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The splicing error of FOXP1 in type I myotonic dystrophy

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The splicing error of FOXP1 in type I myotonic dystrophy

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THE SPLICING ERROR OF FOXP1 IN TYPE I MYOTONIC DYSTROPHY

by

Xinyi Wang

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
MOLECULAR MICROBIOLOGY AND IMMUNOLOGY

August 2020

Copyright 2020 Xinyi Wang
ii

ACKNOWLEDGEMENTS
I would like to express the deepest appreciation to my committee chair, Professor Lucio Comai,
for his instructive advice and useful suggestions not only on my project but also on my thesis. I
am deeply grateful for his help in my research.
I would like to thank my committee members, Professor Sita Reddy and Professor Weiming Yuan
for generously offering their time, support, guidance, and goodwill during the preparation and
review of the dissertation.
I am deeply grateful to Dr. Jongkyu Choi for his great help in my research. I would express my
gratitude to all lab members: Xiandu Li, Nicole Friedlich, Se Jung Lee and Chenyu Zhou, with
whom I have shared moments of anxiety and also of big excitement.
I owe my sincere gratitude to my beloved parents for their love and support throughout my life. I
would also like to give my thanks to all my friends for their love and help in the past two years.

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TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABSTRACT vii
CHAPTER 1. Introduction 1
Symptoms of Myotonic Dystrophy 1
1.1.1 Muscle Abnormalities 1
1.1.2 Non-Muscle Abnormalities 3
Epidemiology 5
Pathogenic Mechanisms of Myotonic Dystrophy 6
1.3.1 The Genetics of Myotonic Dystrophy Type 1 6
1.3.2 RNA Pathogenesis Theory of Myotonic Dystrophy Type 1 7
1.3.3 Lowered DMPK Level Model of Myotonic Dystrophy Type 1 8
1.3.4 The Cis-Effect of Expanded CTG Repeats 10
1.3.5 Myotonic Dystrophy Type 2 11
Muscleblind-like Protein (MBNL) in Myotonic Dystrophy 12
Forkhead Box Protein P1 (FOXP1) 13
Summary 14
CHAPTER 2. Materials and Methods 15
Molecular Cloning 15
2.1.1 Plasmid Constructs 15
2.1.2 Plasmid Maps pCMV10-mFoxP1 and pCMV10-mFoxP1-ES 15
2.1.3 Gene Cloning Process 15
2.1.4 PCR Program 16
2.1.5 Gel Purification / Gel Extraction 16
2.1.6 Ligation 17
2.1.7 LR Reaction 17
2.1.8 Bacterial Host Transformation 18
2.1.9 Plasmid Isolation 18
2.1.10 Insert Confirming Process 19
Lentivirus Production 19
Lentiviral Infection and Selection 20
Western Blot Technique 20
2.4.1 Preparation of Lysates from Cell Culture 20
2.4.2 Protein Separation by Gel Electrophoresis 21
2.4.3 Transferring the Protein to the Membrane 21
2.4.4 Antibody Incubation 21
2.4.5 ECL Development 21
C2C12 Cell Culture and Differentiation Treatment 22
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2.5.1 Cell Culture for Cycling 22
2.5.2 Cell Differentiation Treatment 22
DAPI Staining 23
CHAPTER 3. Results and Discussion 24
Expression of Foxp1 and Foxp1-ES Plasmid in C2C12 Cells 24
Detection of Morphological Changes During Cell Differentiation 25
The Detection of OCT4 and NANOG in C2C12 cells expressing Foxp1-ES 30
Quantitative Analysis of C2C12 Cell Differentiation 31
3.4.1 Cell Fixation and DAPI Staining 31
3.4.2 Fusion Index Calculating 32
Future Plan: Western Blot Analysis 33
Conclusion 33
REFERENCES 36

v

LIST OF TABLES
Table 1 Calculation of the fusion index 32

vi

LIST OF FIGURES
Figure 1 pCMV10-mFoxP1 and pCMV10-mFoxP1-ES Plasmid map 15
Figure 2 The expression of FLAG-tagged Foxp1-pLenti CMV Puro
DEST and FLAG-tagged Foxp1-ES-pLenti CMV Puro DEST
in C2C12 cells
25
Figure 3 Gross changes in cell morphology in response to
differentiation medium and expression of different proteins
27
Figure 4 SDS-PAGE analysis and Coomassie staining 30
Figure 5 DAPI staining 31

vii

ABSTRACT
Myotonic dystrophy (DM) is a genetic disorder that affects the normal function of muscles. There
are two types of DM, DM1 and 2, which are caused by mutations in distinct genes (DMPK or
ZNK9) but share common symptoms and pathogenic mechanisms. There are several models to
explain the mechanism of myotonic dystrophy type 1. According to the RNA pathogenesis model,
the expanded CUG or CCUG repeats sequester the Muscleblind-like (MBNL) family of proteins,
which results in the loss of normal functioning MBNL. MBNL proteins are key regulators of
mRNA metabolism in mammals. The sequestration of MBNL proteins leads to mRNA splicing
aberrations. Foxp1, a gene encodes Forkhead box protein P1, is regulated by MBNL. FOXP1 is a
transcription factor that regulates a large number of genes involved in cell differentiation.
Alternative splicing may change the DNA-binding preference of the FOXP1. The embryonic stem
cell (ESC)-specific isoform of FOXP1, which is called FOXP1-ES, stimulates the expression of
many genes, including pluripotency related genes like OCT4 and NANOG. This FOXP1 isoform
may contribute to the reprogramming of differentiated cells. Here, I seek to gain an understanding
of the role of FOXP1 splicing aberration in myotonic dystrophy. My results suggest that FOXP1
may promote the differentiation of mouse myoblast cells, and FOXP1-ES may prevent the
differentiation of mouse myoblast cells. These results reveal that type 1 myotonic dystrophy may
be related to the ESC specific alternative splicing of Foxp1, which is caused by the sequestration
of MBNL1.

