MATRIX ATTACHMENT REGIONS IN THE HUMAN DYSTROPHIN GENE

Stephanie Doreen Ditta

A tbesis submitted in confordty witb tbe reguirements for the degree of Doctor of Pbilosophy Graduate Department of Molecular and Medical Genetics University of Toronto

O Copyright by Stepbanie Doreen Ditta 200 du="-?l?na- Cana

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A bstract

Duchenne muscular dystrophy @MD) is a lethal degenerative muscle disorder caused by mutations in the dystrophin gene. Dystrophin is a cytoskeletal protein associated with the plasma membrane. .dystrophin gene is exceptional with mspect CO its size and complexity. It is the largest gene identified to date, containing 79 exons across 2300 kb and at least seven distinct tissue-specific promoters. This X-linked gene is characterized by a high mutation rate, with most affected individuals having chromosomal rearrangernents. Approximately 60% of males with the disease have deietions in the dysimpbin gene and 6% have duplications.

In this study, the role of chmatin organization of the dystrophin gene in nuclear functions is investigated. The interactions of DNA with proteins of the nuclear rnatrix rnay be involved in such functions as defining chnimaiin loops or domains, DNA replication, transcriptional reguiation and iiiegitimate recombination. The DNA regions involved in these interactions are specific DNA sequences dedma& attachment regions (MARS). This work examines the possible involvernent of dystrophin gene MARS in both diseasecausing chromosomai rearrangements and transcriptional regulation.

Specifically, points of amchment of tbe dystrophin gene to the nuclear matrix have been identifieci in this study at the breakpoints of a duplication hma DMD patient and in the muscle- specfic promoter region. MARS have been identifieci at both the intmn 7 and inîron 9 breakpoints of a dystmphin gene duplication which resulted hm a non-homologous recombination event. This hding indicates the possible positionhg of these breakpoints in close proximity to one another on the nuclear matrïx, proviiàing the opportunity for

recombination to occw. Both bceakpoints contain topoisoncerase 1 consensus cleavage sites, suggesting a molecular mechanism for DNA exchange in tbis recombination event. In addition, two MARS have been found to flank the intemal muscle-specific promoter of the dystrophin gene. The proximity of these two MARS to this cis-acting mgulatory element indicates a possible role for these MARS in transcriptional replation hmthis promoter, perhaps through interactions with transcription factors on the nuclear matrix.

These results suggest important des for dystrophin gene MARS in both illegitimate recombination and transcriptional regdation in this gene.

iii Acknowledgements

1wodd like to thiuik the Canadian Institutes of Health Research (formerly the Medical Research Councii of Canada), tbe Ontario Graduate Scholarship Program and the Hospital for Sick Children Research Training Centre (Toronto) for the financial support of the work ptesented in this thesis.

1 acknowledge my supervisors, Drs. Ronald Worton and Peter Ray, as well as Drs. Brenda Andrews, Paul Sadowski and Lap-Chee Tsui, for serving on my supervisory cornmittee. My thanks to Dr. Arthur Roach for his guidance and support.

1am grateful to Dr. Amira Klip for her kindness, support and valuable advice.

1would like to thank my laboratory colleagues, pst and present, for their help and friendship, especiaily Ramona Cooperstock, Peny Howard, Xiuyuan Hu, Henry Klarnut and Christine Tennyson. 1also thank my friends Carol Freund, Geoff Clarke and Mandy Lowe.

My very special thanks to Fîora Krasnoshtein for her constant friendship and encouragement.

1wish to thank my mother, Doreen Ditta, my sisters, Susan Lightstone, Mary Louise Diua and Joan Frawley, and my extended family rnembers for their love and support. Many thanks to my aunt, Jean Dunham, who has made my stay in Toronto enjoyable. Special thanks to my cousins, Cameron Jenkins, who first sparked my interest in genetics, and Kirnberly Gray, who remindecl me of who 1 am. 1 also th& my father, Frank L. Ditta, for al1 that he taught me so long ago. Table of Contents .. Abstract ...... u Acknowledgements ...... iv Table of Contents ...... v List of Figures ...... vii Chapter 1 - Introduction Overview ...... 2 Dystrophin ...... -3 Duchenne and Becker Muscular Dystrophy ...... 3 Dystrophin Gene ...... 3 Dystrophin Structure...... 6 Dystrophin Function ...... 8 Dystrophin Isoforms...... 9 Matrix Attachment Regions ...... 12 Nuclear Matrix ...... -12 MAR Structure...... 16 MARS in Replication ...... -17 MARS in Transcription ...... 18 MARS in Illegitimate Recombination ...... 20 Rationale ...... 21 MARS and Illegitirnate Recombination in the Dystrophin Gene...... 21 MARS and Transcription in the Dystrophin Gene...... 22 Hypothesis ...... -24 Chapter 2 .Partid Gene Duplication in the Dystrophin Gene due to Recombination Between Matrix Attachment Regions Abstract ...... 26 Introduction ...... -27 Materials and Metbods ...... 29 Patient ...... 29 DNA clones ...... 30 Ce11 Culture...... 30 in vitro DNA-Binding Assay ...... 30 in vivo MAR Isolation Method ...... 32 Isolation of Total Genomic DNA ...... 33 Results ...... -34 Identification of MAR at intron 9 breakpoint ...... 34 Identification of MAR at intron 7 breakpoint ...... 40 DNA sequence-based MAR predictions ...... -43 Discussion ...... 46 Cbapter 3 .The Role of Matrix Attachment Regions in Transcriptional Regulation fmm the MusckSpecilic Promoter in the Dystrophin Gene

Introduction ...... -31 Materials and Methods ...... 53 DNA clones ...... 53 Ce11 Culture ...... 54 in vitro DNA-Binding Assay ...... 54 in vivo MAR Isolation Method ...... 54 Isolation of Total Genomic DNA...... 55 Results ...... -55 Identification of MARS using the in vitro method ...... 55 Refinement of... MARS using the in vivo method ...... 57 Tissue spec~ficity of MARS...... 63 Sequence analysis of the muscle promoterlexon 1 region ...... 65 Discussion ...... 68 Cbapter 4 .MPeusion and Fu- Dinetions Discussion ...... 72 Future Directions ...... -73 Binding of Proteins to Dystrophin Gene MARS...... 73 Transcriptional Regulation by Muscle-Specific Promoter Region MARS...... 74 MARS at Other Breakpoints in the Dystrophin Gene ...... 75 References ...... 77 List of Figures

Chapter 1 .Introduction 1-1 Dystrophin Gene...... 5 1-2 Dystrophin ...... 7 1-3 Matrix Attachment Regions...... 14 1-4 Duplication Junction of Patient HSC 689 ...... 23

Cbapter 2 .Partial Gene Duplication in the Dystrophin Gene due to Recombination Between Math Attachment Regions in vitro Method of MAR Identification ...... 36 Identification of MAR in Intron 9 O in vitro Study...... 37 Apolipoprotein-B 3' MAR .in vitro Study ...... -.. 38 in vivo Method of MAR Identification ...... 39

Identification of MAR in Intron 9 O in vivo Study...... 41 Identification of MAR in Intron 7 .in vitro Study ...... 42 Intron 9 MAR: Experimentally âetennjlled position coincides with sequence-based analysis...... 44 htmn 7 MAR: Experimentally detemirml position coincides with sequence-based analysis...... 45 Mode1 of Chromatin Organization in the Dystmphin Gene...... 47 Chapter 3 .The Role of Math AtWcbwnt Regions in Transcriptional Reylation from the Muscle-Specific PMmoter in the Dystrophin Gene in vitro Binding Assay of the Muscle PromoterExon 1 Region ...... 56 in vivo MAR Isolation Study of the Upstream 6.6 kb HindIII Fragment ...... 58 Apolipoprotein-B 3' MAR .in vivo Smdy...... 59 in vivo MAR Isolation Study of the Pmmoter-containing 3.4 kb HindIII Fragment ...... 61 in vivo MAR Isolation Study of the Ptomoter-containing 3.4 kb HindIII Fragment ...... 62 in vivo MAR Isolation Study of the Downstream 1.7 kb HindIIUXbaI Fragment...... 64 in vivo MAR Isolation Study of the Tissue Specificity of the Muscle Promoter MAR...... 66 Romoter Region MARS: Experimentally detennined positions coincide with sequence-based analysis ...... -67

vii CHAPTER 1 introduction Duchenne and Becker muscular dystrophy are caused by mutations in the gene encoding dystrophin, a cytoskefetal protein expressed in muscle. The dystrophin gene is extraordinary with respect to its size and complexity. Covering a physical distance of 2.3 Mb, it is the largest known gene, spanning 79 exons. The gene also contains at least seven promoters from which distinct dystmphin isoforms are produceci in a tissue-specific pattern.

