DMPK and myotonic dystrophy:
effects of CTG trinucleotide expansion upon DMPK
and their contribution to DM pathogenesis
by
Brigid Michele Davis A. B. Biochemistry Harvard University, 1988
Submitted to the department of biology in partial fulfillment of the requirements for the degree of
Doctor of Philosophy at the
Massachusetts Institute of Technology April, 1998
copyright 1998 Massachusetts Institute of Technology. All rights reserved
Signature of Author:
Department of Biology
April 24, 1998
Certified
by:-Accepted by:
David Evan Housman Novartis Professor of Biology Thesis Supervisor
Frank Solomon
Professor or Biology
Chairman, Graduate Committee
OF TEC 4KLOGY
APR
2 81998
LIBRAIES---- -1,---L7 _____ ___
DMPK and myotonic dystrophy:
effects of CTG trinucleotide expansion upon DMPK
and their contribution to DM
pathogenesis
by
Brigid Michele Davis
Submitted to the Department of Biology
on April 24, 1998 in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy in
Biology
ABSTRACT
Myotonic dystrophy (DM), an autosomal dominantly inherited form of muscular
dystrophy, results from expansion of a CTG trinucleotide repeat within the 3'
untranslated region of the gene myotonic dystrophy protein kinase (DMPK). The mutant
DMPK allele is transcribed in cultured DM myoblasts, and mutant transcripts are
spliced and polyadenylated normally, despite the presence of long CUG tracts. However,
mutant DMPK transcripts do not exit nuclei by standard pathways, and they never reach
the cytoplasm in differentiated, non-mitotic muscle cells. Instead, they form stable
clusters ("foci") that are tightly linked to the nuclear matrix. Nuclear retention of
mutant transcripts prevents their translation; consequently DMPK levels are reduced by
at least 50% in DM myoblasts. This DMPK deficiency is the probable cause of many of
the muscular symptoms of DM; however, it is unlikely to account for all aspects of the
disease.
DMPK is postulated to play a role in excitation/contraction coupling in muscle tissue,
but its precise role there has not been defined, nor has its role been determined within
non-muscle tissues. The predominant alternatively spliced forms of DMPK are
differentially localized within cultured cells: DMPK+4 is found throughout the
cytoplasm, while DMPK-4 is targeted to the periphery of mitochondria. As a result,
these splice forms may have distinct regulators and activities, and each form's absence
in DM cells may induce a subset of DM's symptoms.
Thesis Supervisor: David Evan Housman
Novartis Professor of Biology
Acknowledgements:
Many people have contributed, either directly or indirectly, to this work, and I am truly grateful for their help and support; without it, this thesis would probably never have
been completed. I would particularly like to thank and acknowledge:
* lain Bason, for encouraging me and believing in me all these years, and for listening to me talk about things he didn't understand.
* past and present members of the Housman lab, who provided invaluable suggestions, encouragement, and moral support, as well as for being wonderful people to work with. I am especially indebted to Julian Borrow, Alain Charest, Alex Kazantsev, John Landers, and Elizabeth Preisinger, who gave freely of their time and knowledge, and to Mila McCurrach, who established the experimental system that formed the basis for this work.
* Ben Blencowe, who provided a wealth of technical information and insight into fields that were completely foreign to me, as well as help and encouragement in finally getting the writing done.
* Ken Moberg, who was a patient and always accessible teacher when I made antibodies; I couldn't have done it without him.
* Tony Albino, who gave me my first opportunities in science and showed me the spirit in which it should be done, and who had faith in me long before I had any in myself. * my parents and sister, for being willing listeners and supporters, especially during
hard times.
* all the long-term members of my thesis committee - David Housman, Phil Sharp, and Tyler Jacks - for helping me to grow up as a scientist and for making excellent suggestions along the way.
* the newest members of my thesis committee - Frank Solomon and Alan Beggs - who gave their time to make this final rite of passage possible.
* my advisor, David Housman, for consistently supporting my research and for providing me with invaluable opportunities for learning to be independent.
The help of these people was central to my graduate school experience, and I feel very fortunate to have encountered and interacted with them all.