1
CHAPTER 1. INTRODUCTION
Myotonic dystrophy is a genetic disorder that affects the function of many organs including
muscle, heart and brain. It was first described by Steinert (Pilz et al., 1974) and Batten and Gibb
(Batten and Gibb, 1909), and became known as myotonic dystrophy, Steinert's disease, dystrophia
myotonica, or myotonica atrophica (Hilton-Jones, 2002). Myotonic dystrophy is a multi-systemic
disorder. It is also the most common form of muscular dystrophy and affects more than 1 in 8,000
people worldwide.
Symptoms of Myotonic Dystrophy
1.1.1 Muscle Abnormalities
One of the most common clinical features of myotonic dystrophy is myotonia. Symptoms also
include gradually worsening muscle loss and weakness. Some patients also suffer from muscle
atrophy and hypertrophy. There are two main types of myotonic dystrophy, type 1 and type 2,
which are caused by an expansion of a trinucleotide repeat (CTG) or tetranucleotide repeat (CCTG)
repeat in the DMPK or ZNF9 genes, respectively. Classical features of myotonic dystrophy are
found in both types, though some symptoms are found only in one type and absent in another.
1.1.1.1 Myotonia
Myotonia is a symptom of a small handful of certain neuromuscular disorders characterized by
delayed relaxation (prolonged contraction) of the skeletal muscles after voluntary contraction or
electrical stimulation (Gutmann and Phillips, 1991). It is thought to be
2
due to increased excitability of muscle fibers, leading to the discharge of repetitive action
potentials in response to stimulation (Hudson et al., 1995). In myotonic dystrophy type 1, myotonia
can be easily induced by making a tight fist or by direct percussion of the thenar eminence in the
hands (Ashizawa and Harper, 2006), but it is also present in many other muscles, including forearm
extensors, facial muscles, jaw or even in the tongue (Hahn and Salajegheh, 2016). Myotonia in
myotonic dystrophy type 1 shows the warm-up phenomenon, in which repeated contraction of the
muscle gradually alleviates the severity of myotonia (Ashizawa and Harper, 2006).
Symptoms of myotonic dystrophy type 2 are similar to those of myotonic dystrophy type 1 but
milder (Gadalla et al., 2011). Clinical myotonia is often absent, and type 2 patients can
occasionally present with asymptomatic hyperkalemia (Hahn and Salajegheh, 2016). Thus,
myotonic dystrophy type 2 is also more difficult to detect. (Dalton et al., 2006).
1.1.1.2 Muscle Weakness
One of the most conspicuous features of myotonic dystrophy is muscle weakness (Harper, 2009).
Myotonic dystrophy type 1 can cause muscle weakness and wasting in a characteristic distribution,
such as distal limb muscles, respiratory muscles, trunk muscles, and facial muscles. Myotonic
dystrophy type 1 patients have unique facial features because all cranial muscles are potentially
affected, producing a characteristic appearance of ptosis (Thornton, 2014). However, there is much
less cranial and respiratory muscle weakness in myotonic dystrophy type 2 compared with type
1(Thornton, 2014). Weakness is seen in the elbow extensors and the hip flexors and extensors
(Dalton et al., 2006). Early muscle weakness usually presents in neck flexors and finger flexors,
and later weakness often involving hip-girdle muscles. Muscle wasting is less pronounced in
Myotonic Dystrophy type 2; instead, some patients show calf and thigh muscle hypertrophy.
3
1.1.1.3 Muscle Atrophy and Hypertrophy
Atrophy and hypertrophy may be observed during muscle pathological examinations in patients
with myotonic dystrophy (Liu et al., 2015). Selective atrophy of type 1 fibers (slow-twitch muscle
fibers) is a common feature in individuals with type 1 myotonic dystrophy, while preferential type
2 fiber atrophy has been observed in individuals with type 2 myotonic dystrophy (Vihola et al.,
2003). Muscle atrophy is normally milder in type 2 myotonic dystrophy. However, muscle
hypertrophy is more often observed in type 2 patients. Myotonic dystrophy type 2 patients develop
significant enlargement of their calves (Meola and Moxley, 2004) and the hypertrophy of type 2
fibers in biceps is also evident (Vihola et al., 2003).
1.1.2 Non-Muscle Abnormalities
Multisystemic features of myotonic dystrophy including myopathy and non-muscle abnormalities.
Both type 1 and type 2 disorders have autosomal dominant inheritance and multisystem
phenotypes (Thornton, 2014).
1.1.2.1 Ocular Disease
Cataracts are ocular diseases that make the eyesight worse, in which the lens of the eye becomes
cloudy. The development of cataracts relatively early in life is a characteristic of myotonic
dystrophy, in both type 1 and type 2 (Norwood et al., 2009). Cataracts before the age of 55 or a
family history of premature cataracts suggest that patients with muscle symptoms may have
myotonic dystrophy (Thornton, 2014). Christmas tree cataract, also called polychromatic cataract,
is a cataract which forms shiny, colored crystals in the lens. This type of cataract is a typical non-
muscle phenotype of myotonic dystrophy.
4
1.1.2.2 Cardiac Disease
Systemic features of myotonic dystrophy include cardiac disease, such as cardiac conduction
defect and cardiomyopathy (Lund et al., 2014). Arrhythmia, especially cardiac conduction defect,
is the second leading cause of death after respiratory failure in patients with myotonic dystrophy.
The effect of myotonic dystrophy type 1 on the heart lies primarily in the conduction system. In
contrast, few studies have examined the cardiac effects of myotonic dystrophy type 2 patients. One
study found that the frequency of conduction disease was lower in type 2 than in type 1 (Wahbi et
al., 2009), but the left ventricular systolic dysfunction was more common (Bhakta et al., 2010).
Thus, both myotonic dystrophy type 1 and type 2 are associated with a high risk of sudden death.
1.1.2.3 Central Nervous System
The central nervous system features of myotonic dystrophy vary from patient to patient, but they
can share some common symptoms and signs. Myotonic dystrophy type 1 is commonly connected
with behavioral effects such as anxiety or avoidant behavior, and cognitive abnormalities
(Thornton, 2014). Besides, patients may suffer from sleep disturbance, such as daytime
hypersomnolence, sleep apneas, and periodic leg movements during sleep (Gourdon and Meola,
2017). Rapid eye movement sleep deregulation and longer habitual nocturnal sleep are also found
in myotonic dystrophy type 1 patients (Laberge et al., 2009) (Heatwole et al., 2012). The cognitive
abnormalities in myotonic dystrophy type 2 patients can involve visuospatial dysfunctions and
executive dysfunctions, and memory impairments in severe phenotypes (Gourdon and Meola,
2017). Lack of executive function is reported in many type 2 patients, which reduces initiative,
planning abilities, and decision making, leading to apathy and inactivity as well (Gourdon and
5
Meola, 2017). Myotonic dystrophy type 2 patients have milder central nervous system phenotype
in comparison to type 1 patients. The pathomolecular mechanism of this remains unknown.
1.1.2.4 Endocrine Abnormality
Patients with myotonic dystrophy have a chance of having diabetes, androgen deficiency,
hyperparathyroidism and adrenocortical dysfunction (Jenkins, 2015). Besides, painful or irregular
menses occurs in both type 1 and type 2 female patients. Myotonic dystrophy type 1 male patients
may suffer from erectile dysfunction, while myotonic dystrophy type 2 patients may have
fluctuating levels of pain and fatigue.
1.1.2.5 Gastrointestinal Symptoms
Gastrointestinal symptoms are very common in myotonic dystrophy type 1 patients (Thornton,
2014), and they may be the initial symptom in myotonic dystrophy (Rönnblom et al., 1996). Both
myotonic dystrophy type 1 and type 2 patients may have problems with chewing or swallowing,
gastroesophageal reflux, abdominal pain, irritable bowel syndrome, and diarrhea.
Gastroesophageal reflux may cause dysphagia and chest pain in myotonic dystrophy type 1
patients.
Epidemiology
Myotonic dystrophy is the most common adult-onset muscular dystrophy with an estimated
prevalence of 1 in 8,000 (Suominen et al., 2011). The prevalence of myotonic dystrophy type 1