Chromatin is organized within the nucleus by its attachment to the nuclear matrix at DM sequences dedrnatrix attacbment regions. investigations of these anchorage sites at other gene loci have previously revealed possible roles for them in such nuclear metabolic events as illegitimate recombination and the regdation of transcription.

This thesis examines the impact thaî the chromatin organization of this large and wplex gene may have on the occurrence of a disease-causing chromosomal rearrangement within the dystrophin gene and on the transcriptional regdation of this gene.

In Chaper 2, the question of the possible involvement of chmatin organization on illegiimate recombination in the dystrophin gene is addressed. Previously, a duplication within this gene, arising as a result of non-homologous recombination, was identifieci in a patient with Duchenne muscular dystrophy. The breakpoint regions of this duplication had DNA sequence characteristics observed in known matrix attachment regions. The experiments presented in this chapter were ched out to investigate the existence of functional ma& attachment regions at these b-nts in order to develop a mode1 of the rnechanism of this recombination event.

The work presented in Chapter 3 of this thesis focusses on the role of chmatin organization on transcriptional regulation in the dystmphin gene. The extraordinary size and compiexity of this 3 gene present a unique pmblem with respect to the organization of its chn>matîn for transcription. Here, an examination of the muscie-specific promofer region of this gene for matrix attachent regions begins to investigate this important issue.

In order to provide the required background, reviews of the dystrophin gene, its mutations, regdatory elements and protein proàucts, as weU as the structure and function of matrix attachent regions, are presented below.

DYSTROPHIN

Ducbenne and Becker Muscular Dystiapby

Duchenne muscuiar dystrophy @MD) is a common lethal genetic disorder. It is characterized prirnatiiy by the degeneration of skeletal and cardiac muscle. Signs of muscle weakness are fmt observed in patients with DMD at 2 to 5 years of age. Progressive muscle degeneration results in the loss of the ability to walk at approximately 12 years of age and death by the age of 20. This X-linked ipcessive disease is caused by mutations in the dystrophin gene, resulting in the absence or alteration of its protein product. The dystrophin gene is characterized by a high mutation rate. DMD affects 1 in 3500 male births, with one third of al1 cases resulting hm new mutations. Becker muscular dystrophy (BMD) is a milder alielic fonn of DMD. BMD patients experience a milder course of the disease with a later onset of syqptoms and longer survival.

Dystrophin Gene

The gene encoding dystrophin is the largest known gene, spanning over 2.3 Mb (Coffey et al., 1992; Monaco et al.. 199î). This enormous gene, which is approxirnately 100 times larger than 4 typical mammahn genes, is located on the short arm of the X chromosome at p21.2. It contains 79 exons which encode a 14 kb muscle franscnpt (Roberts et al., 1993; Koenig et al., 1988). Introns accwnt for more than 99% of the dysimphin gene and vary in size up to 280 kb (CoRey et al., 1992).

The dystrophin gene is also exceptionai with respect to the compkxity of its expression. The gene contains at least 7 different pmmoters/exon 1 regions fiom wbich dystrophin isofonns arie expressed in a tissue-specific manner (Fig. 1-1). Tbree of these fmt exons, from which the brain, muscle and Purkinje isoforms are transcribed, are spIiceâ to exon 2 (Boyce et al., 1991; Klamut et al., 1990, Gorecki et al., 1992). Four other fmt exons, located in introns 29, 44, 55 and 62, are spliced directly to exons 30, 45, 56 and 63, respectively (D'Souza et al., 1995; Lidov et al., 1995; Byers et al., 1993; Hugnot et al., 1992). These four transcripts encode the retinal, central nervous system, peripheral nerve and "non-muscle" isoforms of dystrophin.

Mutations in the dystrophin gene lead to the absence or alteration of dystrophin, a cytoskeletal protein. Approximaîely two tbirds of boys aff&cted with DMD/BMD have large chromosomal rearrangements in the dystrophin gene. The remaining one third may have smailer mutations, such as point mutations, small deletions or small insertions, present in the gene. Approximately 60% of afkcted individuals have a large interstitiai deletion involving one or more exons. These large deletions are not distributeci tandomly throughout the gene, but axe clustered in the central region of the gene. In fact, about 40% of aii deletions have breakpoints which map to intron 44 (den Dunnen et al., 1989; Forrest et al., 1987; Forrest et al., 1988). Deletions which shift the reading irame of the transcnpt, resulting in truncateù proteins with an expccted loss of function, are found mostly in patients with the morie severe DMD, while those deletions which preserve the reading fiame are seen mostly in BMD patients (Monaco et al., 1988; Malhotra et al., 1988; Koenig et al., 1989; Gillard et al., 1989). About six percent of DMDBMD patients have duplications of one or more exons (Hu et al., 1990; den Dunnen et al., 1989; Bmin central NeWou. systm Muscle Pwipheral Newous System

Figum 14. Dy.bophin Gene PiomatdExon 1 Regions Schematic diagram of the dystrophin gene. Amws indicate the positions of the prornoter/exonl regions. Boxes show the positions of the nearest downstream exons. (Not drawn to scale.) 6 Angelini et al., 1990). Unlike dystmphin gene deletions, no clustering of duplications has been observed in t& gene. However, the rrading fnuae hypothesis pmposed for dystrophin deletions appears to hold true for duplications as weil (Hu et al., 1990, Angelini et al.. 1990). DMD and BMD have also been noteù in fende patients. In these rare cases tbe disease has been shown to be a result of bai- X:autosome translocations with breakpoints occwriog in the dystrophin gene. In these patients, non-random inactivation of the nomial X chromosome occurs and the severity of the disease likely depends on the fraction of nomial X chromosomes that are active in muscle tissue (Kean et al., 1986; Boyd et al., 1986; Bodmg et al. 1989).

Dystrophin Structure

Based on cornparisons of the amino acid sequence of the full-length dystmphin isoform (427 kD) with known proteins, a mode1 of dystrophin structure was developed which predicts a number of distinct domains (Fig. 1-2). The N-terminai actin-binding domain has similarity to the actin-b'mding domain of a-actinin, and has been shown to bind to actin (Corrado et al., 1994). Following the N-terminal dornain is the rod domain which shows similarity to the repeat dornains of spectrin. It contains 24 repeat units which are predicted to fold into triple-helices, fonning a rod structure (Ah et al., 1993). Adjacent to the rod domain is the WW domain, whose protein-binding motif has been identifieci in other proteins. This motif is known to interact with probe-rich regions of proteins (Bork et al., 1994). Foilowing is a dornain nch in cysteine residues and, finally, the C-terminal domain which has similarity to utrophin. These 1st two domains are essential to the hction of dystrophin. They interact with a membrane- associated complex of proteins and glycopmteins, the dystrophin-associated glycoprotein complex, which includes dysttoglycans, sarcoglycans, dystrobrevins and syntrophins (Campbell et al., 1989; E~astiet al., 1991; Suzuki et al., 1994; Jung et al., 1995). rod min

Figum 1-2. Dystrophin Schematic diagram of the dystrophin protein, indicating the actin-binding, rod, WW, cysteine-rich and C-terminal domains. (Not drawn to scale.) Dystropbin Function

Two cellular functions have been proposed for dystrophin. The fmt is a de in muscle membrane stabiion during muscle contraction and relaxation. Dystrophin has been shown to associate with the cytoplasmic surface of the sarcolemma (Zubryzycka-Gaam st al., 1988) through the dystrophin-associated glycoprotein complex, This transmembrane complex, in tum, intetacts with merosin, a subunit of laminin, in the basal lamina (Ibraghimov-Beskrovnaya et al., 1992). It is also known that dystrophin can bind to cytoskeletal actin. Together dystrophin and the dystrophin-associateci glycoprotein complex are thought to forrn a structural link between the actin cytoskeleton inside the œil and the extracellular matrix. In DMD patients, the absence of dystrophin hm muscle results in the concomitant absence of the dystmphin-associated glycoprotein complex, dismpting the link between the cytoskeleton and extracellular matrix (Ervasti et al., lm). In dystrophie muscle tissue, the separation of the sarcolemma hmthe basal lamina has been observed, suggesting that the loss of the structural link leads to muscle tissue daniage (Carpenter et al., 1979). Increased intracellular calcium levels have been observed in mdx: mice, a murine mode1 for DMD. These increased levels may be caused by alterations in sarcolernmal calcium channel activities, and may induce muscle necrosis by activating calcium-dependent proteases (Fong et al., 1990).