TABLE OF CONTENTS Abstract 2 Acknowledgements 3 Table of contents 4 List of figures 6 List of tables 7 Chapter 1: Introduction 8
Myotonic dystrophy: introduction to the disease 8
Congenital DM 9
DMPK: the DM-associated gene 9
DMPK mutation 16
Other trinucleotide disorders 16
Mechanisms of trinucleotide instability 19
DM disease models 22
DMPK and DM: experimental results 24
DMPK transcription and transcript processing 24
DMPK protein 25
DMPK splicing 26
DMPK transcript aggregation 27
CUG RNA-binding proteins 27
Mouse models of disease 28
Expression of genes adjacent to DMPK 31
Summary 32
References 34
Chapter 2: Expansion of a CUG trinucleotide repeat in the 3' untranslated region of myotonic dystrophy protein kinase
transcripts results in nuclear retention of transcripts 42
Abstract 43
Introduction 44
Results 45
Generation of "myoblasts" byMyoD retroviral infection of fibroblasts 45 Mutant DMPK transcripts are less abundant than wt transcripts in DM
cells 48
Mutant DMPK transcripts are sequestered within myoblast nuclei 48
In situ localization of DMPK transcripts 53
Mutant nuclear transcripts are associated exclusively with the nuclear
matrix 57
Transcripts are released during cell division 60
Mutant transcripts are polyadenylated and spliced 60
Mutant transcripts and nuclear foci are not unstable 61
Discussion 66
References Chapter 3: in cultured Abstract Introduction Results
DMPK expression and splice form-specific localization human myoblasts
Antibody characterization
DMPK expression levels in DM and control myoblasts Subcellular distribution of endogenous DMPK
Alternative splice forms of DMPK Discussion
Materials and methods References
Chapter 4: Discussion
Probable mechanisms of DM pathogenesis Congenital DM: are different models necessary? Analysis of the DM phenotype
Ca** channels Na+ channels
Na*/K' ATPase K+ channels
DMPK: the significance of subcellular localization Mitochondria
Cytosolic DMPK
DMPK mutation: long term effects Concluding remarks
References
Appendix: Generation and characterization of anti-DMPK monoclonal antibodies
Introduction
Results and discussion
Detection of overexpressed DMPK
Detection of endogenous DMPK in human myoblasts Species specificity
Epitope mapping Materials and methods References Biographical sketch 76 77 81 81 89 89 92 99 109 114 118 118 121 123 124 124 125 125 126 127 128 129 131 132 135 136 136 136 139 146 146 159 164 166
List of figures:
Chapter 1:
Homology of DMPK and DMPK-related genes within their kinase domains Alternative splicing of DMPK
Slipped strand models for trinucleotide instability Chapter 2:
Northern analysis of DMPK and myogenin transcription in normal and MyoD infected fibroblasts
Northern analysis of DMPK transcripts in representative normal and myotonic myoblasts
Comparison of nuclear and cytoplasmic DMPK RNAs isolated from control and myotonic cells
Distribution of triplet repeat transcripts in myoblasts and dividing cells Northern analysis of association of DMPK transcripts with the nuclear matrix Northern analysis of polyadenylation of wt and mutant DMPK transcripts Northern analysis of oligonucleotide-directed cleavage of DMPK transcripts with RNAse H
Northern analysis of DMPK mRNA stability Chapter 3:
Characterization of anti-DMPK antibody 21F5 reactivity against various protein sources on Western blots
Immunofluorescence labeling of transverse cryosections of normal adult skeletal muscle with anti-DMPK (20G8) and anti-skeletal myosin (fast) Western blots showing DMPK expression in control and DM cultured myoblasts Detection of endogenous DMPK in cultured wt human myoblasts by
immunofluorescence microscopy
RT-PCR analysis of alternative splicing of DMPK transcripts Subcellular distribution of transfected DMPK splicing isoforms Schematic depiction of the structure of myofibrils
Appendix:
Immunoprecipitation of 35S Met labeled HADMPK from lysates of transfected Cos-7 cells by potential anti-DMPK hybridoma supernatants
Detection of HADMPK on Western blots of pHADMPK-transfected Cos-7 cells Detection of endogenous DMPK on a Western blot of an extract from cultured human myoblasts by anti-DMPK monoclonal antibodies
Immunoprecipitation of endogenous DMPK from cultured human myoblasts Anti-DMPK antibodies do not detect DMPK in lysates of murine muscle
Fragments of DMPK used for identification of epitopes recognized by the anti-DMPK antibodies
Small regions of DMPK subcloned, using PCR, into bacterial expression vectors to produce GST-DMPK fusion proteins
The anti-DMPK antibodies detected fusion proteins composed of GST and small fragments of DMPK on Western blots
12 14 21 50 52 56 59 59 63 65 83 86 88 91 94 97 106 138 141 151 153 155
List of tables: Chapter 1:
Diseases caused by expansion of trinucleotide repeats 18 Appendix:
CHAPTER 1: INTRODUCTION
Myotonic dystrophy: introduction to the disease
Myotonic dystrophy (DM) is a complex, multisytemic disorder, generally classified as an adult onset form of muscular dystrophy (1). DM patients suffer from a wide variety of muscular dysfunctions, including skeletal muscle weakness and myotonia, cardiac conduction defects and cardiomyopathy, and smooth muscle contractile abnormalities and myotonia. Skeletal muscle weakness is notable especially in superficial facial muscles and in distal limb muscles, while smooth muscle abnormalities are especially prominent within muscles of the gastrointestinal tract. Cardiac symptoms are detected only in a subset of DM patients; however, electrocardiograms of asymptomatic DM patients
frequently reveal cardiac dysfunction, and 30% of deaths among adult DM patients result from cardiac arrhythmia (2). DM's muscular disorders are frequently accompanied by non-muscular abnormalities, such as a disease-specific form of cataracts, macular degeneration, endocrine disturbances (e.g., testicular atrophy and glucose intolerance), and mental retardation. These symptoms permit discrimination between myotonic dystrophy and several other forms of myotonia which lack non-muscular features. The age of onset and severity of all symptoms are quite variable among the affected
population; however, they can be predicted to some degree for the individual, as the disease is characterized by anticipation: the tendency for symptoms to appear earlier and with greater severity in successive generations. DM shows autosomal dominant
inheritance, and its incidence is estimated at 1:8000 in European populations.
DM can be distinguished from other forms of muscular dystrophy by histological analyses as well as by the wide spectrum of symptoms it induces (1). DM skeletal
muscle is marked by type I fiber atrophy, abundant central nuclei, sarcoplasmic masses, ringed fibers, and slight fibrosis. Split fibers, probably resulting from hypersensitivity to mechanical stress, are also present. At the ultrastructural level, disorganized fibers with distorted Z lines, sarcolemmal folding, and abnormal
mitochondria are detected. Degeneration of cardiac and smooth muscle is also found, frequently coupled with fatty infiltration; however, these changes do not occur in all patients. The cardiac symptoms of DM are thought to result primarily from disruption of conductive tissues within the heart, rather than from generalized cardiomyopathy.
