olig2, are expressed exclusively by oligodendrocytes and important for development. While the role of Olig2 in the lineage is well established, the role of Olig1 is still unclear. Understanding the function and regulation of Olig1 in oligodendrocyte development will provide mechanistic insight into the differentiation failure of oligodendrocytes in chronic demyelinated lesions.

The function of Olig1 in oligodendrocyte differentiation and developmental myelination in brain was analyzed. Both oligodendrocyte progenitor cell commitment and oligodendrocyte differentiation were impaired in the corpus callosum of Olig1 null mice, resulting in hypomyelination throughout adulthood in the brain. As seen in previous studies with this mouse line, while there was an early myelination deficit in the spinal cord, essentially full recovery with normal spinal cord myelination was seen. We demonstrate a unique role for Olig1 in promoting oligodendrocyte progenitor cell commitment, differentiation and subsequent myelination primarily in brain, but not spinal cord.

Subcellular translocation of Olig1 is observed during both oligodendrocyte development and remyelination. Translocation of Olig1 seems to be critical for remyelination, but the mechanism regulating this process has not been established. We identified three functional nuclear export

i sequences (NES) localized in the bHLH domain and one specific acetylation site at Lysine 150 (human

Olig1) in NES1. Acetylation of Olig1 decreased its chromatin association, increased its interaction with Id2 (inhibitor of DNA binding 2) and facilitated its retention in the cytoplasm of mature oligodendrocytes. We establish that acetylation of Olig1 regulates its chromatin dissociation and subsequent translocation to the cytoplasm, and is required for oligodendrocyte maturation.

In summary, these results solve the discrepancy of Olig1 function in oligodendrocyte development by unraveling its brain-specific role in multiple stages of early oligodendrocyte differentiation but not myelinogenesis. This work also provides mechanistic insight into the finely tuned regulation of Olig1 translocation and function by acetylation.

The form and content of this abstract are approved. I recommend its publication.

Approved: Wendy B. Macklin

DEDICATION to my wife Jing Wang and my mom Liqin Wang

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ACKNOWLEDGEMENTS

I would like to thank my family for their support throughout the years in the USA. Specifically, I would like to my wife, Jing, whose never-ending love encourage me through this long lasting process of graduate study with all the highs and lows.

I would like to thank my supervisor, Dr. Wendy Macklin, for her tremendous support in many years in the lab. She has provided amazing guidance and I am very lucky to have been in her lab. She always gives me freedom to pursue any interesting scientific questions. Just as one of her kids, she watch me through the process of learning how to speaking in English, written in English, doing research and presenting my work in public. I appreciate for the time she had spent on editing my

Chinese style English and tolerating many troubles I made for the lab. As a senior scientist, your persistent devotion to research is always inspiring to me. I always enjoy the time discussing science with you and have you pulling out the printed papers from your secret drawers. I feel as a spoiled lucky graduate student in Wendy’s lab for many years, and I cannot expect a better mentor than

Wendy.

I would like to thank all the members of Macklin lab, past and present, for their efforts to make the lab as a big familiar. Here are my brothers and sisters, who care about each other and work together to make the lab as the best place to stay. Specifically, I would like to thank Hilary Sachs for sharing her knowledge and intelligence about any aspect of science. I would thank Katie Bercury for helping through all stages of my graduate training. Without you, my English may still poor, and my papers may be still in my hand as awful written manuscripts. As my best friend you take care of so much of my lab life to make sure I will survive. I would like to thank Marnie Preston for her patience to listen to me and help me with a lot molecular cloning techniques. I thank rest of the lab for their always support , patience and encouragement.

I would like to thank my committee members for their constant support, especially my chair Dr.

Linda Barlow. I still remember my first rotation in her lab, and how she would be able to understand what I mean in poor English. Her enthusiasm for science really encourages me for doing research, and her unwavering support always helps me get through the graduate training.

TABLE OF CONTENTS

CHAPTER l. INTRODUCTION------1

Temporal waves of OPC specification and maintenance------3

Extracellular cues guiding oligodendrocyte differentiation and myelination------4

The many faces of BMP and Wnt: Dual mode regulation of OPCs differentiation------4

Growth factors: OPC maintenance and myelinogenesis------5

Extracellular ligands: non-axonal cues leading the way------7

Axonal ligands: local determinants------8

Neuronal activity: old concept and new evidence------10

Intrinsic factors regulate oligodendrocyte differentiation and myelination------11

Transcriptional regulation------12

Epigenetic regulation of oligodendrocyte development------15

Post-transcriptional regulation of oligodendrocyte differentiation: non-coding RNAs------18

Interplay between the extrinsic signalings and transcriptional regulation of oligodendrocyte development------20

Intracellular signaling transduction: the bridge from cell surface to nucleus------21

Post-translational modification of transcription factors------23

Dynamic epigenetic regulation: converging on the chromatin------26

Olig1 and Olig2: molecular and functional divergence------28

II. OLIG1 FUNCTION IS REQUIRED FOR OLIGODENDROCYTE DIFFERENTIATION IN THE MOUSE BRAIN------31

Abstract------31

Introduction------31

Results------33

OPC production, differentiation and early morphogenesis were reduced in Olig1 null mice----33

Oligodendrocyte differentiation was reduced, but not the initiation of axon wrapping in P8 Olig1 null mice------38

Oligodendrocyte deficit persists in the brain of adult Olig1 null mice------39

Developmental recovery and normal myelination in the spinal cord of adult Olig1 null mice------46

Expression and subcellular location of Olig1 is highly coordinated with oligodendrocyte morphogenesis and the onset of myelination------47

Olig2 expression was not altered in Olig1 null brain------50

Olig1 is important for initial oligodendrocyte differentiation in vitro------52

Discussion------56

Materials and Methods------63

III. OLIG1 ACETYLATION AND NUCLEAR EXPORT MEDIATE OLIGODENDROCYTE DEVELOPMENT------68

Abstract------68

Introduction------68

Results------70

Three Nuclear Export Sequences (NES) of Olig1 regulate its export to the cytoplasm ------70

Acetylation of Olig1 is correlated with its cytoplasmic localization ------71

Olig1 lysine (K) 150 acetylation controlled Olig1 cytoplasmic localization ------77

The histone acetyltransferase CBP acetylates Olig1 Lysine 150------78

Acetylation of Olig1 is critical for its role in morphological differentiation and lineage progression ------81

During oligodendrocyte differentiation, increased acetylation of Olig1 decreased its chromatin binding ------82

Acetylation of Lysine 150 on Olig1 increased its binding affinity for Inhibitor of DNA binding 2 (Id2) ------87

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Discussion------93

Materials and Methods------100

IV. CONCLUSIONS AND FUTURE DIRECTIONS------108

REFERENCES------113

APPENDIX

A.Validation of rabbit anti-Olig1 antibody ------141

B.Oligodendrocyte lineage markers------142

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LIST OF TABLES TABLE

3.1: Predicted Olig1 acetylation sites------78

LIST OF FIGURES

FIGURE

2.1 Deficit of pre-OPC to OPC commitment in P1 Olig1 null mice.------35

2.2 Enhanced progenitor cell proliferation and apoptosis in P1 Olig1 null mice.------36

2.3 Oligodendrocyte differentiation and myelination initiation in P8 Olig1 null mice------40

2.4 Oligodendrocyte maturation and myelination in adult brain of Olig1 null mice------43

2.5 The corpus callosum is hypomyelinated in Olig1 null mice------44

2.6 Major myelin proteins expression in cerebrum of P15 and 2 month old Olig1 null mice.------45

2.7 Adult Olig1 null spinal cord had fewer oligodendrocytes, but apparently normal myelination------48

2.8 Major myelin proteins expression in spinal cord of P15 and 2 month old Olig1 null mice------50 … 2.9 Expression and subcellular location dynamics of Olig1 upon myelination onset------52

2.10 Olig2 expression in the CNS of developing Olig1 null mice------54

2:11 Oligodendrocyte differentiation in the absence of Olig1 in vitro------57

3.1 Three Nuclear Export Sequences (NES) of Olig1 regulate its export------72

3.2 Acetylation of Olig1 is correlated with its cytoplasmic localization both in HEK293T cells and in developing mouse brain------75

3.3 CBP is the Histone Acetyltransferase (HAT) responsible for Olig1 Lysine 150 acetylation------79

3.4 Olig1 is acetylated at lysine 150 in vivo------84

3.5 Acetylation of Olig1 is critical for its role in morphological differentiation and lineage progression------88

3.6 Increased acetylation of Olig1 decreased its chromatin binding as oligodendrocytes differentiated------90

3.7 Acetylation of Lysine 150 on Olig1 increased its binding affinity for Inhibitor of DNA binding 2 (Id2)------94

3.8 Model of the mechanism by which Olig1 acetylation regulates its nuclear to cytoplasmic translocation and function------96

LIST OF ABBREVIATIONS

CBP CREB binding protein CC1 Adenomatous polyposis clone CC-1 CNP 2’3’-Cyclic Nucleotide Phosphodiesterase CNS Central Nervous System ERK Extracellular-Regulating Protein Kinase E47 E2A immunoglobulin enhancer-binding factor 47 FGF Fibroblast Growth Factor GCN5 General control of amino-acid synthesis 5 Id2 DNA binding protein Inhibitor 2 Id4 DNA binding protein Inhibitor 4 L1-CAM L1 cell adhesion molecule MAG Myelin Associated Glycoprotein MAPK Mitogen-Activating Protein Kinase MBP Myelin Basic Protein MOG Myelin Oligodendrocyte Glycoprotein MRF Myelin Regulatory Factor NG2 Neural/glial antigen 2 Olig1 Oligodendrocyte transcription factor 1 Olig2 Oligodendrocyte transcription factor 2 OPC Oligodendrocyte Progenitor Cell PCAF P300/CBP-associated factor PDGF-AA Platelet-Derived Growth Factor-AA PDGFRα Platelet-Derived Growth Factor Receptor alpha PI3K Phosphoinositide 3-Kinase PLP Proteolipid Protein PKA Protein Kinase A PKC Protein Kinase C PSA-NCAM Polysialylated-neural cell adhesion molecule Sox2 Sex Determining Region Y (SRY)-Box 2 Sox10 Sex Determining Region Y (SRY)-Box 10 Tcf4 Transcription Factor 4 TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling YY1 Ying Yang 1 Zfp191 Zinc finger protein 191

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CHAPTER I

INTRODUCTION

Rapid conduction of action potentials in the vertebrate central nervous system (CNS) is heavily reliant on myelin(Hildebrand et al., 1993). Myelin is a specialized multilayered lipid-rich membrane wrapped around numerous axons(Bullock et al., 1984). Oligodendrocytes are the myelin forming glia of the central nervous system. The essential role of myelin in maintaining the structural integrity and function of the axons is evidenced by its loss or dysfunction in a variety of devastating demyelinating diseases including multiple sclerosis (MS), leukodystrophies, Pelizaeus-Merzbacher disease and periventricular leukomalacia(Love, 2006; Franklin and Ffrench-Constant, 2008; Nave, 2010c).

Although myelin repair is a comparably robust process in response to demyelinating insults in CNS, its inevitable failure is a common feature of disease progression in MS (Franklin, 2002; Frohman et al., 2006; Franklin and Ffrench-Constant, 2008). Theoretically, the limitation of the remyelination process could be due to a primary deficiency in oligodendrocyte progenitor cells (OPCs), a failure of

OPC recruitment, or a failure of OPC differentiation and maturation(Wolswijk, 1998; Chang et al.,

2000; Chang et al., 2002; Franklin, 2002; Kuhlmann et al., 2008). However, very little is known about the signaling pathways and how these pathways are disturbed in the multi-step process of successive remyelination(Franklin and Ffrench-Constant, 2008). The idea that developmental myelination and the remyelination process share many conserved mechanisms is widely accepted based on accumulating supportive evidence from both experimental demyelinating models and clinical disease(Franklin and Hinks, 1999; Fancy et al., 2011a). Understanding the fine mechanism of oligodendrocyte development has relevance for translating myelin biology into treatments for human myelin disorders. Developmentally, OPCs are specified from subventricular neural progenitors in both brain and spinal cord (Rowitch, 2004; Kessaris et al., 2008). These OPCs appearing in successive waves, divide and migrate throughout the CNS after birth (Kessaris et al.,

2006; Richardson et al., 2006). Given the appropriate environmental cues, OPCs exit the cell cycle and differentiate into premyelinating oligodendrocytes (Emery, 2010a, b; Zuchero and Barres, 2013).

Finally, in line with the maturation of the nervous system, the premyelinating oligodendrocytes will either further mature and myelinate nearby receptive axons or undergo programmed cell death

(Trapp et al., 1997; Hughes et al., 2013). Each step of progression along the oligodendrocyte lineage is tightly controlled by both intrinsic determinants and extracellular cues in a spatial and temporal manner (Emery, 2010a; He and Lu, 2013; Zuchero and Barres, 2013).

Insight into the molecular mechanism of oligodendrocyte differentiation initially came from early experiments on purified OPCs derived from the postnatal CNS (Temple and Raff, 1986; Gao and Raff,

1997; Gao et al., 1998). The intrinsic nature of oligodendrocyte behavior has long been appreciated mainly based on the work of Martin Raff and his colleagues. It was proposed that there is an internal

“clock” that determined the number of divisions of cultured OPCs in the presence of growth factors

(Raff, 2006, 2007). Furthermore, this view of oligodendrocyte differentiation as a default or passive process upon relief of inhibitory signaling was supported by both in vitro and in vivo observations(Raff et al., 1983; Wang et al., 2001). However, this simplified view of OPC differentiation does not take into account the complex regulatory mechanisms that need to be coordinated to achieve myelination in a tight temporospatial manner. In the last decade or so, much effort has been devoted to understanding the transcriptional programs that promote or inhibit the differentiation of oligodendrocytes (Wegner, 2008; Li et al., 2009; Emery, 2010b). Although the process of OPC differentiation and myelination can be uncoupled in vitro(Sarlieve et al., 1983;

Baumann and Pham-Dinh, 2001), terminal differentiation of oligodendrocytes and the wrapping of receptive axons are intimately associated in vivo(Simons and Trajkovic, 2006). Moreover, in recent years, it has become apparent that there is a multitude of regulatory factors, both environmental and intrinsic, that directs the behavior of oligodendrocytes through various stages of myelination

(Emery, 2010a; He and Lu, 2013; Zuchero and Barres, 2013). In addition to the presence or absence of these regulatory factors, the precise timing of action of these factors is also critical for effective myelination and remyelination (Franklin, 2002; Huang et al., 2013). Here, I summarize the current knowledge on the signaling mechanisms that mediate oligodendrocyte differentiation and myelination, with an emphasis on recent discoveries of oligodendrocyte biology.

TEMPORAL WAVES OF OPC SPECIFICATION AND MAINTENANCE

OPCs are developmentally specified from neural progenitors in the ventral neural tube in response to the secreted morphogen sonic hedgehog (Shh) (Pringle et al., 1996; Orentas et al.,

1999).Several transcription factors are induced by Shh, including Nkx6.1, Nkx6.2, Olig1 and Olig2

(Richardson et al., 1988; Rowitch, 2004). Of these factors, Olig2 is the only one absolutely required for OPC specification, with nearly complete loss of OPCs seen in Olig2 null mice (Lu et al., 2002; Zhou and Anderson, 2002). However, in the absence of Nkx6.1 and Nkx6.2, the dorsally derived OPCs are less impaired than their ventral counterparts (Cai et al., 2005). Moreover, loss of Olig1 only mildly reduces OPC generation in brain, leading to a slight increase of interneuron production (Silbereis et al., 2014). OPCs are generated in multiple waves, initially from more ventral sources. The later dorsally-derived cortical OPCs widely spread out and largely replace the early ventral OPCs.

Moreover, when one source is genetically ablated, the other one can fully compensate for its loss(Kessaris et al., 2006). This indicates the functional redundancy and plasticity of OPCs from these two sources. In contrast, recent fate mapping studies showed that ventral and dorsal OPCs preferred different axon tracts, suggesting their functional difference (Tripathi et al., 2011). The regional heterogeneity of oligodendrocytes has not been confirmed yet (Vigano et al., 2013).

Oligodendrocyte transplantation experiments either weaken or support this idea (Fanarraga et al.,

1998; Vigano et al., 2013). Emerging tools such as single cell expression profiling will be useful to further elucidate this question (Usoskin et al., 2015; Zeisel et al., 2015). Once OPCs are specified,

3 they proliferate and migrate multi-directionally from ventricular zone to distinct regions of the CNS in response to both attractive and repulsive cues(Rowitch, 2004). OPCs are overproduced to meet the end goal of sufficient myelination (Barres et al., 1992; Calver et al., 1998). Not surprising given the fact that axons are selectively myelinated in the CNS, OPC differentiation is highly coordinated with the functional maturation of brain(Baumann and Pham-Dinh, 2001). In the developing CNS, a balance of positive and negative signaling determines the timing of oligodendrocyte maturation and myelination. In the absence of receptive axons, terminal differentiation of the OPCs is actively opposed by many different cues, both environmental and intrinsic.

EXTRACELLULAR CUES GUIDING OLIGODENDROCYTE DIFFERENTIATION AND MYELINATION

The many faces of BMP and Wnt: Dual mode regulation of OPC differentiation

Bone morphogenetic proteins (BMPs) belong to the TGFβ superfamily that regulates cell proliferation and differentiation in most cell types (Chen et al., 2004). Dorsally derived BMPs inhibit

OPCs specification by antagonizing the inductive Shh signaling from the ventral neural tube (Mekki-

Dauriac et al., 2002). Transgenic overexpression of BMP4 in the neural progenitor cells largely promotes astrogenesis at the expense of OPCs specification (Gomes et al., 2003). After OPC specification, sustained BMP signaling is required for OPC maintenance, and this acts through upregulating negative regulators of OPC differentiation, such as inhibitor of DNA binding 2/4

(Id2/4)(Samanta and Kessler, 2004). A recent study demonstrated that BMP induced the formation of Smad/P300 complex that directly binds and activates Id2/4 gene promoter activity (Weng et al.,

2012). This brake on OPC differentiation is relieved by Smad-interacting protein-1 (Sip1), whose expression is induced by Olig1 and Olig2 upon OPC differentiation. Sip1 disrupts the association of

P300 with activated Smad complex by recruiting NuRD co-repressor complex. Specific knockout of

Sip1 in the oligodendrocyte lineage causes a severe failure of myelin formation by blocking the differentiation of OPCs (Weng et al., 2012).

Interestingly, another major dorsal derived morphogen, Wnt, acts similarly to BMP by antagonizing OPC specification, while promoting astrocyte development (Shimizu et al., 2005). The canonical Wnt pathway acts to stabilize β-catenin protein, which then translocates to the nucleus and forms a complex with T-cell factor/lymphoid enhancer factor (Tcf/LEF) to regulate the expression of target genes (MacDonald et al., 2009). Tcf4, also known as Tcf7L2, was one of 50 genes that are dynamically regulated during remyelination (Fancy et al., 2009). Successive work by

Fancy and his colleagues demonstrates that the Wnt pathway is a critical inhibitory pathway for OPC differentiation (Fancy et al., 2011b; Fancy et al., 2014). Transgenic overexpression of the constitutively active form of β-catenin in oligodendrocyte lineage cells results in delayed differentiation and myelination. In line with this, Tcf4, the transcriptional effector of β-catenin, is transiently expressed in OPCs and subsequently down-regulated in mature oligodendrocytes (Fancy et al., 2009). However, deletion of Tcf4 actually causes a differentiation block of OPCs, totally in contrast with the expected precocious differentiation phenotype (Fu et al., 2009; Ye et al., 2009).

These studies imply a complex role of Wnt signaling in modulating the precise timing of OPC differentiation. Although Wnt signaling was considered as a negative regulator of the OPC differentiation, a recent study indicates that baseline activation of the Wnt pathway is important for

OPC differentiation (Fancy et al., 2014). However, unrestricted pathological high-activity of Wnt signaling impedes differentiation of oligodendrocytes (Fancy et al., 2014).

Growth factors: OPC maintenance and myelinogenesis

Platelet-derived growth factor (PDGF) was first identified as a potent mitogen for O-2A progenitor cells in vitro (Pringle et al., 1989). In the CNS, its receptor PDGFRα is restricted to developing and adult OPCs. As shown by transgenic overexpression and loss-of-function studies, PDGF signaling is essential for OPC proliferation and maintenance (Calver et al., 1998; Fruttiger et al., 1999). However, at least in culture, sustained PDGF signaling inhibits terminal differentiation of OPCs in vitro (Baron

5 et al., 2000). Consistent with this, although overexpressing PDGF induces ectopic overproduction of

OPCs, the number of myelinating oligodendrocytes is normal because of apoptosis of superfluous oligodendrocytes (Calver et al., 1998).

Similar to PDGF, Fibroblast Growth Factor (FGF) family members are also critical for OPC proliferation, but they function at distinct stages (McKinnon et al., 1990; Wolswijk and Noble, 1992;

Bansal et al., 1996). Many members of the FGF family growth factors stimulate rapid OPC division, but inhibit further differentiation into mature oligodendrocyte (Goddard et al., 2001). Besides its role in OPC proliferation and survival, FGF receptors were found to be enriched in myelin sheets, implying their role in myelination. Indeed, loss of FGFR1/2 in oligodendrocytes inhibits the rapid growth of myelin and leads to thinner myelin throughout adult (Furusho et al., 2012).

In line with the role of PDGF and FGF in OPC proliferation and survival, the insulin-like growth factor 1 (IGF1) not only promotes OPC expansion, but also drives OPC differentiation (Ye et al., 2002).

Oligodendrocyte specific deletion of the IGF1 receptor results in reduced OPC production(Zeger et al., 2007). Ubiquitous overexpression IGF1 causes hypermyelination in brain, but it is unclear whether oligodendrocyte differentiation is under the direct impact of IGF1 (Ye et al., 1995; Luzi et al.,

2004).

It has long been appreciated that these different growth factors interact with each other to a certain extent. PDGF, FGF and IGF-1 have been shown to cooperate to promote OPC expansion

(Goddard et al., 1999; Baron et al., 2000; Jiang et al., 2001). Once they bind their receptors, they activate the Erk1/2 or PI3K/Akt signaling pathway to trigger downstream effectors (Baron et al.,

2000; Frederick et al., 2007). However, their oligodendrocyte specific downstream targets are largely unknown.

Extracellular ligands: non-axonal cues leading the way

After OPCs are specified, they migrate out from the original niche to populate the whole CNS

(Rowitch, 2004; Richardson et al., 2006). This process is mediated largely by various extracellular matrix (ECM) components that provide both instructive and repulsive cues (Barros et al., 2011;

Colognato and Tzvetanova, 2011). Migration of OPCs to their appropriate localizations precedes their terminal differentiation. ECM substrates such as fibronectin, vitronectin and Laminin-2 have been shown to promote OPC proliferation, migration and survival(Buttery and ffrench-Constant,

1999; Gil et al., 2009; Hu et al., 2009c; Relucio et al., 2009; Stoffels et al., 2015). On the other hand, the ECM substrate Tenascin-C and hyaluronan are inhibitory to OPC migration (Frost et al., 1996;

Back et al., 2005). OPCs also respond to some guidance cues utilized by migrating neurons and pathfinding axons, both attractive and repulsive, including members of both the semaphorin and netrin family (Spassky et al., 2002; Cohen et al., 2003; Tsai et al., 2003; Rajasekharan et al., 2009;

Barros et al., 2011; Franco and Muller, 2011).

Precise coordination of OPC migration and survival is also mediated by differential expression of receptors for ECM substrates(Milner and Ffrench-Constant, 1994). Integrin family receptors are the most intensively researched receptors for ECM components (Hynes, 2002), and their dynamic regulation during OPC development has long been appreciated(O'Meara et al., 2011). In addition to their role in OPC migration, major work from the Ffrench-Constant laboratory has shown that integrins are also critical for target-dependent survival of newly differentiated oligodendrocytes(Colognato et al., 2002). Given the fact that the number of oligodendrocytes are proportional to their receptive axons, it is not surprising that the axon- associated ECM substrate

(Laminin-2) is critical for oligodendrocyte survival (Colognato et al., 2002; Yang et al., 2005).

Furthermore, the synergy between ECM substrates and growth factors allows oligodendrocytes to achieve robust signal transduction for differentiation and survival (Baron et al., 2003; Decker and

7 ffrench-Constant, 2004). The cooperation between ECM substrates and other growth factors can potentially regulate the precise timing of oligodendrocyte differentiation and myelination.

In addition to the above mentioned ECM factors, a number of extracellular ligands that modulate oligodendrocyte differentiation and myelination remain to be identified. Recently, the G- protein coupled receptors (GPCRs) have been shown to play a crucial role in glia differentiation and myelination (Chen et al., 2009; Monk et al., 2009; Ackerman et al., 2015). The GPCRs constitute a large protein family of receptors characterized by seven-transmembrane domains. They are involved in cell-cell and cell-ECM interaction and they transduce extracellular signaling into a cellular response through their coupled G-protein(Gilman, 1987). From a large forward genetic screen in zebrafish, Gpr126 was identified to be essential for the peripheral myelination by Schwann cells

(Monk et al., 2009). Although the myelin promoting function of Gpr126 is not conserved in the CNS, the function of other GPCRs in oligodendrocyte development is starting to be unraveled. Gpr17 is transiently expressed in differentiating OPCs, and is downregulated in mature oligodendrocytes.

Overexpression of Gpr17 inhibits the terminal differentiation of oligodendrocytes and results in dysmyelination. In the absence of Gpr17, OPCs precociously mature but make normal myelin (Chen et al., 2009). Recently, an adhesion G-protein coupled receptor Gpr56, was identified in zebrafish as an important regulator of oligodendrocyte development. Loss of Gpr56 results in decreased OPC proliferation and reduction of myelination(Ackerman et al., 2015). However, the potential ligands for both Gpr17 and Gpr56 have yet to be identified.