ranging from 1 in 100,000 in Japan to 3-15 in 100,000 in Europe (Turner and Hilton-Jones, 2010),
and maybe as high as 1 in 500 in Quebec, Canada (Mathieu et al., 1990). There are fewer myotonic
dystrophy type 2 epidemiological studies. Type 1 myotonic dystrophy is thought to be more
6
common than type 2, but recent research suggested that type 2 may be as common as type 1 in
Finland (Suominen et al., 2011). In the United States, clinical experience shows that myotonic
dystrophy type 2 is five times less common than myotonic dystrophy type 1 (Thornton, 2014).
Pathogenic Mechanisms of Myotonic Dystrophy
The pathogenesis of type 1 and type 2 myotonic dystrophy is based primarily on RNA toxicity
(Ashizawa and Harper, 2006). Myotonic dystrophy type 1 is caused by a tri-nucleotide expansion
(CTG) in the 3’ non-coding region of DMPK (myotonic dystrophy protein kinase gene), and
myotonic dystrophy type 2 is caused by the expansion of a tetranucleotide (CCTG) in intron 1 of
the ZNF9 gene (Lee and Cooper, 2009).
1.3.1 The Genetics of Myotonic Dystrophy Type 1
Myotonic dystrophy type 1 (DM1) is caused by the mutation in the myotonic dystrophy protein
kinase (DMPK) gene, located on chromosome 19 (19q13.2–q13.3) (Zinkernagel et al., 2012). The
CTG trinucleotide repeat expansion is located in the non-coding region of DMPK, therefore it does
not affect the protein-coding region. There is little evidence that the repeat sequence blocks the
synthesis or processing of DMPK RNA even with extremely large repeat expansions (Thornton,
2014). However, these expanded CTG repeats are proposed to disrupt normal cellular processes at
the RNA, protein or chromatin level (Cho and Tapscott, 2007).
There are several theories to explain the mechanism of DM1. Among them, a hypothesis has been
strongly corroborated that myotonic dystrophy is primarily an RNA-mediated disease. In addition
to the RNA pathogenesis theory, there are two other major models. One proposes the effects of
7
lowered DMPK levels in causing myotonic dystrophy, and the other model explains the cis effects
of DMPK, which affects the neighboring genes that cause myotonic dystrophy type 1.
1.3.2 RNA Pathogenesis Theory of Myotonic Dystrophy Type 1
The repeat expansion in DMPK is located in non-coding genomic segments. RNA pathogenesis
theory states that the DMPK (CUG)n and also the ZNF9 (CCUG)n repeat (in myotonic dystrophy
type 2) results in a generalized RNA metabolic defect. Initial studies demonstrated that myotonic
dystrophy type 1 cells are associated with multiple nuclear foci of mutant DMPK RNA (Sonnhof
et al., 1975). These unusual transcripts are not exported to the cytoplasm but are retained in the
nucleus instead (Thornton, 2014). The discrete clumps or foci of the mutant RNA were first
revealed by staining tissue with probes that hybridize to the repeat sequence(Thornton, 2014), and
were found most conspicuous in cells with large expansions and high levels of DMPK expression,
such as muscle fibers, smooth muscle cells, cardiomyocytes, and neurons(Mankodi et al., 2005,
Jiang et al., 2004, Mankodi, 2001). The nuclear retention has a toxic gain-of-function effect (Davis
et al., 1997) (Wang et al., 1995), which disrupting RNA splicing and other normal cell functions.
Proteins in the muscleblind-like (MBNL) protein family bind to the CUG repeat RNA sequences
with high affinity (Miller, 2000 This protein family members normally serve as splicing regulators
of many transcripts and also play a role in regulating other aspects of RNA metabolism (Thornton,
2014). MBNL proteins bind to the nuclear CUG repeat RNA and are trapped in nuclear foci.
Therefore, their normal functions are severely affected. As the level of normal regulators declines,
erroneous splicing events occur and abnormal protein isoforms are expressed. For example, Rahul
N. Kanadia and colleagues reported that disruption of the mouse Mbnl1 gene leads to the ClC-1
chloride channel splicing abnormalities, which are characteristics of myotonic dystrophy
(Kanadia, 2003). Mis-splicing of the ClC-1 chloride channel results in reduced chloride
8
conductance in muscle fibers (Thornton, 2014), leading to myotonia. Other splicing defects, such
as insulin receptor, BIN1, dystrophin, and L-type calcium channels, can also cause and myopathy
(Nakamori et al., 2013) (Savkur et al., 2001) (Fugier et al., 2011) (Tang et al., 2012).
1.3.3 Lowered DMPK Level Model of Myotonic Dystrophy Type 1
DMPK gene encodes a serine/threonine protein kinase (Amano et al., 1999). The most abundant
isoform of DMPK is an 80 kDa protein expressed almost exclusively in smooth, skeletal, and
cardiac muscles (Lam, 2000). The specific function of this protein remains unknown, but it may
involve in communication within cells and may play an important part in muscle, heart and brain
cells.
The CTG repeat sequence expansion disrupts the normal RNA cellular processes, therefore the
mutant DMPK transcripts are retained in the nucleus and not efficiently translated. This leads to a
partial reduction in DMPK protein levels. The dysfunction or reduction of DMPK may contribute
to the pathogenesis of myotonic dystrophy type 1.
1.3.3.1 DMPK and Skeletal Muscle Defects
Initial studies of myotonic dystrophy have focused on the role of the DMPK protein since the
decrease in cytoplasmic DMPK was observed in myotonic dystrophy type 1 patients.
Studies in mice have suggested that the loss of Dmpk expression leads to muscle weakness and
mild myopathy (Reddy et al., 1996) (Jansen et al., 1996). Dmpk homozygous knockout mice
developed a progressive myopathy, which is milder than the muscular phenotype observed in
myotonic dystrophy type 1 patients. Besides, histological changes are also observed in mature
mouse muscles, such as increased fiber degeneration and variation in fiber size (Reddy et al.,
9
1996). On the other hand, overexpression of DMPK in mouse myogenic C2C12 cell line inhibits
the terminal differentiation (Okoli et al., 1998). These studies suggested that the level of DMPK
protein is related to the proper structure and function of skeletal muscle.
1.3.3.2 DMPK and Cardiac Abnormalities
Cardiovascular disease is one of the most prevalent causes of death in DM patients (Kaliman and
Llagostera, 2008). DMPK is thought to play a role in myotonic dystrophy cardiac conduction
defects. Electrophysiological studies of skeletal and cardiac muscles in myotonic dystrophy
patients and in Dmpk homozygous knockout mice have shown defects in intracellular Ca2 +
cycling (Pall et al., 2003). In addition, Dmpk homozygous knockout mice exhibited a cardiac
phenotype that reproduces many of the cardiac conduction defects observed in DM patients,
including varying degrees of atrioventricular block (Berul et al., 1999) (Kaliman and Llagostera,
2008). Heterozygous Dmpk mice also had a conduction defect, which was similar to the cardiac
phenotype in myotonic dystrophy patients (Berul et al., 1999).
1.3.3.3 DMPK and Neurons
Myotonic dystrophy type 1 is also a brain disorder that can cause cognitive and behavioral
abnormalities. DMPK expression is detectable in neurons and astrocytes. Studies have shown that
DMPK plays a role in synaptic plasticity, which means DMPK may be relevant to the cognitive
dysfunction associated with myotonic dystrophy (Schulz et al., 2003). Later studies have suggested
that DMPK expression in astrocytes increases during terminal brain development (Oude Ophuis
et al., 2009).
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1.3.4 The Cis-Effect of Expanded CTG Repeats
RNA pathogenesis theory is about the gain of toxic function caused by the expanded CUG repeat,
while the Cis-effect model focuses on the loss of function of related genes.
The CTG tri-nucleotide expansion in DMPK non-coding region might influence the expression of
adjacent genes: the mutation leads to the loss of function of genes near the CTG repeats, including
DMWD and SIX-5.
Researches show that adjacent chromatin structure is altered due to the CTG expansion, which
affects the transcription of vicinal genes. A nuclease resistant region was found downstream of the
CTG triplet expansion, indicating the chromatin region is condensed. (Otten and Tapscott, 1995).
SIX-5 gene is a homeodomain coding gene located in this chromatin region. SIX-5, also known as