Congenital DM. DM is primarily an adult onset disorder; however, a congenital variant of the disease also exists (1). Like adult onset DM, congenital DM induces profound muscular weakness, especially of facial muscles. However, myotonia is never detected in congenital DM patients; in fact, congenital muscle is hypotonic, rather than hypertonic. Development of this muscle appears to be delayed: it contains small and
immature muscle fibers and abundant satellite (precursor) cells. Congenital DM patients who survive gradually develop both the classical muscular symptoms of adult onset DM and the non-muscle symptoms, especially mental retardation. The cause of this alternate disease manifestation is not clear, nor is it known why congenital DM
results almost exclusively from maternal transmission of the disease.
DMPK: the DM-associated gene
DM was the first human disease to be linked to genetic markers (3,4). By 1983 these markers had been localized to chromosome 19 (5-7) and in 1992 a disease-specific restriction fragment length polymorphism was identified (8-10). Cloning and
which was contained within the gene now known as myotonic dystrophy protein kinase (DMPK) (11-13).
The newly identified DMPKgene was predicted, based on sequence homology, to encode a serine-threonine protein kinase (13). At that time, DMPK was most closely related to members of the cAMP-dependent subclass of kinases, in particular to the S. cerevisiae protein TPK2/YKR1. In subsequent years, a number of more closely related proteins, frequently referred to as "DMPK-related" kinases, have been identified, including D. melanogaster Warts, N. Crassa COT-1, S. cerevisiae DBF2 and DBF20, human PK428, and human/bovine Rho-kinase. These proteins show great similarity within the classical kinase domain (Figure 1); however, their flanking sequences diverge significantly, and no data currently suggests that they have overlapping functions.
The sequence encoding DMPK spans approximately 13kb and contains 15 exons (14). Comparisons of human and murine DMPK reveal significant sequence conservation throughout exons 2-8 (92.6% similarity of amino acids), which encode the kinase domain and are contained within all known isoforms of DMPK. Exons 9-12, likewise not subject to alternative splicing, are expected to produce an a helical, coiled-coil-related structure. No specific function for this region has been identified, and the greatest disparity between the human and murine sequences is found in this portion of the
protein. Exons 13-15 allow for the production of diverse isoforms of DMPK via alternative splicing (outlined in Figure 2). Through the selection of different splice sites and the resultant changes in reading frame, both hydrophobic and hydrophilic
carboxyl termini, containing several distinct potential transmembrane domains, can be generated for DMPK. Some analyses suggest that regulation of this splicing is
tissue-Figure 1. Homology of DMPK and DMPK-related genes within their kinase domains. The kinase domain of DMPK is similar to the kinase domains of a number of kinases from a variety of species. The human kinase PK428, whose function has not yet been identified, appears to be particularly closely related to DMPK. However, neither PK428 nor the other genes shown show significant sequence conservation outside of the kinase domain. No evidence currently suggests that DMPK and the DMPK-related genes have overlapping functions.
60 66 65 225 702 59 119 125 124 284 762 118 179 185 182 343 821 177 222 228 225 402 901 223 254 258 254 457 932 242 314 318 313 515 990 296 386 372 369 569 1043 356 RN S WEE --- -T TQL Y S - N K S V S RR C A F TDR SSR-CAF TDR-R C NT-R CASA-D L 5K IT ADT V D D C K DL EDKG PTD IDQU
BP
Qg Q Y -DPEK-:- -P --- lcrq --- KEM C NRAQINDWRRSRRLMA ERRRM---RDHQRMLA --- W---J N EMIKKER N VEEIKR KNDQ A - HEIKSA RIVEF-K Q S-E RVI K E VNF-LQSM5RMIKA W SEVVgEE DHPK D K F II A PK428 R LL 5 DS A Rho-kinaseI SLI KDQLAHV Cot1
NH L T R A VU N QIA H V K A warts RY K QQ VV VVL K Q EH TND TPKZ/YKR1 DMPK E DIE D PK428 V N M N YD - - V E KW Rho-kinase MM I Y E EI-FSEDI EY T~ Cot 1
MS I LNI- FEE L I warts E11E JIF- R -SQ IFN-PV K A TPK2/YKR1
- DMPK M--- PK428 T M M N - - - - Rho-kinase LSTGFHKLHDNNYYTQLLQG K S N Cot 1 LNT G FW T H N S K Y Y Q E N G N H S R Q warts --- FAKEVQ TVT- TPKZ/YKR DMPK S I M E DE- - KE PK428
DTI V K Q - - -KE llDn Rho-kinase
YST IA T G H - - - - Cot 1 H S L A V E - - - - warts -TLC I V ITTKP--- TPK2/YKR1 DMPK MNH RFQF AQVTDS NN K L PK428 N H NS TF - DNDISK K NL Rho-kinase NWRHS YF -D ITLG V ENL Cot V N W E K T HI -PQAELSR T L warts
LQ - - G K V VY P Y - - - F H P D V V L TPK2/YKR1
-N IN C E Y I V S S P PK428 WET TA V VV LLSSDI - Rho-kinase -ES RIRA~E RLTS E- Cot1
- A D M~Q K A Y I E I K H Pa- warts R L L A K I E T Y E P ITS G I G I SL TPK2/YKR1 DMPK PK428 Rho-kinase Cot 1 warts TPK2/YKR1
Percentage similarity with DMPK (kinase domain region only) PK428 Rho-kinase Cot-1 Warts TPK2/YKR1 66.25 45.5% 37.7% 36.1% 27.9% NO I B~IQI HPIU~ sH M M V A V IAI SM F I V M I L I K KL G II L Ur KH F IR)1 K l ) L IT ) V KN V TCV CM I KF II D ONKT V I L LEYLA H N I I YI Ll E H 1 Ml) Nlm K IT PRD SME - - D
Figure 2. Alternative splicing of DMPK. Alternative splicing of the exons at the carboxyl terminus of DMPK allows for the production of many diverse isoforms of DMPK. The final two exons can be translated in all three reading frames; therefore, alternate forms of DMPK contain mutally exclusive series of amino acids at their carboxyl termini. The two forms of DMPK containing all 15 exons both have hydrophobic regions containing potential transmembrane domains. However, a hydrophilic region can also be generated, via deletion of exon 13.