Axonal ligands: local determinants

In developing CNS, axons are selectively myelinated (Hildebrand et al., 1993; Baumann and Pham-

Dinh, 2001). In the upper layer cortex, individual axons have distinct longitudinal distribution of myelin. Intermittent myelination is characteristic of these cortical axons, within which myelinated segments are interspersed with long unmyelinated tracts (Tomassy et al., 2014). To achieve the

8 complex diversity of myelination, expression of permissive or inhibitory cues on axons is likely the simplest strategy for selective axon myelination (Waxman and Sims, 1984; Barres and Raff, 1999;

Bozzali and Wrabetz, 2004). This mechanism allows the myelinating oligodendrocyte to discriminate axons that need to be myelinated at a subcellular level (Simons and Trajkovic, 2006; Piaton et al.,

2010). In fact, axonal diameter has long been thought to be a crucial regulator of myelination based on ultra-structural observation of CNS myelin (Duncan, 1934; Voyvodic, 1989; Chomiak and Hu,

2009). Intriguingly, only axons above 0.2μm are myelinated and myelin thickness is consistently proportional to axon size (Waxman and Bennett, 1972; Berthold et al., 1983; Graf von Keyserlingk and Schramm, 1984; Voyvodic, 1989). More recent studies imply that axonal size only represents one of the axonal factors that regulate myelination. Other axon expressed ligands have been found to be inhibitors of myelination including Jagged for Notch pathway(Wang et al., 1998), Lingo1(Mi et al., 2005), PSA-NCAM(Charles et al., 2000) and axonal adhesion molecular L1(Itoh et al., 1995;

Barbin et al., 2004), all of which inhibit either OPC terminal differentiation or myelination. The prominent expression of these endogenous inhibitors of developmental myelination on axons implies that cell-cell contact inhibition is required to prevent the precocious differentiation of OPCs and myelination. Clearly, in order to achieve myelination for all relevant axons, both permissive and inhibitory axonal cues are necessary. In the peripheral nervous system (PNS), the relationship between axon size and myelin thickness is regulated by the level of neuregulin expressed on axonal surface (Michailov et al., 2004). However, in contrast, in CNS the neuregulin signaling is largely dispensable for myelination by oligodendrocytes (Brinkmann et al., 2008). One explanation for this striking difference is that other unknown promyelination signaling may compensate for the loss of neuregulin in CNS.

Neuronal activity: old concepts and new evidence

A fully functioning brain is reliant on proper myelin. There is evidence that the myelination process is partly modulated by electrical activity in axons (Barres and Raff, 1993; Demerens et al.,

1996; Zalc and Fields, 2000). This concept resulted from studies of OPC survival and myelination in optic nerve from the natural blind cape mole rat(Omlin, 1997) or experimental manipulation of neuronal activity(Gyllensten and Malmfors, 1963; Tauber et al., 1980; Fukui et al., 1991).

A number of mechanisms have been proposed to explain how neuronal activity may regulate OPC differentiation and myelination (Stevens et al., 1998; Wake et al., 2011). Synapse-like structures between axons and OPCs were first identified in hippocampus and have since also been found in other grey and white matter regions (Bergles et al., 2000; Ziskin et al., 2007). OPCs express a variety of ionotrophic glutamate receptors and ion channels that they could depolarize in response to neurotransmitter release from axon terminals without generating action potential(Barres et al.,

1990; Butt, 2006; Karadottir et al., 2008; De Biase et al., 2010). This synaptic coupling of OPC with axon is quickly lost as OPCs differentiate into mature oligodendrocyte (Kukley et al., 2007; Ziskin et al., 2007; Kukley et al., 2010). In vitro, glutamate treatment blocks both OPC division and differentiation (Gallo et al., 1996). These results imply that glutamatergic signaling may function to inhibit the differentiation of OPCs and maintain a progenitor pool. However, strong in vivo experimental evidence to support this concept is lacking. Additionally, it has long been proposed that axonal action potentials could modulate expression of ligands on the axonal surface (Itoh et al.,

1995; Itoh et al., 1997). However, this attractive mechanism has only limited support. One alternative mechanism of action involves axonal release of adenosine or ATP which may promote

OPC differentiation and myelination (Stevens et al., 2002; Ishibashi et al., 2006). Other neurotransmitters have also been shown to modulate OPC differentiation in vitro (Yuan et al., 1998;

Karadottir and Attwell, 2007; Cavaliere et al., 2013). However, the direct link between neuronal

10 activity and oligodendrocyte differentiation or myelination in vivo has not been proved until recently.

By using optogenetic stimulation of premotor cortex of awake adult mice, Monje and her colleagues provided the first evidence in vivo that directly manipulating neuronal activity promoted oligodendrogenesis and myelination(Gibson et al., 2014). However, the exact mechanisms by which neuronal activity modulates oligodendrocyte differentiation and myelination are largely unknown.

Recent studies from zebrafish provide some intriguing mechanistic insights into this unsolved problem by manipulating the activity and vehicle release in single axons in spinal cord of living fish(Hines et al., 2015). In developing zebrafish, development of oligodendrocytes is not disrupted by blocking the neuronal activity by tetrodotoxin (TTX). Interestingly, myelination starts with excessive and indiscriminate wrapping of axons followed by extensive refinement based on activity- dependent vesicle release from individual axons. Although the nature of the secreted molecule(s) is unknown at this point, these studies challenge the unique role of electrical activity in OPC proliferation and differentiation and further divide the myelination process into a two step process: initial wrapping of axons and stabilization, and refinement of growing internodes. Based on these studies, neuronal activity-dependent axonal cues seem functionally distinct from the above mentioned activity-independent axonal cues. How these two related but distinct axonal-derived signals cooperate to achieve the fine control of myelin onset spatiotemporally remains mysterious.

INTRINSIC FACTORS REGULATE OLIGODENDROCYTE DIFFERENTIATION AND MYELINATION

The early studies on purified OPCs provide many mechanistic insights into the intrinsic nature of oligodendrocyte behavior (Gao and Raff, 1997; Gao et al., 1998; Raff, 2006). In vitro, after withdrawal of mitogens, OPCs are “de-repressed” and undergo a defined series of steps to differentiate into mature oligodendrocytes, with or without pro-differentiation factors such as thyroid hormone (Knapp et al., 1987; Pfeiffer et al., 1993). Furthermore, this de-repression model of oligodendrocyte differentiation is supported by both in vitro and in vivo evidence. The molecular

11 mechanism underlying this unique behavior of oligodendrocytes has been intensively researched in the past decade (Li et al., 2009; Emery (2010b)), focusing on the intrinsic factors involved both in activation of genes to promote differentiation and repression of genes that inhibit differentiation.

These factors regulate genes through transcriptional, post-transcriptional and epigenetic mechanisms (Li et al., 2009; Emery, 2010b) .

Transcriptional regulation

The emergence of the transgenic technique in the early ‘80s boosted the research of myelin biology (Capecchi, 1980; Gordon et al., 1980; Capecchi, 2001). Most major myelin proteins have been knocked out in mice, which provided insight into the myelin structure at a molecular level(Chernoff, 1981; Klugmann et al., 1997; Griffiths et al., 1998; Yin et al., 1998; Delarasse et al.,

2003; Lappe-Siefke et al., 2003; Nave, 2010b). These studies also opened the question about how this sophisticated structure is assembled and how the oligodendrocyte controls this process. The idea of transcriptional control of development took its place in the ‘90s, when many key transcription factors were identified as essential regulators of development of specific cell types(Yamada et al., 1991; Ericson et al., 1992; Rudnicki et al., 1992; Weintraub, 1993). In the early

2000s, great efforts were made to identify the oligodendrocyte-specific transcription factors responsible for OPC development, encouraged by the success in identifying transcriptional regulators of neurogenesis in chick spinal cord(Jessell, 2000; Lu et al., 2000; Novitch et al., 2001;

Vetter, 2001). Two closely related basic helix-loop-helix (bHLH) transcription factors, Olig1 and Olig2 were identified as oligodendrocyte-specific regulators of OPC specification and differentiation by two independent groups (Lu et al., 2002; Zhou and Anderson, 2002). Meantime, the SRY-related

HMG-box family member Sox10 was found to be essential for the terminal differentiation of oligodendrocytes(Stolt et al., 2002). These findings opened broad interest in searching for transcription factors required for oligodendrocyte maturation. A number of transcription factors

12 were identified in the past decades(Wegner, 2008), most notably Mash1(Battiste et al., 2007),

Nkx2.2(Qi et al., 2001), YY1(He et al., 2007), ZFP191(Howng et al., 2010), ZFP488(Wang et al., 2006),

Tcf4(Fu et al., 2009) and Sip1(Weng et al., 2012). Surprisingly, most of these factors are expressed in

OPCs as well as in mature oligodendrocytes. None of these factors is strictly required for the initial specification, proliferation and migration of OPCs. Instead, all of them are necessary for promoting oligodendrocyte differentiation. The differential function of these factors in OPCs vs. myelinating oligodendrocytes suggests that their role in promoting differentiation may be regulated by additional factors, such as binding partners and chromatin co-regulators etc. There are a number of transcription factors, including Id2, Id4(Wang et al., 2001), Hes5(Liu et al., 2006) and Sox5/6(Stolt et al., 2006), which actively maintain OPCs in their undifferentiated state by antagonizing the function of other pro-differentiation factors. A handful of inhibitory extracellular ligands exert their function by regulating the expression or localization of these inhibitory factors (Stolt et al., 2002; Samanta and Kessler, 2004). Relieving the repression of these negative transcriptional regulators is required for the subsequent action of pro-differentiation factors.

Identification and characterization of the afore mentioned transcription factors substantially changed the way we think of oligodendrocyte development and myelinogenesis. However, it is almost certain that many intrinsic factors remain to be identified. Lack of efficient high throughput genetic screens for oligodendrocyte differentiation and myelination really slow the progress in identifying novel genes involved in this process. Two ENU mutagenesis-based forward screens were conducted in order to search for new genes responsible for myelin formation in zebrafish with limited success (Kazakova et al., 2006; Pogoda et al., 2006). This low efficacy may potentially result from two factors: functional redundancy of genes and the limitation of the selection marker used for the screen. Both of these factors could be improved and large-scale screens will provide essential information about oligodendrocyte differentiation and myelination in the future (Amsterdam and

Hopkins, 2006; Wang et al., 2007).

Alternatively, gene expression analysis by microarray and RNA-seq has proven to be particularly useful to identify oligodendrocyte specific or regulated genes that likely play roles in regulating differentiation and myelination (Cahoy et al., 2008; Zhang et al., 2014). Many genes that are differentially expressed in OPCs vs. mature oligodendrocytes have been identified by transcriptome analysis (Dugas et al., 2006). Among them, myelin gene regulatory factor (MRF) is a potential transcription factor that is enriched in post-mitotic oligodendrocytes in CNS, whose expression is induced upon terminal differentiation. Conditional ablation of MRF does not impair the specification of OPCs and their early differentiation. However, the differentiated oligodendrocytes stall at the premyelinating stage and undergo apoptosis due to failure of myelination initiation(Emery et al.,

2009). In other studies, in addition to its developmental function, MRF has also been shown to be vital for adult myelin maintenance (Koenning et al., 2012). It has been controversial whether MRF functions as a transcription factor to activate expression of myelin genes, because MRF is predicted to be a membrane bound protein with no nuclear localization signaling. Interestingly, MRF has been found to be an ER membrane-bound transcription factor that undergoes autoproteolyic cleavage to generate a nuclear-targeted N terminal domain (Bujalka et al., 2013; Li et al., 2013). Furthermore,

ChIP-seq analysis of the N-terminal domain proves direct binding of this domain to enhancers of oligodendrocyte-specific and myelin genes(Hornig et al., 2013). The terminal differentiation deficit seen in the absence of MRF within the oligodendrocyte lineage recapitulates those seen after Sox10 deletion. This observation raises the possibility that MRF and Sox10 may cooperate to regulate oligodendrocyte terminal differentiation and myelination. Indeed, recent findings from the Wegner laboratory show that MRF is induced by Sox10 concurrent with terminal differentiation, and they physically interact and synergistically activate several myelin-specific genes(Hornig et al., 2013).

Another transcription factor, ZFP191, has also been also identified as a key regulator of late stage

14 oligodendrocyte differentiation. In the absence of ZFP191, abundant CC1+ mature oligodendrocytes are generated and they extend their process to contact axons but fail to fully myelinate the axons

(Howng et al., 2010). This is highly reminiscent of the shiverer mouse phenotype in which the MBP protein is absent(Matthieu et al., 1980). An array of major myelin genes is dramatically downregulated in the ZFP191 mutant mice which have normal oligodendrocyte differentiation. Thus, the oligodendrocyte differentiation and myelination processes are uncoupled in the absence of

ZFP191. These findings add to the de-repression model of oligodendrocyte differentiation by demonstrating that in addition to the down-regulation of inhibitory factors, the successive induction of pro-differentiation factors (Sox10, MRF) and pro-myelination factors (ZFP191) are indispensable for normal myelination.

Epigenetic regulation of oligodendrocyte development

Multiple stages of oligodendrocyte lineage progression are regulated by interplay between genetic and epigenetic determinants (Copray et al., 2009; Yu et al., 2010). Progressive chromatin compaction is a unique feature of oligodendrocyte differentiation concurrent with its dynamic cytoskeletal and transcriptional changes. At the ultra-structural level, the myelinating oligodendrocyte contains an electron-dense nucleus with heavy clumps of heterochromatin layered beneath the nuclear envelope (Mori and Leblond, 1970; Menn et al., 2006). The organization of chromatin within the nucleus is crucial for regulation of gene expression, and the resulting nuclear position of chromosome territories may poise specific genomic loci to active transcription or repression on a large scale(Cremer and Cremer, 2001; Schneider and Grosschedl, 2007; Finlan et al.,

2008; Therizols et al., 2014). This suggests a mechanism by which oligodendrocyte differentiation and myelination are regulated at the level of chromatin remodeling. However, genome-wide mapping and analysis of chromatin states of differentiating oligodendrocytes are just starting (Yu et al., 2013; Liu et al., 2015a). Histone modification and ATP-dependent large scale chromatin

15 remodeling have emerged as key regulators of cell identity (Vignali et al., 2000; Clapier and Cairns,

2009). Work by Casaccia and colleagues first established the essential role of HDAC-mediated histone deacetylation in OPC differentiation (Marin-Husstege et al., 2002). Pharmacological inhibition of Class I HDAC activity in rats causes a delay of oligodendrocyte differentiation and myelination (Shen et al., 2005). Furthermore, oligodendrocyte-specific deletion of both HDAC1 and

HDAC2 impairs both OPC specification and differentiation. Mechanistically, HDAC1/2 is required for inhibition of the canonical Wnt signaling pathway and HDAC1/2 cooperate with YY1 to inhibit the expression of Id4 and Tcf4 (He et al., 2007; Ye et al., 2009). Taken together, the major role of class I

HDACs is to repress “brakes” on oligodendrocyte differentiation by cooperating with a variety of transcriptional regulators. At present, the exact functions of other major HDACs that are known to be expressed in oligodendrocytes remain to be determined. Among them, both HDAC11 and Sirtuin

2 have been shown to regulate oligodendrocyte differentiation in vitro (Li et al., 2007b; Liu et al.,

2009). However, their role in vivo is largely unknown, let alone the mechanism by which they regulate this process. In PNS, Sirt2 has been demonstrated to regulate Schwann cell myelination by deacetylating the cell polarity regulator Par3, but whether this function of Sirt2 is conserved in oligodendrocyte is unknown (Beirowski et al., 2011). Other known epigenetic modifications, including histone methylation and DNA methylation are also implicated in regulating oligodendrocyte differentiation or they are altered in MS lesions (Huynh and Casaccia, 2013; Huynh et al., 2014; Zhao et al., 2014), but their exact function remains elusive.

Chromatin remodeling is a dynamic modification of chromatin architecture that facilitates the access of condensed genomic DNA to transcription machinery. Such remodeling is primarily carried out by an ATP-dependent chromatin remodeling complex that restructures nucleosomes. There are five major families of chromatin remodelers: SWI/SNF, NuRD, ISWI, INO80 and SWR1, with the first two complexes being the most well studied so far (Luo and Dean, 1999; Vignali et al., 2000; Clapier

16 and Cairns, 2009). The specificity of different remodeling complexes comes from their binding to specific transcription factors and recognition of unique histone modifications (Kadam and Emerson,

2003). Recently, it has been shown that SWI/SNF and NuRD complexes are critical for neural stem cell specification and differentiation in conjunction with other transcription factors (Juliandi et al.,

2010; Narayanan and Tuoc, 2014). However, their role in oligodendrocyte specification and differentiation is just being revealed (Yu et al., 2013).

Although they were initially considered primarily to be major histone modifiers, the abovementioned HDAC1 and HDAC2 also serve as essential components in complexes like NuRD,

Sin3 and co-REST(Xue et al., 1998; Andres et al., 1999; Wolffe et al., 2000). These repressor complexes are recruited by many different signaling pathways, such as Wnt, BMP and Notch, to repress the expression of oligodendrocyte differentiation inhibitors (Kao et al., 1998; Ye et al., 2009;

Wu et al., 2012). However, the broad function of these repressor complexes in oligodendrocyte development is largely unknown. It will be necessary to identify their oligodendrocyte-specific genomic binding sites at distinct stages of oligodendrocyte differentiation. Fortunately, next generation high-throughput sequencing technology paves the way for analyzing the genome wide impact of chromatin regulatory complexes on gene transcription(Grada and Weinbrecht, 2013). By integrating information from whole genome sequencing, ChIP-seq and RNA-seq analysis, the gene regulatory landscape in T cell development can be deciphered with unprecedented detail(Zhang et al., 2012). Recently, this technology has been applied to oligodendrocyte research to address the question how the stage specific role of Olig2 is achieved during oligodendrocyte development (Yu et al., 2013). In this case, the core subunit of SWI/SNF complex, Smarca4/Brg1, was found to be enriched in mature oligodendrocyte both in vitro and vivo. Conditional deletion of Brg1 in oligodendrocyte lineage cells led to a differentiation block and subsequent hypomyelination.

Furthermore, ChIP-seq analysis of Brg1 and Olig2 co-occupancy on chromatin at multiple stages of

17 oligodendrocyte differentiation revealed that Olig2 serves as a pre-patterning factor to recruit Brg1- containing SWI/SNF chromatin remodeling complex to enhancers of myelin regulatory genes. This study provides an intriguing example of how key transcription factors could interact with the chromatin remodeling machinery and transcription-linked chromatin modifications to precisely initiate the differentiation process. However, recent work from Michael Wegner’s laboratory indicates other unknown chromatin remodeling complexes may also play important roles in oligodendrocyte differentiation(Weider et al., 2012).

Post-transcriptional regulation of oligodendrocyte differentiation: non-coding RNAs

It has been established over the past few years that oligodendrocyte differentiation and myelination are tightly regulated at the post-transcriptional level by microRNAs in addition to the abovementioned transcriptional regulation(Shin et al., 2009; Budde et al., 2010; Dugas et al., 2010;

Zhao et al., 2010b). miRNAs are small non-coding RNAs that are initially transcribed as long primary miRNAs, which are successively processed by Drosha and Dicer to produce 19-23nt double-stranded

RNA duplexes(Ha and Kim, 2014). Once assembled in a miRNA-induced silencing complex (RISC), it will target the 3’UTR region of mRNAs to repress their expression either through mRNA degradation or translational block. miRNAs are widely used by various cell types to regulate complex cellular processes that require coordinated expression of large numbers of genes(He and Hannon, 2004;

Treiber et al., 2012). Conditional ablation of Dicer within oligodendrocyte lineage cells has been independently conducted by three different groups using Cre recombinase driven by different oligodendrocyte specific promoters (Shin et al., 2009; Dugas et al., 2010; Zhao et al., 2010b). In all cases, loss of Dicer in oligodendrocytes results in profound blockage of OPC differentiation and severe dysmyelination. For unknown reasons, mice with Dicer deletion driven by Olig2-Cre somehow slowly recover and survive into adulthood (Dugas et al., 2010). Furthermore, using microarray profiling of miRNAs that are regulated during oligodendrocyte differentiation, mir-219

18 and mir-338 were independently identified as the most notable miRNAs that are induced upon OPC differentiation. Computational prediction of potential mir-219 and mir-338 targets reveals a number of common targets including PDGFRα, Hes5 and Sox6, which are critical for maintaining OPC proliferation and inhibiting their differentiation. Less conclusively, some other miRNAs have also been shown to play subtle roles in regulating oligodendrocyte development at multiple stages.

Based on these findings, it seems that a collection of miRNAs may function as a brake that sharpens the transition from OPC to differentiated oligodendrocytes by repressing the expression of differentiation inhibitors(Nave, 2010a).

Discoveries over the past decade suggest an essential function of non-coding RNAs in regulation of genomic organization and gene expression at many different levels(Morris and Mattick, 2014;

Palazzo and Lee, 2015). The list of functional non-coding RNAs, including tRNAs, rRNAs, snoRNAs, snRNAs, eRNAs, piRNAs and long non-coding RNAs, continues to grow(Eddy, 2001; Huttenhofer et al., 2005). The ENCODE Project reports that about 80% of the genome transcribes non-coding RNAs

(Washietl et al., 2007). These different groups of regulatory RNAs are involved in many cellular processes, such as protein translation, DNA replication, RNA splicing, chromosome remodeling and gene regulation(Mattick and Makunin, 2006). In particular, some non-coding RNAs are active players in the epigenetic regulation of differentiation and development (Lee, 2012; Li and Chang, 2014). The role of non-coding RNAs in CNS development and function is just emerging as novel regulatory machinery (Qureshi and Mehler, 2012; Fenoglio et al., 2013; Wu et al., 2013; Ziats and Rennert,

2013). Hundreds of long non-coding RNAs (lncRNAs) have been shown to be highly expressed in the brain in a regional and subcellular specific manner (Mercer et al., 2008). However, in brain, the exact function of most of them remains elusive. Interestingly, the expression of several lncRNAs has been associated with oligodendrocyte specification and maturation in vitro (Mercer et al., 2010; Mills et al., 2015). RNA-seq and more sophisticated analytic tools will facilitate the characterization of novel

19 lncRNAs in oligodendrocyte lineage. The emerging new genome manipulation technologies such as

TALEN and CRISPR/Cas9 will make the genetic manipulation of lncRNAs more effective and affordable. This will open the door to investigate physiological role of lncRNAs in oligodendrocyte development and brain development more generally.

INTERPLAY BETWEEN THE EXTRINSIC SIGNALING AND TRANSCRIPTIONAL REGULATION OF

OLIGODENDROCYTE DEVELOPMENT

Single cell organisms sense and detect the change of nutrient availability in their surrounding environment and respond by up- or down-regulating selected genes. This phenomenon is well illustrated in the classic model of Lac operon regulation in E.coli (Jacob and Monod, 1961). In higher eukaryotes, most if not all cells have retained the ability to adapt to environmental changes by reprogramming their transcriptional profiles (Finnegan et al., 2004; Ospina-Alvarez and Piferrer,

2008). The environment an individual cell experiences is determined by the physiological condition and metabolism of the organism and by its immediate neighboring cells(Niklas, 2014). The local environment modulates gene expression of resident cells and determines their cell fate, either by cell-cell contact or soluble factors, which depends on cell type and developmental context(Li and Xie,

2005; Riquelme et al., 2008; Ehninger and Trumpp, 2011). This principle of cell and environment interaction applies to most cell types. However, the molecular mechanisms by which extrinsic signals impact the functioning of the genome of multi-cellular organisms remains elusive. The emerging evidence from studies on myogenesis and neurons highlights the role of adaptive change of transcriptional machinery in response to developmental and environmental cues(Guasconi and

Puri, 2009; Ma et al., 2010; Fong et al., 2012; Jobe et al., 2012). The development and function of oligodendrocytes is achieved by regulated expression of large numbers of specific genes coordinated with brain maturation (Dugas et al., 2006). The list of extrinsic cues and intrinsic regulators for oligodendrocyte development has been growing(Zuchero and Barres, 2013). The mechanisms by

20 which extracellular signals regulate the activity of both transcription factors and chromatin- modifying enzymes have just begun to be characterized.

Intracellular signaling transduction: the bridge from cell surface to nucleus

The binding of specific ligands to their receptors triggers a conformational change of the intracellular domain of these receptors. This results in either promoting the enzymatic activity of the receptor or exposure of docking sites for other intracellular signaling proteins, which eventually propagates the signal through the cytoplasm(Klein, 1984; Pawson and Nash, 2000). Most of the former mentioned extracellular signals functioning in oligodendrocyte development have their known receptors expressed concurrently in oligodendrocytes in a stage specific manner. Receptor tyrosine kinases, G-protein coupled receptors, integrins, neurotransmitter receptors and nuclear receptors are among the most important and well studied receptors for oligodendrocyte development. When bound with their specific ligands, they all respond by activating downstream signaling cascades, which could function in parallel or synergistically to modulate gene expression in the nucleus.