The second fûnction which has been proposed for dystrophin is a role in the clustering of receptors in the plasma membrane. Both dystrophin and utrophin, a protein which has similarity to dystrophin, have been found in muscle at neuromuscular junctions and are known to interact with the dystrophin-associated glycoprotein complex (Matsumura et al., 1992). Agrin, a protein with similarity to laminin, has been shown to bind to a component of the dystrophin-associated glycoprotein complex, a-dystroglycan, and can induce clustering of acetylcboline receptors (Campabelli et al., 1994; Gee et al., 1994). As dystrophin isoforms have been identifieci in brain and retina, it is possible that dystrophin may be involved in synapse formation in neural tissues as well.

Dystropbin Isoforms

With at least seven distinct promoters, the dystrophin gene is a locus of great complexitty. The transcripts produced from each of the seven known promoters have a unique exon 1. Each of the dystrophin isofom expressed from these seven promoters has a unique amino terminus, with the exception of the central nervous system isofonn. Translation of the CNS isoform starts in exon 5 1 rather than in its unique exon 1 (Lidov et al., 1995). Aitemative splicing also contributes to the diversity of dystrophin isoforms, and likely affects kirconformation and function. Alternative splicing primariiy involves exons 71 to 74 and 78 of the dystrophin gene, which encode amino acid residues of the C-terminal dotnain. Splicing involving exons 7 1 to 74 yields in-frame deleted transcripts, while the splicing of exon 78 results in a shift of the reading fiame and an alternative carboxy temiinus in the protein.

The brain isofom is expressed fiom a promoter which is located 137 kb upstream of ttie muscle-specific promoter and about 400 kb upstearn of exon 2 (Boyce et al., 1991; Wood and Whittaker, 1994). Expression hmthis promoter produces a 14 kb full-length dystropbin transcript and a 427 kD full-length protein product. The brain isoform is expressed in neurons of the cerebrai cortex and the bippocampus (Barnea et al., 1990, Chelly et al., 1990, Gorecki et al., 1992). Common cis-elernents involved in transcription, including a TATA box, have not been found at this promoter.

A full-length dystrophin transcript and bill-length isofonn are also expressed from the muscle- specific promoter (Klarnut et al., 1990). The muscle isoform of dystmpbin is expressed in skeletal aad cdacmuscle, and at lower leveb in smooth muscle and brain (Chelly et al. 1988). Dystrophin can be detected at the membranes in human skeletal muscle from about 9 weeks 10 gestation, and increases over the course of developmnt (Clerlc et al., 1992). The expression of dystrophin hm also kenstudied during the course of differentiation in cultured myogenic cells. When serum. wbich contains growth fpctors, is removed hmthe media of growing myoblasts, thea ceils undergo a differentiation process like that which occurs in vivo. The myoblasts stop dividing, elongate and fuse to form multinucleated myotubes. Dystrophin îranscript levels arr not detectable in growing myoblasts, but can be detected afier the induction of differentiation (Klamut et al., 1990; Lev et al., 1987). (The sequence characteristics of the muscle-specific promoter are discussed below.)

The nirkinje isoform is also a 427 kD isoform which is translaîed from a full-length transcnpt expressed in the Purkinje ceils of the cerebellum. Its promoter lies downsbeam of the muscle- specific promoter, in intron 1, and its unigue fmt exon is spliced directly to exon 2 (Gorecki et

al., 1992).

The retinal isoform of dystrophin is a 260 kD protein expressed primarily in the outer plexiform layer of the retina and at low levels in brain. Abnormal neural transmission across the mtina in some DMD patients and the correlation of such transmission anomalies with the absence of 260 kD dystrophin (the 427 kD and 7 1 kD isoforms are also âetected here) indicates that dystrophin may have an important function in this neural tissue @'Souza et al., 1995; Piliers et al., 1993). The promoter of the tetinal isoform is located in intron 29, and its unique exon 1 is spliced ditectly to exon 30. This isofonn lacks the actin-binding domain of the full-length isoforms, but contains 15 spectrin repeat units of the rod domain dong with the complete WW,cysteine- rich and C-terminal domains.

The central nervous system isoform of dystrophin is expressxi primarily in the glial cells of the brain (Lidov et al., 1995). It has also been obsemd in the developing kidney (Lidov and Kunkel, 1997). Its pmmoter is located in intron 44 of the dystrophin gene and its unique exon 11 1 is spliced to exon 45. Translation begins at a =thionine codon in exon 51 and results in a 140 lcD pduct.

The peripheral nerve isoform is expressed in the Schwann =Us of peripberal nerves (Byers et al., 1993). This 1 16 kD isoform is expressed hma pmmoter in intron 55. Its exon 1 is spliced to exon 56 of the dystmphin gene. The CNS and peripheral nerve isoforms contain four and two spectrh repeats of the rod domain, respectively, as well as complete WW,cysteine-rich and C-terminai domains.

The "non-muscle" isoform of dystrophin is expressed from a promoter in intron 62, and has a unique exon 1 which is spliced to exon 63 (Hugnot et al., 1992). This isoform is 7 1 kD in size and contains only the cysteine-rich and C-temiinal domains, dong with its unique amho terminal. It is expressed in many tissues, including brain, liver, kidney, lung and retina (Bar et al., 1990, Lederfein et al., 1992; Howard et ai., 1998a; Hugnot et al., 1992). Evidence of expression has aiso been observed in HepG2, HeLa and ES ceii lines, as well as cultured neuronal and glial cells (Rapaport et al., 1992; Rapaport et al., 1993; Lambert et al., 1993). The transcript of the "non-muscle" isoform has been detected in fetai rat muscle but decreases after birth (Lambert et al., 1993). This is opposite to the expression pattern of the full-length isofom which increases in muscle during development. Similarly, expression of the "non- muscle" isofonn occurs in cultuced myoblasts but decreases during diffemntiation into myotubes (Schofield et al., 1994; Tennyson et al., 1996). AIthough the "non-muscle" isoform is known to localize to the membrane in some cell types (Rapaport et al., 1993), it may play a different role from the full-length isoform in muscle. This isofonn can restore the dystrophin- associated glycoprotein cornplex to the membrane in both myogenic ceils that do not express full-length dystrophin and in rndx mice expressing a "non-muscle" isoform Éransgene (Cox et al., 1994; Greenberg et al., 1994). However, the muscle phenotype of the transgenic rndx mice was more severe than that of their rndx counterparts. Recently, the "non-muscle" isofom of 12 dystmphin has ben shown to interact witb actin in myogenic celis. It was obsemd to local& to stress fibrie-like structures and to cosediment with actin bundles upon fiactionation of myogenic cells (Howard et al., 1999). An actin-binding motif at the unique amino terminal of the "non-muscle" isofonn is thought to be responsible for its direct interactions with actin (Howard et al., 1998b). The pmmoter of the "non-muscle" isoform has characteristics of a housekeeping promoter, including a high GC content and no TATA box, which is consistent with the wide tissue distribution observed.

MATRIX ATTACHMENT REGIONS

Nuclear Mat*

The nuclear matrix was fmt identifid by Berezney and Coffey in 1974 as a nuclear protein framework which remained subsequent to the removal of chromatin (Berewiey and Coffey, 1974). Following mntsof nuclei with high salt (2M NaCl) and DNaseI to remove histones and DNA, they observed stable residual structures by electron microscopy. These structures could be disrupted by protease digestion, indicating tbat this fiamework was composed primarily of proteins. Tightly bound DNA and RNA also remained.

Using resinless section electron microscopy, improved images of the nuclear matrix were achieved by Fey et al. (1986), revealing a network of 10 nm core filaments covered with 20 to 30 nm particles, enclosed within the nuclear lamina The composition of the core filaments has yet to be determineci. Large dark masses, which are present within the nuclear matrix, may represent remnant nucleoli.

Despite nuclease treatment, some DNA sequences cosediment with the proteins of nuclear matrices. These DNA sequences, calied maaix attachment regions (MARS), anchor DNA to the 13 nuclear matrix and, likely, the chromosome scaffold. The interaction of MARS with proteins of the nuclear maaU is thought to organize cbromatin into loops or domains (Fig 1-3). Support for this hypothesis cornes hmobservations by electron microscopy of DNA loops extending from histone-depleted nuclei and chromosomes (Paulson and Laemmli, 1977). In vivo, these DNA loops or domains, defined by MARS, can undergo varying levels of condensation.