DMPK+4 DMPK-4 DMPKA13 DMPKA13A14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 .. .CAT---CTAGATGGCCCCCCGGCC... L D G P P A ...
III
[II-
m-
m-
El
E-
EI
m m m
I
m
~z
zz
... CAT---ATGGCCCCCCGGCCGTGG... M A P R P W ...I-II
IF-II
LI
E
] II
]
I]
W-
II]
II
I
. .CAG---CTAGATGGCCCCCCGGCC ...
R W P P G ...
exon 14 deleted reading frame 1 predicted size: 65 kD
kinase domain * last 5 amino acids sometimes spliced out
reading frame 1 hydrophobic exon 15
transmembrane domain exon 15? predicted size: 69 kD
Xba I cleaves 13/14 junction
4 bases spliced out reading frame 2
transmembrane domains exons 14, 1 predicted size: 69 kD
Nde I cleaves 13/14 junction
exon 13 spliced out reading frame 3 hydrophilic exon 14 predicted size: 62 kD brain specific transcript
exon 13 and 14 spliced out reading frame 3
predicted size: 59 kD
specific (15); however, little is known about the relative levels of these isoforms and the different functions they may perform. DMPK is most highly expressed in the major tissues affected by myotonic dystrophy, namely skeletal, cardiac, and smooth muscle (16). Within skeletal muscle, DMPK is most abundant within type I (slow) muscle fibers. DMPK expression in skeletal muscle is likely controlled, in part, by a MyoD-responsive element located within its first intron (17). Transcripts can be detected early in development (mouse embryonic day 10.5) in cells of the myogenic lineage and they remain abundant throughout life. Transcripts are also detectable in brain, though at several-fold lower levels than in most muscles (16). In mice, brain transcripts are not present in embryos or newborns, but they are found in 14 day old pups. In rat brain, DMPK protein is found within the choroid plexus and the apical membrane of the ependyma. In addition, truncated forms may be made within the hippocampus, frontal cortex, and other regions; however, these smaller immunoreactive species may not be authentic forms of DMPK (18). Low levels of DMPK transcripts have also been
observed in fibroblasts, lymphoblastoid cells, retina, testis, seminal vesicle, liver, and lung.
DMPK's role within these tissues is still largely a subject for speculation. The protein has been shown to have serine-threonine kinase activity, but its natural substrate(s) has not been identified. It can phosphorylate histone H1 and the DHPR P subunit in vitro
(19, 20) and it can phosphorylate and regulate the tetrodotoxin (TTX)/R-conotoxin sensitive skeletal muscle sodium channel when coexpressed with the channel in Xenopus
oocytes (21). However, endogenous DMPK has not been shown to interact with or phosphorylate any of these proteins, nor has their phosphorylation status been shown to be altered in DMPK-deficient mice. Electrophysiological data from DMPK -/- mice
suggests that DMPK may be involved in excitation/contraction coupling and/or the maintainance of Ca"+ homeostasis (22, 23). The subcellular distribution of the kinase -neuromuscular junctions and triads in skeletal muscle, intercalated disks in cardiac muscle - is consistent with this theory but not a sufficient basis for conclusions (18, 24-26). Even less data exists concerning the role of DMPK in brain and other tissues in which it is expressed at low levels. This lack of knowledge about DMPK's normal
function has hindered efforts to understand the relationship between DMPK mutation and development of DM.
DMPK mutation
Only one class of mutation within DMPK has been identified in DM patients: the expansion of a CTG trinucleotide repeat within the 3' untranslated region of the gene (11-13). Unaffected individuals have DMPK alleles containing 5-35 CTGs; in contrast, DM patients carry an allele with 50-1000s of CTGs. Expanded alleles are both
mitotically unstable, resulting in somatic mosaicism, and unstable upon transmission between generations (27-30). In general, the number of CTGs within a mutant allele
increases when transmitted. This finding, coupled with the observation that disease symptoms tend to be more severe in patients with large expansions, is thought to
underly the phenomenon of anticipation.