Recently, intracellular kinase signaling pathways have emerged as important players in oligodendrocyte differentiation and myelination (Flores et al., 2008; Chew et al., 2010; Ishii et al.,

2012; Ishii et al., 2013; Mayes et al., 2013; Bercury et al., 2014; Chung et al., 2015). Among them, the mitogen-activated protein kinase (MAPK)/extracellular related kinase (ERK) pathway and

PI3K/Akt/mTOR pathway are two major point of convergence of many different extrinsic signals in oligodendrocytes, including neuregulins, PDGF, FGF, IGF1, Integrin and neurotrophins (Baron et al.,

2000; McKinnon et al., 2005; Lee et al., 2006; Frederick et al., 2007; Van't Veer et al., 2009;

Guardiola-Diaz et al., 2012); and the crosstalk between these two pathways has been found in various cell types including oligodendrocytes (Carracedo et al., 2008; Dai et al., 2014). Loss and gain of function studies of these two pathways in oligodendrocyte development demonstrate their

21 critical role in myelinogenesis , although they appear not to be as important in oligodendrocyte differentiation(Flores et al., 2008; Ishii et al., 2012; Ishii et al., 2013; Bercury et al., 2014). Both Erk and Akt pathways have been implicated in regulating myelin specific gene transcription and translation. It has been learned from many cancer studies that both pathways play important roles in cell proliferation and prevention of apoptosis(De Luca et al., 2012). Many down-stream transcription factors involved in the pro-survival function of Erk and Akt pathways have been identified, including NF-kB, CREB, AP-1 (c-Jun/c-fos), Ets, Elk1 and c-myc for the Erk pathway(Chang et al., 2003) and FOXO, NF-kB, p53 and CREB for the Akt pathway (Manning and Cantley, 2007). The list of transcription factors regulated by Erk and Akt pathway is still growing in many cell types.

However, almost nothing is known about the transcription factors directly regulated by Erk and Akt pathways that are involved in oligodendrocyte differentiation and myelination. It is not clear whether some of the formerly mentioned critical oligodendrocyte transcription factors (Olig1, Sox10,

MRF and ZFP191) could be directly phosphorylated through the Erk or Akt pathway. If this is not the case, the pro-survival transcription factors targets of these two pathways may also play a role in oligodendrocyte differentiation and myelination. Yet how their specificity of myelin gene regulation is achieved is still an open question. High throughput phosphoproteomic analysis of oligodendrocyte specific phosphorylation of transcription factors will be a powerful tool to answer this question in the future (Daub et al., 2008).

G-protein coupled receptors provide another example of how the extracellular signal could transduce signals through the cytoplasm through generation or release of small molecules called second messengers, such as cyclic adenosine monophosphate (cAMP) and calcium(Marinissen and

Gutkind, 2001; Wettschureck and Offermanns, 2005). In Schwann cells, elevation of cAMP can mimic axon contact in vitro(Lemke and Chao, 1988). Recently, one G-protein coupled receptor Gpr126 was identified as an upstream driver of increased cAMP in Schwann cells. Gpr126 is essential for

Schwann cell myelination both in zebrafish and mice (Monk et al., 2009). G-protein coupled receptors can also trigger an increase of intracellular calcium level (Dolphin, 2003), but how intracellular calcium regulates gene expression in glia is largely unknown. One study of Schwann cell myelination identifies Nuclear factor of activated T-cells (NFAT) as the downstream effector of calcium. The calcium level in Schwann cells increases after exposure to neuregulin, which activates calcineurin and transcription factor NFATc3 and c4. As a result, NFAT4c physically binds to Sox10 and synergistically activates Krox20 and myelin gene expression (Kao et al., 2009). cAMP and calcium are also induced in oligodendrocytes in response to neurotransmitter treatment through G-protein coupled neurotransmitter receptors(Butt, 2006; Karadottir and Attwell, 2007), but their function in vivo is not clear. The question of whether oligodendrocytes use similar mechanisms as Schwann cells to regulate myelin gene expression needs to be addressed in the future.

Post-translational modification of transcription factors

Post-translational modification of transcription factors represents a mechanism frequently used by cells in rapid response to environmental changes (Turner, 2009; Prabakaran et al., 2012). For instance, multiple signaling transduction pathways coordinate transcriptional responses by phosphorylating transcription factors (Jackson, 1992; Whitmarsh and Davis, 2000). Acetylation and ubiquitinylation are the other two common post-translational modifications of transcription factors

(Kerscher et al., 2006; Choudhary et al., 2009; Verdin and Ott, 2015). These modifications are conducted by the same enzyme families that are responsible for the histone modifications, e.g. acetyltransferase CBP/P300, GCN5, PCAF can act on the same factor with distinct functional outcomes. Androgen receptor, P53 and c-myc can all be phosphorylated at specific serinine and threonine sites, or acetylated at specific lysine sites(Glozak et al., 2005; Vervoorts et al., 2006; Popov et al., 2007). These modifications have either positive or negative impact on the activity of different transcription factors and result in appropriate changes in cell behavior (Soutoglou et al., 2000). The

23 abovementioned modifications can regulate different aspects of transcription factor function, including subcellular localization, protein interactions, DNA binding and protein stability. A number of transcription factors shuttle between the nucleus and cytoplasm, which is an active process that is determined by nuclear localization signals (NLS) and nuclear export signals (NES)(Hood and Silver,

1999). Both phosphorylation and acetylation can regulate the accessibility of NLS and NES to the nuclear import and export machinery (Zhu et al., 1998; Li et al., 2012). Phosphorylation or acetylation of NFAT, STAT, FOXO and SMAD family transcription factors can regulate their nuclear import in response to specific stimuli(Chow et al., 1997; Moustakas et al., 2001; Kramer et al., 2009;

Tzivion et al., 2011). The interaction between transcription factors and other transcription factors, epigenetic coregulators and basal transcription complexes is potentially regulated by phosphorylation and acetylation. For example, the transcription factor cAMP response element binding protein (CREB) can be phosphorylated by multiple kinases on ser133(De Cesare et al., 1999).

This phosphorylaiton facilitates its interaction with co-activator CREB binding protein (CBP) and leads to increased transactivation of specific gene targets(Shiama, 1997). The G1/S cell cycle checkpoint retinoblastoma (RB) protein is phosphorylated by a combination of CDKs and this phosphorylation causes its dissociation from E2F preceding the G1 to S phase transition(Harbour and Dean, 2000). Moreover, specific modifications have a selective impact on transcription factor activity. Under genotoxic stress, the DNA binding ability of P53 is regulated by multiple mechanisms

(Gu and Roeder, 1997; Siliciano et al., 1997; Luo et al., 2004). Phosphorylation or acetylation on specific sites in the N-terminal of p53 both enhance its DNA binding, but activate distinct subsets of target genes in response to different types of stress. Post-translational modification of cell type- specific transcription factors has been emerging as an important regulator of glia development. In

Schwann cells, HDAC1/2 mediates the deacetylation of NF-kB, which is essential for Schwann cell development and myelination (Chen et al., 2011). Many of the mechanisms mentioned above have

24 been employed by oligodendrocytes to control the function of key regulators of their development, but the modification of most oligodendrocyte-specific transcription factors remains to be determined. In oligodendrocytes, site specific modifications have selective effects on Olig2 function.

Olig2 phosphorylation on serine 30 regulates its nuclear export and is required for astrogenesis from neural progenitor cells (Setoguchi and Kondo, 2004). In contrast, the proliferation promoting function of Olig2 in neural progenitor cells and OPCs is regulated developmentally by phosphorylation on a conserved triple serine (S10/S13/S14) motif within its N-terminal domain (Sun et al., 2011). Furthermore, phosphorylation of Olig2 on serine 147 is found to regulate its binding factor preference and to facilitate the switch from motor neuron specification to OPC specification

(Li et al., 2011). Interestingly, the equivalent serine is also phosphorylated in Olig1, but has a distinct impact on Olig1, where it promotes its nuclear export and membrane expansion as oligodendrocytes mature in culture (Niu et al., 2012). Low cellular abundance of phoshorylated or acetylated proteins has resulted in their underrepresentation in most mass spectrum based proteomic analysis, including most post-translational modification studies on transcription factors(Oppermann et al.,

2009; Zhao et al., 2010a). In recent years, new enrichment methods have been developed for enriching phospho-peptides by small chemical compound-based selective affinity and acetyl- peptides by anti-lysine antibody affinity. In combination with quantitative mass spectroscopy after peptide enrichment, the large scale analysis of transcription factor modification became feasible and affordable recently(Daub et al., 2008; Choudhary et al., 2009). There is almost no doubt that the post-translational modification of many transcription factors important for oligodendrocyte development will be identified in the near future.

Regulation of chromatin modifier activity: converging on the chromatin

Chromatin is a complex of DNA, RNA, histone and non-histone proteins which undergoes constant change in response to the changing environment. Epigenetic changes in histone modification, DNA

25 methylation and chromatin architecture have long-lasting impacts on cell behavior (Guasconi and

Puri, 2009; Riccio, 2010). However, the mechanism for transducing cytoplasmic signaling cascades to the chromatin remodeling remains elusive. In the past two decades, the nature of the majority epigenetic modifiers has been revealed, but their interplay and regulation by multiple signaling pathways is largely unknown. Most chromatin modifying enzymes use substrates or cofactors derived from cellular metabolites to transfer different chemical moieties to their targets. It is not surprising that fluctuation of metabolites could regulate the activity of chromatin modifiers and therefore influence epigenetic dynamics(Lu and Thompson, 2012). For example, mammalian histone acetyltransferases (HATs) transfer the acetyl group of acetyl-CoA to the lysine residue of histone and non-histone proteins. In cancer cells, the elevated acetyl-CoA is sufficient to drive cell growth by promoting histone acetylation and expression of growth-related genes(Cai et al., 2011). The NAD+- dependent class-III deacetylase Sirtuin-1 is another protein that senses the metabolic status in cells.

A high NAD/NADH ratio enhances Sirt-1 activity and facilitates the deacetylation of Androgen receptors and reduces its growth-promoting activity (Fu et al., 2006). The metabolic regulation of the epigenetic landscape may also occur during oligodendrocyte development and myelination. It has long been known that myelinating oligodendrocytes are high energy demanding cells which generate huge amounts of protein and lipid in a short period of time. Intriguingly, recent work from the Nave laboratory demonstrates the glycolytic nature of oligodendrocyte metabolism in supporting axonal integrity (Funfschilling et al., 2012). Enhanced glycolysis in tumors has been linked to an increase of acetyl-CoA production and promotion of histone acetylation (Morrish et al., 2010).

It will be interesting to test whether the epigenetic landscape in myelinating oligodendrocytes is highly regulated by their metabolic status.

In addition to the direct regulation by metabolic intermediates of enzymatic activities that modify chromatin, these chromatin regulatory enzymes themselves can be further regulated by post-

26 translational modifications. For example, class I HDACs can be phosphorylated, acetylated, nitrosylated and ubiquitinated by different signaling pathways(Brandl et al., 2009). Whereas all these modification influence HDAC function, the physiological stimuli and intracellular signaling pathway responses driving these changes are largely unknown. The identification of signaling pathways that modulate the activity of histone modifiers will be the key to understanding how the transcriptional response integrates extracellular stimuli. Evidence of cooperation between distinct signaling pathways in regulating common epigenetic targets is emerging. In both neurons and cardiomyocytes, one of the major HATs, CBP, can be phosphorylated by multiple kinases in response to neural activity or the regenerative environment (Guasconi and Puri, 2009; Riccio, 2010). It seems likely that finely tuned activation of CBP or other histone modifiers may be orchestrated by multiple phosphorylation events, which could regulate the transition between short and long term epigenetic changes that underlie the development of many cell types. Long-term stable chromatin modifications are necessary to enforce the inhibition of early development related genes that need to be repressed in differentiated cells. Interestingly, differentiated oligodendrocytes favor a repressive chromatin landscape, with the early neural response genes marked and repressed by

H3K9 methylation (Liu et al., 2015a). On the other hand, rapid epigenetic changes are required for the transcriptional response to extracellular cues, which is well illustrated in the neural activity regulation of gene expression in neurons (Hardingham et al., 1999; Chawla et al., 2003; Ma et al.,

2009). OPCs receive synaptic input from unmyelinated axons, and they can respond by generating miniature electrical potential. Although evidence for the posttranslational modification of chromatin modifiers in oligodendrocyte is lacking, it is reasonable to speculate that similar mechanisms may also occur in oligodendrocytes as they differentiate.

Vast chromatin reconfiguration is a unique feature of cell differentiation in the CNS(Manuelidis,

1984). As oligodendrocytes differentiate, condensed chromatin forms and becomes apparent

27 beneath the nuclear envelope. The nature of this chromatin compaction is completely unknown.

Lessons from neurons and other cell types provide some hint of the mechanism by which this process may work. Hippocampal neurons undergo large nuclear infolding and chromatin re- arrangement in response to repeated pulses of action potentials. This reconfiguration of nuclear structure is triggered by calcium influx into the nucleus and concurrent activation of the Erk signaling cascade (Wittmann et al., 2009). The three-dimensional view of the nucleus is starting to be more appreciated, with the advance of new molecular and microscopy techniques. In the nucleus, distinct transcriptionally active genomic domains have been identified as “transcription factories” that are enriched with cis-regulatory elements and transcriptional and chromatin modifier complexes (Fraser and Bickmore, 2007; Schneider and Grosschedl, 2007). Changes in nuclear architecture can not only ensure the long lasting expression or suppression of specific genes in terminally differentiated cells like myelinating oligodendrocytes and neurons, but also for more rapid transcriptional responses. Understanding the nature of chromatin reorganization and the signaling that controls this process in myelinating oligodendrocytes will be exciting and challenging for future research.

OLIG1 AND OLIG2: MOLECULAR AND FUNCTIONAL DIVERGENCE

Olig1 and Olig2, are closely related basic helix-loop-helix (bHLH) transcription factors that play critical roles in oligodendrocyte specification and differentiation (Lu et al., 2002; Zhou and Anderson,

2002).Besides its essential role for oligodendrocyte lineage specification, Olig2 is also a modulator of various aspects of cell specification in the CNS, including neurogenesis and astrogenesis (Lee et al.,

2005; Marshall et al., 2005; Cai et al., 2007; Liu et al., 2015b). Olig2 complete knockout in mice are embryonic lethal due to a lack of motor neurons. Conditional deletion of Olig2 in oligodendrocyte lineage cells revealed its stage-specific role in postnatal oligodendrocyte differentiation (Mei et al.,

2013). However, despite the fact that multiple Olig1 knockout mice have been developed, the

28 function of Olig1 during oligodendrocyte lineage development is still unclear (Lu et al., 2002; Xin et al., 2005; Paes de Faria et al., 2014). The original Olig1 knockout mouse made in Charles Stiles’ laboratory shows a delayed myelination phenotype in spinal cord. The mild phenotype of this original Olig1 null mice has been speculated to be due to compensatory up-regulation of Olig2 by the cis-acting regulatory effect of the Pgk-Neo cassette retained in the Olig1 locus (Lu et al., 2002).

When that cassette was removed from the original line, the new Olig1 null line had greater dysmyelination and died postnatally at two weeks (Xin et al., 2005). It has been speculated that in the Olig1 knockout line without the Pgk-neo cassette, an unintended recombination event may have taken place and somehow disrupted both Olig1 and Olig2 expression to certain extent(Paes de Faria et al., 2014). Indeed, when two additional Pgk-neo cassette free Olig1 null mouse lines were generated by different strategies recently, only mild developmental delay is found in these mice

(Paes de Faria et al., 2014). However, Olig2 levels were never investigated in either of these Olig1 null lines. At present, the discrepancy of the different phenotypes of the above mentioned multiple

Olig1 null lines has not been resolved. At least the different phenotypes of different Olig1 knockout cannot be simply explained by the presence or absence of the Pgk-Neo cassette. Interestingly, one recent study from the Rowitch laboratory demonstrates the impairment of OPC specification in brain of the original Olig1 null mice, on a relatively pure C56BL/6 genetic background (Silbereis et al.,

2014). This study brings up two concerns about the phenotypes of the different Olig1 null mice, the genetic background variance and the potential phenotype differences between brain and spinal cord.

Nevertheless, Olig1 has been shown to be essential for remyelination in Olig1 knockout mice, using different demyelination models. Thus, loss of Olig1 in the original Olig1 null mouse causes an absolute failure of remyelination (Arnett et al., 2004). It is conceivable that Olig1 and Olig2 may have some overlapping function during development. Perhaps Olig2 could compensate for some of Olig1 function in development but not in remyelination.

Subcellular translocation of Olig1 is observed during both oligodendrocyte development and remyelination. Olig1 is first identified in the nucleus during oligodendrocyte specification and it moves out of the nucleus to the cytoplasm after P14. Importantly, during remyelination, Olig1 relocates back to the nucleus, suggesting a nuclear function for Olig1 that appears essential for remyelination (Arnett et al., 2004). Although translocation of Olig1 seems to be critical for remyelination, the mechanism regulating this process has not been established. Post-translational modification of proteins, particularly phosphorylation and acetylation, regulates the subcellular location of many transcription factors (Kramer et al., 2009; Meek and Anderson, 2009). Based on its essential role in remyelination, agonists of Olig1 activity has been proposed as a potential therapy for MS(Frohman et al., 2006). However, transcription factors are usually not ideal pharmaceutical targets.

The function of Olig1 in oligodendrocyte development is still elusive. Two important questions remain to be addressed: 1. Whether Olig1 plays a unique role in oligodendrocyte development in brain, in contrast to its mild function in spinal cord? 2. What is the role of Olig1 at distinct stages of oligodendrocyte differentiation? To solve these problems, the first part of my thesis study aims to characterize the phenotype of the original Olig1 null mice in brain on a pure C57BL/6 background.

The subcellular translocation of Olig1 is developmentally regulated and has been suggested to be critical for its function during remyelination. However, the mechanism that regulates its subcellular translocation is largely unknown. The second part of my thesis aims to investigate the hypothesis that post-translational modification of Olig1 regulates its subcellular translocation and function.

Understanding the developmental function of Olig1 will provide insight into its essential role in remyelination. Identification of the mechanism underlying its subcellular translocation may provide a new target for enhancing Olig1 activity during remyelination.

CHAPTER II

OLIG1 FUNCTION IS REQUIRED FOR OLIGODENDROCYTE DIFFERENTIATION IN

THE MOUSE BRAIN1

ABSTRACT

Oligodendrocyte differentiation and myelination are tightly regulated processes orchestrated by a complex transcriptional network. Two bHLH transcription factors in this network, Olig1 and Olig2, are expressed exclusively by oligodendrocytes after late embryonic development. While the role of

Olig2 in the lineage is well established, the role of Olig1 is still unclear. The current studies analyzed the function of Olig1 in oligodendrocyte differentiation and developmental myelination in brain.

Both oligodendrocyte progenitor cell commitment and oligodendrocyte differentiation were impaired in the corpus callosum of Olig1 null mice, resulting in hypomyelination throughout adulthood in the brain. As seen in previous studies with this mouse line, while there was an early myelination deficit in the spinal cord, essentially full recovery with normal spinal cord myelination was seen. Intriguingly, this regional difference may be partially attributed to compensatory upregulation of Olig2 protein expression in the spinal cord after Olig1 deletion, which is not seen in brain. The current study demonstrates a unique role for Olig1 in promoting oligodendrocyte progenitor cell commitment, differentiation and subsequent myelination primarily in brain, but not spinal cord.

INTRODUCTION

Myelin ensheathment of axons dramatically increases axonal conduction velocity and provides valuable trophic support to axons (Bullock et al., 1984; Hildebrand et al., 1993; Nave, 2010c). In demyelinating diseases such as multiple sclerosis (MS), remyelination occurs, but the myelin is

1 This chapter was published : Dai J, Bercury KK, Ahrendsen JT, Macklin WB (2015) Olig1 function is required for oligodendrocyte differentiation in the mouse brain. The Journal of neuroscience : the official journal of the Society for Neuroscience 35:4386-4402.

31 thinner than normal and many axons remain demyelinated (Franklin and Ffrench-Constant, 2008).

Oligodendrocyte progenitor cells (OPCs) and premyelinating cells are present in MS lesions but in general, remyelination is greatly impaired in MS tissue (Chang et al., 2000; Chang et al., 2002;

Kuhlmann et al., 2008). Thus, understanding the molecular mechanisms driving oligodendrocyte maturation and myelination remains an extremely important research question.

Over the past decade, much of the transcriptional regulatory network of oligodendrocyte lineage progression has been elucidated (Emery, 2010a). Two transcription factors, Olig1 and Olig2, are closely related basic helix-loop-helix (bHLH) transcription factors that play critical roles in oligodendrocyte specification and differentiation (Lu et al., 2002; Zhou and Anderson, 2002).

Although it appears to have an essential role in remyelination (Arnett et al., 2004), the function of

Olig1 during oligodendrocyte development is still unclear. This results from the fact that several

Olig1-null lines have been developed that have quite different phenotypes (Lu et al., 2002; Xin et al.,

2005; Paes de Faria et al., 2014). Oligodendrocyte differentiation in developing spinal cord is delayed in the original Olig1-null mouse, yet there is no long term developmental problem (Lu et al.,

2002). It was hypothesized that this mild phenotype might result from compensation by Olig2 (Lu et al., 2002), and a second Olig1-null mouse was designed to reduce any Olig2 compensation. This animal has greater dysmyelination and dies by postnatal day 14 (Xin et al., 2005). Two additional

Olig1 null mouse lines have been described recently, which were generated by different strategies, and only mild developmental delay of spinal cord myelination is found in these mice (Paes de Faria et al., 2014).

In ongoing studies using the original Olig1-Cre mice for gene deletion in oligodendrocytes, we noted unexpected changes in myelination in the brains of homozygous null Olig1-Cre mice. In general, the studies on the different Olig1 null mouse lines have focused on spinal cord development. However, differential regulation of oligodendrocyte differentiation and myelination

32 has been seen in different brain regions (Fruttiger et al., 1999; Sperber et al., 2001; Bercury et al.,

2014; Wahl et al., 2014). We therefore began investigating the role of Olig1 in oligodendrocyte development outside the spinal cord. These studies demonstrate that in the brain, Olig1 has a critical role in the commitment of pre-oligodendrocyte progenitor cells (pre-OPCs) to oligodendrocyte progenitor cells (OPCs) and for subsequent OPC differentiation. The data from the current study demonstrate a unique role of Olig1 in promoting OPC commitment and differentiation, which appears more important in brain than spinal cord OPCs.

RESULTS

OPC production, differentiation and early morphogenesis were reduced in Olig1 null mice.

The impact of Olig1 on OPC specification and number in the corpus callosum during the early postnatal period was investigated. In Olig1 null mice at P1, there was a 40% reduction in the total number of oligodendrocyte lineage cells (Olig2+ ) in subcortical white matter compared to control

(Fig2.1A,B,C). This reduction is consistent with a recent study that showed reduced oligodendrocyte lineage cells in Olig1 null mice and ectopic production of interneurons (Silbereis et al., 2014). Within the Olig2+ cell population in the Olig1null mice, the percent that were NG2+ OPCs was only approximately half that in control animals (Fig2.1A,B,D). Thus, compared to control P1 animals, where 61.9% of Olig2+ cells are NG2+, intriguingly, in the Olig1 null mice, only 29.5% of the Olig2+ cells were also NG2+ (Fig3.1D). In order to identify the Olig2+/NG2- cell population, we analyzed expression of a pre-OPC marker, Sox2. Sox2 is expressed in the subventricular zone (SVZ) by neural progenitors and by pre-OPCs (Olig2+/Sox2+ cells) migrating out of the SVZ (Ellis et al., 2004; Menn et al., 2006; Hu et al., 2009b; Wang et al., 2013). Once pre-OPCs reached the subcortical white matter, they reduced Sox2 expression and transitioned into Olig2+/NG2+ OPCs (Fig2.1E, F). At P1, this reduction in Sox2 expression was still in progress. Thus, there were cells with high Sox2 expression, presumably pre-OPCs, and cells with low Sox2, presumably cells beginning to differentiate to OPCs.

The total number of Sox2+ cells was reduced in Olig1 null mice (Fig2. 1G), which may reflect that the reduction in total oligodendrocyte lineage cells in Olig1 null mice occurs at early specification stages

(Silbereis et al., 2014). The pre-OPC population was quantified as the number of Olig2+/Sox2+/NG2- cells in control or Olig1 null mice, including both high and low-expressing Sox2+ cells. Interestingly, there was a significant increase in the percent of Olig2+ cells that were Olig2+/Sox2+/NG2- pre-OPCs in Olig1 null mice, relative to control mice (Fig2.1G). Furthermore, as can be seen in Figure 1E,F, while Sox2 levels were very low in NG2+ cells in control samples (Figure 1E and H, merged image; 1E’,

Sox2 image, compare arrowheads to arrows), in Olig1 null cells, numerous NG2+ cells retained strong Sox2 stain (Figure 1F and H, merged image, arrowheads). This apparent delay/deficit in the downregulation of Sox2 and the transition from pre-OPC to OPC in Olig1 null mice suggest an important role of Olig1 in this very early stage of oligodendrocyte development, a stage that has not been investigated extensively in vivo previously.

Despite being a smaller population, the Olig2+ cells in Olig1 null mice were highly proliferative

(Fig2. 2A, B). P1 pups were injected with BrdU and sacrificed 2 hours later; tissue was analyzed for

BrdU incorporation coupled with Ki67 immunohistochemistry. BrdU permanently labels cells during

S phase, and their progeny retain the label, while Ki67 is a marker of proliferating cells currently in the cell cycle (Kee et al., 2002). The number of Olig2+ cells that were either BrdU+ or Ki67+ was quantified in the subcortical white matter. The percentage of Olig2+ cells that were BrdU+ or Ki67+ in P1 Olig1 null mice was essentially twice that of control (Fig2.2C). Both the pre-OPCs (Olig2+/NG2-) and the OPCs (Olig2+/NG2+) had far more Ki67 expression (52.7% and 83.2%, respectively) than control cells (18.6% and 59.2% respectively) (Fig2.2D, E, F, G). These data suggested that a significant segment of the Olig2+ cell population remained in the cell cycle in Olig1 null mice at P1.