The original protocol for nuciear matrix preparation, developed by Berezney and Coffey (1974), employed nuclease (DNaseI) digestion and high io~cstrength (2nd NaCI) to remove chromatin from isolated rat ber nuclei. The hi@ saIt concentration of the extraction procedure was considered to be relatively harsh, and did not cornpletely maintain the nuclear matrix ultrastructure. Other modifications of this protocol, using different salts, different ionic strengths and different enzymes, were developed. However, Mirkovitch et al. (1984) used another approach to remove chromatin. This group used the mild ionic detergent, lithium diiodosalicylate, under low sait conditions to remove histones, followed by digestion by restriction enzymes to remove most of the DNA. MARS identified using this protocol have also been called scaffold attachment regions (SARs). This protocol includes a heat stabilization step pior to the extraction procedure. Concems have been raised about the possibility that this siabilkation step creates artefactual attachments of DNA to the nuclear ma& proteins. Yet another protocol used electrophdc fields to remove chromatin from the nucleus at isotonic salt concentrations following restriction endonuclease digestion (Jackson and Cook, 1985a).

Many proteins of the nuclear maûix have now been identified. Topoisornerase II, for exampie, has been shown to be a major component of the nuclear matrix and chromosome scaffold (Berrios et al., 1985; Eamshaw et al., 1985), and to bind cooperatively to MARantaining DNA in vitro (Adachi et al., 1989). Topoisomcrase II is an enzyme that can coattrol DNA Figure 13. MaMx Attichment Regions Schematic diagram of MARS bound to the proteins of the nuclear rnatrix, defining chromatin loops or domains. 15 supercoiling, altering DNA topology, through the double-stranded cleavage and ligatim of DNA (Wang. 1985). Although a major constituent of the nuclear mahix in many œil types, topoisornerase II is not essential to nuclear matrix structure or chromatin orgsniuuion, which are maintained in differentiated cells lacking topoisonierase II (Heck and Earnshaw, 1986; Phi-Van and Stratling. 1988). Topoisornerase 1, which controls supercoiling through the cleavage and ligation of a single-strand of DNA, may only associate with the nuclear mai& in a facultative rnanner (Boulikas, 1995; Nishizawa et al., 1984).

Other enzymes involved in the metabolic events of the nucleus have been found in the nuclear matrix. Both DNA plprase a and B activities have been observed in nuclear matrix fractions (Smith and Berezney, 1980; Smith et al., 1984). Nuclear rnatnx preparations also retain most of the transcriptional activity of the nucleus, suggesting that RNA polymerases a~ immobilized on the nuclear matrix (Jackson and Cook, 1985). Human RNA polymerase II subunit 11 has been shown to interact with keratin, a nuclear matrix component (Bruno et al., 1999). Most of the histone acetyltransferase and histone deacetylase activities are xetained by the nuclear matrix fraction of fmctionated nuclei (Hendzel et al., 199 1; Hendzel et al., 1994).

Nuclear rnatrix DNA-binding proteins include the lamins, which represent the major components of the nuclear lamina at the perïphery of the nucleus. These filamentous polypeptides are thought to be largely responsible for anchoring chromatin loops to the nuclear lamina. Studies indicate that only B lamins are present in undifferentiated cells, while A lamins appear as differentiation proceeds (Lanoix et al., 1992; Mattia et al., 1992). ARBP (attiichrnent region binding protein) has been shown to bind to MAR DNA in a cooperative mode and, by dimerization, can cause looping of DNA in vitro, suggesting a similar role in the nucleus (von Kries et al., 1991). Similarly, SAF-A (scaffold attachent factor A) can bind DNA in a cooperative rnanner and bnngs SAF-A sites together, causing looping of DNA (Romig et al., 1992). Interestingly, NUMA (nuclear mitotic apparatus protein) is found in the nuclear maûix 16 of interphase nuclei but is localized at the spindle poles of the rnitotic apparatus during œil division (Lyderson and Pettijohn, 1980). Rather than forming fdaments, NUMA has been observed to fom multi-ann oligomers in vitro and three-dimensional lattice structures when overexpressed in cells (Harôorth et al., 1999; Gueth-Hallonet et ai., 19%).

Transcription fxtors have also been identifiecl as components of the nuclear matrix. It has been suggested that the nuclear matrix may act to concentrate and localize transcription factors within the nucleus, thereby playing a role in gene transcription (Stein et al., 1991). Transcription factors which have been found in the nuclear matrix include NMP-2,c-Myc, NF-1,RFP and corticosteroid receptors (Bidwell et al., 1993; Waitz and Loidl, 1991; Sun et al., 1994; Isomura et al., 1992; van Steensel et al., 1991). As weîl, van Wijnen et al. (1993) used oligonucleotide sequences of transcription factor binding sites SP- 1, ATF, CCAAT, C/EBP, OCT-1, and AP-1 to show that the binding activities of these transcription faictors are enriched in the matrix versus non-matrix nuclear fractions. They also discovered that îhe distribution of activity between the nuclear htions is cell type-specific.

MAR Structure

Matrix attachment regions are specific DNA sequences which anchor chromatin to the proteins of the nuclear matrix. They have been isolated from many different eukaryotic species including plants, animals and insects. The interactions of MARS with proteins of the nuclear matrix are thought to be involved in many metabolic functions in the nucleus, including DNA replication and repair, transcription, and RNA splicing and transport.

MARS are usually several hundred base pairs in length and, on average, occur at 90 kb intervals in the mammalian genome (Vogelstein et ai., 1980). Their distribution in the genome is non- random. They are often located in the 5' and 3' flanking regions of genes (Gasser et al. 1986; 17 Phi-Van et al. 1988; Bode et al. 1992; Levy-Wilson et al. 1989). and at elements involved in transcriptional control. MARS have not been defïnitively identifid in coding sequences (Beggs and Migeon, 1989; Brotherton et al., 1991; Ellis et al, 1988; Schuchard et al., 1991). However, some have been found in introns (Awamova and Paneva, 1992; Beggs and Migeon, 1989; Brotherton et al., 1991; Cockerill aad Garrard, 1986; Cockerill et al., 1987; Ellis et al, 1988; Sykes et al., 1988).

Although tùey share little sequence homology with one another, most MARS are A+T-rich regions of DNA. Most MARS also contain consensus binding sites for topoisornerase II

(Boulikas, 1993). MARS can contain oligo(dA)/oligo(dT) tracts which fom a nmow minor groove (Gasser et al., 1986). Some MARS contain the ATTA, ATïTA and ATTITA motifs also seen in homeotic protein binding sites and origins of replication, while some have TG-rich sequences (Boulikas, 1992). DNA sequences characteristic of cwed or kinked DNA have been observed in MARS (Boulikas, 1993). DNA bending (Anderson et al., 1986; von Kries et al., 1990) and unpairing (Kohwi-Shigematsu and Kohwi, 1990; Cockerill et al., 1987; Bode et al., 1992) properties are also associated with MARS. In addition, MARS are capable of binding to matrices prepared hmother species (IzaUflalde et al., 1988). These characteristics suggest that the DNA topology rather than the DNA sequence of MARS is probably important for recognition by nuclear matrix proteins.

MARS in Replication

The mle of the nuclear matrix in replication has been suspected for many years. The fmt evidence was published in 1975 by Bereniey and Coffey, who demonstrateci, by injecting [3~-thymidineinto regenerating rat liver, that newly synthesized DNA was associated with the matrix fissction of the nucleus. Analyses of DNA replication intennediates, using two- dimensional gel el8ctrophorcsis has confirmed that replication forks are associated with the 18 nuclear matrix (Vaughn et al., 1990). Along with the fmding of DNA polymerase activity in matrix tiactions of nuclei (Smith and Bertzney, 1980; Smith et al., 1984), this evidenœ indicates thaî replicaîion occurs at fmed sites on the nuclear ma&. Using fluorochrome- conjugated triphosphates, DNA synthesis has been observed as nzplication foci within the nucleus, further supporthg the proposed localization of replication machinery at specinc sites on the nuclear matrix (Hassan and Cook, 1993).