myotonic dystrophy locus-associated homeodomain protein (DMAHP), is part of the SIX gene
family. SIX proteins are transcription factors that regulate genes related to normal development.
The mRNA level of SIX-5 is decreased in DM1 patients, reported by Thornton and colleagues
(Thornton et al., 1997). Klesert and colleagues found that the SIX-5 knockout mice developed
premature cataracts (Klesert et al., 2000). Personius and colleagues found that the loss of SIX-5
resulted in testicular atrophy and cardiac conduction defects (Personius et al., 2005). Therefore,
some features related to myotonic dystrophy type 1 may be caused by CTG expansion-induced
alterations in chromatin organization (Cho and Tapscott, 2007).
The DMWD gene is located upstream of DMPK and encodes the Dystrophia myotonica WD
repeat-containing (DMWD) protein. Since the CTG expansion repeats prefer to associate with
histones at the nucleosomal dyad (Godde and Wolffe, 1996), protein expression of genes adjacent
11
to DMPK may be affected. Alwazzan and colleagues reported that the level of the myotonic
dystrophy associated DMWD allele in the cytoplasm of myotonic dystrophy cell lines was reduced
by 20–50% compared with the wild type DMWD allele (Alwazzan et al., 1999). DMWD expresses
predominantly in the brain and testis (Alwazzan et al., 1999), thus it may be responsible for the
testicular atrophy and mental deficiency in myotonic dystrophy patients.
1.3.5 Myotonic Dystrophy Type 2
Myotonic dystrophy type 2 was previously named “proximal Myotonic Myopathy” or “PROMM”.
The clinical phenotype of myotonic dystrophy type 2 is similar to myotonic dystrophy type 1,
though the conditions of type 2 are generally milder. The similarity suggests that myotonic
dystrophy type 1 and type 2 may share some common pathogenic mechanisms.
Myotonic dystrophy type 2 (DM2) results from unstable CCTG repeats within intron 1 of Zinc
Finger Protein 9 (ZNF9), which locates in chromosome 3q21 (Cho and Tapscott, 2007). Like
myotonic dystrophy type 1, the CCUG repeat expansion in RNA also forms nuclear foci and
sequesters muscleblind-like (MBNL) proteins (Liquori, 2001). Intranuclear RNA foci containing
CCUG repeat expansion and MBNL proteins are detected in myotonic dystrophy type 2 cells
(Mannkodi et al., 2001). These studies suggest that the RNA pathogenesis model may be
applicable to myotonic dystrophy type 2.
Despite the largely similar clinical syndromes, there is an important phenotypic difference between
myotonic dystrophy type 1 and type 2. Only myotonic dystrophy type 1 has a congenital form.
Therefore, due to the lack of myotonic dystrophy type 2 congenital phenotype, further research
and new models are needed.
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Muscleblind-like Protein (MBNL) in Myotonic Dystrophy
Muscleblind-like (MBNL) proteins are key regulators of mRNA metabolism in mammals
(Konieczny et al., 2014). As discussed above, proteins in the muscleblind-like (MBNL) protein
family bind to the CUG and CCUG repeat RNA sequences with high affinity (Miller, 2000) and
specificity (Warf and Berglund, 2007). When MBNL proteins bind tightly to CUG or CCUG
repeats and are trapped in the nucleus, they are prevented to perform their normal functions.
Muscleblind-like proteins (MBNL) are encoded by three genes: MBNL1, MBNL2, and MBNL3
(Fardaei, 2002). All three family members have similar structures, among which the four zinc-
finger (ZnF) domains are essential for identifying mRNA targets (Konieczny et al., 2014). MBNL1
and MBNL2 are ubiquitously expressed, and they have compensatory roles. MBNL1 is expressed
in most tissue except the brain, where MBNL2 is predominantly expressed (Konieczny et al.,
2014). The human MBNL3 is only predominantly expressed in the placenta, and the mouse Mbnl3
is expressed at a low level in all tissues (Kanadia, 2003).
Researches show that disruption of the mouse Mbnl1 gene leads to myotonia, cataracts, and RNA
splicing abnormalities (Kanadia, 2003). These diseases are the characteristics of myotonic
dystrophy. As discussed above, CUG or CCUG repeat expansion may sequester the MBNL
proteins, these experimental results indicate that myotonic dystrophy may be caused by MBNL
protein sequestration.
Human MBNL1 is an alternative splicing regulator with dual functions, serving as both a repressor
and an activator for muscle differentiation (Teplova and Patel, 2008). The sequestration of human
MBNL1 leads to RNA-splicing defects related to myopathy (Yadava et al., 2019).
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Forkhead Box Protein P1 (FOXP1)
As discussed above, MBNL proteins are alternative splicing regulators that can function as
repressors or activators of splice sites. Studies have shown that MBNL 1 and MBNL2 are direct
negative regulators of many alternative splicing events related to embryonic stem (ES) cell
differentiation (Han et al., 2013). Han and colleagues reported that the knockdown of MBNL
proteins in differentiated cells resulted in ES-cell-like alternative splicing, while overexpressing
MBNL proteins in ES cells lead to differentiated-cell-like alternative splicing (Han et al., 2013).
They also reported another striking discovery that the knockdown of a protein called forkhead box
protein P1 (FOXP1) enhanced the expression of pluripotency genes and the formation of induced
pluripotent stem cells (iPSCs) (Han et al., 2013). Interestingly, MBNL regulates the splice site
choices of Foxp1 transcripts. FOXP1 has the ES-cell-specific alternative splicing switch that
controls pluripotency.
Forkhead box protein P1 (FOXP1) is encoded by the FOXP1 gene in humans. It contains a C-
terminal forkhead domain, with N-terminal zinc finger and leucine zipper domains (Gabut et al.,
2011). The forkhead family members are transcription factors that recognize particular DNA
sequences through the C-terminal forkhead domain. FOXP1 is a member of the forkhead box P
(FOXP) subfamily and is widely expressed (Gabut et al., 2011) in humans.
FOXP1 has several splice variants of unknown function (Brown et al., 2008). Dasen and colleagues
found that the knockout of Foxp1 in mice disrupted the establishment of specific cell types (Dasen
et al., 2008). Gabut and colleagues identified a conserved alternative splicing event in FOXP1
transcripts that are only activated in embryonic stem cells (Gabut et al., 2011). The alternative
splicing event changes one exon, which is located in the C-terminal forkhead domain (Graveley,
14
2011). This finding suggests that the alternative splicing alters the DNA-binding specificity of
FOXP1, and switches the transcriptional output (Gabut et al., 2011). The ES-cell-specific
alternative splicing isoform is called FOXP1-ES. FOXP1-ES activates pluripotency genes such as
OCT4 and NANOG, and represses genes related to cell-lineage specification and differentiation
(Gabut et al., 2011).
Summary
In summary, myotonic dystrophy is a genetic disorder that affects the normal function of muscles
and other organs. Myotonic dystrophy type 1 and type 2 share common symptoms and pathogenic
mechanisms, although there are differences in the severity of these two diseases. There are several
models to explain the mechanism of myotonic dystrophy type 1, including the RNA pathogenesis
model, the cis-effect model and the reduced DMPK levels level model. According to the RNA
pathogenesis model, the expanded repeats RNAs sequester the MBNL family of proteins, which
result in the loss of their normal function. The sequestration of MBNL proteins leads to the mis-
splicing of many transcripts including FOXP1, which is thought to enhance the expression of
pluripotency genes in differentiated cells. My project seeks to understand the role of FOXP1 in
myotonic dystrophy.
15
CHAPTER 2. MATERIALS AND METHODS
Molecular Cloning
2.1.1 Plasmid Constructs
Plasmid constructs of Foxp1 and Foxp1-ES were generated by the following methods. The
cDNA sequence information for Foxp1 was obtained from the National Center for Biotechnology
Information (NCBI) DNA database. All enzymes for molecular cloning were purchased from NEB
unless otherwise indicated.
2.1.2 Plasmid Maps pCMV10-mFoxP1 and pCMV10-mFoxP1-ES