Despite their increased proliferation, the number of Olig2+ cells remained lower in Olig1 null mice, and it seemed possible that the reduced number of cells resulted from increased cell death

(Reid et al., 1999; Castedo et al., 2004). Indeed, cell death essentially doubled in the Olig1 null mice

(Fig2.2H, I, J). Although many dying cells had lost their cell-specific markers, a number of these apoptotic cells in Olig1 null mice expressed NG2 proteoglycan, which was rarely seen in control mice

(Fig2. 2K, L, arrows). Thus, the increased cell death in Olig1 null mice was likely in OPCs that failed to differentiate properly. These data indicate that OPCs were delayed in exiting the cell cycle in Olig1 null mice, and the number of both

Figure 2.1 :Deficit of pre-OPC to OPC commitment in P1 Olig1 null mice. (A, B) Representative images of cells stained with NG2+(green) and Olig2(red) in the corpus callosum of P1 Olig1 heterozygous (A,A’ and A”) vs. Olig1 null (B,B’ and B”) mice. Scale bar: 50μm.(C, D) Quantification of total Olig2+ cells (C) and proportion of NG2+ cells within Olig2+ cells (D).(E, F) Representative images of cells stained with NG2+(green), Sox2+(red) and Olig2(blue) in the corpus callosum of P1 Olig1 heterozygous (F, F’ and F’’) vs. Olig1 null (G, G’ and G’’) mice. Arrow marked NG2-Sox2highOlig2+ pre-OPC and arrowhead marked the NG2+Sox2lowOlig2+ OPC. Scale bar: 50μm.(G) Quantification of the proportion of Sox2+NG2- pre-OPC population within Olig2+ cells. (H) Quantification of the total Sox2+ cells, and cells with high and low Sox2 staining, respectively. For all quantifications, data represent Mean ± SEM; the number of animals analyzed was indicated in each panel;****p < 0.00005, *****p < 0.000005; two sample Student’s t test

Figure 2.2: Enhanced progenitor cell proliferation and apoptosis in P1 Olig1 null mice.

Figure 2.2: Enhanced progenitor cell proliferation and apoptosis in P1 Olig1 null mice. (A, B) Representative images of the BrdU+ (green, [A and B]), Ki67(red, [A’ and B’]) and merged with Olig2+ (A’’ and B’’) cells in the subcortical white matter of P1 Olig1 heterozygous(A) or Olig1 null (B) mice pulse injected with BrdU for 2 hours. Scale bar: 50μm.(C) Quantification of the proportion of BrdU+ and Ki67+ cells within the Olig2+ OPCs population. (D and E) Representative images of cells stained with NG2+(green), Ki67+(red) and Olig2(blue) in the corpus callosum of P1 Olig1 heterozygous (D) vs. Olig1 null (E) mice. High magnification images (D’, E’) highlight less complex OPC morphology in Olig1 null mice. Scale bar indicates 50μm in (D, E) and 15μm in (D’, E’).(F, G) Quantification of the proportion of NG2-Ki67+ pre-OPCs within Olig2+ cells (F) and NG2+Ki67+ proliferating OPCs out of NG2+ OPC population (G). (H and I) Low-magnification images of the cerebrum stained with TUNEL (red) and Olig2 (blue) of the P1 Olig1 heterozygous (H) vs. Olig1 null (I) mice. (H’ and I’) High magnification images of the corpus callosum corresponding to the box insets in panel (H) and (I). Arrow marked TUNEL+ Olig2- cells and arrowhead marked the TUNEL+Olig2+ dying OPC. Scale bar 500μm in (H) ad (I) and 50μm in (H’) and (I’).(J) Quantification of TUNEL+ cells within the midline of corpus callosum in Olig1 heterozygous vs. Olig1 null mice. (K, L) Representative images of cells stained with NG2 (green), TUNEL (red) and Olig2 (blue) within the corpus callosum of control (K) vs. Olig1 null (L) mice. Arrows mark TUNEL+ NG2+ cells; arrowheads mark the TUNEL+NG2- cells. Scale bar: 10μm.For all quantifications, data represent Mean ± SEM; the number of animals analyzed was indicated in each panel; ***p < 0.0005, *****p < 0.000005, ******p < 0.0000005; unpaired Student’s t test).

37 proliferative (Ki67+) and dying cells (TUNEL+) in the Olig1 null mice was twice that in control mice.

Thus it appears that in the absence of Olig1, Olig2+ cells failed to effectively exit cell cycle and continued to proliferate, resulting in increased cell death.

Finally, it must be noted that the NG2+ cells in Olig1 null mice were morphologically far simpler than in control mice, with fewer processes (Fig2.2D’,E’), suggesting a role for Olig1 in early OPC differentiation. In summary, these findings provide evidence that Olig1 is important for pre-OPC to

OPC commitment and subsequent OPC differentiation and morphogenesis.

Oligodendrocyte differentiation was reduced, but not the initiation of axon wrapping in P8 Olig1 null mice.

We next investigated the impact of Olig1 loss on the initial stages of myelination in P8 mice. At this age, myelination had already begun in the striatum and lateral corpus callosum of Olig1 control mice (Fig2.3A), but it was significantly reduced in Olig1 null mice (Fig2. 3B). The tissue was stained

d CC1, and as at earlier ages, there were 43% fewer oligodendrocyte

and a greater reduction in the number of CC1+ mature cells (Fig2.3C,D, E,F). Thus, as a percentage of the total Sox10+ oligodendrocyte lineage cells, the progenitor cell population was increased by 27%, while the differentiated cell population was reduced by 32% (Fig2.3F), which suggested that, in addition to the delay in cell cycle exit, loss of Olig1 also delayed differentiation of the progenitor cells.

As OPCs differentiate, they move through a premyelinating stage to the myelinating stage (Trapp et al., 1997). It was important to assess whether the reduction of CC1+ cells resulted from an inability to begin to differentiate, or rather an inability to interact with axons and start myelination.

were premyelinating PLP+ cells (Fig2.3G, H). Thus, PLP+ premyelinating oligodendrocytes only

38 accounted for 7.2% of total Sox10+ cells in Olig1 null mice, far less than the 20.6% seen in control tissue (Fig2.3I). Thus, the initiation of differentiation from OPC to premyelinating oligodendrocyte was impaired in these mice.

As noted above, by P8, myelination was occurring rapidly in the subcortical white matter of control animals, but this was rarely seen in Olig1 null mice, where only limited myelination was seen in the lateral corpus callosum (Fig2.3A, B). This likely resulted only from the reduced numbers of

CC1+/Sox10+ mature oligodendrocytes, since those that did differentiate had no deficit in initial axonal contact. Thus, the initial axonal contact, which appeared to involve PLP-expressing membranes (Figure 3J’, 3K’ arrowhead), and further wrapping and segment elongation, which involved membranes containing both PLP and MBP (Fig2.3J’’, J’’’, K’’, K’’’, arrowheads), were relatively normal in Olig1 null (Figure 3K), compared to control littermates (Figure 3J). Indeed, as these cells continued to myelinate, they appeared relatively comparable to control cells (Fig2. 3L, M).

These data indicate that, in addition to a role of Olig1 in early OPC cell cycle exit, the loss of Olig1 impacted terminal differentiation of oligodendrocytes, but for cells that had terminally differentiated, Olig1 was not essential for myelination initiation per se.

Oligodendrocyte deficit persists in the brain of adult Olig1 null mice.

An important question was whether the reduced oligodendrocyte number and impaired differentiation in Olig1 null corpus callosum eventually recovered as the animals matured.

Oligodendrocyte maturation and myelination were therefore investigated in 2 month Olig1 null corpus callosum. Consistent with the early developmental stages, the number of Sox10+ cells at the midline of the corpus callosum of Olig1 null mice was reduced by approximately 60% relative to control. This reduction resulted primarily from a reduction in mature oligodendrocytes, since there was a 68% reduction of the total number of CC1+ mature oligodendrocytes in Olig1 null mice, while

Figure 2.3: Oligodendrocyte differentiation and myelination initiation in P8 Olig1 null mice.

Figure 2.3: Oligodendrocyte differentiation and myelination initiation in P8 Olig1 null mice. (A, B) Low magnification images of cerebrum of Olig1 heterozygous (A) vs. Olig1 null (B) at P8 stained for PLP (green), MAG (red) and neurofilament protein(blue), indicating impaired myelination initiation both in corpus callosum and striatum. Scale bar: 400μm.(C) Quantification of total number of oligodendrocyte lineage cells (Sox10+), OPCs (PDGFRα+) or mature oligodendrocytes (CC1+) as shown in panel (D) and (E).(D, E) Representative image showing reduced mature oligodendrocytes (CC1+) in P8 Olig1 null brain (E) relative to control (D), corresponding to region of the box insets labeled (D) and (E) in panel (A) and (B) respectively. Scale bar: 50μm.(F) Quantification of the percentage Sox10+ cells that are OPCs (PDGFRα+), mature oligodendrocytes(CC1+) and immature oligodendrocytes (PDGFRa-CC1-) as shown in (D) and (E) in lateral corpus callosum of Olig1 heterozygous vs. Olig1 null mice, demonstrating a blockage of oligodendrocyte differentiation in Olig1 null mice.(G, H) Representative images showing fewer premyelinating oligodendrocytes with bushy morphology (PLP+) in Olig1 null mouse corpus callosum. Scale bar: 50μm; Insets scale bar: 10μm. (I) Quantification of percentage of OPCs (PDGFRα+), premyelinating oligodendrocytes (PLP+) and immature oligodendrocytes (PDGFRα-PLP-) as shown in (G) and (H).(J, K) High magnification image of myelinating oligodendrocytes(PLP+, green) from corpus callosum of P8 Olig1 heterozygous (J) vs. Olig1null (K), showing that initial axon contact (J’ and K’, open arrowhead); wrapping (J’’ and K’’, arrowhead) and myelin elongation (J’’’ and K’’’, arrowhead) were not disrupted. Scale bar 20μm in (J, K); 5μm in ( J’,J’’,J’’’)and (K’,K’’,K’’’). (L, M) High magnification merged images of myelinating oligodendrocytes marked with PLP (green), MBP (red) and neurofilament (blue) showing that the myelin processes appeared relatively normal was not reduced in Olig1 null (M) vs. control (L) mice. Scale bar: 30μm.For all quantifications, mean ± SEM; *p < 0.05, ***p < 0.0005, ****p < 0.0005,******p < 0.0000005; unpaired Student’s t test).

41 the PDGFR

Within the oligodendrocyte population in Olig1 null mice, the percentage of mature oligodendrocytes was reduced approximately 18% in Olig1 null mice compared to control, while the population that remained OPCs doubled (Fig2.4D). This sharp reduction of mature oligodendrocytes had a profound impact on myelination in Olig1 null mice. Immunofluorescent quantification of the major myelin proteins PLP and myelin basic protein (MBP) indicated that both PLP and MBP were decreased by 60% in Olig1 null mice, compared with control (Fig2.4E, G, I), and there appeared to be far more unmyelinated axons in Olig1 null mice, in contrast to the intensively stained myelin in control samples (Fig2.4 F, H).

In order to establish whether there were in fact more unmyelinated axons and to further assess the impact of Olig1 loss, samples were analyzed by electron microscopy, and consistent with the immunofluorescence data, the number of unmyelinated axons in the Olig1 null animal was increased approximately twofold (Fig2. 5A, B, F), and the number of myelinated axons was reduced by 47% in midline corpus callosum of Olig1 null mice relative to control mice (Fig2.5A, B, E).

Furthermore, this deficit remained in 6 and 15 month old Olig1 null mice (data not shown), excluding the possibility that over time there was long term recovery. However, as with the data indicating that the few cells that terminally differentiated in Olig1-null mice had no problem beginning myelination (Fig2.3J,K), the g-ratios for those axons that were myelinated were essentially normal (Fig2.5C,D). Despite the persistent hypomyelination in the Olig1-null mouse, no axonal degeneration at the ultrastructural level was observed (Fig2. 5B) and the total number of axons was not statistically different compared to control (Fig2. 5G). In addition, axonal degeneration was further evaluated by SMI32 antibody staining of the nonphosphorylated form of neurofilament, and no significant accumulation of SMI32 was observed in Olig1 null mice at 2 months of age (Fig2.5H-K).

Figure 2.4: Oligodendrocyte maturation and myelination in adult brain of Olig1 null mice. (A, B) Representative images showed staining of oligodendrocyte lineage cells (Sox10+,red), OPCs (PDGFRα+, blue) and mature oligodendrocytes (CC1+,green) in midline corpus callosum of 2 month Olig1 null (B) vs. heterozygous mice (A) . Scale bar: 50μm.(C, D) Quantification of total number (C) or percentage (D) of mature oligodendrocytes(CC1+), OPCs (PDGFRα+) and immature oligodendrocytes (PDGFRa-CC1-) as shown in (A) and (B), demonstrated a persisted blockage of oligodendrocyte maturation in Olig1 null mice.(E, G) Representative images showed reduced myelin protein: PLP (green) and MBP (red) staining in midline of corpus callosum of 2 month Olig1 null (G) vs. heterozygous mice (E). Scale bar: 50μm.(F, H) High magnification merged images corresponding to box insets in (E) and (G) with PLP (green), MBP (red) and neurofilament (blue) showing many unmyelinated axons in Olig1 null (H) vs. Olig1 heterozygous (F) mice. Scale bar: 20μm.(I) Quantification of corrected total cell fluorescence (CTCF) of PLP and MBP staining in Olig1 heterozygous (E and E’) vs. Olig1 null (G and G’) mice.For all quantifications, mean± SEM; n=8 (+/-) vs. n=6 (-/-); ***p < 0.0005, ****p < 0.00005, ******p < 0.0000005; unpaired Student’s t-test).

Figure 2.5: The corpus callosum is hypomyelinated in Olig1 null mice. (A, B) Electron micrographs of midline corpus callosum in sagittal sections of Olig1 heterozygous (A) vs. null (B) mice at 2 month. The reduction in the number of myelin sheaths is evident. Scale bar: 1μm. (C, D) Quantification of the g-ratio as a scatter plot with axonal diameter on the x-axis and g- ratio on the y-axis(C) or average value (D); no overall difference of g-ratio was noted. (E-G) Myelinated, unmyelinated and total axons were counted in at least 10 randomly selected, non- overlapping fields from midline sagittal sections of the corpus callosum. The number of myelinated axons (E), umyelinated axons (F) and total number of axons (G) per square millimeter of Olig1 heterozygous vs. Olig1 null was calculated corresponding to panel (A) and (B). For all quantifications, mean± SEM; n=5 (+/-) vs. n=6 (-/-);**p < 0.005; unpaired Student’s t test).(H-K) Representative images showed axon bundles in striatum stained with SMI32(green), Neurofilament H (red), MAP2 (blue) 2 month Olig1 null (H and I) vs. heterozygous mice (J and K). Scale bar: 50μm for H and J. (I, K) High magnification images corresponding to (H) and (J). Scale bar: 10μm.

Figure 2.6: Major myelin proteins expression in cerebrum of P15 and 2 month old Olig1 null mice. (A, B) Representative western blot of major myelin proteins (MAG, CNPase, MBP, PLP and MOG) from P15 (A) and 2 month (B) cerebral lysates of Olig1 (+/-, black) vs. (-/-,red) mice. Three samples from each genotype are shown. (C, D) Quantification of the intensity of western blot bands of P15 (C) and 2 month (D) cerebral lysates of Olig1 heterozygous (black column) vs. Olig1null, red column) mice shown in (A) and (B). Individual myelin protein intensity of Olig1 (+/-) was set to 1 based on averaging the actually numeric values, and that of Olig1 null values were graphed as percentage of control. For all quantifications, mean± SEM; n=6 (+/-) vs. n=6 (-/-); *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005; paired Student’s t test).

Consistent with the immunofluorescent studies, total myelin protein was reduced in the Olig1 null mice. Myelin proteins were quantified by western blot of whole cerebrum, and at P15, there was already reduced myelin in Olig1 null mice (Fig2.6A, C), which persisted in 2 month Olig1 null mice (Fig2.6B, D).

Developmental recovery and normal myelination in the spinal cord of adult Olig1 null mice.

The original report of these Olig1 null mice indicated that loss of Olig1 did not result in significant dysmyelination in embryonic spinal cord (Lu et al., 2002). In order to confirm that these mice retained that phenotype, we analyzed oligodendrocyte development and myelination in spinal cord. By P8, the majority of the axons both in the gray and white matter of cervical spinal cord in control mice were myelinated (Fig2.7 A). However, myelination was delayed in the gray matter of

Olig1 null spinal cord, and numerous axons were not yet myelinated in both the dorsal and ventral columns (Fig2.7B). Strikingly, however, PLP and MBP immunofluorescence in Olig1 null spinal cord appeared relatively normal by 2 month (Fig2.7C, E), suggesting recovery from the early deficit in the spinal cord. At this time point, the majority of axons in the dorsal column were myelinated both in

Olig1 null and control mice (Fig2.7 D, F). Electron micrographs indicated that myelination appeared normal in the 2 month of Olig1-null spinal cord (Fig2.7 K,L ), which is consistent with the Arnett et al.

(2004) study of Olig1-null spinal cord (Arnett et al., 2004). Interestingly, despite the apparently normal myelin in Olig1 null spinal cord, there was still a 35% reduction of total Sox10+ oligodendrocytes. This was attributable primarily to a nearly 40% loss of CC1+ mature oligodendrocytes in Olig1 null spinal cord, compared to control littermates (Fig2.7 G, H, I). Thus, in contrast to brain, where less mature oligodendrocytes resulted in reduced myelination, in spinal cord, there was some loss of oligodendrocytes, but the percentage of OPCs and mature oligodendrocytes was only modestly different between Olig1 null and control mice (Fig2. 7J) and myelin appeared normal.

The relatively minor impact on myelination from the loss of Olig1 in spinal cord was quantified by western blot analysis of total spinal cord. The myelin deficit had almost recovered by P15 (Fig2.8A,

C), and had recovered even more so by 2 month (Fig2.8B, D). These data dramatically contrasted with the major difference in myelin proteins in cerebrum (Fig2.6).

Collectively, these data indicate that although the number of mature oligodendrocytes was sharply reduced in both brain and spinal cord of Olig1 null mice, OPC differentiation in spinal cord was relatively normal, and the remaining mature oligodendrocytes generated essentially normal amounts of myelin in the spinal cord. These data are consistent with the initial report of these mice

(Lu et al., 2002).

Expression and subcellular location of Olig1 is highly coordinated with oligodendrocyte morphogenesis and the onset of myelination.

Olig1 and Olig2 are highly conserved at the molecular and functional level, yet they have distinct subcellular localization in mature oligodendrocytes (Arnett et al., 2004). Olig1 is initially found in the nucleus of OPCs and as oligodendrocytes mature, Olig1 translocates to the cytoplasm of myelinating oligodendrocytes. The role for Olig1 in the cytoplasm is unclear, but our studies suggest that it may be a regulated event. We analyzed Olig1 expression in wild-type mice during the transition from premyelinating to myelinating oligodendrocytes. Strikingly, Olig1 was undetectable in premyelinating oligodendrocytes right before they contacted axons and in oligodendrocytes that were initiating axon contact (Fig2. 9A, B, D, E, arrowheads). Olig1 expression then gradually increased in the cytoplasm of myelinating oligodendrocytes as myelinogenesis proceeded (Fig2. 9C,

F, arrowheads). This dynamic Olig1 expression and localization was even more apparent in single cells co-stained for both Sox10 and Olig2 (Fig2.9 G-J). Furthermore, co-labeling with PDGFRα (OPC) and PLP (premyelinating oligodendrocytes) confirmed stage-specific reduction of Olig1 in the premyelinating oligodendrocyte population (Fig2. 9K, L).

Figure 2.7: Adult Olig1 null spinal cord had fewer oligodendrocytes, but apparently normal myelination.

Figure 2.7: Adult Olig1 null spinal cord had fewer oligodendrocytes, but apparently normal myelination. (A, B) Low magnification images of P8 whole cervical spinal cord stained with PLP(green), MBP(red) and neurofilament (blue) showing delayed myelination in Olig1 heterozygous (A) vs. null mice (B). Scale bar: 200μm.(C, E) Representative images of PLP (green) and MBP (red) staining of dorsal column of cervical spinal cord from 2 month Olig1 heterozygous (C ,C ’,C’’) vs. null (E,E’,E’’) mice. Scale bar 50μm.(D, F) High magnification merged images corresponding to labeled box inset in (C’’) and (E’’) showing most axons were myelinated both in Olig1 heterozygous (F) vs. null (D) mice. Scale bar : 10μm.(G, H) Representative images showing staining of mature oligodendrocytes (CC1, green), oligodendrocyte lineage cells (Sox10, red) and OPCs (PDGFRα, blue) in cervical spinal cord of 2 month Olig1 null (H) vs. heterozygous (G). Scale bar: 50μm. Small inset (right) adjacent showing the total Sox10+ oligodendrocytes (G’ and H’) and PDGFRα+ OPCs (G’’ and H’’) correspondingly. (I, J) Quantification of total number (I) or percentage (J) of OPCs (PDGFRα+), mature OLs(CC1+) as shown in (G) and (H) in lateral CC of Olig1 heterozygous vs. null) mice. (K, L) Electron micrographs of coronal section of dorsal funiculus of cervical spinal cord of Olig1 heterozygous (K) vs. null (L) mice at 2 month.. Scale bar: 2μm. For all quantifications, mean± SEM; n=5 (+/-) vs. n=8 (-/-);**p < 0.005, ***p < 0.0005; two sample Student’s t test.

Figure 2.8: Major myelin proteins expression in spinal cord of P15 and 2 month old Olig1 null mice. (A, B) Representative western blot of major myelin proteins (MAG, CNPase, MBP, PLP and MOG) from P15 (A) and 2 month (B) spinal cord lysates of Olig1 (+/-, black) vs. (-/-,red) mice, three blots from each genotype were shown. (C, D) Quantification of the intensity of western blot bands of P15 (C) and 2 month (D) spinal cord lysates of Olig1 (+/-,black column) vs. (-/-, red column) mice shown in (A) and (B). For all quantifications, individual myelin protein intensity of Olig1 (+/-) was set to 1 based on averaging the actually numeric values, and that of Olig1 (-/-) values were graphed as percentage of control.For all quantifications, mean± SEM; n=6 (+/-) vs. n=6 (-/-); *p < 0.05; paired Student’s t test).

Olig2 expression was not altered in Olig1 null brain.

In earlier reports, it had been suggested that the mild impact of Olig1 loss might result from compensatory increases in Olig2 expression (Lu et al., 2002). We therefore analyzed Olig2 expression in P1, P8, P15 and 1 month Olig1 null and control mice (Fig2.10 A-D). No obvious dysregulation of Olig2 was seen in Olig1 null oligodendrocytes in subcortical white matter by immunohistochemical analysis (Fig2.10 A-D). To confirm this, Olig2 in cerebrum and spinal cord was quantified by western blot at P15 and 2 month. Olig2 protein expression was reduced by 60% in the

50 cerebrum of both P15 and 2 month Olig1 null mice compared with control littermates (Fig2.10E), which was consistent with the approximately 60% reduction in total oligodendrocytes. Thus, on a per cell basis, these data suggest little change in Olig2 expression, and in particular no compensatory increase of Olig2 in cerebrum. In contrast, although we observed a 40% reduction of Olig2 in spinal cord of P15 Olig1 null mice by western blot (Fig2.10F), Olig2 expression in Olig1 null spinal cord was comparable to control spinal cord by 2 months (Fig2.10F). Since the total oligodendrocyte number was decreased around 35% in Olig1 null spinal cord, this finding suggests that by 2 months, the Olig2 level may actually be up-regulated in individual oligodendrocytes in the Olig1 null spinal cord.

Collectively, these results suggest that there was no compensatory change in Olig2 expression in cerebrum, where there was severe dysmyelination, but intriguingly there was potentially compensatory up-regulation of Olig2 in spinal cord, where less dysmyelination was seen.

Olig1 is important for initial oligodendrocyte differentiation in vitro

In order to expand on the in vivo studies of Olig1 null OPCs, we examined the impact of Olig1 loss on proliferation and differentiation of purified OPCs in vitro. OPCs were generated from neurospheres derived from embryonic 12.5 to E14.5 day cerebrum of Olig1 null or control mice.

OPCs were plated and maintained in proliferation medium containing PDGF and FGF for 3 days. In contrast to the in vivo data (Fig2.1), Olig1 null OPCs in vitro had similar proliferation rates to control

(Fig2.11D, D’, E), which suggested that Olig1 did not directly regulate the proliferation of OPCs. In contrast with the increased cell death in Olig1-null mice in vivo analysis, the number of apoptotic cells did not increase as OPCs differentiated in the absence of Olig1 in vitro (Fig2. 11G).

After switching to differentiation media containing T3 for 3 days in vitro, 22.5% of control OPCs differentiated to O4+ immature oligodendrocytes, but only 11.4% of Olig1 null OPCs differentiated

(Fig2.11F,F’, H). Thus, in culture, these cells had normal proliferation rates, but reduced ability to differentiate, relative to control cells.Olig2 was not up-regulated in the cultured

Figure 2.9: Expression and subcellular location dynamics of Olig1 upon myelination onset.

Figure 2.9: Expression and subcellular location dynamics of Olig1 upon myelination onset. Representative images of premyelinating and myelinating oligodendrocytes were labeled with PLP (green), Olig1 (red) either co-stained for neurofilament (A to C, blue) or Olig2 (D to E, blue) in cortex of P8 wild type mice. Box insets showed Olig1 protein disappeared in premyelinating OLs ([A] and [D], arrowhead) and re-appeared in cytoplasm of active myelinating OLs ([C] and [F], arrowhead), whereas it is localized in the nucleus in surrounding OPCs (bar-arrow). Scale bar: 20μm (A-E); 10μm (F). (G to J) Representative images of a single nucleus showed dynamic Olig1 expression and localization in maturing oligodendrocytes within the corpus callosum of P8 wild type mice. Scale bar: 1.5μm.(K, L) Representative images of Olig1 (green) localization in OPCs (PDGFRα+, blue, K) and premyelinating oligodendrocytes (PLP+, blue, L) corresponding to cells labeled in (G, H). Arrow:OPCs; arrowhead:premyelinating oligodendrocytes. Scale bar: 10μm

Figure 2.10: Olig2 expression in the CNS of developing Olig1 null mice.