Origins of replication have been localized at or near known MARS. Such origins have been identifkd in the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary oells (Dijkwel et al., 1988; Pemov et al., 1998), the human hypoxanthine-guanine phospho~bosyltransferase (HPRT)gene (Sykes et al., 1988; shown to be an autonomously replicating sequence (ARS) in S. cerevisiae), and the bovine papilloma virus (Adom et al., 199 1). Two ongins of replication have been found withia the MARs of the Epstein-Barr vim (Jankelovich et ai., 1992; Mattia et al., 1999). OriP acts as the origin of replication during the latent cycle and is contained within a MAR which is bound to the matrix during this cycle. After induction of the early events of the lytic cycle, the virus is attached to the nuclear matrix at a mgion encompassing OriLyt, the replication origin used during the lytic cycle.

MARS in Transcription

Interactions between chromatin and the nuclear matrix proteins may impact gene transcription in different ways. Some MARS have been suspected of serving a boundary function, isolating one

DNA region hmthe effects of cis-acting elements in a neighbouring region. Other MARS arie located at or near cis-acting regdatory elements and may be positioned at sites on the nuclear rnaîrix wbere transcription factors are concenrated. Dynamic associations between active chmatin and the nuclear matrix might occur through direct interactions of active chromatin with the transcriptionai machinery and transcription factors. It bas been observed in 19 ktionation studies that transcriptionally-active DNA is found associateci with the nuclear matrix, while inactive genes are not (Gerdes et al., 1994).

MARS often flank genes or gene clusters, and have been fond to CO-locaiizewith the DNase I- sensitive chromath domain borders of genes (Gasser et al., 1986; Phi-Van et al., 1988; Bode et al., 1992; Levy-Wiîson et al., 1989). Due to this observed positioning of MARS at gene loci, MARS have been examined for a suspected dein boundary function. MARS hmthe chicken lysozyme, the human Sinterferon, and the human apoigopprotein-B genes have each been shown to confer a position-independent and copy number-dependent increase in transcriptional activity of a reporter gene flanked by these elements in stably-transfected cells, indicating that each serves a boundary function in the integrated state (Stief et al., 1989; Phi-Van et al., 1990,

Klehr et al., 1991 ; Kalos and Fournier, 1995). &interferon MARS have been shown to elevate transcription levels only in the integrated state and not in transiently-transfected cells (Klehr et al., 1991). The chicken lysozyme gene MAR has also been shown to confer a position- independent effect on transcriptional regdation of a reporter gene in transgenic rnice (McKnight et al., 1992; Bonifer et al., 1994; McKnight et al., 1996). Deletion mutants of this MAR displayed ectopic expression of the reporter gene, indicating that this MAR may function to suppress ectopic expression (Bonifer et al., 1994). MARS hmthe human apolipoprotein-B and alphal-anitrypsin genes showed position-independent effects on transgene expression in Drosophila (Namciu et al., 1998).

Many MARS have been found at or near cis-acting regdatory sequences. Rornoter region MARS have been identifiecl in the human histone H4, hwnan osteocalcin and Drosophila alcohol dehydrogenase genes (Dworetzky et al., 1992; Stein et al., 1991; Gasser and Laemmli, 1986). The interactions of nuclear matrix proteins 1 and 2 (NMP- 1, NMP-2),transcription factors aiso known as W1 and AML, with the osteocalcin promoter are thought to play a rok in transcriptional mgulation hmthis promoter (Stein et al., 1997). MARS found at or near enhancers include those in tbe muse IgH, mouse Igic, human &globia and chicken bglobin genes, and the Epstein Barr virus (Cockerill et al., 1987; Cockerill et al., 1986; Jarman and

Higgs, 1988; Brotherton et al., 199 1; Jankelevich et al., 1992). Deletion of the I~KMAR led to reduced IgK gene expression in transgenic mice (Xu et al., 1989). One of the MARs flanking the Igp gene enhancer has been shown to extend chromatin accessibility hmthis enhancer (Jenuwein et al., 1997). Tbe human &-globingene contains a silencer which associates with nuclear matrices (Yan and Qian, 1998).

As well, tissue-specific differences in matrix binding could have an impact on gene expression. MARS hmthe flanking regions of the avian beta-globin, avian maüc enzyme, and tk human apolipoprotein-B genes show tissue-specific binding to the nuclex matrix, suggesting a tissue- specifk influence on gene expression (Levy-Wilson et al., 1989; Bmtherton et al., 1991). In each case, the tissue-spccific MAR is matnx-bowid in cells where the gene is expressed, but unbound where the gene is not expressed.

MARS in Illegitimate Recombination

A dysfimction of some MARS rnay be a dein illegitimate recombination. MARS might be susceptible to the topological strains of DNA loops or to cleavage by enzymes. MARS have been found at breakpoints of chromosoma1 rearrangements in the human myeloid-lymphoid leukemia @LI,)gene, mouse immunoglobulin whain gene, human ring chromosome 21 and human $-globin gene cluster (Anand et al., 1988; Sperry et al., 1989; Strissel Broeker et al., 1996; Vanin et al., 1983), some of which are known to be a result of non-homologous recombination (Strissel Broeker et al., 1996; Sperry et al., 1989; Vanin et al., 1983). As well, the dihydrofolate reductase (DHFR) gene MAR is located at junction between amplification units (Dijkwel et al., 1988). Topoisornerase II cleavage has been demonstrated at the breakpoints in the mouse immunoglobuün u-chain gene and the human Mgchromosome 21 21 (Sperry et al.. 1989). in addition, topoisomnse II consensus cleavage sites have been identifid withùi the MLL gene MAR (Stnssel Bmeker et al., 1996). Tberapy-dated acute myeloid kukemia (t-AML) bas been obsemd in some patients who naived topoisomrarc II inhibitors as treatment for a primary neoplasm (GU Super et al., 1993). Translocation breakpoints found in some patients with either t-AML or de novo AML have been mapped to the MLL gene MAR (Sbissel Broeker a al., 1996). These observations suggest a mo1ecular mechanism for tbese illegitimate recombination events involving topoisornerase ïï. Topoisornerase II can mediate illegitirnate recombination in vitro (Bae et al., 1988; Ikeda, 1986).

RATIONALE OF THJB?S

The organizattion of chmatin within the dystrophin gene may involve aîtachments to the nuclear matrix at specific DNA sequences. However, due to its extraordinary size and complexity of the dystrophin gene, its chrornatin organization may differ greaîly from that of other genes. Generally, MARS are found in the flanking regions of genes. However, the size of the dystrophin gene (2.3 Mb) greatly exceeds typical inter-MAR distances of 90 kb, indicating that many MARS might exist within this gene.

MARS and Ulegitimate Recombination in the Dystrophin Gene

MARS may be involved in some of the many disease-causing chromosomal rearrangements observed in DMD patients. Some patients, stuclied in out laboratory, were found to have chromosomal rearrangements resulting hmnon-homologous recombination events (Hu et al., 1991; Bodnig et ai.. 199 1). Tbe breakpoints regions also showed chaiacteristic featuries of MARS. 22 One DMD patient, HSC689, had a duplication of exons 8 and 9 of the dystrophin gene. involving breakpoiits in i-s 7 and 9 (Hu et al., 1988). RFLP (restriction fiagrnent length polymorphism) analysis showed that the duplication liicely arose in the patient's materna1 grandfather by an unequal cross over between the sister chromatids of the X chromosome (Fig.

. MA-, Hu a al., 1989). 'Ibe duplication event pmbably occdduring spermatogenesis, although not necessariiy during meiosis. The bre-nts of this duplication are, like MARS, AT-rich (Hu et al., 1991). The AT

MARS and Transcription of the Dystrophin Gene

MARS may play a role in the regulation of transcription of the dystrophin gene. MARS coincide

with cis-acting regulatory sequences at any or ail of the pmmoter regions within this complex gene.

Klamut et al. (1990) examined the dystrophin gene promoter hmwhich the muscle isoform is transcribed. They identified an 850 bp sequence upstream of tb transcription initiation site which can direct the muscle-specific expression of a reporter gene. They also found binding site motifs which am known to be involved in expression of other muscle-specific genes. These motifs include a muscle-CAAT consensus sequence and a CArG box, both of which have been found in the promoters of other muscle genes. The CASbox is known to be a binding site for

the senun response factor (SRF), a transcription ' factor involved in the activation of transcription in muscle genes (Walsh, 1989). In addition, three MEF-1 (myocyte- Fiaure 1-4. Duplication Junction of Patient HSC689 A)-schematic dikgram of the mechanism of duplication in patient HSC6û9. Boxes represent exons of the dystrophin gene. B) DNA sequence from the patient duplication junction and from intron sequence fmm a normal individual. Asterisks indicate the position of the duplication breakpoints. Horizontal ams indicate topoisornerase I consensus sequences. 24 specific enhancer-binding nuclear factor 1) consensus sequeme motifs were identified, two of which contain an E box (CANNTG). MEF-1 is expresseà only in differientiated myotubes and shares antigenicity with MyoDl which can transfonn fibmblasts into myoblasts (Buskin et al., 1989).