2.1.3 Gene Cloning Process
PCR (Polymerase chain reaction) was used to amplify the Foxp1 and Foxp1-ES sequences. The
following PCR program was used as the first step to construct Foxp1 and Foxp1-ES plasmids.
Figure 1. Plasmids purchased from Addgene. pCMV10-mFoxP1: Plasmid map size:
8515 bp. Backbone size without insert: 6400 bp. Insert size: 2115 bp. pCMV10-
mFoxP1-ES: Plasmid map size: 8523 bp. Backbone size without insert: 6400 bp.
Insert size: 2163 bp.
16
2.1.4 PCR Program
Step 1: 94 °C, 2 min
Step 2: 94 °C, 10 sec
Step 3, 60 °C, 30 sec
Step 4, 68 °C, 2 min 30 sec
Step 5, repeat steps 2-4 for 35 times
Step 6, 72 °C, 5 min
Step 7, hold at 10 °C
Foxp1 and Foxp1-ES were purified by 0.8% Agarose gel after PCR and were extracted to insert
into pENTR Dual Selection Vector.
2.1.5 Gel Purification / Gel Extraction
PCR products were gel-purified after digesting by restriction enzyme, to separate the gene of
interest from other fragments. The following procedure was the gel extraction kit protocol.
Excise the DNA fragment from the agarose gel (0.8% Agarose) with a clean, sharp scalpel. Weigh
the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel. Incubate the
mixture at 50 °C for 10 min and mix by vortexing the tube every 2 min during the incubation.
After the gel slice has dissolved completely, add 1 gel volume of isopropanol to the sample and
mix by inverting the tube several times. Place a spin column in a provided collection tube. To bind
DNA, pipet the sample into a column and centrifuge for 1 min. discard the flow-through and place
the column back in the same collection tube. Add 0.5 ml of buffer QG to column and centrifuge
17
for 1 min. Add 0.75 m; of buffer PE to column and centrifuge for 1 min. Discard the flow-through
and centrifuge the column for an additional 1 min at 13,000 rpm. Place the column into a clean 1.5
ml microcentrifuge tube. To elute DNA, add 30 μl of Buffer EB (10 mM Tris-Cl, pH 8.5) or water
(pH 7.0-8.5) to the center of the membrane, let the column stand for 1 min, and then centrifuge for
1 min.
This gel purification procedure was used to purify PCR products and empty vectors after restriction
enzyme digestions.
2.1.6 Ligation
The following procedure was the ligation method used for both Foxp1 and Foxp1-ES plasmids.
For ligation, insert DNA (100-130 ng) was added to 200 ng pENTR Dual Selection Vector (1:1)
in a 16 μl reaction mixture containing 2X ligation buffer (60mM Tris-HCl, pH 7.8, 20mM MgCl2,
20mM Dithiothreitol, 2mM ATP and 10% Polyethylene glycol) and T4 ligase. The mixtures were
then incubated at 16 °C overnight in a dry bath. This procedure was repeated with control groups
with only vector, T4 ligase, and the buffer.
2.1.7 LR Reaction
This procedure was to transfer the gene of interest from the Gateway entry clone pENTR Dual
Selection Vector to the destination vector pLenti CMV Puro DEST.
1 μl entry clone, 1 μl destination vector, and 8 μl TE Buffer were added to a clean tube at room
temperature and mixed. The LR Clonase II enzyme mix (purchased from Invitrogen) was thawed
on ice for 2 min and was vortexed briefly. 2 μl LR Clonase II enzyme mix was added to the tube
and was mixed well by vortexing briefly twice. The reaction was incubated at 25 °C for 1 hour. 1
18
μl Proteinase K (purchased from Invitrogen) solution was added to terminate the reaction. The
sample was incubated at 37 °C for 10 min.
2.1.8 Bacterial Host Transformation
MAX Efficiency DH5α Competent Cells were the bacterial hosts. Agar plates with kanamycin
were used for pENTR Dual Selection Vector selection and agar plates with ampicillin were used
for pLenti CMV Puro DEST selection. Plates were pre-warmed at 37 °C before transformation.
The ligation reaction mixture or LR reaction sample was added to DH5α competent cells (25 μl)
and incubated on ice for 30 min. After that, cells were subjected to heat shock for 20 sec in a 42
°C water bath without shaking and were re-incubated on ice for 2 min. 125 μl pre-warmed LB
medium was added to each tube. Incubated on a shaker at 37 °C for 1 hour at 225 rpm. Competent
cells (150 μl) were then spread to pre-warmed selective agar plates. Plates were incubated at 37
°C, overnight.
2.1.9 Plasmid Isolation
To isolate plasmids for screening, mini prep plasmid isolation kits (QIAquick) were used
according to manufacturer’s (QIAprep) protocols.
After selection, colonies were picked up and cultured in LB medium separately. Bacterial
overnight cultures were then pelleted by centrifugation at 13,000 rpm for 3 min at room
temperature. Pelleted bacterial cells were resuspended in 250 μl Buffer P1 and transferred to
microcentrifuge tubes. 250 μl Buffer P2 was added in each microcentrifuge tube and mixed
thoroughly by inverting the tube 10 times until the solution became clear. 350 μl Buffer N3 was
added to each tube and mixed thoroughly by inverting the tube 10 times. All the tubes were
19
centrifuged for 10 min at 13,000 rpm. All the supernatant from centrifugation was applied to the
spin column by pipetting, followed by a 1 min centrifugation and discarded the flow-through.
Every spin column was washed by 750 μl Buffer PE and centrifuged for 1 min and discarded the
flow-through. 1 min additional centrifugation was applied to remove residual wash buffer. Clean
microcentrifuge tubes were used to place the spin column. 30 μl Buffer EB was added to the center
of every spin column. After 1 min incubation at room temperature, spin columns were centrifuged
for 1 min to collect the DNA.
2.1.10 Insert Confirming Process
All clones were screened using an antibiotic resistant strategy. Agar plates with kanamycin were
used for pENTR Dual Selection Vector selection and agar plates with ampicillin were used for
pLenti CMV Puro DEST selection. Restriction enzymes were used to verify inserts. All clones
were verified by sequencing.
Lentivirus Production
HEK-293T cells were plated in a 200 mm tissue culture plate. Before transfection, cells were