Figure 2.10: Olig2 expression in the CNS of developing Olig1 null mice. (A to D) Olig2 expression in developing corpus callosum of Olig1 heterozygous (A, B, C,D) vs. null (A’, B’, C’,D’) mice at P1 (A), P8 (B), P15 (C) and 1 month (D). The absence of Olig1 expression is confirmed in Olig1 null mice at varies stages. Scale bar: 50μm.(E, F) Representative western blot and quantification of Olig1 and Olig2 proteins from cerebrum (E) and spinal cord (F) whole tissue lysates of P15 and 2 month Olig1 (+/-, black) vs. (-/-,red) mice, three blots from each genotype were shown. Olig2 protein intensity was set to 1 for control samples, based on averaging the actual numeric values, and Olig2 protein in Olig1 null mice was graphed as a percentage of control. An estimation of Olig2 level per cell was also quantified in 2 month spinal cord by dividing Olig2 protein values by the number of total oligodendrocytes in control and Olig1-null samples. For all quantifications, mean ± SEM; n=6 (+/-) vs. n=6 (-/-); **p < 0.005, ***p < 0.0005, ****p < 0.00005; paired Student’s t test).

55 oligodendrocytes derived from Olig1 null cerebra (Fig2.11 B, C), which was consistent with the lack of upregulation of Olig2 in Olig1 null cells in cerebrum in vivo (Fig2.10). As in the in vivo context, in the differentiating oligodendrocytes in vitro, Olig1 loss compromised oligodendrocyte morphological differentiation. Analysis of process complexity showed a 40% reduction of total branch points of

Olig1 null O4+ cells compared to control (Fig2.11I).

In order to confirm that the deficit of OPC differentiation in vitro resulted only from loss of

Olig1 and not from unknown compensation during early commitment of the oligodendrocyte lineage in Olig1 null mice, we analyzed rat oligodendrocytes after transient siRNA knockdown of

Olig1. These cells are well past the pre-OPC to OPC transition. Acute knockdown of Olig1 in primary rat OPCs derived from postnatal mixed glia was validated by reduction of Olig1 protein after knockdown (Fig2.11 J, K, L). Consistent with the finding from Olig1 null mouse OPCs, knockdown of

Olig1 in rat OPCs did not impair their proliferation but had a similar impact on OPC initial differentiation and morphogenesis (Fig2.11 M-Q). In summary, consistent with our in vivo findings,

Olig1 impacted initial differentiation of cultured rat and mouse OPCs and their subsequent morphological development.

DISCUSSION

Developmental phenotypes of different Olig1 null mouse lines.

In this study, we observed the developmental delay and long term recovery in the spinal cord first reported for this Olig1 null mouse (Lu et al., 2002), but as discussed below, we noted dramatic oligodendrocyte deficits in the brain. The initial report analyzed mice that were still on a mixed genetic background, and in the ensuing 12 years, they have been backcrossed to C57Bl/6J, perhaps making the phenotype more robust.

The mild phenotype of the original Olig1 null mice had been speculated to result from compensatory up-regulation of Olig2 by the cis-acting regulatory effect of the Pgk-Neo cassette

Figure 2.11: Oligodendrocyte differentiation in the absence of Olig1 in vitro.

Figure 2.11: Oligodendrocyte differentiation in the absence of Olig1 in vitro. (A) Representative images showing absence of Olig1 in the OPCs derived from neurospheres generated from cortical progenitors of Olig1 heterozygous (A’) vs. Olig1 null (A). Scale bar: 20μm.(B) Representative western blot of Olig1 and Olig2 protein from cultured OPCs derived as shown in (A), three blots from each genotype were shown. (C) Quantification of the intensity of western blot bands of Olig2 protein from lysates of Olig1 (+/-,black column) vs. (-/-, red column) mice derived OPCs shown in (B). (D) Representative images of the cultured Ki67+(red) OPCs derived from Olig1 heterozygous (D) vs. Olig1null (D’) mice. Scale bar indicates 20μm. (E) Quantification of the proportion of Ki67+ cells within the Olig2+ OPCs population cultured in proliferation medium for 3 days in vitro.(F) Representative images of O4+ (green) immature oligodendrocytes derived from Olig1 heterozygous (F) vs. Olig1 null (F’) mice after differentiation 3 days in vitro. Scale bar: 70μm.(G- I) Quantification of total TUNEL+ cells (G), the percentage of O4+ immature oligodendrocytes(H) and the total branch points/cell (I) as shown in heterozygous (F) and null (F’).(J) Immunoblot of Olig1 and Olig2 expression in OPCs electroporated with control or Olig1 smart pool siRNAs and cultured as OPCs for 2d or shifted to differentiation conditions for 1d after 1d as OPCs.(K, L) Quantitative analysis of Olig1 (J) or Olig2 (K) protein expression relative to beta-tubulin shown in (I).(M, O) Impact of Olig1 knockdown on OPC proliferation (M) and differentiation (O). Rat OPCs were electroporated with control (M,O) or Olig1 siRNAs (M’,O’) and cultured in proliferation media for 2 days and stained with Ki67 (red) and Olig2 (green) in (M, M’) or in differentiation media for 1d and stained with O4(green) and Olig2(red) in (O,O’). Scale bar: 20μm.(N) Quantitative analysis of the percentage of Ki67+ cells within Olig2+ OPCs. (P, Q). Quantitative analysis of the percentage of O4-positive cells (P) and the total branch points per cell (Q). Data in all graphs represent the mean ± SEM from three independent experiments. *p < 0.05, **p < 0.005; paired Student’s t test.

58 retained in the Olig1 locus (Lu et al., 2002), and when that cassette was removed from the original line, the new Olig1 null mice were very severely affected, dying around third postnatal week with severe dysmyelination (Xin et al., 2005). Unfortunately Olig2 expression was not directly examined in the earlier reports on these lines, so it was unclear whether that explained the difference in phenotype. One recent study attempted to resolve the discrepancy of these two early Olig1 null mouse lines by generating two new Olig1 null mouse lines (Paes de Faria et al., 2014). As in the initial study, much analysis was done in the spinal cord of these two new lines, and developmental delay and subsequent recovery of oligodendrocyte differentiation was observed in spinal cord for both new lines. In neither line was compensation by Olig2 noted (Paes de Faria et al., 2014). These differences among studies have led to significant speculation on the role of Olig1. Paes de Faria et al.

(2014) discuss potential reasons for the dramatic differences of the Xin et al. (2005) study relative to the original Lu et al. (2002) and

Paes de Faria et al. (2014), but clearly in several Olig1 null lines, spinal cord myelination is delayed but not permanently changed.

Our studies in spinal cord are consistent with these earlier studies on spinal cord development in the absence of Olig1. Nevertheless, despite apparently normal myelination in the spinal cord of

Olig1 null mice, our studies do demonstrate that oligodendrocyte production and differentiation were also impaired in spinal cord. However, despite this loss of oligodendrocytes, there was no reduction in Olig2 expression (Fig2.10), which suggested upregulation of Olig2 on a per cell basis in spinal cord. The compensatory upregulation of Olig2 may explain some aspects of the relatively normal myelination in the spinal cord of Olig1 null mice.

Temporo-spatial control of OPC differentiation by Olig1 in brain.

Oligodendrocyte lineage progression and myelin initiation are tightly controlled both temporally and spatially in the developing mouse brain (Trapp et al., 1997; Baumann and Pham-Dinh,

2001). Both extrinsic and intrinsic cues play critical roles in oligodendrocyte differentiation and myelination (Emery, 2010b; Wood et al., 2013). Olig1 and Olig2 play critical roles in oligodendrocyte specification (Lu et al., 2002; Zhou and Anderson, 2002; Silbereis et al., 2014) and early OPC differentiation (Mei et al., 2013). (Silbereis et al., 2014) demonstrated that early commitment to the oligodendrocyte lineage relative to interneuron lineage is regulated by Olig1. Furthermore, within the oligodendrocyte lineage, the current studies show that Olig1 is critical for OPC cell cycle exit and subsequent differentiation of OPCs into premyelinating oligodendrocytes in cerebrum, but not spinal cord.

The pre-OPC to OPC transition has largely been studied in vitro in studies generating oligodendrocytes from human embryonic stem cells or iPS cells (Hu et al., 2009a; Wang et al., 2013).

Pre-OPCs express Sox2 and Sox9 transcription factors, and the transition to OPCs involves Sox10, which is required for maintaining the differentiated state of neural progenitors by repressing the expression of Sox2 and Sox9 (Castelo-Branco et al., 2014). Based on the known interaction of Olig1 and Sox10 (Li et al., 2007a), Olig1 may promote the pre-OPC to OPC commitment by interacting with

Sox10 to repress Sox2 expression in newly formed OPCs, since the Sox2 level remains high in OPCs from Olig1 null mice, in contrast to the sharp down-regulation of Sox2 in OPCs from control mice.

Considering the large set of common target genes downstream Olig1 and Olig2, including most major myelin proteins (Meijer et al., 2012; Weng et al., 2012) and key regulators of differentiation, such as GPR17 (Chen et al., 2009) and Sip1 (Weng et al., 2012), Olig1 and Olig2 may coordinate to regulate early OPC differentiation. The similar phenotype observed in these Olig1 null mice and the

Olig2 conditional knockout mice, in which Olig2 is selectively deleted in OPCs (Mei et al., 2013), implies the potential interplay of these two related transcription factors, which may function simultaneously in OPC differentiation in the developing mouse brain. However, in cerebrum, it appears that neither protein can fully compensate for the loss of the other protein. While Olig2 has

60 been shown to promote pre-OPC to OPC commitment in vitro (Hu et al., 2009b), this process was impaired in Olig1 null mice, even in the presence of Olig2.

Region-specific regulation of oligodendrocyte differentiation and myelination by Olig1 in the CNS

Both regional oligodendrocyte heterogeneity and diverse local environmental cues have been suggested to result in different myelination patterns in different CNS regions (Almeida et al., 2011;

Vigano et al., 2013). Indeed, regional differences in dysmyelination have been seen in several knockout mice lines, including conditional Fyn knockout mice (Sperber et al., 2001) knockout mice (Fruttiger et al., 1999), and mTOR, Rictor or Raptor knockout mice (Bercury et al.,

2014; Wahl et al., 2014). The current studies are consistent with these reports. Thus, in contrast to the mild phenotype in the Olig1 null spinal cord, we observed a severe phenotype without long term recovery in the brain of Olig1 null mice. Despite the increased proliferation of OPCs in Olig1 null mice, there was a permanent deficit of oligodendrocyte number, maturation and myelination in

Olig1 null cerebrum, which lasted through adulthood. It is likely that distinct local environmental cues result in the dramatic myelin deficit in the brain of Olig1 null mice but not in the spinal cord.

Whether this difference results simply from the lack of compensatory up-regulation of Olig2 in the brain or from other aspects of Olig1 function that lead to the severe reduction of myelinating oligodendrocytes is unclear at this point.

Importance of the subcellular localization of Olig1 in oligodendrocytes

In contrast to the constant expression and nuclear localization of Olig2 throughout oligodendrocyte development, dynamic expression and subcellular translocation of Olig1 was found to be highly coordinated with oligodendrocyte lineage progression and myelination initiation.

Intriguingly, in contrast to the positive role of nuclear Olig1 in early OPC differentiation, Olig1 was downregulated in premyelinating oligodendrocytes before they started ensheathing axons. Olig1 then reappeared in the cytoplasm of myelinating oligodendrocytes as the myelin segments

61 elongated. The downregulation of Olig1 in premyelinating oligodendrocytes prior to initiating axon contact suggests that sustained nuclear Olig1 might play an inhibitory role in this process. However, the link between Olig1 subcellular location and function is not clear. As a transcription factor, nuclear Olig1 location in the OPCs fits its role as a positive regulator of early OPC differentiation.

However, while fewer in number, Olig1 null oligodendrocytes can generate apparently normal myelin (Fig2.3, 4, 6). If the cytoplasmic Olig1 does have de novo function in myelinogenesis per se, one might predict that myelinating oligodendrocytes in Olig1 null mice either produce less myelin segments or bear thinner myelin. In contrast to this prediction, in the neocortex of P8 mice when the growing myelin segments can be distinguished, there was no apparent difference between the number of segments generated by individual myelinating oligodendrocytes of Olig1 null and heterozygous mice (Fig2.3 L, M). In addition, the myelin thickness assessed by g-ratio was normal in the Olig1 null mice (Fig2.6C, D).

Unique function of Olig1 in myelination and remyelination

Olig1 is downregulated in premyelinating cells, suggesting that it might have an inhibitory role in the myelination initiation process, in contrast to its positive role in early OPC differentiation. Yet it returns as a cytoplasmic protein in myelinating cells. There may be an active function for Olig1 in the cytoplasm, but an interesting alternative explanation could be to prevent Olig1 entry into the nucleus during myelinogenesis, yet maintain it as a store potentially available for remyelination.

Although Olig1 loss apparently can be partially compensated for during developmental myelination, its function is essential for remyelination (Arnett et al., 2004; Whitman et al., 2012).

The differentiation block in demyelinated lesions of adult Olig1 null mice resembles MS lesions where OPCs are present but many either fail to differentiate (Chang et al., 2000; Kuhlmann et al.,

2008) or are blocked as premyelinating oligodendrocytes unable to differentiate further (Chang et al., 2002). Considering the role of Olig1 in both early OPC differentiation and myelination onset, it is

62 plausible that dysregulation of Olig1 expression and localization may result in the differentiation block of OPCs and premyelinating oligodendrocytes in chronic inactive MS lesions. The current study demonstrates the critical role of Olig1 in oligodendrocyte differentiation in the brain, in parallel with the role of Olig2 in this process. In addition, phosphorylation has been shown to modify the function of both Olig1 and Olig2 in oligodendrocyte maturation and specification respectively (Li et al., 2011;

Niu et al., 2012). Ongoing studies in this laboratory suggest that posttranslational modification may also modulate Olig1 stability and localization. With further mechanistic insight into the regulation of

Olig1 function during development, it may be possible to modulate Olig1 expression and activity to promote oligodendrocyte differentiation and subsequent myelination in MS patients.

MATERIALS AND METHODS

Animals

The B6;129S4-Olig1tm1(cre)Rth/J strain of Olig1 null mice was obtained from the Jackson

Laboratory (stock#011105). The original mixed background mouse strain has been backcrossed to

C57BL/6J mice over numerous generations during the last 12 years. The total number of oligodendrocyte lineage cells and mature cells was quantified at both P15 and 1 month of age and no differences were observed between Olig1+/- and wildtype mice. Therefore, we used male and female Olig1 +/- and Olig1-/- littermates for further analysis. Genotypes of all mice were determined by PCR analysis of tail genomic DNA based on the suggested Jackson Laboratory protocol with modified PCR primer. PCR primers specific for wild-type alleles were: Olig1-WT-F, 5'-

AAACGCTGCGCCCCACCAAG-3’, and Olig1-WT-R, 5'-TCACTTGGAGAACTGGGCCT-3’; for mutant alleles, they were: Olig1-Mut-F ， 5’-CGCCCCAGATGTACTATGC-3’, and Olig1-Mut-R,5’-

AATCGCGAACATCTTCAGGT-3’. All animal procedures were performed in an AAALAC-accredited facility in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the University of Colorado Denver Institutional Animal Care and Use Committee.

Immunohistochemistry, immunocytochemistry and TUNEL assay

Mouse perfusion and immunohistochemistry was performed as described previously (Trapp et al.,

1997) , with some modifications. Free-floating cortex and cervical spinal cord sections (30μm) were analyzed, with antigen retrieval in 10mM sodium citrate (pH6.0) at 65°C for 10 min as needed, using a Pelco Biowave Pro tissue processor (Ted Pella, Redding, CA). For immunocytochemistry, oligodendrocytes were cultured on coverslips (see below) and fixed with 4% paraformaldehyde for

15 min at RT. Cells were permeabilized with 0.1% Triton X-100 for 10 min, blocked with 3% BSA in

PBS for 60 min at RT, and incubated with primary antibodies overnight at 4°C. For detection of O4 cell surface antigens, O4 antibody was diluted with media and incubated with live cells on coverslips for 1 h prior to fixation.

Cell death was analyzed by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay. Sections were permeabilized with 3% Triton-X 100 for 30 min, and labeled with in situ cell death detection kit following the manufacturer's instructions (Roche applied science#11684795910, Indianapolis, IN).

The following primary antibodies were used: guinea pig anti-NG2 (gift of Dr. W. Stallcup,

Burnham Institute, La Jolla, CA), rabbit anti-Olig2 and Olig1 (a gift of Dr. Charles Stiles, Harvard

University, Cambridge, MA), rat anti-PLP/DM20 (Clone AA3), O4 hybridoma (gift of Dr. Rashmi

Bansal, University of Connecticut Health Sciences Center, Farmington, CT), rat anti-BrdU (Accurate chemical# YSRTMCA2060GA, Westbury, NY),rabbit anti-Sox2 (Millipore#ab5603,Gibbstown,

NJ),goat-anti-Sox2 (Santa Cruz biotech#sc-17320, Santa Cruz, CA), mouse anti-Olig2

(Millipore#MABN50A4, Gibbstown, NJ), rabbit anti-Ki67 (Abcam#16667, Cambridge, MA), mouse anti-CC1 (Millipore#OP80, Gibbstown, NJ),goat anti-Sox10 (Santa Cruz biotech#sc-17342, Santa Cruz ,

CA), rabbit anti-PDGFRα (Santa Cruz biotech#sc-338, Santa Cruz, CA), chicken anti-

64 neurofilament(Neuromics#CH22105, Minneapolis, MN), rabbit anti-MBP (Millipore#ab980,

Gibbstown, NJ).

Primary cell culture and electroporation

Mouse neural progenitor cells were isolated from Olig1 heterozygous or null neocortex E12.5–

E14.5 embryos to generate neurospheres and OPCs as previously described (Pedraza et al., 2008).

Rat mixed glial cultures were generated from P0-3 day old Sprague Dawley rat pups as described previously (Dai et al., 2014). Oligodendrocyte cultures were typically greater than 90% pure as assessed by immunocytochemistry for the oligodendrocyte lineage markers PDGFRα/NG2 and Olig2 and the astrocytic marker glial fibrillary acid protein (GFAP). Rat OPCs were shaken from mixed cultures after 10 days and 5x106 cells were electroporated (Amaxa nucleofection apparatus, Lonza,

Mapleton, IL) in 100μl Nucleofection solution (Amaxa basic glial cells nucleofector kit #VPI-1006, IL) with siRNAs (10 μl of 20 μM rat Olig1 siRNAs (#L100044-01) or siControl non-targeting siRNA pool

(#B002000-UB) from Dharmacon (Thermo Scientific, Marietta, OH). After electroporation, cells were resuspended and seeded onto poly-D-lysine/Laminin coated dishes or round 12mm coverslips in

DMEM supplemented with N2 (Life technologies #17502-048,Grand Island, NY) , Fibroblast growth factor(FGF, 10ng/ml) and Platelet-derived growth factor(PDGF, 10ng/ml) for 24h, after which they were incubated in differentiation media for 1d.

Cell counts and immunofluorescence quantification

For oligodendrocyte lineage cell number quantification of Olig1 mutant mice, images at 40X magnification were obtained either at the midline of the corpus callosum or in the dorsal column of the cervical spinal cord on a Leica SP5 confocal microscope. Cells within the field of each image for the corpus callosum or spinal cord were counted with the ImageJ cell counting plugin. Three sections per animal were quantified from at least 5 animals per group. In order to quantify the fluorescence intensity of proteolipid protein(PLP) and myelin basic protein (MBP) immunoreactivity

65 in corpus callosum, the corrected total cell fluorescence (CTCF) was calculated as the Integrated

Density - (Area of selected cell X mean fluorescence of background readings)(Burgess et al., 2010).

For cultured primary oligodendrocytes, images were taken on a Zeiss Axio Imager M2. A total of 6 microscopic fields per coverslip with two coverslips per condition were sampled in each experiment.

The total number of cells counted per condition averaged 700-1500. The total branch points/cell of

O4+ cells was analyzed with Imaris filament tracer module (Bitplane, Zurich, Switzerland).

Western blot

Cultured cells, brain or spinal cord tissue were lysed in RIPA buffer (25mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% DOC) supplemented with complete mini-protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor cocktail set II

(Calbiochem # 564652, Cambridge, MA). Samples were analyzed by a standard western blot protocol.

Protein bands were detected using the LICOR Odyssey infrared scanner (LI-COR Bioscience, Lincoln,

NE) and proteins were quantified using the Odyssey Scanner Software 2.0. The following primary antibodies were used: rabbit anti-myelin associated glycoprotein (MAG) (Cell signaling tech#8043,

Danvers, MA), rabbit anti-2’,3’-Cyclic-nucleotide 3’-phosphodiesterase (CNPase) (Cell signaling tech#5664, Danvers, MA), rabbit anti-myelin oligodendrocyte glycoprotein (MOG) (abcam# 32760,

Cambridge, MA), mouse anti-myelin basic protein (MBP) (Covance#SMI-94,San Diego, CA), mouse anti-β-tubulin (Sigma#T8328, St Louis, MO), rabbit anti-glyceraldhyde-3-phosphate dehydrogenase

(GAPDH)(Cell signaling tech#2118, Danvers, MA). Samples were loaded at 50μg protein/lane in order to detect myelin proteins in P15 cerebral samples and at 20μg/lane for others. In order to detect

Olig1 and Olig2, 100μg protein/lane was loaded.

Electron Microscopy

Animals were perfused with cold PBS followed by modified Karnovsky’s fixative (2% paraformaldehyde/2.5% glutaraldehyde). The brain and spinal cord were removed and post-fixed

66 overnight in the same fixative. Corpus callosum was isolated from 1 mm coronal slices of brain between -.94 and -2.18 of Bregma. All tissue was post-fixed in 1% osmium tetroxide, dehydrated in graded acetone, and resin embedded in Embed 812 (Electron Microscopy Sciences, Hatfield, PA) using a Pelco Biowave Pro tissue processor (Ted Pella, Inc., Redding, CA). The corpus callosum samples were oriented such that sections could be cut midline in a sagittal plane. Ultra-thin sections

(80 nm) were mounted on copper grids, stained with uranyl acetate and lead citrate and viewed at

80kV on a Technai G2 transmission electron microscope (FEI, Hillsboro, OR). Electron micrographs of the corpus callosum were imaged at midline.

Statistical analysis

For cell counts, the mean number of immunoreactive cells per image field was determined.

Student’s t-test, paired or unpaired, was used to calculate statistical significance and graphed using

OriginPro (Origin lab corporation, Northampton, MA) or Graphpad Prism (Graphpad software Inc, La

Jolla, CA). Values represent the means ± SEM.

CHAPTER III

OLIG1 ACETYLATION AND NUCLEAR EXPORT MEDIATE OLIGODENDROCYTE DEVELOPMENT

ABSTRACT

The oligodendrocyte transcription factor, Olig1, is critical for both oligodendrocyte development and remyelination. Nuclear to cytoplasmic translocation of Olig1 protein occurs during mouse and human brain development and in multiple sclerosis, but the detailed molecular mechanism of this translocation remains elusive. Here, we report that Olig1 acetylation and deacetylation drive its active translocation between the nucleus and the cytoplasm. We identified three functional nuclear export sequences (NES) localized in the bHLH domain and one specific acetylation site at Lys 150 (human Olig1) in NES1. Olig1 acetylation and deacetylation are regulated by the acetyltransferase, CBP, and the deacetylases, HDAC1, 3 and 10. Acetylation of Olig1 decreased its chromatin association, increased its interaction with Id2 (inhibitor of DNA binding 2) and facilitated its retention in the cytoplasm of mature oligodendrocytes. These studies establish that acetylation of Olig1 regulates its chromatin dissociation and subsequent translocation to the cytoplasm, and is required for oligodendrocyte maturation.

INTRODUCTION

Myelin ensheathment of axons provides valuable trophic support to axons and it is physiologically critical to dramatically increase electrical conduction (Bullock et al., 1984). Human demyelinating diseases in the central nervous system (CNS), such as multiple sclerosis (MS), result in oligodendrocyte cell death, axonal degeneration and cognitive and motor deficits (Franklin and

Ffrench-Constant, 2008; Trapp and Nave, 2008). Oligodendrocyte progenitor cells (OPCs) are present in MS lesions, suggesting that demyelinated lesions should be able to remyelinate (Chang et al.,

2000). Nevertheless, only minimal remyelination occurs, and it appears that OPC maturation is particularly vulnerable in MS (Wolswijk, 1998, 2000; Chang et al., 2002; Kuhlmann et al., 2008). The

68 molecular mechanisms of oligodendrocyte differentiation and myelination during development and after demyelination are being actively investigated, and the current studies address one element of this program.

Two closely related basic helix-loop-helix (bHLH) transcription factors, Olig1 and Olig2, function in the well-established transcriptional regulatory network of oligodendrocyte lineage progression

(Emery, 2010a, b), and are critical for oligodendrocyte specification and differentiation (Lu et al.,

2002; Zhou and Anderson, 2002; Mei et al., 2013). However, the function of Olig1 during oligodendrocyte development has been unclear, with differing evidence based on the generation of different Olig1 null lines (Lu et al., 2002; Xin et al., 2005; Paes de Faria et al., 2014). Our recent analysis of brain development in Olig1 null mice reveals an essential role for Olig1 in OPC differentiation in brain, but less so in spinal cord (Dai et al., in press, 2015).The current studies focus on elements of the regulation of Olig1 function in oligodendrocyte development in brain.