A muscle-specific enhancer has been identifid appmximately 6 kb downstream hm the dystrophin gene muscle exon 1 (Klamut et al., 1996). Located in inîmn 1, the core enhancer is 195 bp in length (Klamut et al., 1997). Like the muscle-specific promoter, the enhancer aiso contains three MEF-1/E box consensus binding sites. In addition, two MEF-2 consensus sites have been identified. MEF-2 aiso skssimitarity with MyoD 1. An overlapping MEF- I/MEF- 2 site within this enhancer was shown, by DNaseI fwtprinting assays, to bind factors from myotube nuclear extracts. Mutations in this overlapping site decreased expression of a reporter gene in myotubes.

Hypothesis

1 hypothesized îhat MARS might exist in the dystrophin gene, and rnight participate in DMI, causing chromosomal rearrangements and in transcriptional regdation hmthe promoters of this gene. To test this hypothesis, 1 asked whether matrix attachent regions c~localizewith known sites of iliegitixnate recombination in the dystrophin gene or with known regdatory regions. To analyze the involvement of MARS in illegitimate recombination in the dystrophin gene, 1 have assayed the recombinaiion breakpoints fiom a DMD patient having a partiai gene duplication resdting hma non-homologous reçombiion event. To test for possible MAR involvement in transcriptional =Nation in the dystrophin gene, 1 exafnined a 24 kb DNA region at the muscle-specific pmrnoter region for nuclear matrix-binding. CHAPTER 2

Partial Gene Duplication in the Dystrophin Gene due to Recombinatjon Between MatRn Attachment Regions.

Xiuyuan Hu. a former nsearch associate in the laboratory, camed out the experiments involving intron 9 of the dystmphin gene. The author ducted experiments involving intmn 7 and the apoüpoprotein-B MAR,and examinai DNA sequence data. Duchenne muscular dystrophy @MD) is caused by genetic alteration in the 2300 kb dystrophin gene. Approximate1y 60% of the alterations are intragenic deletions while another 6% are intragenic duplications. In a study of a patient with a dystrophin gene duplication, it was shown previously that sister chmatid exchange took place by non-homologous recombnation between DNA regions containing SeQuence motifs that are ofien associateci with matrix attachment regions (MARS). Both duplication breakpoints in this patient also containeci topoisomerase 1 consensus cleavage sites. This study shows experimentaüy the existence of functional MARs at both breakpoints of the duplication in this patient. These are the first MARS to be idcntified in the dystrophin gene, and represent the fîrst example of a non-homologous ncombinaîion event involving MARS at both breakpoints. These results suggest a molecuiar mechanism for duplication in DMD whereby binâing of the two bmùcpoiit regions to the nuclear matrix brings them into close physical proximity witbin the nucleus, providing the opporninity for recombination to occur via topoisomerase 1cleavage. The largest gene identined to date is that encoding dystrophin, the cytoskeletal protein that is defective in Duchenne and Becker musçular dystrophy @MD, BMD). Its enormous size, estimated at 230kb, and its complexity, with 79 exons and multiple promoters, make it unique in many respects.

The dystrophin gene has, for example, at least seven tissue-specific promoters (Boyce et al., 1991; Byers et al., 1993; D'Souza et al., 1995; Gorecki et al., 1992; Hugnot et al., 1992; KIamut et al., 1990, Lidov et al., 1995). Three promoters are located at the 5' end, all upstream of exon 2, but spaced over 100 kb apart (Wood et al., 1994). Within the gene, four additional promoters initiate transcription hmwithin intmns 29,44,55 and 62.

Io addition to the large intrms at the 5' end, hee additional inmns withh the dystrophin gene am over 100 kb and many others are over 30 kb in size. Because of its size, transcription of a single message takes in excess of 16 hours, and the full 2300 kb mRNA is never reahed, as splicing occurs to remove introns at the 5' end long before the 3' end of the message is transcribed (Tennyson et ai., 1995)

Another consequence of such a large gene is the frequency and type of mutations that disnipt the gene. Approximately one third of DMD cases arise fiom de novo mutations. Unlike a typid gene where genetic alterations are normally point mutations or deletions or duplications of a few nucleotides, the majority of alterations responsible for DMD are deletions or duplications that span mdtiple exons and involve hundreds of kilobases of the genome. Sixty percent of afkcted boys have large interstitial deletions involving one or more exons, while another 6% have duplications (Blonden et al., 199 1; Hu et al., 1990, den Dunnen et al., 1989). Mkcted femaies have been show to have X:autosome translocations, resulting in non-random inactivation of 28 the nordX chromosome (Bodrug et ai., 1987).

In previous stuàies of the molecuiar mechanisms of duplications and translocations in DMD patients (Hu et PI., 1991; Bod~~get al., 1991). sequence anaiysis of the breakpoints revealed that many of ther chromosod rearrangements resuited hmnon-homologous recombiaation events. Som of the breakpoiit regions containeci sequence elements charâcteristic of matrix attachent regions (MARS; Boulikas, 1993), suggesting that MARS may be involved in the recombination events leading to these rearrangements.

As described in Chapter 1, cwrent models of chromatin organization suggest that the genome is attacfied to the nuclear matrix by DNA sequences, called matrix attachment regions. MARS are specific DNA SeQuences which bind to the proteins of the nuclear matrix. Although they share little sequence homology with one another, most MARS are A+T-rich and contain consensus binding sites for topoisornerase II (Boulikas, 1993). While MARS are often found in the 5' and 3' flanking regions of genes, they have also been identified within introns in some genes (Gasser et al., 1986; Levy-Wilson et ai., 1989; Cockerill et al., 1986). Evidence suggests tbat they participate in such fûnctions as defining chromatin domains, transcriptional regulation and DNA replication (Phi-Van et al., 1988; Levy-Wilson et al., 1989; Stief et al., 1989; Phi-Vau et al., 1990, Klehr et al., 1991; Kalos et al., 1995; McKnight et al., 1992; Bonifer et al., 1994; Berezney et al., 1975; Jackson et ai., 1986; Razin et ai., 1986; Vaughn et al., 1990). A few MARS have been found at breakpoints of chromosornai rearrangements and have, therefore, been hplicated in non-homologous recombination (Anand et al., 1988; Strissel Broeker et al., 1996; Sperry et al., 1989).

Because of its size, complexity and numrous chromosomal rearrangements, the dystrophin gene represents a unique mode1 for the study of MARS and their potential desin transcriptional regulation and non-homologous recombination. Given the fact that MARs typically occur at 29 intemals of approximately 90 kb in tk genorne (Vogelstein et al., 1980), one would expect many MARS io be positioned throughout the 2300 kb dystrophh gene. This funher raises the possibility that the many chromosoma1 nanangements tbat owur in this gene m@ht be mdisicd by recombination between MAR sequences located in the nuclear matrix.

To test this hypothesis, the DNA at the breakpoints of a partial gene duplication occufring in a DMD patient have been examined for the presence of MARS. Utilizing two functional assays and sequence anaiysis for MAR identification, the presence of MARS. located at both breakpoints of the duplication, has been conflmed. These findings demonstrate that non- homologous recombination i&g to chromosoma1 reacrangement has occurred at the sites of MARS within the dystrophin gene. The additional fmding of consensus sequences for topoisornerase 1 cleavage sites within these MARS Mer supports the concept that enzymaticaily mediated exchange between chromatids at the bases of chromosomal lwps can be responsible for disease-causing mutations in Duchenne muscular dystrophy.