incubated at 37°C, 5% CO2 overnight. A mixture was made for both Foxp1 and Foxp1-ES plasmid.
42 μl PEI was added to 2400 μl plain medium to make PEI-solution. DNA solution was made by
7 μg DNA (containing transfer vector, envelope vector and packaging vector) and 2467 μl plain
medium. PEI- the solution was added to the DNA solution and mixed well. The mixture of the two
solutions was incubated at room temperature for 15 min. After incubation, the mixture was added
into complete medium in cell culture plates. Cells were then incubated overnight at 37 °C and 5%
CO2. The medium was changed on the following day, and the virus supernatant was harvested
after 24 h.
20
Lentiviral Infection and Selection
C2C12 cells were plated in 100 mm cell culture plates and were incubated at 37 °C and 5% CO2
overnight. The infection was done along with killing controls. Polybrene was added to cells at a
final concentration of 8 μg/ml after changing the medium. 1 ml lentivirus was added to each 100
mm plate except the killing controls. Puromycin was added to cells at a final concentration of 2
μg/ml. Fresh media with puromycin was added every day until all the cells in the killing control
plates are dead. Cell colonies were then collected.
Western Blot Technique
To verify the proteins, SDS-PAGE and Western blot analysis were carried out after whole-cell
lysis. The identity of the proteins was verified using antibodies targeted against FLAG. All
antibodies for Western blot were purchased from Abcam unless otherwise indicated. Protein
samples were prepared along with negative controls (C2C12 cells without virus infection). Whole-
cell lysates were run on SDS-PAGE to separate proteins based on the molecular weight.
2.4.1 Preparation of Lysates From Cell Culture
10 cm cell culture dishes were placed on ice. Cells were washed with ice-cold PBS. After aspirating
PBS, an ice-cold RIPA buffer was added to cells, 1 ml per 100 mm dish. C2C12 cells were scraped
off the dish using cold plastic cell scrapers. The cell suspension was gently transferred into pre-
cooled microcentrifuge tubes. Constant agitation was maintained for 30 min at 4 °C. Cell lysates
were spun at 13,000 rpm for 30 min at 4 °C and the supernatants were transferred to a fresh tube.
100 μl supernatant was taken from each sample and was boiled in sample buffer at 95 °C for 5
min.
21
2.4.2 Protein Separation by Gel Electrophoresis
Protein samples were loaded in equal amounts along with molecular weight markers into the wells
of a 10% SDS-PAGE gel. The gel was run at 100 V for 10 min, and at 150 V for 1 h.
2.4.3 Transferring the Protein to the Membrane
SDS-PAGE gel was placed in the transfer buffer for 10 min. The transfer sandwich was assembled
and bubbles were removed. The cassette was placed in the semi-dry transfer tank, run at 25 V for
30 min. The proteins were transferred to the PVDF membrane.
2.4.4 Antibody Incubation
After transferring, the blot was rinsed in TBST and was blocked in 5% skim milk in TBST at room
temperature for 1 h. The membrane was incubated in primary anti-FLAG antibody solution
(1:4,000) overnight at 4 °C, on a shaker. The blot was rinsed 3 times for 10 min with TBST. After
rinsing, the membrane was incubated in HRP-conjugated secondary antibody solution for 1 h at
room temperature and was rinsed 3 times for 10 min with TBST.
2.4.5 ECL Development
After antibody staining, a working solution of the ECL Western blotting substrate is prepared by
mixing Agents 1 and 2. Membrane was incubated in the solution for 1 min at room temperature.
To capture the signals, the membrane was removed from the solution and was placed in the
ChemiDoc Gel Image System. The X-ray film and the photo-developing cassette were used for
exposure.
22
C2C12 Cell Culture and Differentiation Treatment
The cell line C2C12 is an immortal line of mouse skeletal myoblasts originally derived from
satellite cells from the thigh muscle of mouse (Yaffe and Saxel, 1977), which can be induced to
differentiate into myotubes. Mononucleated C2C12 myoblasts proliferate rapidly under high
serum conditions and differentiate into multinucleated myotubes under low serum conditions,
which also known as starvation. Yaffe and Saxel reported that the C2C12 cells derived from
dystrophic muscle formed very few multinucleated fibers (Yaffe and Saxel, 1977). This is a sign
of muscle wasting. In cultures prepared from normal mouse muscle, cell fusion and multinucleated
fiber network formation was observed (Yaffe and Saxel, 1977). C2C12 cells are considered to be
a good model for studying myoblast differentiation and myogenesis. They are also used to express
and study various target proteins.
2.5.1 Cell Culture for Cycling
Cells were grown at 37 °C, in a humidified incubator with 5% CO2.
Myoblast growth medium: 79% Dulbecco's modified eagle medium (DMEM), with high glucose
and glutamine, no Sodium Pyruvate. 20% fetal bovine serum (FBS). 1% 1X
Penicillin/Streptomycin, with a final concentration of 100 units/ml and 100 μl/ml.
2.5.2 Cell Differentiation Treatment
C2C12 cells were differentiated for 5 days by rinsing fully confluent cells once with PBS and
adding a low-serum differentiation medium. Cells are fed with fresh differentiation medium every
24 hours.
23
Differentiation medium: 97% Dulbecco's modified eagle medium (DMEM), with high glucose and
glutamine, no Sodium Pyruvate. 2% Donor equine serum. 1% 1X Penicillin/Streptomycin, with a
final concentration of 100 units/ml and 100 μl/ml.
DAPI Staining
Cells were carefully with ice-cold PBS twice and were fixed with paraformaldehyde for 30 min at
room temperature.
3ml DAPI (diluted to 300nM in staining buffer) was added to each 100 mm petri dish. Fixed cells
were completely covered by DAPI solution and were incubated for 10 min at room temperature.
The cell samples were rinsed 3 times in ice-cold PBS. After rinsing, samples were viewed using a
fluorescence microscope.