Subcellular translocation of Olig1 during both oligodendrocyte development and remyelination is unique to Olig1, relative to Olig2, which is only found in the nucleus. Olig1 is present in OPC nuclei, and it progressively translocates to the cytoplasm as oligodendrocytes mature in the developing mouse and human brain (Arnett et al., 2004; Jakovcevski and Zecevic, 2005; Kitada and Rowitch,

2006; Niu et al., 2012). Interestingly, during remyelination, Olig1 relocates back to the nucleus, suggesting a nuclear function for Olig1 that appears essential for remyelination (Arnett et al., 2004).

The mechanism regulating this intriguing Olig1 translocation has not been established. Post- translational modification of proteins, particularly phosphorylation and acetylation, regulates the subcellular location of several transcription factors (Kramer et al., 2009; Meek and Anderson, 2009), and a recent in vitro study indicates that Olig1 phosphorylation at ser149 (ser138, mouse Olig1) can regulate its cytoplasmic location and cell membrane extension (Niu et al., 2012).

In the current study, we focused on the role of acetylation in regulating Olig1 localization. We established that Olig1 is acetylated by CREB-binding protein (CBP) on lysine150 (Lys139, mouse Olig1) as oligodendrocytes mature, both in vitro and in vivo. Acetylation of Olig1 increased its binding with inhibitor of DNA binding 2 (Id2), decreased its association with chromatin, and facilitated its nuclear to cytoplasmic relocation during oligodendrocyte development. Our data show that Olig1 acetylation is important for oligodendrocyte lineage progression and morphological differentiation, and reveal the mechanism by which its acetylation regulates its nuclear to cytoplasmic translocation.

RESULTS

Three Nuclear Export Sequences (NES) of Olig1 regulate its export to the cytoplasm.

Olig1 and Olig2 are highly conserved at the molecular and functional level, yet they have distinct subcellular localization during oligodendrocyte development (Arnett et al., 2004; Kitada and Rowitch,

2006; Niu et al., 2012). Consistent with previous in vivo findings, Olig1 localized only to the nucleus of OPCs that expressed PDGFRα at postnatal day 1 (P1) (Fig2. 1A). At P10, Olig1 was present in both the nucleus and the cytoplasm in maturing oligodendrocytes that expressed CC1 (Fig2. 1B), and it was completely cytoplasmic in myelinating oligodendrocytes (Fig2.1C), suggesting this was a period of Olig1 relocalization to the cytoplasm. Thus, we predicted that Olig1 must have nuclear export sequences (NES) not found in Olig2, and analyzed the Olig1 protein sequence for potential NES.

Three consensus NES were identified either within or adjacent to the bHLH domain of Olig1 (Fig2.

1D). The sequence of NES1 was fully conserved with Olig2, but there were two additional NES sequences in Olig1 (NES2 and NES3) (Fig2. 1D, E). The three Olig1 NES were conserved across different species from mouse to human but not zebrafish (Fig2.1E).

To test the role of the conserved Olig1 NES in regulating nuclear export, GFP- tagged human

Olig1 was transfected into multiple cell lines. Strikingly, Olig1-GFP was localized in the nucleus less than the cytoplasm (N

L). To test active export of Olig1-GFP after overexpression in HEK293T cells, cells were treated with the nuclear exportin inhibitor, leptomycin. By contrast to untreated transfected cells (Fig2. 1F), at

24h post-treatment, the majority of GFP localization was nuclear for cells transfected with Olig1-GFP in 67.24% of transfected cells (Nuclear>Cytoplasm [N>C], Fig3.1G and L). In order to investigate the role of the three Olig1 NES, they were deleted individually or collectively. After NES1 or NES3 deletion, Olig1 was more evenly distributed between the nucleus and the cytoplasm (N=C in 86.4% or 60.5% transfected cells, Fig3.1H, J, L), although NES3 deletion did result in slightly more nuclear

Olig1 (N>C in 39.5% transfected cells, Fig3.1J and L). Deletion of Olig1 NES2 severely blocked Olig1 export to the cytoplasm, with 92.5% of transfected cells having Olig1 predominantly localized in the nucleus (Fig3.1I and L). Consistent with this, when all three Olig1 NES were collectively deleted,

Olig1 was retained in the nucleus (96.5% of cells, N>C, Fig3. K and L). These data suggest that the three predicted NES regulate Olig1 export together and eliminating any one alters Olig1 export out of the nucleus. When wild type (WT) Olig1 or the different Olig1 NES deletion mutants were co- expressed in HEK293T cells with a major nuclear exportin protein, CRM1, all mutants displayed decreased interaction with CRM1, relative to WT Olig1, and deletion of just NES2 or all three NES essentially eliminated that interaction (Fig3. 1M). From these results, we concluded that all three

NES work together and are required for complete nuclear export of Olig1.

Acetylation of Olig1 is correlated with its cytoplasmic localization.

Olig1 translocation to the cytoplasm in non-neural HEK293T cells suggested that Olig1 translocation may be intrinsic to the protein sequence. Phosphorylation and acetylation are well known post-translational modifications that can regulate the nuclear-cytoplasmic shuttle of proteins

(di Bari et al., 2006; Kramer et al., 2009). In order to test the role of phosphorylation or acetylation in regulating Olig1 nuclear to cytoplasmic translocation, we applied a variety of kinase inhibitors

Figure 3.1: Three Nuclear Export Sequences (NES) of Olig1 regulate its export.

Figure 3.1: Three Nuclear Export Sequences (NES) of Olig1 regulate its export. (A- (mature oligodendrocytes, blue, A”, B”, C”) in corpus callosum of P1 (A), P10 (B) or P30 (C) mice. -positive OPCs at P1 (A); switches from the nucleus to cytoplasm of CC1-positive oligodendrocytes at P10 (B) and is exclusively localized to the cytoplasm in CC1- positive cells at P30 (C). Scale bar: 20μm.(D) Structural features of human Olig1 protein, showing the conserved bHLH domain. Three predicted nuclear export sequences (NES), as determined by analysis with NetNES1.1 are highlighted in red.(E) Alignment of the bHLH domain of human Olig1 and Olig2 (upper panel). Note high conservation of NES1, with less for NES2 and NES3. Sequence of this Olig1 region among different species with the three NES domains boxed in blue (lower panel). Note unique Danio rerio sequence in blue highlight that disrupts NES3 in that species. (F-K) Subcellular location of GFP-tagged Olig1 or its mutants deleted for indicated NES sequences (dNES) 24h after transfection into HEK293T cells. (F) Olig1-GFP; (G) Olig1-GFP plus Leptomycin (20nM); (H) Olig1- dNES1-GFP; (I) Olig1-dNES2-GFP; (J) Olig1-dNES3-GFP; (K) Olig1-dNES1/2/3-GFP. Scale bar: 50μm. Inset scale: 50μm.(L) Quantitative analysis of the subcellular distribution of Olig1-GFP or mutants imaged in F to K. Data were quantified as nuclear localization less than cytoplasm (NC, blue). Data represent the mean ± SEM from at least three independent experiments, and in each experiment at least 150 cells were counted. (M) Interaction of Olig1 or the NES-deleted mutants with exportin1 (CRM1) in HEK293T cells. Flag-CRM1 and Olig1-GFP or its NES deletion mutants were expressed in HEK293T cells. Olig1 and its mutants were immunoprecipitated with anti-Olig1 antibody, and flag-CRM1 interactions with Olig1 or its mutants were detected by western blot with an anti-Flag antibody (red box).

(protein kinase A, protein kinase C or Akt) and histone acetyltransferase (HAT) inhibitors (C646, MB-

3) to HEK293T cells that were transfected with Olig1-GFP. Only C646, which inhibits CBP and/or

P300, blocked the nuclear to cytoplasmic translocation of Olig1-GFP (Fig3. 2A-F). This suggested that

Olig1 acetylation could regulate its nuclear to cytoplasmic translocation.

Olig1-GFP shifted localization over time in transfected cells. It was localized to the cytoplasm 24h post transfection and was in the nucleus at 48h (Fig3. 2K). We investigated whether acetylation might regulate this by immunoprecipitating acetylated proteins from transfected cells with a pan- acetyl lysine antibody followed by immunoblotting for Olig1 (Fig 2G, box). At 24 h, Olig1, which was localized in the cytoplasm (Fig3. 2G,K) was predominantly acetylated, but at 48 hr, when Olig1 was localized in the nucleus (Fig3. 2K’), it was deacetylated (Fig3. 2G).This change of acetylation was specific to Olig1 since the histone H3 acetylation did not change over time (Fig3. 2G). Comparable data were obtained in the reverse experiment, when Olig1 was immunoprecipitated from transfected cells and immunoblotted with pan-acetyl lysine antibody (Fig3. 2H).

In order to determine whether acetylation played a role in Olig1 translocation in vivo, we investigated whether Olig1 translocation from the nucleus to the cytoplasm correlated with increased Olig1 acetylation in the brain. At P1, when Olig1 was in OPC nuclei (Fig3. 1A), it was very minimally acetylated (Fig3. 2I red arrowhead, not asterisk; note endogenous mouse Olig1 migrates very near normal IgG, and is often the minor band seen above the nonspecific IgG band in blots of immunoprecipitated mouse tissue); the peak of Olig1 acetylation was at P10, when the Olig1 nuclear to cytoplasmic translocation occurred (Fig3. 1B). At P30, Olig1 acetylation was reduced compared to

P10 (Fig3. 2I). Thus, Olig1 acetylation increased during oligodendrocyte maturation in vivo, particularly during the translocation period, which suggested that Olig1 acetylation could regulate its cytoplasmic translocation, but it was not required for maintenance in adult oligodendrocyte cytoplasm.

Figure 3.2: Acetylation of Olig1 is correlated with its cytoplasmic localization both in HEK293T cells and in developing mouse brain.

Figure 3.2: Acetylation of Olig1 is correlated with its cytoplasmic localization both in HEK293T cells and in developing mouse brain. (A-F) GFP-tagged WT Olig1 was transfected into HEK293T cells. Five hours later (A) DMSO or (B-F) inhibitors were added to media: (B) Protein kinase A inhibitor: 10μM H89; (C) Protein Kinase C inhibitor: 10μM Gö6976; (D) PI3K inhibitor: 25μM LY294002, or different histone acetyltransferase inhibitors: (E) CBP/P300 inhibitor: 40μM C646; (F) GCN5 inhibitor: 100μM MB-3. Olig1 subcellular locations were analyzed 16h after treatment. Scale bar: 50μm.(G) Olig1-myc was transfected into HEK293T cells, and 24h or 48h after transfection, cell lysates were immunoprecipitated with acetylated-lysine antibody (acetyl-K) and detected with Olig1 antibody (red box). (H) Olig1-myc was transfected into HEK293T cells, and 24h or 48h after transfection, cell lysates were immunoprecipitated with anti-Olig1 antibody and detected with acetylated-lysine antibody.(I) Olig1 was acetylated in developing mouse brain. Endogenous Olig1 was immunoprecipitated from subcortical white matter homogenates of different postnatal ages and Olig1 acetylation was detected with acetylated-lysine antibody. Arrowheads (red):Olig1; Asterisks: Rabbit IgG.(J). Schematic illustrating the predicted acetylated lysine sites in the Olig1 bHLH domain and NES sequences, including K109, K116, K147, K150.(K-M) Acetylation of lysine (K) 150 on Olig1 regulated its cytoplasmic localization in HEK293T cells. Olig1-GFP (K, K’), Olig1-K150R (L, L’) or Olig1-K150Q (M, M’) were overexpressed in HEK293T cells and the subcellular location (nuclei: DAPI, blue) was determined 24h or 48h post transfection. Scale bar: 50μm. Inset scale: 50μm.(O-Q) Acetylation of lysine (K)150 on Olig1 regulated its cytoplasmic localization in cultured rat OPCs. Olig1-GFP (O, O’ and O”), Olig1-K150R (P,P’ and P”) or Olig1-K150Q (Q,Q’ and Q”) mutants were electroporated into the rat OPCs and Olig1 subcellular location was analyzed 48h later in transfected OLs, using DAPI (blue) to localize nuclei. Scale bar: 20μm.

Olig1 lysine (K) 150 acetylation controlled Olig1 cytoplasmic localization.

The Olig1 protein sequence was analyzed for potential acetylated lysines using the Prediction of

Acetylation on Internal Lysines (PAIL) program (Li et al., 2006), and four (K109, K116, K147, K150) were identified in the Olig1 bHLH domain (Fig3.2J, Table 1). Analysis of Olig1 protein sequences from different species revealed that the predicted lysine sites are conserved. Each lysine was mutated to arginine (R) to generate individual mutants that mimic the deacetylated status and a mutant Olig1 with all four R mutations at K109, K116, K147, K150 (4KR) was generated. These deacetylation- mimic mutants were transfected into HEK293T cells, and their subcellular location was determined at 24 h. K150 was the only lysine in the Olig1 NES (Fig3.2J) and K150R was the only single mutation that showed nuclear retention (data not shown).

To further evaluate the role of K150 acetylation in regulating Olig1 subcellular localization, the

K150 was mutated to glutamine (Q) to mimic acetylation. WT-Olig1-GFP, Olig1-K150R-GFP and

Olig1-K150Q-GFP were transfected into HEK293T cells, and their cellular localization was compared.

At 24h, the Olig1-K150R-GFP mainly localized to the nucleus (Fig3.2L), while both WT-Olig1-GFP and

Olig1-K150Q-GFP showed predominant cytoplasmic localization (Fig3. 2K , M). At 48h, WT-Olig1-GFP and Olig1-K150R-GFP were in the nucleus (Fig3.2K’ , L’), in contrast to the acetylated mimic Olig1-

K150Q-GFP, which remained in the cytoplasm (Fig3. 2M’). When these Olig1 lysine mutants were electroporated into primary cultured rat OPCs, WT Olig1 was present in both the nucleus and cytoplasm (Fig3. 2O, note nucleus location by DAPI stain in Fig3. 2O”), which was similar to endogenous Olig1 in cultured OPCs (data not shown). Nuclear retention of Olig1-K150R was found in rat OPCs as in HEK293T cells (Fig3. 2P). Strikingly, the acetylated Olig1 mimic, Olig1-K150Q, was frequently found to be exclusively localized to rat OPC cytoplasm (Fig3. 2Q). These results indicated that Olig1 acetylation at lysine 150 regulates its cytoplasmic localization, both in cell lines and primary oligodendrocytes.

Table 3.1: Predicted Olig1 acetylation sites.

Peptide Position Score

TAPLLPKAAREKP 67 1.15 1.15

PKAAREKPEAPAE 72 1.91

QQQLRRKINSRER 109 0.81

INSRERKRMQDLN 116 1.07

QGAPGRKLSKIAT 147 2.10 2.10

PGRKLSKIATLLL 150 2.23 2.23

DALRPAKYLSLAL 218 1.80 1.80

CTCAVCKFPHLVP 251 1.51 1.51 Potential acetylation sites of human Olig1 were predicted by PAIL server (http://bdmpail.biocuckoo.org/prediction.php) , with 0.75 threshold cut-off.

The histone acetyltransferase CBP acetylates Olig1 Lysine 150.

To identify the histone acetyltransferase (HAT) responsible for Olig1 acetylation at K150, the expression of four major HATs, p300 (E1A-binding protein, 300 kDa), CBP (cAMP response element- binding protein [CREB]-binding protein), PCAF (p300/CBP-associated factor), and GCN5 (KAT2A), was determined in mouse P10 corpus callosum. All were expressed in Sox10-positive oligodendrocytes in vivo (Fig3. 3A), and Olig1 interacted with all of them in HEK293T cells (Fig3. 3B-E). The individual

HATs were expressed with either Olig1-myc or Olig1-K150A-myc (de-acetylated mimic) in HEK293T cells to test their acetylation activity on Olig1 at K150. Both CBP and p300 overexpression increased

Olig1 acetylation (Fig3.3F,G), while PCAF or GCN5 overexpression did not (data not shown). However, while p300 acetylates Olig1, it is not at lysine 150, since Olig1-K150A acetylation by p300 was comparable to WT-Olig1 (Fig3.3G), i.e, p300 is acetylating other lysines. Importantly, Olig1 acetylation by CBP overexpression was largely abolished by the single mutation at lysine 150 in Olig1

(Fig3. 3F). The relevance of Olig1 acetylation for oligodendrocyte development was also studied in

Figure 3.3: CBP is the Histone Acetyltransferase (HAT) responsible for Olig1 Lysine 150 acetylation.

Figure 3.3: CBP is the Histone Acetyltransferase (HAT) responsible for Olig1 Lysine 150 acetylation. (A) CBP, p300, GCN5 and PCAF are expressed in oligodendrocytes in P10 mouse corpus callosum. P10 mouse corpus callosum was co-stained with CBP, p300, GCN5, PCAF (green), Sox10 (red) and DAPI (blue). Scale bar: 10μm.(B-E) Olig1 and CBP, p300, GCN5, PCAF interact in HEK293T cells. Olig1- myc and HA-CBP (B), p300-myc (C), Flag-GCN5 (D) or Flag-PCAF (E) were co-transfected into HEK293T cells and cell lysates were immunoprecipitated with CBP, p300, GCN5 or Flag antibody, respectively, and detected with Olig1 antibody. (F and G) Olig1 is acetylated at lysine 150 by CBP in HEK293T cells. Olig1-myc or Olig1-K150A-myc was co-transfected with HA-CBP plus/minus CBP shRNA (F) or with p300-myc or p300DY-myc (acetylase-deficient p300,G) into HEK293T cells. Note increased acetylation in the presence of HA-CBP, which is reduced for Olig1_K150A-myc (F), but that increased acetylation by P300 is comparable for WT Olig1 and Olig1 K150A-myc (G). (H) Olig1 is acetylated in cultured rat oligodendrocytes. Cultured rat OPC or oligodendrocyte cell lysates were immunoprecipitated with an Olig1 antibody and detected with an acetyl-lysine antibody (upper panel) or Olig1 antibody (middle panel). Merged western bands were shown in the bottom panel.(I) Olig1 interacted with CBP in nuclei of cultured rat oligodendrocytes, and this interaction increased as oligodendrocytes differentiated. Nuclear lysates from rat OPCs or differentiated oligodendrocytes were immunoprecipitated with CBP antibody and detected with Olig1 antibody. Note the increase in Olig1 association with CBP in oligodendrocytes detected with two different Olig1 antibodies.

80 primary cultured rat OPCs. Olig1 acetylation increased as OPCs differentiated to oligodendrocytes in vitro (Fig3. 3H), and the interaction between Olig1 and CBP increased in the nucleus of differentiated cells compared to that in OPCs (Fig3. 3I). Collectively, these results indicate that CBP is the HAT responsible for Olig1 K150 acetylation.

Olig1 is acetylated at lysine 150 in vivo.

In order to investigate this post-translational modification of Olig1 further, antibodies specific to the acetylated lysine 150 of Olig1 were generated. The acetyl-Olig1-K150 (acetyl-Olig1) antibody specificity was confirmed by its ability to recognize acetylated but not unacetylated peptide (Fig3.4A, left column). When the unacetylated peptide was incubated with purified CBP, p300 or GCN5 in vitro, the acetyl-Olig1 antibody specifically recognized the peptide acetylated by CBP or P300 (to a lower degree), but not GCN5 or control (Fig3.4A, middle column). In addition, the acetyl-Olig1 antibody specificity was further confirmed by its ability to recognize purified WT Olig1 that was acetylated by CBP in vitro, but not the Olig1-K150R mutant that could not be acetylated at K150(Fig3.

4B). Overexpression of CBP in HEK293T cells increased WT Olig1 acetylation at K150 but not that of the K150 de-acetylated mimic Olig1-K150R (Fig3.4C).Thus, Olig1 was acetylated by CBP at K150 both in vitro and in vivo, and this acetylated K150 could be specifically recognized by the acetyl-Olig1 antibody.

To identify Olig1 K150 acetylation in developing mouse brain, endogenous Olig1 was immunoprecipitated from P3, P10 or 3M subcortical white matter lysates or subcellular fractions.

Consistent with our earlier results (Fig3. 2I), studies with acetyl-Olig1 antibody demonstrated that

Olig1 K150 acetylation peaked in the P10 fraction, when Olig1 translocation was occurring (Fig3.4D, red arrow). Furthermore, acetylated Olig1 K150 was enriched in the cytoplasmic fraction of the tissue, not the nuclear fraction. Acetylated Olig1 K150 was almost undetectable at P3 or 3M in the cytoplasmic fraction, when Olig1 was either predominantly nuclear (P3) or cytoplasmic (3M),

81 respectively. Weak Olig1 K150 acetylation was detected in the P10 nuclear fraction at the time Olig1 would be translocating to the cytoplasm, but interestingly, at 3M, there was increased nuclear Olig1

K150 acetylation compared to P10 (Fig3. 4D). Since Olig1 is not nuclear in mature oligodendrocytes in the adult, the acetylated Olig1 protein is likely in adult OPCs (Fig3. 1A). Overall, these studies suggest that Olig1 is acetylated at K150 in the developing mouse brain, when it is actively translocated from the nucleus to the cytoplasm.

Presumably, Olig1 acetylation must be highly regulated both by acetyltransferases and by deacetylases. In order to identify the Olig1 deacetylase, we co-expressed Olig1 with HDAC1 through

10, Sirt1 or Sirt2 and immunoprecipitated samples to assess potential interactions. Olig1 interacted with HDAC1, 2, 3, 4, 10 and Sirt2 (Fig3. 4E). When Olig1 was co-expressed with these candidate

HDACs, excess CBP, HDAC1, 3 and 10 decreased Olig1 acetylation but HDAC2, 4 and Sirt2 did not

(Fig3. 4F). HDAC1, 3 and 10 deacetylated Olig1 K150, the validated CBP acetylation site (Fig3.4G).

Since HDAC1, 3 and 10 were also highly expressed in oligodendrocytes in the corpus callosum in vivo

(Shen et al., 2005), these results suggested that HDAC1, 3 and/or 10 are present in oligodendrocytes, and could potentially deacetylate Olig1 specifically at lysine 150 in vivo.

Interestingly, Olig1 interaction was higher with HDAC1 in P3 brain (Fig3. 4H) when deacetylated

Olig1 was greater than acetylated Olig1 (Fig3. 4D). On the other hand, the increased interaction of

Olig1 and CBP in P10 brain correlated with elevated Olig1 acetylation (Fig3.4H). Collectively, these data suggest interaction between Olig1 and CBP or HDACs is developmentally regulated, and this interaction could be responsible for the increased Olig1 acetylation as oligodendrocytes mature in vivo.

Acetylation of Olig1 is critical for its role in morphological differentiation and lineage progression.

Our earlier studies (Dai et al., in press 2015) establish that Olig1 is critical for initial OPC differentiation both in vitro and in vivo in brain. Since Olig1 acetylation at K150 correlated with

82 oligodendrocyte maturation both in vitro and in vivo, we examined whether Olig1 K150 acetylation had a direct impact on oligodendrocyte differentiation. In order to introduce the WT Olig1 or Olig1 lysine 150 mutants into differentiating OPCs, we treated cells with HA-tagged Olig1 protein linked to the cell-penetrating peptide TAT. The proteins were taken up by OPCs after one day growth in differentiation medium plus purified TAT-tagged proteins, as demonstrated both by immunostaining and western blotting (Fig3. 5A and 5B). This allowed us to study overexpression of WT Olig1 or its lysine 150 mutants during oligodendrocyte differentiation. Control cells or Olig1-knockdown cells were generated by electroporating OPCs with control siRNA or Olig1 siRNA. Differentiation of control cells or Olig1-knockdown cells was then studied after incubation with TAT-tagged WT or mutant Olig1 proteins. Initial studies were done to analyze the effect of WT Olig1 overexpression in control cells that express endogenous Olig1. This did not influence their initial lineage progression, as measured by the number of O4-positive cells (Fig3. 5K, compare TAT-GFP to TAT-Olig1 treated cells). However, WT Olig1 overexpression in control cells significantly increased their morphological differentiation, as measured both by total covered area (Fig3. 5D and L). The overexpression of the

K150 acetylation mimic Olig1 K150Q in control cells significantly promoted both initial OPC differentiation to O4-positive cells (Fig3. 3E and K), and their morphological differentiation (Fig3. 5E and L). In contrast, overexpression of the K150 deacetylation mimic Olig1-K150R impaired OPC lineage progression, and reduced the total covered area of O4-positive cells by 36%, despite the presence of endogenous Olig1 (Fig3. 5F, K and L). Interestingly, while overexpression of the K150 acetylation mimic Olig1-K150Q had no impact on oligodendrocyte survival, overexpression of either

WT Olig1 or deacetylation mimic Olig1-K150R increased oligodendrocyte survival, as measured by total cell number after 1d differentiation in vitro (Fig3. 5M). These results suggested that Olig1 acetylation at K150 is critical for promoting oligodendrocyte lineage progression and for morphological differentiation, whereas deacetylated Olig1 in the nucleus is important for

Figure 3.4: Olig1 is acetylated at lysine 150 in vivo.