Other trinucleotide disorders. DM was thus one of the founding members of a new class of disorders, frequently characterized by anticipation, caused by expansion of trinucleotide repeats, or "allelic expansion" (Table 1). Other members of this family
now include fragile X syndrome (31), Huntington's disease (32), spinal and bulbar muscular atrophy (33), spinocerebellar ataxia types 1, 2, 3, 6, and 7 (34-40), and
Table 1. Diseases caused by expansion of trinucleotide repeats. DM is the only disease caused by expansion of a CTG trinucleotide, as well as the only disease caused by a mutation in the 3' untranslated region of the mutated gene. Most cases of allelic expansion involved an enlarged CAG repeat within the coding region of a gene, which generates an extensive polyglutamine tract. These mutations cause a variety of
neurodegenerative disorders. Chromosomal fragile sites, which can be associated with mental retardation syndromes, have been linked to the presence of expanded CGG repeats. CGG expansions are subject to aberrant methylation and can result in transcriptional silencing.
Region Mechanism *
Myotonic dystrophy
Fragile X syndrome
Huntington's Disease
Spinocerebellar ataxia Type 1 (Sca 1)
Spinocerebellar ataxia Type 2
Spinocerebellar ataxia Type 3 (Machado Joseph Disease) Spinocerebellar ataxia Type 6
Spinocerebellar ataxia Type 7
Spinal and bulbar muscular atrophy (SMBA) (Kennedy's Disease) Dentatorubral pallidoluysian atrophy (DRPLA) Friedreich's ataxia CTG CGG CAG CAG CAG CAG CAG CAG CAG CAG GAA DMPK FMR-1
3' UTR loss of function + ?? (dominant)
5' UTR loss of function
(transcriptional silencing) Hdh
Sca 1
Sca 2
Sca 3
alA Ca++ channel (CACNI1A4) Sca 7 Androgen receptor (AR) DRPLA frataxin coding poly(gln) coding poly(gln) coding poly(gln) coding poly(gln) coding poly(gln) coding poly(gln) coding poly(gln) coding poly(gln)
intron loss of function (recessive)
* The presence of extended polyglutamine tracts [poly(gln)] is thought to confer novel functions and properties upon these mutant proteins, as might be expected from the dominant inheritance pattern of most of
these disorders. In some cases the mutant proteins have been shown to form cytoplasmic aggregates and/or nuclear inclusions.
Friedreich's ataxia (41). However, DM is unique among these disorders in that it is due to a CTG expansion and that the expansion is within the 3' untranslated region of the mutated gene. Thus, the mechanisms that seem to explain disease pathogenesis for these other disorders are not appropriate for DM (42-44).
Mechanisms of trinucleotide instability. It is theorized that trinucleotide instability results from the capacity of these repetitive DNA sequences to assume atypical conformations. In vitro, both CTG and CGG repeats are unusually flexible, and they can sustain higher levels of supercoiling than can random sequence DNA (45).
Repeats also migrate anomolously on polyacrylamide gels, reflecting their atypical secondary structures (46). Formation of novel structures probably induces the polymerase pausing observed during in vitro replication of long repeats, which presumably occurs in vivo as well (47). Polymerase pausing seems to allow for the formation of slipped DNA structures, containing hairpin loops of repetitive DNA, which promote replication errors (Figure 3). Formation of slipped structures is also fostered within regions of single stranded DNA, such as those generated during lagging strand
replication (48). Since slipped structures are more likely to form during lagging strand replication, and since, at least in E. coli, lagging strand polymerase complexes
more readily continue DNA synthesis when faced with abnormal replication
intermediates than do leading strand polymerases (48, 49), it is likely that repeat size alterations are created primarily during lagging strand synthesis. Slipped structures can faciliate both expansion and contraction of repeats, depending upon whether the hairpin is within the template (contraction) or newly synthesized (expansion) strand. CTG repeats form much more stable hairpins than do CAG repeats; it is therefore not surprising that CTG expansions occur, both in E. coil and in S. cerevisiae, primarily
Figure 3. Slipped strand models for trinucleotide instability. Both CTG and CAG repeats can form hairpins; however, CTGs are believed to assume more stable secondary
structures, so they are most likely to catalyze changes in repeat length. a. Formation of a CTG hairpin as the CTG repeat is synthesized from the lagging strand template allows for CTG expansion. b. Formation of a CTG hairpin within the lagging strand template facilitates CTG contraction.
lagging template leading template agging template
ileading
template
hairpin formation CTG in new strand hairpin formation CTG in template continued synthesis, repair trinucleotide expansion continued synthesis, repair trinucleotide contractiontemplate DNA strands newly synthesized DNA trinucleotide repeats
~~i~f---4LI-- 14~
when the CAG repeat is in the lagging strand template (48, 50). Hairpin formation is also dependent upon the length of the repeat sequence (51); this probably accounts for the stability of short repeats (i.e., wt DMPK alleles) relative to expanded repeats. Most analyses of repeat instability have utilized E. coli or S. cerevisiae, rather than
mammalian cells, so it is possible that models do not accurately reflect the mammalian replication process. However, the fact that the most common disease-causing expansion in humans - CTG/CAG expansion - is also the most frequent trinucleotide expansion in E.
coli (52) suggests that similar mechanism of expansion may operate in these organisms.
DM disease models
Many models were initially proposed to explain how the untranslated mutation in DMPK could be responsible for DM. Since the coding region of DMPK was not directly altered by the trinucleotide expansion and normal protein could theoretically be produced from the mutant allele, most models assumed that DMPK's expression and/or function must somehow be deregulated. Increases and decreases in DMPK levels both seemed possible consequences of the expansion, as the trinucleotide might alter determinants of
transcript stability or translation and thereby disrupt regulation of DMPK synthesis. Such regulatory motifs had previously been detected in 3' untranslated regions (53-57). However, it was difficult to envision DMPK deficiency as the sole cause for DM, as
patients still had one normal allele of DMPK. In addition, the absence of other kinds of dosage-deregulating mutations (i.e., deletions, promoter mutations) cast doubt upon this
hypothesis. Mislocalization of the protein, with a gain-of-function due to exposure to a new substrate, seemed somewhat more plausible, as did altered splicing of the 3' exons due to the nearby expansion.