24
CHAPTER 3. RESULTS AND DISCUSSION
Expression of Foxp1 and Foxp1-ES Plasmid in C2C12 Cells
As mentioned before, FLAG-tagged Foxp1-pLenti CMV Puro DEST plasmid and FLAG-tagged
Foxp1-ES-pLenti CMV Puro DEST plasmid were constructed by the gateway cloning method.
Plasmids were transferred into C2C12 cells via lentivirus.
To test the expression of Foxp1-pLenti CMV Puro DEST and Foxp1-ES-pLenti CMV Puro DEST
plasmid in C2C12 cells after transduction, C2C12 cells were lysed and the proteins in the cell
lysates were separated on SDS-PAGE. The identities of the proteins were determined by Western
blot analysis stained with anti-FLAG antibody (DYKDDDDK Tag Antibody, MA1-91878, mouse
monoclonal antibody, dilution factor: 1:4,000) and secondary antibody (goat anti-mouse IgG-
HRP, sc-2031, dilution factor: 1:10,000) as shown in Figure 2.

25

As can be seen in lane 2 and lane 3, Foxp1-pLenti CMV Puro DEST plasmid and Foxp1-ES-pLenti
CMV Puro DEST plasmid were expressed in C2C12 cells. Besides, non-specific bands were
observed in each line. To determine the cause of multiple bands, more experiments are needed,
such as the use of positive controls in Western blot analysis or antibody titers before Western blot.
Detection of Morphological Changes During Cell Differentiation
C2C12 cells were grown in the myoblast growth medium and differentiated in differentiation
medium. To determine whether the expression of Foxp1 and Foxp1-ES in C2C12 cells results in
morphological changes during cell differentiation, cell images were taken daily after
differentiation medium was added. C2C12 cells without virus infection were used as negative
Figure 2. The expression of FLAG-tagged Foxp1-pLenti CMV Puro DEST and
FLAG-tagged Foxp1-ES-pLenti CMV Puro DEST in C2C12 cells. Lane 1: the
Western blot marker. Lane 2: expression of FLAG-tagged Foxp1. Lane 3: expression
of FLAG-tagged Foxp1-ES. Tagged proteins are seen at 76 kDa. FLAG was detected
when stained with anti-FLAG monoclonal antibody (monoclonal anti-FLAG primary
antibody 1:4,000 and goat-anti-mouse anti-IgG conjugated hRP 1:10,000). Lane 4:
C2C12 cell lysate without virus infection. The GAPDH served as control and are
present at 37 kDa.

26
controls in this experiment, although I will use an empty vector construct in future experiments.
The empty vector is the recombination product of Gateway entry clone pENTR dual selection
vector and the pLenti CMV Puro DEST destination vector, but without Foxp1 or Foxp1-ES gene.
Cells were fed with fresh differentiation medium every 24 hours, for the differentiated cells deplete
and acidify the medium more quickly. Changing medium is stopped 6 h before fixation to avoid
serum-response effects.
27
Figure 3. A. 0 h in differentiation medium.

Figure 3. B. 24 h in differentiation medium

Figure 3. C. 48 h in differentiation medium

28
Figure 3. D. 72 h in differentiation medium

Figure 3. E. 96 h in differentiation medium

Figure 3. F. 120 h in differentiation medium

Figure 3. Gross changes in cell morphology in response to differentiation medium and
expression of different proteins. Panel A: an image of undifferentiated (day 0) proliferating
C2C12 myoblast cells. The image was taken right after changing the growth medium into a
differentiation medium. Panel B-F: images of differentiating C2C12 cells at various time
points (24 h, 48 h, 72 h, 96 h, 120 h). Images labeled Foxp1 are C2C12 cells expressing Foxp1.
29
Images labeled Foxp1-ES are C2C12 cells expressing Foxp1-ES. Images labeled Control are
C2C12 cells without virus infection.
As shown above, the C2C12 cells expressing Foxp1 and Foxp1-ES differ in the rate of
differentiation as compared to the control group. In Panel C (48 h in differentiation medium),
myotube formation is observed in the control group, but cell differentiation in Foxp1 group and
Foxp1-ES group is not obvious. In Panel D (72 h in differentiation medium), there are more
myotubes formed in the FOXP1 group compared with the FOXP1-ES group or the control group.
There was no significant difference in the number of myotubes between the Foxp1-ES group and
the control group. In Panel E (96 h in differentiation medium) and Panel F (120 h in differentiation
medium), the FOXP1 group has the most myotubes in the three groups, while Foxp1-ES group has
the least.
Foxp1 is reported to play a role in regulating embryonic neural stem cell differentiation (Braccioli
et al., 2017). FOXP1-knockdown was found to reduce neural stem cell differentiation during
corticogenesis (Braccioli et al., 2017). The difference in C2C12 cell differentiation status shown
above also suggest that Foxp1 may play a role in C2C12 cell differentiation. Cells expressing
Foxp1 have more myotubes, compared with the control group. More interestingly, cells expressing
Foxp1-ES have fewer myotubes than the control group. FOXP1-ES is found to have the function
of enhancing the expression of many pluripotency genes, such as OCT4 and NANOG (Graveley,
2011), and represses genes related to cell differentiation (Gabut et al., 2011). Since the C2C12
cells expressing Foxp1-ES show fewer myotubes, FOXP1-ES may also inhibit C2C12 cell
differentiation.
Myotonic dystrophy type 1 patients have muscle weakness and wasting in many muscles, like
distal limb muscles, respiratory muscles, trunk muscles, and facial muscles. Studies indicate that
30
satellite cells have decreased activity and density in muscle wasting (McKenna and Fry, 2017).
When muscle wasting occurs, satellite cells may differentiate and contribute to muscle repair or
regeneration. If FOXP1-ES suppresses genes related to cell differentiation in satellite cells and
inhibits satellite cell differentiation, the satellite cells may not able to repair atrophic muscles.
Foxp1 mis-splicing caused by MBNL1 sequestration may play an important role in muscle wasting.
The Detection of OCT4 and NANOG in C2C12 Cells Expressing Foxp1-ES
Gabut and colleagues reported that FOXP1-ES stimulates the expression of transcription factor
genes related to pluripotency, such as OCT4 and NANOG (Gabut et al., 2011). To confirm the
C2C12 cells expressing Foxp1-ES also express OCT4 and NANOG, proteins in the cell lysates
were analyzed by SDS-PAGE and Western blot. Coomassie staining of protein lysates was used
to visualize total proteins and assure equal loading.

Figure 4. SDS-PAGE analysis and Coomassie staining. Lane 1&6: cell lysates of
C2C12 cells without virus infection. Lane 2&7: cell lysates of C2C12 cells expressing
Foxp1. Lane 3&8: cell lysates of C2C12 cells expressing Foxp1-ES. Lane 4&5: the
Western blot marker.
31
To measure the optimal loading volume, the polyacrylamide gel was stained with Coomassie blue
solution. The next step is SDS-PAGE analysis under the same load, followed by Western blot
analysis. Huh7 cell lysates will be used as the positive control for OCT4 and NANOG expression,
which has been proved to express NANOG (Chen et al., 2016).
Quantitative Analysis of C2C12 Cell Differentiation
Myoblast fusion can be assessed in vitro via the fusion index. The fusion index is calculated as the
percentage of nuclei associated with multinucleated myotubes. Before measuring the fusion index,
cells need to be fixed and stained by DAPI solution.
3.4.1 Cell Fixation and DAPI Staining
To calculate the fusion index, cells need to be fixed by paraformaldehyde and stained by DAPI
solution, protected from light. DAPI is a fluorescent stain that binds strongly to adenine–thymine
rich regions in DNA and stains nuclei specifically. It makes the nuclei visible to calculate the
fusion index.