Figure 3.4: Olig1 is acetylated at lysine 150 in vivo. (A) The anti-acetyl-mOlig1-K139 antibody specifically recognizes mouse Olig1 peptide acetylated on lysine 139. Varying amount (0.1ng to 1ug) of the peptide corresponding to non-acetylated peptide or Olig1 aceetylated on lyine 139 were spot on a PVDF membrane and the membrane was immunoblotted with the anti-acetyl-Olig1-K139 antibody(left column); Varying amount (0.1ng to 1ug) of non-acetylated peptides were in vitro acetylated by purified CBP, P300 or GCN5 respectively and the reaction mixes were spot on a PVDF membrane and the membrane was immunoblotted with the anti-acetyl-Olig1-K139 antibody(middle column) or anti-pan-Olig1 antibody (right column). (B)The acetyl-Olig1-K150 antibody specifically recognized recombinant human Olig1 protein acetylated on lysine 150. One microgram of recombinant WT Olig1 or acetylation-deficient Olig1 mutant protein (Olig1-K150R) was incubated in vitro with purified CBP, P300 or GCN5, and their acetylation was detected by immunoblotting with acetyl-Olig1-K150 antibody, acetylated lysine antibody or GST antibody. The activity of the different HATs was indicated by self-acetylation of the GST-acetylase detected by acetylated lysine antibody (top rows). The GST-Olig1 proteins were detected by GST immunostaining (lower rows) and quantification of the ratio of acetylated Olig1 to total Olig1 is presented in red, normalized to the WT control. (C) Olig1 was acetylated at lysine 150 by CBP in HEK293T cells detected by acetyl-Olig1-K150 antibody. Olig1-myc, Olig1-K150R-myc or Olig1-K150Q-myc was co-transfected with HA-CBP into HEK293T cells. 24 hours later, cells were treated with 500nM TSA for 10h before the cell lysate was immunoprecipitated with Olig1 antibody and detected with acetyl-Olig1-K150 antibody. (D) Olig1 was acetylated at lysine 150 in developing mouse brain. Subcortical white matter from P3, P10 or adult samples was fractionated and endogenous Olig1 was immunoprecipitated from whole lysates, cytoplasmic or nuclear fractions and immunoblotted with acetyl-Olig1-K150 antibody. Arrowheads (red): Olig1; Asterisks (black): Rabbit IgG. (E). Olig1 interact with different HDACs. Olig1-myc were co-transfected with different HA tagged or Flag tagged HDACs as indicated into 293T cells. 48h after transfection the cells were lysed, then the cell homogenate was immunoprecipitated with Olig1 antibody detected with anti-Flag and anti- HA antibody, red asterisk indicates binding HDACs. (F). HDAC1/3/10 is the HDACs which could potentially deacetylate Olig1. Olig1-myc, HA-CBP were co-transfected with different HA tagged or Flag tagged HDACs into 293T cells. 6h after transfection the cells were treated with 500nM TSA overnight, then the fresh media were added for another 10h, then the cell homogenate was immunoprecipitated with Olig1 antibody detected with anti-acetylated lysine antibody. (G) HDAC1, HDAC3 and HDAC10 were potential HDACs responsible for Olig1 acetyl-K150 deacetylation in vivo. The indicated constructs were co-transfected into HEK293T cells. Six hours after transfection, cells were treated with 500nM TSA overnight, and fresh media was then added for another 10h. Cell homogenates were immunoprecipitated with Olig1 antibody and detected with acetyl-Olig1-K150 antibody. Quantification of the ratio of acetylated Olig1 to total Olig1 is presented in red. (H) Olig1 preferentially interacted with HDAC1 or CBP in developing mouse brain. Endogenous Olig1 was immunoprecipitated from subcortical white matter homogenates from different postnatal developmental ages and bound CBP was detected with CBP antibody (middle row). Endogenous HDAC1 was immunoprecipitated from the same sample, and bound Olig1 was detected with Olig1 antibody as indicated (top row).

85 oligodendrocyte survival upon withdrawal from cell cycle.

The role of Olig1 K150 acetylation in regulating oligodendrocyte differentiation was further examined in Olig1 knockdown cells. As noted above, oligodendrocyte differentiation was significantly reduced upon Olig1 knockdown (compare Fig3. 5C and G). Lineage progression (the percentage of O4-positive cells) and morphological differentiation were rescued by re-introduction of either WT Olig1 (Fig3. 5H) or the acetylation mimic Olig1-K150Q (Fig3. 5I), and the impact of

Olig1-K150Q on morphological differentiation was more dramatic than that of WT Olig1 (Fig3.5O). In contrast, the deacetylation mimic Olig1-K150R only partially rescued the reduction in O4-positive cells (Fig3. 3J and N) but to a significantly lesser extent when compared with that of WT Olig1 (Fig3.

5H, J and N). In addition, Olig1-K150R failed to rescue the morphological differentiation defect with

Olig1 knockdown (Fig3.5J and O). Both WT Olig1 and Olig1-K150R were able to rescue the reduction in the total number of cells induced by Olig1 knockdown, but Olig1-K150Q could not (Fig3. 5P).

These results confirmed the essential role of K150 acetylation in oligodendrocyte differentiation and membrane expansion, and further support the concept of a role of non-acetylated Olig1 in oligodendrocyte survival upon cell cycle withdrawal.

During oligodendrocyte differentiation, increased acetylation of Olig1 decreased its chromatin binding.

For many transcription factors or chromatin binding complexes, dissociation from the chromatin precedes their inactivation or nuclear export (Niida et al., 2007; Nakamura et al., 2012). In the developing mouse brain, complete nuclear extrusion of Olig1 is seen in actively myelinating oligodendrocytes (Arnett et al., 2004). However, we saw only partial cytoplasmic translocation of

Olig1 in fully differentiated oligodendrocytes in vitro (see Fig3.7F below, where Olig1 is found both in the nucleus and cytoplasm). To test if the chromatin dissociation of Olig1 and its subsequent nuclear export was partially uncoupled in cultured oligodendrocytes, chromatin binding of Olig1 in

86 proliferating OPCs and mature oligodendrocytes cultured in vitro was analyzed. Endogenous Olig1 was immunoprecipitated from OPCs and differentiating oligodendrocytes, and analyzed for histone association. Histone H3 was strongly associated with Olig1 in OPCs, but not in mature oligodendrocytes, which had elevated Olig1 acetylation (Fig3. 6A, T3-treated cells). These data indicated that although some Olig1 was present in the nucleus in mature oligodendrocytes in vitro, most nuclear Olig1 in these differentiating cells did not bind to the chromatin. Additionally, using a modified chromatin immunoprecipitation protocol, more histone H3 interacted with Olig1 in OPCs than in mature oligodendrocytes (Fig3. 6B), and less Olig1 was found associated with insoluble chromatin in the mature oligodendrocytes than in OPCs (Fig3. 6C). The impact of Olig1 acetylation on its association with chromatin in vivo was also studied. At different postnatal time points, there was increased nuclear Olig1 acetylation at K150 (Fig3. 6D top panel). Endogenous Olig1 was immunoprecipitated from the nuclear fraction of subcortical white matter obtained from P3, P10 or

3M brains. More histone H3 interacted with Olig1 in P3 brain nuclei than in P10 nuclei, and much less association was seen in 3M brain nuclei (Fig3. 6D). Despite reduced binding to chromatin in adult brain, Olig1 from all ages tested had comparable binding to DNA itself (Fig3. 6E). This indicated that changes in the direct DNA binding of Olig1 cannot account for the dissociation of Olig1 from chromatin in differentiating oligodendrocytes, and that additional mechanisms must be involved.

Collectively, these data suggest that as oligodendrocytes mature, Olig1 acetylation facilitates its dissociation from chromatin, which precedes its subsequent nuclear export.

Acetylation of Lysine 150 on Olig1 increased its binding affinity for Inhibitor of DNA binding 2 (Id2).

Olig1 can dimerize with other bHLH transcription factors, such as Olig2, Olig1, Id2, Id4 and E47, or the non bHLH transcription factor Sox10 (Samanta and Kessler, 2004; Li et al., 2007a). We therefore investigated whether Olig1 acetylation changes its binding affinity for these known binding partners.

We analyzed the interactions of WT Olig1, Olig1-K150R or Olig1-K150Q with Olig1, Olig2, E12/E47,

Figure 3.5: Acetylation of Olig1 is critical for its role in morphological differentiation and lineage progression.

Figure 3.5: Acetylation of Olig1 is critical for its role in morphological differentiation and lineage progression. (A) Cultured rat oligodendrocytes were electroporated with smart pool control siRNA and recovered in proliferation media for 1d, and then shifted to differentiation medium with addition of either purified HA tagged TAT-GFP,TAT-Olig1,TAT-Olig1-K150R or TAT-Olig1-K150Q protein (0.4uM). After differentiation for 1d, the cells were co- stained with O4 and anti-HA tagged antibodies. Scale bar: 50μm.(B) Cultured rat oligodendrocytes were grown and treated as in (A), collected and immunoblotted with HA antibody and Olig1 antibody. Arrows: Olig1 degradation products, Arrowhead: TAT-protein, Asterisks: non-specific bands.(C-F) Cultured rat oligodendrocytes were electroporated with smart pool control siRNA and recovered in differentiation media for 1d. Cells were then switched to differentiation medium and either purified TAT-GFP (C), TAT-Olig1 (D), TAT- Olig1-K150Q (E) or TAT-Olig1-K150R(F) protein (0.4uM) was added. After differentiation for 1d, the cells were live stained with O4 antibody (green) and co-stained with Olig2 antibody (red).(G-J) Cultured rat oligodendrocytes were electroporated with smart pool Olig1 siRNA as in (C-F) and either purified TAT-GFP (G), TAT-Olig1 (H), TAT-Olig1-K150Q(I) or TAT-Olig1-K150R(J) protein (0.4uM) was added during differentiation. After 1d, the cells were fixed and co-stained with O4 (green) and Olig2 (red) antibody. Scale bar: 50μm.(K-M) Quantitative analysis of the impact of TAT-GFP, TAT- Olig1, TAT-Olig1-K150R or TAT-Olig1-K150Q protein transduction on OPC differentiation with control siRNA as in (C -F). The percentage of O4-positive cells (K), average covered area of O4-positive cells (L) and total cell numbers (M) were quantified. The data were obtained from three independent experiments and in each condition at least 300 cells were visually examined and counted. (N-P) Quantitative analysis of impact of TAT-GFP, TAT-Olig1, TAT-Olig1-K150Q and TAT-Olig1-K150R protein transduction on OPC differentiation after Olig1 siRNA knockdown, as in (G-J). The percentage of O4-positive cells (N), average covered area of O4-positive cells (O) and total cell numbers (P) were quantified as indicated. The data were obtained from three independent experiments and in each condition at least 300 cells were visually examined and counted. Data represent the mean ± SEM from at least three independent experiments. *p < 0.05, **p < 0.01 (Student’s t test).

Figure 3.6: Increased acetylation of Olig1 decreased its chromatin binding as oligodendrocytes differentiated.

Figure 3.6: Increased acetylation of Olig1 decreased its chromatin binding as oligodendrocytes differentiated. (A) Binding of Olig1 with Histone H3 decreased as Olig1 acetylation increased in cultured oligodendrocytes. Mouse OPCs were maintained in PDGF/bFGF for 5d or differentiated for 5d. Cell lysates were then immunoprecipitated with Olig1 antibody and immunoblotted for acetyl-lysine or histone H3 association (note boxed area). (B) Association of Olig1 with chromatin decreased as oligodendrocytes differentiated in vitro. Mouse OPCs were maintained in PDGF/bFGF for 3d or differentiated for 5d, then cells were fixed with 1% formaldehyde. Subsequent chromatin protein immunoprecipitation was conducted using Olig1 antibody, and samples were immunoblotted with Histone H3 antibody. Note reduced Histone H3 association with Olig1 in cells after 5 days in T3 (boxed area).(C) Olig1 dissociated from chromatin as oligodendrocytes differentiated in vitro. The chromatin fraction of mouse cultured OPCs (PDGF/bFGF, 3d) or differentiated oligodendrocytes (T3, 3d) was isolated, and the presence of Olig1 or Olig2 in either the soluble or pellet chromatin fraction was determined by western blot. Note presence of Olig1 primarily in the chromatin pellet in OPCs, but primarily in the soluble fraction, dissociated from chromatin, in oligodendrocytes.(D) In developing mouse brain, Olig1 binding with histone H3 decreased as oligodendrocytes matured. Subcortical white matter from different postnatal ages was fractionated into nuclear and cytoplasmic fractions. Endogenous Olig1 was immunoprecipitated from the nuclear fraction with Olig1 antibody. Olig1 acetylation was detected with acetyl-Olig1-K150 antibody (top panel), and Olig1-bound Histone H3 was detected with both acetyl-histone H3 and histone H3 antibodies. Arrowheads: Acetyl-Olig1; Asterisks: Rabbit IgG. Note increase in acetylated Olig1 with age, at the same time as the Olig1 association with histone decreased. (E) DNA binding affinity of Olig1 did not change as oligodendrocytes matured in the developing mouse brain. The same nuclear fractions as in (C) were incubated with a streptavidin-coupled tandem E-box consensus DNA probe. The endogenous Olig1 was pulled down with DNA probes and detected with Olig1 antibody. Note comparable association of Olig1 with DNA, irrespective of the age of the samples.

Id4 or Id2 in co-immunoprecipitation assays in HEK293T cells. All proteins interacted with WT Olig1 or the acetylation mimic Olig1-K150Q, and Olig1, Olig2, and E12/E47 bound comparably to WT and the deacetylation mimic Olig1-K150R (data not shown). Sox10 had slightly increased binding to

Olig1-K150R and reduced binding to Olig1-K150Q (Fig3.7A), suggesting it binds preferentially to deacetylated Olig1. However, Olig1-K150R had reduced binding affinity for Id2 (Fig3.7B) but not Id4

(Fig3. 7C), suggesting that Id2 might be a specific partner for acetylated Olig1.

Id2 is a negative regulator of oligodendrocyte differentiation and undergoes nuclear to cytoplasmic translocation upon oligodendrocyte differentiation in vitro (Wang et al., 2001). The subcellular localization of Id2 in the developing mouse brain was predominantly in the nucleus of

OPCs at P4, and it either translocated to the cytoplasm or was downregulated in mature oligodendrocytes at P15 (Fig3. 7D). The nuclear to cytoplasmic translocation of Id2 and Olig1 occurred over the same time period in vivo. In order to assess possible interactions of these proteins in vivo, we analyzed cytoplasmic and nuclear fractions from P3, P10 or 3M mouse subcortical white matter for Id2 localization and interactions. Endogenous Id2 was immunoprecipitated from the cytoplasmic or nuclear fractions and immunoblotted with Olig1 antibody. Olig1 was found to interact strongly with Id2 in the cytoplasmic fraction at P10, but not at 3M (Fig3. 7E). The Olig1/Id2 interaction was not found in the nuclear fraction (Fig3.7E, bottom). The strong Olig1/Id2 interaction in the P10 mouse cerebrum cytoplasm correlated with high acetylation of Olig1 at K150 (Fig3. 7E).

Because Id2 is also expressed in neurons and astrocytes in CNS, we used primary cultured oligodendrocytes to test the role of Olig1 K150 acetylation in regulating the Olig1/Id2 interaction.

Consistent with earlier studies, the acetylated level of Olig1 K150 increased as oligodendrocytes differentiated, and Olig1 acetylation increased in the cytoplasm of differentiated oligodendrocytes in vitro (Fig3. 7F). The interaction between Olig1 and Id2 was strongest in the cytoplasm of differentiated oligodendrocytes, which correlated with the higher acetylation of Olig1 in these cells

(Fig3. 7F). Furthermore, increased Olig1/Id2 interaction was observed when differentiating oligodendrocytes were treated with the general HDAC inhibitor trichostatin A (TSA) (Fig3. 7G), which increased Olig1 acetylation (Fig3.7H). These studies demonstrated that Olig1 acetylation at K150 increased the interaction of Olig1 and Id2. The interactions of Olig1 and Id2 would likely facilitate Id2 retention in the cytoplasm, which would repress its inhibitory impact on oligodendrocyte differentiation.

DISCUSSION

Mechanism of nuclear export of Olig1

In this study, we first investigated protein sequences that could explain the differences in subcellular localization of Olig1 and Olig2 by identifying two additional nuclear export sequences

(NES) in Olig1 not found in Olig2. NES1 was exactly conserved between Olig1 and Olig2, but Olig1 contained two extra NES, which were stronger than NES1. However, given the fact that the localization of Olig1 changes with differentiation, regulatory mechanisms in addition to the primary protein sequence of these three NESs in Olig1 must be involved in the translocation. NES1, although present in Olig2, functions differently in Olig1 and is essential for its cytoplasmic translocation. The charge on amino acids in nuclear export consensus sequences influences the intensity of the export signal (Guttler et al., 2010; Regot et al., 2014), and both phosphorylation and acetylation of targeted amino acids impact their charge (Gauci et al., 2008). By using inhibitors for major protein kinases and acetyltransferases, we found that acetylation had a greater impact on Olig1 subcellular localization than phosphorylation. Based on bioinformatics analysis of potential acetylation sites, lysine 150 in NES1 (lysine 139 in mouse) was identified as the major regulator of Olig1 localization.

Acetylation of lysine 150 could potentially neutralize the positive charge on this lysine and increase the strength of NES1 as a nuclear export signal, which would facilitate nuclear export of Olig1. K150 acetylation was highly correlated with the dynamic in vivo nuclear to cytoplasmic translocation of

Figure 3.7: Acetylation of Lysine 150 on Olig1 increased its binding affinity for Inhibitor of DNA binding 2 (Id2).

Figure 3.7: Acetylation of Lysine 150 on Olig1 increased its binding affinity for Inhibitor of DNA binding 2 (Id2). (A-C) Lysine 150 acetylation increased Olig1/Id2 interaction. Olig1-myc, deacetylation mimic Olig1- K150R-myc or acetylation mimic Olig1-K150Q-myc were co-transfected with pCMV5-Sox10 (A), Id2- GFP (B) or Id4-GFP (C) into HEK293T cells. Protein interactions were detected by co- immunoprecipitation with the indicated antibodies. Note increased association of deacetylated Olig1 with Sox10 (A), strong increase in association of acetylated Olig1 with Id2 (B) and no impact of acetylation on association of Olig1 with Id4 (C). (D) Oligodendrocytes in P4 and P15 mouse corpus callosum were co-stained with Id2 (green) and Sox10 (red). Note cytoplasmic localization of Id2 at P15. Scale bar: 10μm.(E) Id2 interacted with Olig1 at P10 in the cytoplasmic fraction, but not in nuclear fraction. Mouse subcortical white matter from different postnatal developmental time points was fractionated. Endogenous Id2 was immunoprecipitated from the cytoplasmic or nuclear fraction with Id2 antibody and Olig1 association was determined by western blot for Olig1. Arrowheads: Olig1; Asterisks: Rabbit IgG.(F) The Olig1/Id2 interaction increased as oligodendrocytes differentiated in vitro. Primary cultured OPCs or differentiated oligodendrocytes were treated with TSA (500nM) for 10h, fractionated and the subcellular fractions were immunoprecipitated with Id2 antibody and detected with Olig1 antibody (top panel), or immunoprecipitated with Olig1 antibody and detected with acetyl-Olig1-K150 antibody (bottom panel). Arrowheads: Olig1; Asterisks: Rabbit IgG.(G) Excess acetylation increased the interaction of Olig1 and Id2. Mouse OPCs were induced to differentiate for 4d, and treated with DMSO or TSA (100nM) for 1d. Cell lysates were immunoprecipitated with anti-Id2 antibody and detected with Olig1 antibody. (H) Cultured mouse OPCs or differentiated oligodendrocytes were treated with TSA (500nM) for 10h, then immunoprecipitated with Olig1 antibody and detected with general anti-acetyl-lysine antibody (top panel). The ratio of acetylated Olig1 over total olig1 was quantified.

Figure 3.8: Model of the mechanism by which Olig1 acetylation regulates its nuclear to cytoplasmic translocation and function. Deacetylated Olig1 localized in the nucleus is associated with chromatin, and functions as a positive regulator for cell cycle exit of OPCs and of post-mitotic oligodendrocyte survival after cell cycle exit. Acetylation of Olig1 facilitates its dissociation from chromatin and is required for morphogenesis of newly formed oligodendrocytes. Acetylated Olig1 and Id2 interact in mature oligodendrocytes, and the retention of Id2/acetylated Olig2 in the oligodendrocyte cytoplasm could reduce the Id2 inhibition of oligodendrocyte differentiation.

Olig1 in maturing oligodendrocytes in the P10 brain. Interestingly, K150 acetylation decreased in adult mice, suggesting that once translocated, K150 acetylation is dispensable for Olig1 cytoplasmic retention in mature oligodendrocytes.

Potential role of Olig1 acetylation in temporospatial control of myelin initiation

Oligodendrocyte lineage progression and myelin initiation are tightly controlled both temporally and spatially in the developing mouse brain (Trapp et al., 1997; Baumann and Pham-Dinh, 2001).

Both extrinsic and intrinsic cues play critical roles in oligodendrocyte differentiation and myelination

[see reviews (Emery, 2010a, b; Wood et al., 2013)]. However, the mechanism by which oligodendrocytes integrate extracellular signaling with their intrinsic transcriptional program to determine when and where to start myelination is largely unknown. While Olig1 is clearly important for oligodendrocyte specification and OPC survival(Lu et al., 2002; Zhou and Anderson, 2002;

Silbereis et al., 2014), tight temporal control of Olig1 expression and localization as oligodendrocytes initiate myelination implies a potentially negative function of Olig1 when localized in the nucleus of myelinating cells. Acetylation of non-histone proteins is emerging as an important regulator of protein function (Choudhary et al., 2009; Zhao et al., 2010a). In this study we unveiled a novel mechanism by which the nuclear to cytoplasmic translocation of Olig1 is regulated by acetylation at a single lysine site (K150) both in vitro and in vivo. How the acetylation level of Olig1 is temporally regulated during myelinogenesis is still unclear. CBP and HDAC1 expression were constant as oligodendrocytes differentiated in vivo, but before differentiation in P3 brain, Olig1 interacted preferentially with HDAC1, which would likely maintain it in a deacetylated state, and at the peak of

Olig1 acetylation in P10 brain, it interacted more strongly with CBP, which would acetylate it.

Additional mechanisms regulating acetylation may also be important for regulating Olig1 translocation. For example, phosphorylation can modulate the degree of Stat3 acetylation (Kramer et al., 2009), and an Olig1 phosphorylation site that impacts Olig1 translocation in cultured

97 oligodendrocytes is next to the K150 acetylation site (Niu et al., 2012). Thus, it will be important to understand their interactions in regulating Olig1 acetylation and oligodendrocyte lineage progression.

Nuclear vs cytoplasmic function of Olig1

Our studies establish a mechanism for Olig1 translocation (Fig3.8): in OPCs, deacetylated Olig1 binds chromatin along with its potential partners and serves as a positive regulator of OPC cell cycle exit and survival. As OPCs differentiate, Olig1 is acetylated and dissociates from chromatin. This acetylation on Olig1 facilitates its subsequent nuclear export and possible retention of a negative regulator of oligodendrocyte differentiation, Id2, in the cytoplasm of newly formed oligodendrocytes. In many other systems, transcription factors important for early cell development are downregulated at later developmental stages (Jessell, 2000; Isshiki et al., 2001; Braun and

Gautel, 2011). Thus an important question is why Olig1 remains in the cytoplasm as the oligodendrocyte matures. It is possible that Olig1 is important for initial differentiation, but it becomes a negative regulator of later oligodendrocyte differentiation, and must be downregulated or translocated. As OPCs begin to differentiate in vivo, they become premyelinating oligodendrocytes before starting to wrap axons (Trapp et al., 1997). Interestingly, Olig1 is greatly reduced in premyelinating oligodendrocytes (Dai et al., in press 2015), which suggests that sustained nuclear Olig1 may be inhibitory to late oligodendrocyte differentiation and it may need to be eliminated in this stage. Consistent with this concept, deacetylated Olig1 in OPC nuclei was associated with chromatin, and this was important for post-mitotic oligodendrocyte survival upon cell cycle withdrawal and the initiation of differentiation. However, sustained deacetylated Olig1 nuclear retention inhibited subsequent lineage progression (Fig3. 5K). These data support an inhibitory effect of sustained nuclear Olig1 on late oligodendrocyte differentiation.

Intriguingly, while Olig1 is downregulated in the premyelinating cell, it reappears and is retained in the cytoplasm of myelinating oligodendrocytes. Olig1 expression in the oligodendrocyte cytoplasm long after active myelination is finished suggests its importance in mature oligodendrocytes, but not for myelinogenesis per se (Dai et al., in press 2015). It is possible that cytoplasmic Olig1 might serve as a sensor for injury to myelinating oligodendrocytes. In order to further investigate the role of cytoplasmic Olig1 at the molecular level, identification of Olig1 interacting proteins in the cytoplasm will be necessary.

The Olig1/Id2 interaction and the presence of Id2 in the oligodendrocyte cytoplasm has been shown, although those studies also showed interaction of Id2 with Olig2 (Samanta and Kessler, 2004;

Gokhan et al., 2005). Our studies indicate that the OIig1/Id2 interaction is involved in retention of

Id2 in cytoplasm, with increased interaction between Olig1 and Id2 in the cytoplasm of mature oligodendrocytes both in vitro and in vivo, and that the Id2 interaction was preferentially with the acetylated Olig1.

Olig1 translocation and remyelination

Recent studies indicate extensive myelin remodeling in the adult mouse brain, mainly attributed to newly generated oligodendrocytes (Rivers et al., 2008; Young et al., 2013). However, retrospective birthdating of oligodendrocytes with 14C in human brain indicates that oligodendrocyte number does not change with age, and in fact very little turnover of oligodendrocytes occurs through life in human white matter. By contrast, comparable analysis of the myelin itself in these postmortem samples indicated that myelin was generated and remodeled throughout life (Yeung et al., 2014). This raises the question of how mature oligodendrocytes remodel myelin. Whether mature oligodendrocytes with cytoplasmic Olig1 are involved in myelin remodeling in the mouse has not been investigated. Newly generated myelin is laid down by existing mature oligodendrocytes in response to learning (Bengtsson et al., 2005; Scholz et al., 2009), and it

99 is intriguing to consider the possibility that movement of cytoplasmic Olig1 back to the nucleus might reactivate myelinogenesis in these cells.