DMD patient HSC689 has a duplication of a DNA region containhg exons 8 and 9 of the dystrophin gene involving breakpoints in introns 7 and 9 (Hu et al., 1990; Hu et al., 1991). He was diagnosed on the basis of grossly elevated semm crieatine kinase activity, pseudohypertrophy of Uie calf muscles, electromyographic abnormalities typicai of myopathy, and muscle biopsy results consistent with muscular dystrophy. He was a slow learner, has been üeated for primary hypothymidism, and became wheelchair-bound at 11 years of age. Both the patient's mother and sister are carriers of the duplication (Hu et al., 1989). 30 DNA clones

The intron 7 breakpoint region was previously isolated from a Dash (Stratagene, USA) bacteriophage library constmcted from the DNA of patient HSC689 (Hu et al., 1991). This phage clone contained the normal DNA region surrounding the position of the breakpoint in intron 7 (is. from 5' of the actuai breakpoint). Simüarly. a clone containing the normal DNA region surrounding the intmn 9 breakpoint was isolated from a 49,XXXXY genomic Lorist B cosmid libracy (Kïamut et al., 1990; Hu et al., 199 1). Restriction fragments hmboth the phage and cosmid clones werp fùrther subcloned into the plasmid vector pBluescript (Stratagene, USA).

A plasmid clone (p3'MAR) containing the 3' MAR hmthe apolipoprotein B (ApoB) gene was used as a control in botb the in vitro DNA-binding assay and the in vivo MAR isolation method described below (a generous gift from Dr. B. Levy-Wilson).

HeLa celis were grown on plastic culture dishes in aMEM containing 16 mM glucose, 10% fetal bovine serum and 10 pg/ml gentamycin. in vitro DNA-Binding Assay

The in vitro DNA-binding assay, altematively called the exogenous assay (Spiker and William, 1996), is described schematicaiiy in Fig. 2-1. The procedure folîowed the method of Cockerili and Garrard (1986). Ail steps were ched out at 4OC, unless otherwise indicated. Cultured HeLa celis were washed in phosphate-buffered saiine and homogenized, after a IO-minute incubation on iœ in RSB (10 mM NaCl; 3 mM MgC12; 10 mM Tris-HCl; 0.5 mM 31 phenylmethylsuifonylfluoride (PMSF), pH 7.4) with a Dounce homogenizer meaton, USA). Nuclei were pelîeted for 10 min at 750 x g aad washed twice in RSB-0.25 M sucrose. Tbey were then resuspended in RSB-2 M sucrose and centrifugeci through a RSB-2 M sucrose cushion at 25,000 rpm for 30 min in an SW41 rotor. The nuclear pellet was washed in RSB- 0.25 M sucrose, resuspended in RSB-0.25 M sucrose with 1 mM CaC12 at 1 mg/ml nucleic acids, digested with 100 pg/ml DNase 1for 2 h at mmtempera-, and centrifugecl for 10 min at 750 x g. The pellet was resuspended in RSB-0.25 M sucrose, an equal volume of 4 M NaCi,

20 mM EDTA and 20 mM Tris-HC1 (pH 7.4) was added and the suspension was incubated at 0°C for 10 min. Foliowing centrifugation for 15 min at 1500 x g, the nuclei were extracted twice with 2 M NaCl, 10 mM EDTA, 10 rnM Tris-HCl, 0.5 mM PMSF and 0.25 mg/ml bovine senun albumin (BSA) (pH 7.4) and pelîeted for 15 min at 4500 x g. The nuclear matrices were washed in RSB-0.25 M sucrose with 0.25 mg/ml BSA, resuspended in the sarne solution and stored at -20°C with the addition of an equal volume of glycerol. Prior to use in binding assays, the nuclear matrices were washed three times in 50 IDM NaCl, 10 mM Tris-HCl (pH 7.4), 1 rnM MgC12,0.25 M sucrose, 0.25 rnglml BSA and pellet4 &ter each wash.

To study the binding of DNA restriction fiagments to nuclear matrices, matrices from approximately 1.6 x 107 ceils were resuspended in a final volume of 1ûO pi of reaction mixture containhg 50 mM NaCl, 10 mM Tris-HCl (pH 7.4), 2 mM EDTA, 0.25 M sucrose, 0.25 mghnl BSA, 20 nglrnl 32~tnd-labelledDNA restriction fiagments, and 100 pghl unlabelled sonicated Escherichia coli DNA, and incubated for 2 h at room temperature. @NA restriction fragments were radiolabelled using the High Rime DNA Labeling Kit (Boehringer-Mannheim, Gennany): the restriction fragments remaineci double-stranded, random primers were not added, and dCTP was the radiolabelled nucleotide used.) AAer addition of 500 pl of assay buffer (50 mM NaCl; 10 mM Tris-HC1, pH 7.4; 2 mM EIYï'A; 0.25 M sucrose; 0.25 @ml

BSA), the nuclear matrices were pelleted at 10,000 x g for 1 min, and then washed in 1 ml of assay bufEer. The matrices were resuspended in TE witb 0.5% SDS and 0.4 mdml protehase 32 K to release the matrix-bound DNA. Ten rnimgrams of uniabellecl carrier DNA was added, foiiowed by phenoi/chioroform extractions, and ethanol precipitation. The purifiecl matrix- bound DNA was separated by electrophoresis on a 1% agarose gels in 1 x TAE. Gels were dried ont0 Zeta-nobe nylon membranes (BioRad) and exposed to a phosphorimager screen for a minimum of 4 hours. The percentage bound of each DNA fhgment was detennined using a phosphorimager. These experimental conditions, as outlined by Cockerili and Garrard (1986), give specific binding of 2% to 3096 of input montainhgDNA fragments.

in vivo MAR Isolation Methoà

Altematively called the endogenous assay (Spiker and William, 1996), the in vivo MAR isolation method of Mirkovitch et al. (1984) was used in the isolation of MARS hmHeLa cells (Fig. 2-4). HeLa cells were collected in isolation buffer (3.75 mM Tris-HCl, pH 7.4; 0.05 mM spennine; 0.125 mM spermidine; 0.5 mM EDTAWH, pH 7.4; 1% (v/v) thiodyglycol; 20 mM KCI; 0.5 pg/ml aprotinin) with a cell scraper and centrifugai for 5 min at 900 x g. Tbe pelleted cells were then washed twice, resuspended in ice-cold isolation buffer containing 0.1% digitonin, 5 pg/rnl aprotinin and 0.1 mM PMSF, and homogenized with a Dounce homogenizer

(Wheaton, USA). Nuclei were pelleted by centrifugation for 10 min at 900 x g and washed three times with the isolation buffet containing digitonin, PMSF and 5 p@ml aprotinin. The pellet was then resuspended in an isolation bmer containing 0.1% digitonin and 5 pghl aprotinin, but lacking EDTA. Twenty-five Obuni& of nuclei in 250 pl of this isolation buffer were incubated at 37°C for 20 min. The nuclei were exmted for 5 min at room temperatwe by adding 7 ml of extraction buffer (5 mM Hepes/NaOH, pH 7.4; 0.25 mM spennidine; 2 mM EDTAKOH, pH 7.4; 2 mM KCl; 0.1% digitonin; 25 mM 3,5aüodosalicylic acid, Lithium salt). The nuclear haloes were pUeW for 20 min at 2400 x g and then washed four tims in 8 ml digestion buffer (20 mM Tris/HCl, pH 7.4; 0.05 mM spermine; 0.125 mM spennidine; 20 mM KCl; 70 mM NaCl; 10 mM MgCl2; 0.1% digitonin; 5 p@ml apmtinin; O. 1 33 mM PMSF). nie nuclear haloes were digested with restriction enzymes at 10U/ml for 3 h d 37*C, and centrifugeû for 10 min at 2400 x g. Both the pellet ktion, containing nuclear matrix-bound DNA, and the supernatant fraction, containhg mbound DNA, were treaîed with protehase K in the presence of 1% SDS, phenoVchloroform extracted, and etbanol precipitated. DNA samples (2 pg) were separateci by electrophoresis on 1% agarose gels and Southem transferred to Zeta-Probe nylon membranes (BioRad). DNA was cross-linked to membranes with 0.12 J of uitcaviolet irradiation. Membranes were prehybridized for 2 h at 42°C in 6 x SSC, 50 mM NaP04, 40% formamide, 1% SDS, 5 mg/d skim milk powder, 100 pg/ml single-suandeci salmon spem DNA, and hybridized overnight at 42OC in the same solution containing 10% dextran sulfate and single-stranded dolabelled probe, but lacking salrnon sperm DNA. Probes were radiolabeiied with the High Prime DNA Labeling Kit (Boehringer-

Mannheim, Germany). Membranes were washed for 30 min at room tempe- in 2 x SSC, 0.1% SDS and then for 30 min at 50°C in 0.5 x SSC, 0.1% SDS. Autoradiogfaphy was conducted at -70°C using an intensiQing screen for a minimum of 12 hrs.