Deleterious consequences stemming solely from the mutant DMPK transcript,
independent of its protein product, could also be envisioned. The 3' untranslated regions of several muscle-specific genes had recently been shown to be capable of influencing myoblast growth and differentiation (58). Overexpression of these 3' untranslated regions induced expression or upregulation of other muscle genes, possibly by sequestering negative regulators of differentiation. If the CUGs within the 3'
untranslated region of DMPK transcripts normally functioned in a similar manner, then expansion of the CUG tract might mimic overproduction of DMPK transcripts.
Alternatively, the expanded trinucleotide might disrupt such a regulatory region, freeing RNA-binding factors to bind transcripts of other genes. A third possibility was that the poly-CUG tract might have a novel affinity for an RNA-binding protein, and deplete the cellular supply of a critical factor.
DNA-mediated mechanisms of pathogenesis were also considered. The expanded
trinucleotides were found to be extremely strong nucleosome positioning elements (59, 60), and their presence in the DNA of DM patients was predicted to alter the chromatin structure of the region containing DMPK and flanking genes. Since chromatin structure can influence transcription, the expression of many genes in the region could thereby be perturbed by mutation of DMPK. The pleotropic consequences of trinucleotide expansion implied by this mechanism and by the "toxic RNA" model - consequences that would not be induced by other kinds of mutations within DMPK, which had never been detected in
DM patients - strengthened the appeal of these hypotheses. In addition, these theories did not preclude the possibility that some DM symptoms might result solely from changes in DMPK levels. In the years since DMPK was cloned, the validity of many of
these hypotheses has been explored; however, the molecular paths between DMPK mutation and development of DM have still not been conclusively defined.
DMPK and DM: experimental results
DMPK transcription and transcript processing. Analyses of DMPK transcript levels in DM cells and tissues have yielded complex and contradictory results. Sabourin et al. (61) reported increased levels of DMPK mRNA in a subset of tissues from a congenital DM patient, and postulated that excess DMPK in muscle might induce DM
symptoms. Other investigators found decreased levels of DMPK transcripts, but disagreed as to the the cause of this reduction. Krahe et al. (15) observed variable reductions in processed transcripts, but not pre-mRNA, from the mutant DMPK allele relative to the wt allele. However, they observed no changes in the net (wt + mutant) levels of processed transcripts, casting doubt on the quantitativeness of their assays. In contrast, Carango et al. (62) found reductions in pre-mRNA as well as processed RNA, while Wang et al. (63) observed only an insignificant, non-disease-specific reduction
in processed RNA plus a dramatic loss of both wt and mutant transcripts within poly
(A)+ RNA. Fu et al. (64) observed decreased mRNA from the mutant DMPK allele but did not assess pre-mRNA or poly (A)+ transcripts. All these diverse conclusions were based on quantitative PCR assays; in most cases restriction digest polymorphisms were used to discriminate between wt and DM alleles. The experiments that will be described in Chapter 2 were designed to resolve some of these controversies, using techniques less subject to artifactual distortion, in order to assess whether altered DMPK expression might contribute to development of DM.
DMPK protein. The possibility that DMPK mutation disrupts DMPK production has been addressed using anti-DMPK antibodies as well as through analysis of DMPK RNA. Although this question might appear to be fairly straightforward, it has, like research on DMPK RNA, yielded contradictory and confusing results. The first round of
publications on this question are now generally disregarded (64-66). Most of the antibodies used recognized 50-55 kD proteins (the predicted size of DMPK is 69 kD) and it was later shown that these proteins were still present in DMPK -/- mice (16) and hence must be cross-reacting species, not DMPK. More recently generated
antibodies recognize proteins of 64, 70, 71, 72, 74, 80, 82, and 85 kD (18, 19,
24-26, 67). Most of these size discrepancies probably reflect differences in SDS-PAGE
conditions that slightly alter DMPK migration, rather than differences in the species detected.
Surprisingly few new comparisions of DMPK protein levels in DM and control muscle samples have been published. Maeda et al. (24) observed reduced levels of 71 and 80 kD species of DMPK in skeletal and cardiac muscle from DM patients. However, Dunne et al.
(67) detected only a 64 kD species in human muscle with levels that are either constant
(relative to total protein) or increased (relative to slow myosin heavy chain, a type 1 fiber marker) in DM muscle samples. Dunne et al. did not explain why their antibodies only recognized one form of DMPK or how this form might differ from the significantly larger proteins detected by other antibodies. Experiments using a new set of monoclonal antibodies against DMPK that yielded further insight into the question of DMPK dosage
DMPK splicing. Antibodies can also be used to investigate the normal role of DMPK and to characterize the various proteins produced from the DMPK gene, but detailed analyses of these questions have not been performed. Alternatively spliced transcripts for DMPK have been identified and their relative levels in DM patients have been found to be unchanged (15). However, little is known about the different proteins derived from these transcripts. Only one isoform-specific antibody has been produced, against DMPK-4 (see Figure 2) (25). On western blots and in skeletal muscle sections it detects species with similar sizes and distributions as have been seen with other
antibodies (18, 24, 26), although the results are not identical. This could indicate that DMPK-4 is the predominant isoform of DMPK; however, since DMPK+4 is expected to be the same size and may have the same distribution as DMPK-4, this cannot be concluded. The other anti-DMPK antibodies are presumed to detect all forms of DMPK, yet they may preferentially react with a subset of isoforms. In particular, polyclonal antibodies generated against full length forms of DMPK (usually DMPK+4) may be biased towards recognition of these forms, since reactive epitopes may be contained within alternatively spliced exons. Most polyclonal anti-DMPK clearly contains some reactivity against epitopes present in all splice forms of DMPK (namely, exons 1-12), since they can detect artificially truncated forms of the protein. However, no attempts have been reported to determine their precise sensitivity, nor have the species detected by the antibodies on Western blots been conclusively identified as particular splice forms. In addition, potential differences in activity, subcellular distribution, and substrates due to alternative carboxyl termini have not been investigated. Efforts to fill
in some of these gaps, especially regarding the subcellular distribution of DMPK, will be presented in Chapter 3.