Olig1 function is essential for remyelination in several different demyelination models (Arnett et al., 2004; Whitman et al., 2012). This differentiation block in Olig1 null mice during remyelination resembles the finding in MS lesions in which abundant OPCs fail to differentiate (Chang et al., 2000;

Chang et al., 2002; Kuhlmann et al., 2008). Considering the role of Olig1 acetylation in regulating its function in oligodendrocyte differentiation, it is possible that Olig1 acetylation is disrupted in OPCs in MS lesions, and may be increased, affecting its function in oligodendrocytes. This disruption could result from the altered acetylation patterns seen in MS lesions, where increased acetylated histone is seen in oligodendrocyte nuclei and increased CBP is found in some MS lesions (Pedre et al.,

2011)However, Olig1 is still present in the nuclei in MS lesions (Arnett et al., 2004), where our data suggest it should be deacetylated to function. Modulating the Olig1 acetylation might be one approach to promote oligodendrocyte differentiation and subsequent myelination in MS patients.

MATERIALS AND METHODS

Animal Procedures and Primary Cell Culture

All animal procedures were conducted in accordance with the University of Colorado Institutional

Animal Care and Use Committee, and the National Institutes of Health guidelines for the care and use of laboratory animals. Mouse neural progenitor cells were isolated from neocortex of male and female E12.5–E14.5 embryos and converted to OPCs as previously described (Pedraza et al., 2008).

Mixed glial cultures were generated from male and female P0-3 day old Sprague Dawley rat pups as described previously (Dai et al., 2014).

Plasmid vectors

The open reading frame of human Olig1 was amplified from a human embryonic cDNA library

(Origene,Rockville, MD) and subcloned into pCMV-myc vector (Clontech, Mountain View, CA),

100 pEGFP-N1/C3 vectors (Clontech, Mountain View, CA), pGEX-KG vector (for GST tag protein purification) and HA-TAT vector (a gift from Dr. Steven Dowdy, University of California San Diego, CA for TAT tag fusion protein purification). The Olig1-K150R, Olig1-K150Q, Olig1-deltaNES1, Olig1- deltaNES2, Olig1-deltaNES3 and Olig1-deltaNES1/2/3 mutant vectors were generated using

Stratagene’s QuikChange Site-Directed Mutagenesis Kit (Agilent technology, La Jolla, CA), following the manufacturer’s protocol (detailed primers and PCR reaction conditions provided upon request).

The p300-myc and p300DY-myc vectors were kindly provided by Dr.Tso-Pang Yao (Duke University,

North Carolina). The pEGFPC1-mId2 and pEGFPC1-mId4 vectors as described (Kurooka and Yokota,

2005) were kind gifts of Dr. Hisanori Kurooka (University of Fukui, Japan). The HA-tagged human

HDAC1,2,3,4,5,6,7,8,10 vectors were from Dr. Minoru Yoshida and Akihisa Matsuyama (Chemical

Genetics Laboratory, RIKEN, Japan).The Flag-Sirtuin1/2 vectors were from Dr. Eric Verdin (University of California San Francisco, CA). The pCMV6-Sox10 vector was kindly provided by Dr. Michael

Wegner (Lehrstuhl für Biochemie und Pathobiochemie, Germany). The following vectors were obtained from Addgene (Cambridge, MA): pRc/RSV-m CBP-HA(#16701), pAdEasy® Flag

GCN5(#14106), pAd-Track Flag GCN5 Y621A/P622A(#14425), pCI-flag-PCAF(#8941), p3xFLAG- mE47( #34585), Flag-hCRM1(#17647). The GCN5 and GCN5-Y621A/P622A cDNA were digested with

KpnI and XhoI and subcloned into pcDNA3.1+ vector (Life Technologies, Grand Island, NY). CBP shRNA was constructed by Genepharma (Shanghai GenePharma Co., Ltd, Shanghai, China) into a lentivirus expression vector pGLVH1/GFP+Puro using the sequence (5’-gcagcagccagcattgata-3’ and

5’- ctgatgagctgatacccaatg-3’).

Antibodies

Two rabbit anti-mouse Olig1 antibodies targeting different regions were generated by Abgent

(Shanghai, China). The sequences of immunizing peptides were SLLPKPAREKAEAP (residue 55-68 of mOlig1, rabbit anti-Olig1-1) and TKYLSLALDEPPC (residue 206-218, rabbit anti-Olig1-2). The

101 specificity of these two antibodies was validated by western blot, immunoprecipitation (see Appedix

A). Based on their performance, the Olig1-1 antibody was used for western blots and Olig1-2 antibodies were used for immunoprecipitation studies. The K150 acetylation site-specific antibody of Olig1, which is conserved among human, mouse and rat Olig1 proteins, was generated against the modified peptide GAPGRKLS[Ac]KIAT (residues 142-153 of human Olig1/ 131-142 of mouse Olig1) by

Abgent. The antiserum was first absorbed on a column containing non-acetylated peptide and then affinity purified on an acetyl-peptide column. The purified acetyl-Olig1 antibody was validated and used at 1:250 for western blot.

Primary antibodies used for western blot, immunoprecipitation or immunofluorescence were as follows: rabbit anti-Olig2 and Olig1 (a gift from Dr. Charles Stiles, Harvard University, MA); mouse anti-Olig1 (Millipore #MAB5540); the following antibodies were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA ): mouse anti-Olig1 (sc-166257); rabbit anti-Id2 (sc-489); rabbit anti-

CBP (sc-369); goat anti-GCN5 (sc-6303,); rabbit anti-p300 (sc-584); goat anti-Sox10 (sc17342); rabbit anti-Sox10 (Sigma #S8193,St Louis, MO); mouse anti-β-tubulin (Sigma#T8328,St Louis, MO); mouse anti-CC1 antibody (Calbiochem, #OP80, Cambridge, MA); rat anti-PDGFRα (BD PharMingen #55874

San Diego, CA); O4 hybridoma (gift of Dr. Rashmi Bansal, University of Connecticut Health Sciences

Center, CT); mouse anti-myelin basic protein (MBP) (Covance#SMI-94, 1:500 for ICC); mouse anti-

Flag M2 (Sigma#F3165,St Louis, MO); anti-Flag M2 affinity gel (Sigma#A2220,St Louis, MO); mouse anti-myc (9E10) supernatant (Developmental Studies Hybridoma Bank ,Iowa City, IA ). The following antibodies were obtained from Cell Signaling Technology (Danvers, MA): rabbit anti-Id2 (#3431); rabbit anti-CBP (#7389); rabbit anti-GCN5 (#3305); rabbit anti-GAPDH ((#2118); rabbit anti-LaminA/C

(#2032,); rabbit anti-acetylated lysine (#9441);rabbit anti- acetylated lysine (#9814,); mouse anti- acetylated lysine(#9681); rabbit anti- Histone H3 (#4620); Acetyl-Histone H3 antibody sampler Kit

(#9927); mouse anti-GFP(#2955); rabbit anti-HA (#3724).

102

Cell line culture and transfection

HEK293T cells were cultured in DMEM high glucose (Life Technologies, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum at 37°C with 5% (v/v) CO2. Plasmid transfection was performed using either Lipofectamine LTX and Plus reagent (Invitrogen#15338, Grand Island, NY)

(if using 3 plasmids or more) or LipoD 293 DNA transfection reagent (SignaGen # SL100668,

Gaithersburg, MD) (2 plasmids or less), according to manufacturer’s instructions. Total DNA concentrations were normalized with empty vector DNA when required.

OPC transfections

Rat OPCs were collected after shaking and 5x106 cells were electroporated (Amaxa nucleofection apparatus) in 100μl Nucleofection solution (Amaxa basic glial cells nucleofector kit #VPI-1006, Lonza,

Mapleton, IL) with expression plasmids (5μg/transfection) or siRNAs (10 μl of 20 μM rat Olig1 siRNAs

(#L100044-01) or siControl non-targeting siRNA pool (#B002000-UB) from Dharmacon (Thermo

Scientific, Marietta, OH) After electroporation, cells were resuspended and seeded onto poly-D- lysine/Laminin (Sigma #I6758,St Louis, MO) coated dishes or round 12mm coverslips in DMEM supplemented with N2 (Life technologies#17502-048, Grand Island, NY), fibroblast growth factor(FGF, 10ng/ml) and platelet-derived growth factor(PDGF, 10ng/ml), and allowed to recover for

24h.

Expression and purification of recombinant TAT and GST fusion proteins

Plasmids for TAT-HA or GST fusion proteins were transformed into the Escherichia coli strain BL21

(DE3). Protein production was induced with 100 μM isopropyl β-d-1-thiogalactopyranoside (Sigma

#I6758,St Louis, MO) at 16 °C for 18 h, and protein was purified by TALON Metal Affinity Resin

(Clontech # 635501,Mountain View, CA) as described (Mi et al., 2012) or Glutathione-Superflow

Resin (Clontech # 635607,Mountain View, CA), respectively. The size and purity of all the proteins were confirmed by western blot using tag-specific antibodies and Olig1 specific antibodies. Apparent

103 degradation of Olig1 protein was noticed under different purification conditions, likely because Olig1 protein is relatively unstable (unpublished observation). After dialysis and filtration, proteins were aliquoted and stored at −80 °C.

In vitro histone acetyl transferase assay

Recombinant GST tagged human histone acetyl transferase (HAT) proteins were purchased from

SignalChem (Richmond, Canada): cAMP response element-binding protein [CREB]-binding protein

(CBP), #C07-31G, p300 (E1A-binding protein, 300 kDa; #P07-31G), and GCN5 (KAT2; #K311-381G).

Their HAT activity was validated using histone H3 1-21aa peptide as a substrate (data not shown). To perform the in vitro HAT peptide assay, varying amounts (0.1ng to 1μg) of non-acetylated peptide

(GAPGRKLSKIAT, Abgent) were incubated with recombinant HATs (CBP,500ng; p300,500ng;GCN5,200ng) or 200ng glutathione S-transferase (GST) in 20μl of acetyltransferase assay buffer (250 mM Tris-HCl [pH 8.0], 0.5mM EDTA and 2mM dithiothreitol), with 100μM acetyl-CoA

(Sigma#A2506) at RT for 1 hr. The reactions were spotted on polyvinylidene difluoride (PVDF) membrane to detect acetylation of Olig1 peptides with Acetyl-K150-Olig1 antibody. To perform the acetylation assay on Olig1 protein, 40pmol of purified Olig1-GST, Olig1-K150R-GST protein were incubated with recombinant HATs (CBP, 500ng; p300,500ng; GCN5,200ng) in 40μl of acetyltransferase assay buffer at RT for 1 hr. The reactions were stopped by adding 5X Laemmli buffer and boiling for 5min. The reaction product was analyzed by western blot and the acetylation of Olig1 protein was detected by either general acetyl-lysine antibody or Acetyl-K150-Olig1 antibody.

Western Blots and Immunoprecipitation

After transfection or treatment, cultured cells or brain tissue were lysed in RIPA buffer (25mM

Tris-HCl [pH 7.5], 150 mM NaCl; 1 mM EDTA, 1% NP-40, 0.1% DOC,0.1%SDS) supplemented with complete mini-protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitor cocktail set II (CalbioChem # 564652,Cambridge, MA). Samples were analyzed by western

104 blot and protein bands were detected with the LICOR Odyssey infrared scanner (Lincoln, NE).

Protein expression levels in scanned images were quantified using the Odyssey Scanner Software 2.0.

For immunoprecipitations, primary antibodies, or control mouse IgG (Invitrogen#10400C ,Grand

Island, NY) or rabbit IgG (Invitrogen#10500C, Grand Island, NY) were incubated with protein A/G agarose (Pierce#20421,Rockford, IL) or Dynabeads protein G (Invitrogen#10004D, Grand Island, NY) overnight. Antibodies were then cross-linked to beads using the Pierce immunoprecipitation kit

(Thermo Scientific#26147,Rockford, IL) using manufacturer’s instructions. Cell lysates were precleared by incubation with IgG beads for 2h; antibody-crosslinked beads were then added and samples incubated overnight at 4°C while rotating. Beads were washed 3 times in RIPA buffer wash, and then resuspended in 30 μl SDS gel sample buffer, boiled for 5 min, and subjected to SDS-PAGE, followed by western blot.

Immunohistochemistry and Immunocytochemistry

Immunohistochemistry was performed as described previously (Trapp et al., 1997). Free-floating cortex sections (30μm) were analyzed, with antigen retrieval in 10mM sodium citrate (pH6.0) at

65°C for 10 min as needed, using a Pelco Biowave Pro tissue processor (Ted Pella, Inc., Redding, CA).

For immunocytochemistry, oligodendrocytes were cultured on coverslips and fixed with 4% paraformaldehyde for 15 min at RT. Cells were permeabilized with 0.1% Triton X-100 for 10 min, blocked with 3% BSA in PBS for 60 min at RT, and incubated with primary antibodies overnight at

4°C. For detection of O4 cell surface antigens, O4 antibody was diluted with media and incubated with live cells on coverslips for 1 h prior to fixation.

Images were taken on a Zeiss Axio Imager M2 (Muchen, Germany). A total of 6 microscopic fields per coverslip from two coverslips per condition were sampled in each experiment. The total branch points/cell of O4+ cells was analyzed with Imaris filament tracer module (Bitplane, Zurich,

Switzerland).

105

Subcellular fractionation

Subcellular fractionation of cells was performed as described (MacDonald et al., 2004). Cultured cells or brain tissue were suspended in hypotonic buffer (10 mM HEPES 7.9, 10 mM KCl, 2 mM

MgCl2,0.5%NP40, 1 mM dithiothreitol with protease and phosphatase inhibitors). The cells were incubated on ice for 5 min, and cytoplasmic and nuclear fractions were harvested by centrifugation at 1,500 × g for 5 min. The isolated nuclei were then washed in hypotonic buffer, lysed in hypotonic buffer plus 500mM NaCl and incubated on ice for 30 min. The nuclear fraction was used for immunoprecipitations or western blots, or subsequent sub-nuclear fractionation was performed as described in (Niida et al., 2007). The isolated nuclei were then washed in hypotonic buffer, lysed in chromatin extraction buffer (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT), and incubated on ice for 30 min. The soluble nuclear and chromatin fractions were harvested by centrifugation at 1,500 × g for

15 min. The isolated chromatin was then washed in chromatin extraction buffer, centrifuged at

10,000 × g for 1 min, and resuspended in Laemmli sample buffer and boiled for 5min.

Histone association assay

Protein histone association was performed as described with slight modification (Ricke and

Bielinsky, 2005). Primary cultured oligodendrocytes were cross-linked with 1% formaldehyde for 15 min at RT. Reaction was quenched with 125 mM glycine for 5 min at RT. The cells were washed twice with ice-cold PBS, and lysed in buffer L1 (50mM HEPES pH7.5, 140mM NaCl, 1mM EDTA,

10%Glycerol, 0.5%NP40, 0.25% Triton X-100) on ice for 15min. The nuclear pellet was collected by centrifugation at 1,500 × g for 15 min. Then the nuclear pellet was resuspended in buffer L2 (10mM

Tris pH8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) and lysed at RT for 10 min. Pellets were concentrated by centrifugation at 1,500 × g for 15 min. Nuclear pellets were washed in Micrococcal

Nuclease digestion buffer (20mM Tris.HCl [pH7.5], 5mM NaCl and 2.5mM CaCl2) and then resuspended in Micrococcal Nuclease digestion buffer. The chromatin was released by digested with

106

20 U/ml Micrococcal Nuclease ( Takara#2910A, Mountain View, CA) at 37°C for 9min. Digestion was stopped by adding 0.5mM EDTA to a final concentration of 0.2mM on ice. The chromatin preparation was further diluted in chromatin immunoprecipitation (ChIP) lysis buffer ( 50mM

HEPES(pH7.5), 140mM NaCl, 1mM EDTA, 1% Triton X100 and 0.1% DOC) and precleared with 50μl

Dynabeads at 4°C for 1h. For immunoprecipitation, precleared chromatin and Olig1 antibody conjugated Dynabeads were incubated overnight on a rotator at 4°C. Beads were then washed two times in ChIP lysis buffer and two times in ChIP lysis buffer plus 500mM NaCl. Beads were resuspended in 1.5X Laemmli loading buffer with 100mM DTT, and reverse cross-linked by boiling for 30 min.

DNA Pulldown Assay

DNA pulldown assays were performed as described previously (Bujalka et al., 2013) with slight modifications. Briefly, the nuclear fraction was collected as described above, lysates were clarified by centrifugation at 15,000× g for 20 min at 4°C and adjusted to a total protein concentration of 1

µg/µl using buffer H (100mM KCl, 200mM HEPEs,PH7.8, 20% glycerol, 1mM DTT, 0.1% NP40). Five

µg biotinylated, annealed oligonucleotides (5’-BioTEG-AACAGCTGTGAACAGCTGTGAACAGCTGT-

GAACAGCTGTGAACAGCTGTGAACAGCTGTG-3’) were conjugated to the Steptavidin magnetic beads

(Roche#11636502103) following manufacturer’s instruction. The oligonucleotide-conjugated beads were washed twice with 500 µl buffer H and added to cell lysate in the presence of 2ug poly (dIdC)

(Sigma #4929). Beads were incubated with cell lysates overnight at 4°C with rotation and then washed three times in 500 µl buffer H. Bound protein was eluted by boiling the beads in 1.5×

Laemmli buffer and subject to western blot.

107

CHAPTER IV

CONCLUSION AND FUTURE DIRECTION

In the CNS, myelin provides many levels of support to axons, which makes the communication among neurons feasible and energetically affordable. One devastating feature of chronic multiple sclerosis (MS) is the progressive loss of remyelination capacity. This failure of myelin regeneration and consequent axonal loss are attributed to many different pathological obstacles. Despite the abundance of OPCs in many chronic inactive lesions in MS, the failure of OPC differentiation has been observed recently and attracts much attention. Developmental myelination and the adult regenerative process of remyelination share many common mechanisms, despite notable differences between these two processes. Transcriptional regulation of oligodendrocytes is one of the most intensively researched and well understood field of myelin biology, which has already shed much light on oligodendrocyte regeneration and myelin repair in demyelinating disease. Olig1 and

Olig2 sit in the center of this complex transcriptional regulatory network, with many of their downstream and upstream targets also critical for oligodendrocyte differentiation. The demonstration of an essential role of Olig1 in remyelination has evoked a lot of interest in developing cell based small molecule agonists of Olig1 to promote myelin repair. However, transcription factors in most cases are not ideal targets for designing drugs. And the exact role of

Olig1 in oligodendrocyte differentiation is not well characterized. The diverse phenotypes obtained in many different Olig1 knockout mice lines obscure the understanding of Olig1 function and regulation.

My thesis study started with characterizing the developmental phenotype of the original Olig1 knockout mice. This study unravels an unexpected brain specific role of Olig1 in oligodendrocyte development. In contrast to the proposed role of Olig1 in axon contact by one recent report(Xin et al., 2005), our Olig1 null mice exhibit a differentiation block at multiple stages of early OPC

108 development in brain, but not myelination initiation. Specifically, our study is the first to show that in the absence of Olig1, the oligodendrocyte differentiation is impaired at the pre-OPC to OPC commitment stage, early OPC cell cycle exit and it is also impaired at the stage transitioning to premyelinating oligodendrocytes right before wrapping the axons. This failure of differentiation at multiple steps along the oligodendrocyte lineage in Olig1 null mice recapitulates some features of the differentiation block in chronic MS lesions. Olig1 and Olig2 are highly conserved at the molecular and functional level. Accumulating evidence supports the idea that they may interact with each other to regulate OPC differentiation at multiple stages. Olig1 and Olig2 have been shown to be dys- regulated in MS lesions(Arnett et al., 2004; Kuhlmann et al., 2008). Manipulating Olig1 and Olig2 expression or activity may potentially promote remyelination in both active and chronic lesions as a proof of principle in animal demyelinating models(Mei et al., 2013; Wegener et al., 2015). However, it is difficult to target Olig1 and Olig2 molecularly due to the related nature of the transcription factors. Understanding the mechanism how the level and activity of Olig1 and Olig2 are developmentally regulated will have relevance for rational design of drugs to promote myelin repair.

In contrast to the constant expression and nuclear localization of Olig2 throughout oligodendrocyte development, subcellular translocation of Olig1 is observed during both oligodendrocyte development and remyelination. During remyelination, Olig1 relocates back to the nucleus, suggesting a nuclear function for Olig1 that appears essential for remyelination (Arnett et al., 2004). Dynamic expression and subcellular translocation of Olig1 was highly coordinated with oligodendrocyte lineage progression and myelination initiation. However, the mechanism regulating this intriguing Olig1 translocation has not been established. And the link between Olig1 subcellular location and function is not clear.

In the second part of my thesis, by comparing the sequence difference between Olig1 and Olig2,

I identified three functional nuclear export sequences (NES) localized in the bHLH domain of Olig1

109 with two of them not found in Olig2. Based on the fact that Olig1 appears in the nuclei of OPCs even when containing three NESs, some additional mechanism(s) probably exists to regulate the activity of these NESs. Post-translational modification of protein has been known to regulate the subcellular translocation of many transcription factors. Among them, the role of phosphorylation and acetylation in this process is well established. Using various kinase and acetyltransferase inhibitors to block the potential phosphorylation or acetylation events in Olig1, and a subsequent mutagenesis screen, I identified one lysine site, K150, whose acetylation is critical for exporting Olig1 from nucleus of OPC. I further confirmed acetylation of Olig1 in vivo by using an acetyl-specific antibody target acetyled-lysine 150. Using a candidate approach, the acetyltransferase, CREB binding protein

(CBP) and the deacetylases, HDAC1, 3 and 10 were found to be responsible for Olig1 acetylation and deacetylation respectively. The function of Olig1 acetylation was further investigated. And I found that acetylation of Olig1 decreased its chromatin association upon cell cycle exit of OPCs. This acetylation modifcation on Olig1 changes its binding preference for inhibitor of DNA binding (Id2) and facilitates Id2 retention in the cytoplasm of mature oligodendrocytes. I establish that acetylation of Olig1 regulates its chromatin dissociation and subsequent translocation to the cytoplasm. Olig1 acetylation is important for oligodendrocyte lineage progression and morphological differentiation. Acetylation-mediated regulation of Olig1 translocation and function may provide a new target to overcome differentiation failure and reduced myelin repair in chronic lesions of MS.

In summary, the demonstration of a brain-specific role of Olig1 in oligodendrocyte differentiation in my thesis reconciles the debate about a distinct function of Olig1 during remyelination vs. development. Consistent with a previous remyelination study (Arnett et al., 2004), the delayed but mild phenotype of spinal cord myelination was observed using the same Olig1 null mice. In spinal cord, it is conceivable that Olig1 has a mild effect during early development but a marked effect during remyelination. However, in brain Olig1 is required for the early

110 oligodendrocyte differentiation and myelination. The brain specific function of Olig1 in OPC differentiation raises several important questions. 1. Does this difference reflect the heterogeneity of oligodendrocytes in different regions? 2. Are Olig1 binding partners different between brain and spinal cord? 3. Does Olig1 have different gene targets in brain vs. spinal cord? 4. Is the function of

Olig1 differently regulated in brain vs spinal cord? In order to answer the listed questions, it will be a priority to identify the Olig1 genome binding sites using high throughput ChIP-seq(Yu et al., 2013) and determine the Olig1 interactome by combining Olig1 immunoprecipitation and high throughput mass spectroscopy(Meijer et al., 2014). The success of both approaches relies on high quality antibodies raised against Olig1. Several antibodies were generated in this study targeting either N- terminal or C-terminal of Olig1, and we found the Olig1 antibodies raised against its C-terminal had better performance on ChIP and IP. Importantly, the function of Olig1 cytoplasmic retention in myelinating oligodendrocytes is still obscure at this point. Thus, identification of the Olig1 binding proteins in the cytoplasm will provide invaluable information about its cytoplasmic function.

Interestingly, I also observed the downregulation of Olig1 protein in premyelinating cells.

Preliminary data suggest Olig1 is an unstable protein, at least in cultured oligodendrocytes, it undergoes constant degradation. These results suggest Olig1 downregulation can be an actively regulated process. I hypothesize that the continued presence of Olig1 in the nucleus during and after the premyelinating stage might have an inhibitory role in the myelination initiation process, in contrast to its positive role in early OPC differentiation. Given that it reappears in the cytoplasm of myelinating oligodendrocytes, OIig1 may have an active function in cytoplasm. However, an intriguing alternative explanation is that Olig1 is actively retained in the cytoplasm to prevent its reentry into the nucleus during myelinogenesis where it may play an inhibitory role. The finely tuned regulation of Olig1 level and localization may be controlled by signaling from axons, and dysregulation of this process may underlie the myelin initiation deficits observed in chronic lesions

111 of MS. It will be important to determine the mechanism responsible for Olig1 degradation and test whether the continued presence of Olig1 in the nucleus of myelinating oligodendrocytes will be detrimental.

112

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APPENDIX

A. Validation of rabbit anti-Olig1 antibody.

(a) GFP tagged human Olig1, Olig2 and Olig3 were transfected into HEK293T cells, and 48h later the cells were lysed and protein expression were detected by GFP and Olig1-1 or Olig1-2 antibodies. Both Olig1-1 and Olig1-2 specifically detected the Olig1-GFP protein but not Olig2-GFP or Olig3-GFP protein. Based on the specificity and low background of Olig1-1 antibody, it was used for Western Blot to detect overexpressed Olig1 protein. Whithe asterisk indicates the Olig1-GFP protein. (b) Mouse anti-Olig1 (E12) antibody from Santa cruz and rabbit Olig1-1 and Olig1-2 antibodies were used for immunoprecipitating the endogenous Olig1 from 2 month old mice cortex lysate. All of them were able to pull down the endogenous Olig1. Olig1-2 did not pull down any Olig1 from the kidney which noramlly does not express Olig1, indicating its specificity. Both mouse Olig1(E12) and rabbit Olig1-2 antibodies were used for immunoprecipitation.

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B. Oligodendrocyte lineage markers.

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