Figure 5 DAPI staining. To calculate the fusion index, cells were fixed by paraformaldehyde
and were stained by DAPI solution, after 120 hours in the differentiation medium.
32
3.4.2 Fusion Index Calculating
The fusion index was calculated by dividing the number of nuclei within multinucleated myofibers
by the total number of nuclei. For each sample, 4 random areas were selected for the fusion index
calculation. The final fusion index was the average number of the four areas. The fusion index was
calculated daily after feeding cells with the differentiation medium.
Cells in Foxp1, Foxp1-ES, and control group were fixed and stained, and the fusion index was
calculated after culturing cells in the differentiation medium for 120 hours.
Table 1 Calculation of the fusion index.
Control Myotube nuclei All nuclei fusion index average
1 47 211 0.222748815
2 73 215 0.339534884
3 72 211 0.341232227
4 46 184 0.25 0.288378982

FOXP1-ES
1 41 165 0.248484848
2 28 206 0.13592233
3 28 208 0.134615385
4 27 201 0.134328358 0.16333773

FOXP1
1 45 306 0.147058824
2 60 309 0.194174757
3 48 308 0.155844156
4 46 302 0.152317881 0.162348904
As the table shown above, the average fusion index in the control group is larger than the average
fusion index of either the Foxp1 group or Foxp1-ES group. The fusion index represents the ability
33
of myoblasts to differentiate and form multinucleated. A larger fusion index in the control group
means the C2C12 cells in the control group have a higher differentiation capacity.
However, the fusion index between Foxp1 and Foxp1-ES group is not significantly different.
Considering the myotubes observed in Foxp1 group are apparently more than that in Foxp1-ES
group (Figure 3), the nuclei counting and index calculation need to be repeated. The fusion index
calculation in the Foxp1 group can be inaccurate, due to the high density of myotubes and high
total nuclei count. Nuclei in stacked cells are difficult to identify and count, and the fusion index
of Foxp1 group may be underestimated.
Pilot experiments such as differentiation tests of C2C12 cells in vary cell density gradients are
required. The pilot experiments will differentiate cells at different concentration gradients and
perform DAPI staining to calculate the fusion index, in order to find a suitable cell density.
Future Plan: Western Blot Analysis
C2C12 cell lysates were analyzed by SDS-PAGE and Western blot. The data was not shown due
to uncleared bands. SDS-PAGE and Western blot analysis will be performed under the adjusted
load. Huh7 cell lysates will be used as the positive control for OCT4 and NANOG expression,
which has been proved to express NANOG (Chen et al., 2016).
Conclusion
To understand the role of FOXP1 in myotonic dystrophy and the relationship between Foxp1 mis-
splicing and muscle wasting, Foxp1-pLenti CMV Puro DEST plasmid and Foxp1-ES-pLenti CMV
Puro DEST plasmid were constructed and transfected into C2C12 cells. Foxp1 and Fopx1-ES were
confirmed to be expressed in the transfected C2C12 cells by Western blot (Figure 2). As shown in
34
Figure 3, the C2C12 cells expressing Fopx1 and Foxp1-ES differ in the status of differentiation as
compared to the control group. Myotube formation occurred in the control group after culturing
C2C12 cells in differentiation medium for 48 hours (Figure 3. C.). Interestingly, cells expressing
Foxp1 formed more myotubes in the following figures. After 72 hours in differentiation medium,
more myotubes were formed in the FOXP1 group compared with the FOXP1-ES group or the
control group (Figure 3. D.). After cultured in differentiation medium for 96 hours (Figure 3.E)
and for 120 hours (Figure 3. F.), the FOXP1 group formed the most myotubes in the three groups,
while Foxp1-ES group formed the least. Foxp1 was found to play a role in regulating embryonic
neural stem cell differentiation (Braccioli et al., 2017). FOXP1 knockdown reduced the
differentiation of neural stem cells (Braccioli et al., 2017). More myotube formation in Foxp1
expressing C2C12 cells suggests that Foxp1 may also play a role in C2C12 cell differentiation.
Besides, C2C12 cells expressing Foxp1-ES show fewer myotubes, suggests that FOXP1-ES may
inhibit C2C12 cell differentiation. FOXP1-ES was reported having a negative effect on C2C12
differentiation (Gabut et al., 2011). It up-regulates the expression of transcription factor genes
related to pluripotency (Gabut et al., 2011), which may contribute to the myotube reduction in
C2C12 differentiation process. To better understand the role FOXP1-ES plays in C2C12 cell
differentiation process, more experiments are needed.
To quantify the difference in C2C12 cell differentiation, the fusion index calculation is needed to
describe the effect of Foxp1 or Foxp1-ES expression on C2C12 cell differentiation. The fusion
index is calculated as the percentage of nuclei associated with multinucleated myotubes. It
represents the proportion of the total cell population that has fused. By measuring the fusion index,
the efficiency of C2C12 cell differentiation can be evaluated.
35
To better understand the role of FOXP1-ES in C2C12 cell differentiation, the expression of
pluripotency-related transcription factors (like OCT4 and NANOG) in cells expressing Foxp1-ES
needs to be analyzed by Western blot. The Coomassie blue staining helps to find the optimal
sample loading volume in SDS-PAGE and Western blotting. The expression levels of OCT4 and
NANOG are important for studying the role of FOXP1-ES in the differentiation of C2C12 cells,
because FOXP1-ES is reported to up-regulates the expression of transcription factor genes related
to pluripotency in differentiated cells (Gabut et al., 2011). Further studies would explain the effect
of FOXP1 and FOXP1-ES in C2C12 cell differentiation as well as myotonic dystrophy.

36
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Abstract (if available)

Abstract Myotonic dystrophy (DM) is a genetic disorder that affects the normal function of muscles. There are two types of DM, DM1 and 2, which are caused by mutations in distinct genes (DMPK or ZNK9) but share common symptoms and pathogenic mechanisms. There are several models to explain the mechanism of myotonic dystrophy type 1. According to the RNA pathogenesis model, the expanded CUG or CCUG repeats sequester the Muscleblind-like (MBNL) family of proteins, which results in the loss of normal functioning MBNL. MBNL proteins are key regulators of mRNA metabolism in mammals. The sequestration of MBNL proteins leads to mRNA splicing aberrations. Foxp1, a gene encodes Forkhead box protein P1, is regulated by MBNL. FOXP1 is a transcription factor that regulates a large number of genes involved in cell differentiation. Alternative splicing may change the DNA-binding preference of the FOXP1. The embryonic stem cell (ESC)-specific isoform of FOXP1, which is called FOXP1-ES, stimulates the expression of many genes, including pluripotency related genes like OCT4 and NANOG. This FOXP1 isoform may contribute to the reprogramming of differentiated cells. Here, I seek to gain an understanding of the role of FOXP1 splicing aberration in myotonic dystrophy. My results suggest that FOXP1 may promote the differentiation of mouse myoblast cells, and FOXP1-ES may prevent the differentiation of mouse myoblast cells. These results reveal that type 1 myotonic dystrophy may be related to the ESC specific alternative splicing of Foxp1, which is caused by the sequestration of MBNL1.

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Asset Metadata

Creator Wang, Xinyi (author)

Core Title The splicing error of FOXP1 in type I myotonic dystrophy

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 06/22/2020

Defense Date 04/23/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag alternative splice,FOXP1,Myotonic Dystrophy,OAI-PMH Harvest,stem cell pluripotency

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Comai, Lucio (committee chair), Reddy, Sita (committee member), Yuan, Weiming (committee member)

Creator Email wang574@usc.edu,xinyiwang000@gmail.com

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