DMPK transcript aggregation. The subcellular distribution of DMPK in DM cells was studied indirectly by Taneja et al. (68) but, unexpectedly, they gained insight into DMPK transcripts rather than DMPK itself. They examined the subcellular localization of DMPK transcripts in order to assess whether the trinucleotide expansion might
disrupt a determinant of message localization within the 3' untranslated region of DMPK. Such determinants, which can regulate the distribution of protein produced from
transcripts, had been identified for a number of genes expressed in muscle (69-71). Taneja et al. found that some mutant DMPK transcripts were normally distributed within the cytoplasm of DM fibroblasts; however, they also detected nuclear aggregates of mutant transcripts in DM fibroblasts and muscle biopsies. Thus, although expanded repeats did not seem to interfere with the cytoplasmic distribution of transcripts (and
presumably protein), the repeats did confer novel characteristics upon the RNA, with potential significance for pathogenesis. The nuclear transcript "foci" were apparently distinct from other known nuclear structures, such as speckles and coiled bodies. Taneja et al. postulated that these foci might be sites at which other nuclear components were also sequestered, and that nuclear processes might be disrupted in DM cells. Focus formation could also theoretically interfere with processing of DMPK pre-mRNA from the mutant allele. Analysis of this question and comparison of relative levels of the nuclear and cytoplasmic mutant transcripts, aimed at determining whether transcript foci might contribute to DM, will be presented in Chapter 2.
CUG RNA-binding proteins. Transcript foci probably do not contain just DMPK transcripts, and several labs have searched for proteins that preferentially bind CUG
RNA repeats. The most interesting and thoroughly characterized of these proteins is CUG-BP/hNab50 (72). CUG-BP/hNab50 binds transcripts derived from the 3'
untranslated region of DMPK better than it binds actin transcripts, and it interacts preferentially with mutant, rather than wt, DMPK sequence. In addition, the
subcellular distribution of CUG-BP/hNab50 is somewhat shifted into nuclei in DM cells, and it has been speculated that this may be due to binding of the mutant DMPK
transcripts found there. However, this hypothesis is undermined by the finding that CUG-BP/hNab50 also shows nuclear displacement in DMPK -/- mice and in cardiac tissue from a patient with dilated cardiac myopathy (73, 74). Clearly, factors other than poly CUG tracts can regulate CUG-BP/hNab50, and it is not certain that the mutant DMPK transcripts are the cause of nuclear translocation in DM patients.
CUG-BP/hNab50 is not targeted to nuclear foci in DM myoblasts; instead it shows a diffuse nuclear distribution, as it does in control myoblasts. Stronger evidence is clearly needed to show that CUG-BP/hNab50, or other RNA-binding proteins, play a role in DM pathogenesis.
Mouse models of disease. Several disease models for DM have been tested through the creation of knockout and transgenic mice. Many of these mice suffer from a subset of
DM symptoms, but no animal model has managed to recapitulate the full DM pathology. Work is underway to generate a "knock-in" mouse carrying the expanded trinucleotide and some flanking human sequence within the context of endogenous murine DMPK. Thus, it remains to be seen whether even the presence of the human genetic disturbance will induce development of DM in mice. It may be that physiological differences between
mice and humans render mice immune to some of the mutation's human consequences.
Creation of DMPK -/- and +/- mice permitted testing of the hypothesis that DMPK insufficiency induces, or contributes to, DM development. DMPK-deficient mice are
viable and superficially normal, at least at an early age (16, 75). However, on some genetic backgrounds the absence of DMPK causes a late-onset progressive myopathy, marked by degeneration and increased regeneration of muscle fibers, by ultrastructural disturbances, including Z line distortion and mitochondrial abnormalities, and by
decreased force-generating capacity (75). Cultured myoblasts from DMPK -/- mice also have elevated basal Na and Ca++ concentrations in their cytosol and decreased resting membrane potentials (22). In addition, cardiac conduction abnormalities, including a prolonged PR interval and sporadic A-V block, are present in DMPK -/- mice (23). Most of these symptoms are less severe but still detectable in DMPK +/- mice. They are also characteristic of DM patients, suggesting that DMPK deficiency may be responsible for a significant subset of DM symptoms. However, other diagnostic aspects of DM, such as myotonia and congenital disease, have never been detected in -/- mice.
Transgenic mice overexpressing DMPK have also been created. As with the DMPK
-/-mice, variable results have been obtained, perhaps due to the genetic background of the mice. Alternatively, researchers may actually have used different DMPK cDNAs to create their transgenic lines, as "full length" DMPK could refer either to DMPK+4 or
DMPK-4. Mice created by Jansen et al. (16) displayed no skeletal muscle abnormalities despite overexpressing wt DMPK. The mice did develop hypertrophic cardiomyopathy;
however, this was histologically distinct from the fatty infiltration and fibrosis characteristic of DM cardiac tissue. Transgenic females were frequently ill during
pregnancy, and transgenic pups suffered from increased perinatal and neonatal mortality (cause of death not addressed), but male fertility was not impaired by the transgene. Overall, these transgenic mice seemed a poor model of DM.
In contrast, transgenic mice generated by Korneluk and Narang did display some of the muscular symptoms of DM (76). Sarcoplasmic masses, central nuclei, and type I fiber atrophy - all histological markers for DM - were frequently detected. Decreased fusion potential, indicating a defect in myoblast differention, was also observed. However, this may have been due to overexpression of DMPK's 3' untranslated region rather than to overproduction of DMPK, since these authors have blocked differentiation using only 293 bases from the untranslated region (77). In contrast, overexpression of just the coding sequence of DMPK has been shown to promote expression of muscle-specific genes by BC3H1 cells (78). Thus, multiple pathways may have been impacted by introduction
of the DMPK transgene into these mice. It is not currently possible to reconcile the conflicting data regarding overexpression of wt DMPK in mice.
Transgenic mice have also been used for studies of trinucleotide expansion and of the physiological impact of large trinucleotides, although most labs have only preliminary, not yet published, results (76, 79, 80). Transgenes containing 55 or more repeats flanked by varying amounts of the DMPK 3' untranslated region and coding sequences have demonstrated somatic and/or intergenerational instability in several independent studies. In most cases, only small (<6 bp) intergenerational changes in repeat length were observed (81, 82), but one researcher did detect some large changes (76). Monckton and coworkers' transgenics (162 CTG) showed impaired male fertility (80); however, this has not been reported by other researchers using sequences containing up to 320 CTG (76, 79). Only Narang and Korneluk's CTG transgenics had central nuclei and perhaps other muscular features of DM (76). Unfortunately, the formation of
eagerly await publication of experiments upon these mice, as it is currently very difficult to evaluate and interpret the discordant results.
Expression of genes adjacent to DMPK. Since altered DMPK expression in mice
has not yet induced all the symptoms of DM, and since the expanded trinucleotide was predicted to alter chromatin structure (59, 60), the impact of trinucleotide expansion upon the genes flanking DMPK has also been explored. DM patient DNA was found to have an altered chromatin structure, detectable as changed DNAse I sensitivity, surrounding the DMPK locus (83). Neighboring genes are found quite close to both the 5' and 3'
boundaries of DMPK, probably within this region of alternative structure.
DMR-N9(mouse)/59 (human), the 5' flanking gene, encodes a ubiquitously expressed
WD-repeat containing protein of unknown function that is especially abundant in brain and testis. DMAHP, the 3' flanking gene, encodes a homeobox-containing protein expressed at low levels in skeletal muscle, brain, and some regions of the eye. The fact that these two genes, like DMPK, are normally expressed in a number of the tissues affected by DM makes them attractive candidate genes for DM. Recently, two groups reported that steady-state levels of DMAHP transcripts from alleles in cis to DMPK trinucleotide expansions were reduced, presumably due to transcriptional repression (84, 85). In contrast, altered expression of gene 59 was not observed in DM patients (86). Thus, only deficiency of DMAHP is likely to contribute to DM pathology. Interestingly, expression of DMR-N9 was altered in some DMPK -/- mice, due to the targeting construct (87). This raises the possibility that some of the phenotypes previously attributed to DMPK deficiency in mice are in fact due to reduced expression of
neighboring genes. However, since the cellular roles of DMR-N9 and DMAHP, like that of DMPK, have not been defined, and knockout mice have not been generated for these
flanking loci, the precise relationship between DM and DMR-N9 and/orDMAHP insufficiency remains ambiguous.
Summary
The genetic basis for DM is the expansion of a CTG trinucleotide repeat in the 3' untranslated region of DMPK. DMPK encodes a serine-threonine protein kinase that probably contributes to excitation/contraction coupling; however, its precise function is unknown. Investigations into the impact of CTG expansion upon DMPK transcription, transcript processing, and protein production have yielded complex and contradictory results. Various groups have reported both overexpression and underexpression of DMPK in DM tissues. Mouse models of DM - knockouts and cDNA transgenics both with and without expanded trinucleotides - have also yielded confusing data. The similarities between DM patient muscle and the muscle of DMPK -/- mice suggests that DMPK deficiency could be a major etiological factor for DM. However, the surprising absence of non-repeat loss-of-function mutations in DM, coupled with the failure of DMPK
-/-mice to manifest the full range of DM symptoms, indicates that the triplets probably have deleterious effects beyond those on DMPK dosage. Potential effectors of the DM
phenotype include the nuclear transcript foci formed by mutant DMPK transcripts and DMAHP deficiency, due to transcriptional downregulation of this DMPK-flanking gene.
The work presented in Chapters 2 and 3 should help to resolve the controversy over DMPK dosage in DM patients, and thereby aid in the selection of appropriate mouse models for DM. In addition, it sheds some light upon alternative mechanisms of pathogenesis due to expanded trinucleotides by further exploring the formation of trinucleotide-based transcript foci. Finally, it offers new insight into the subcellular
distribution of the various DMPK isforms, which suggests new potential functions for the kinase.
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