Isolation of Total Genomic DNA

HeLa tells were removed from plates in the presence of 0.25% trypsin and centrifiiged at 900 x g. The pellet was then washed in ice-cold PBS and resuspended in O. 1 M NaCl, 10 mM Tris- HCl (pH 8), 25 mM EDTA, 0.5% SDS. Proteinase K was added to a final concentration of 75 pghl with an ovemight incubation at 37°C. Total genomic DNA sample was extracted with phenoYchlorofonn and precipitated with ethanol. DMD patient HSC689 has a duplication of exons 8 and 9 of the dystrophin gene which resulted in a shift in the translational reading frame and priemature tnmcation of the protein (Hu et al., 1990). This duplication occurred by recombination between non-homologous sequences in introns 7 and 9 (Fig. 14A; Hu et al., 1991). DNA sequence analysis of the regions immediately smunding the breakpoints indicated that boih are A+T-rich, a cornmon characteristic of MARS, with the intron 7 and intron 9 breakpoint regions having an A+T contents of 70% and 63.596, respectively. Sequence analysis also identifiai topoisornerase 1 consensus sites (5' AR--T/A-T 3') in these breakpomt regions, with one site occurring precisely at each of the two breakpoints (Fig. 1-4B). These data suggest that these regions may participate in matrix aüachment and that the rearraagement may have taken place by recombination between sequences at the bases of chromosomal loops.

The intron 7 breakpoiit region was isolated hm a phage library constructed from the duplication patient's DNA. A clone containing the normal intron 7 region smunding the position of the breakpoint (i.e. hm5' of the actual breakpoint) was digested with restriction enzymes and further subcloned into plasmid vectors (Hu et al., 1991), providing substrates for the MAR assays.

Similarly, a clone conaining the normal intron 9 region was isolatecl fiom a 49,XXXXY genornic cosmid library. Restriction ftagments from a 19.6 kb cosmid clone were also subcloned into plasmid vectors (Hu et al., 1991).

To investigate funher the mechanism of duplication in patient HSC689, Che intron 9 breakpoint 35 region was assayed for nuclear matrix binding activity using the mthod of Cockeriil and Garrard (1986). In this "in vitro" ("exogenous") method (Fig. 2-l), DNA restriction fragments are tested for their abiity to bind to nuclear matrices, prepared by digestion of isolated nuclei with DNaseI and extraction with high salt to remove histones and other soluble proteins. Specific binding (greater than 2% under conditions specifid, see Materials and Methads) of a restriction fragment to the matrices is considered to indicate thaî the -nt contains a matrix attachment region (Cockerill and Garrard, 1986). Thus, restriction fragments from the cosmid (Lorist B) clone containing at least 19.6 kb fiom intron 9 were assayed for binding to HeLa nuclear matrices (Fig 2-2). The cosmid clone was digested with HindIII to yield ten fragments ranging from 0.57 to 10 kb in size (Fig. 2-2A). These restriction fragments were end-labelled and incubateci with the matrices in the presence of excess unlabelled E. coli cornpetitor DNA. The matrix-bound DNA was purifd and electrophoresed through an agarose gel, alongside a control sample of unincubateci fragments.. The gel was dried ont0 a nylon membrane and autoradiographed in order to identify those fragments demonstrating a high binding affinity to the nuclear matrix. As seen in Fig. 2-2B, only the 1.8 kb Hindm fragment containing the duplication breakpoint bound strongly to the nuclear matrices, indicating the presence of a MAR within this restriction fragment. The binding of this 1.8 kb fragment was confiid in a second in vitro assay using a subclone of the fragment in pBluescript (Fig. 2-2C). As positive and negative controls, respectively, for the "in vitro" assay, we demonstrated nuclear matrix binding for a known MAR-containing mstriction fragment hmthe apolipoprotein B (ApoB) gene, and a lack of binding for the plasmid vector in which it had been cloned (Fig. 2-3A,B; Levy-Wilson et al., 1989).

To confitbis result with an independent method and to refme the position of the MAR within the 1.8 kb fragment, we used the "in vivo" ("endogenous") MAR isolation rnethod (Fig. 2-4). With this method, nuclei isolated hmHeLa ceils were depleted of histones using the ionic detergent lithium diiodosalicylate (LIS)under low sait conditions. These nuclear matrices were DNA DNase l and Histones 1 2M NaCl

- - . - NUCLEAR MATRIX O7 - lncubate SP-end- Restriction .r

Supernatant Pellet

Purify DNA, Electrophorese, Autoradiograph

Figure 2-1. In vltio Whod of MAR kâentmcation in vitro DNA-binding assay of Cockerill and Garrard (1986). (1) input DNA, and (A) matrix-associated DNA. of MAR in Intron 9 - in vitro study sert cantaining the intron 9 breakpoint region. e breakpoint. Hatched box indicates position of MAR. 6)in vitro binding assay. The end-labelleci restriction fragments from a Hindlll digest of the cosmid were incubated with nuclear matrices prepared from Hehcelis. C) in vitro binding assay. A plasmid containing the 1.8 kb Hindlll breakpoint-containing restriction fragment was digested with Hindlll and Avall and incubated with nuclear matrices prepared from Hela cells. The 1.6 and 1.4 kb fragments represent the plasmid vector. The Horizontal amw indites MAR~containingfragment. (1) input DNA, (A) matrix-associateci DNA. The amount of unincubated end-labelled DNA (input DNA) loaded into each lane is indicated as a percentage of that used in the incubation. The ratio by mass of unlabelleci E. di cornpetitor DNA to end-labelled restriction fragments is indicated for incubated samples (rnatrix-associated DNA). %S. vos va Digest with Restriction Ï

Supernatant Pellet

Punfy DNA, Electrophorese, Sauthem Blot & I

Figure 2-4. In VIVOMahod of MAR Ickntiticatkn in vivo MAR isolation method of Mirkovitch et al. (1984). (S) supernatant DNA, (P) pellet DNA, (T) total genomic DNA. 40 tben digested with Hindm and EcoRI, and centrifuged to separafe the nuclear matrix pelIet, containing matrix-bound DNA, hm unbound DNA fragments in the supematant. DNA purified hmboth the peliet and supematant fractions was separated by electrophoresis and transferred to a nylon membrane. The 1.8 kb Hindm fragment, when used as a probe, hybridized to both the 1.O and 0.8 kb Hindm/EcoRI fragments (Fig 2-5). The 1.O kb fragment was detected almost exclusively in the rnatrix-bound fraction, confirming the position of the

M.determined by the in vitro method. This MAR may extend across the EcoRI site into the 0.8 kb fragment since this fragment, too, is observed in the matrix-bomd fraction, although a portion was also found in the supematant fraction. As a control, this membrane, prepared by the in vivo MAR isolation method, was probed with a restriction fiagrnent containing a MAR from another part of the dystmpùin gene. This hybndization detected tbree restriction fragments, one of which was present in the pellet lane (matrix-bound) and two of which were present in the supematant lane (unbound; Fig. 3-4C).

Identrj?cution of MAR ut intron 7 breakpoint

To test for the presence of a functional MAR at the intron 7 breakpoint of duplication patient HSC689, restriction fragments subcloned from the phage clone of the normal inmn 7 region were used in the in vitro assay. Of the six cloned restriction fragments spanning 19 kb across the breakpoint region (Fig. 2-6A;the 0.2 kb XbaI fragment was not cloned), two showed strong matrix binding activity when inçubated with nuclear matrices prepared from HeLa cells (Fig. 2-6B). These fragments corresponded to a 5.1 kb XbaI fragment containing the breakpoint and a 2.6 kb XbaI fragment irnmediaîely 5' of the breakpoint. These data indicate the presence of a MAR within this 7.7 kb region of intron 7 that contains the duplication breakpoint in patient HSC689, and suggest that matrix attachment of the breakpoint maàe possible the duplication event in this patient. SPT

Figure 2-5. Identification of MAR8 in lntron 9 - in vivo midy A) Restriction map of the intron 9 breakpoint region. Hatched box indicateo the restriction fragment used as a probe. Vertical amw indites position of breakpoint 8) Southem Mot containing DNA from HindllVEcoRl digested nuclear haloes prepared frm HeLa cells. Horizontal arrows indicate the MAR-containing fragments. Apparent restriction fragment size diierences reflect variations in migration in the different lanes. MAR

Figura 2-6. Identitication ot MAR in Intron 7 - in vitro study. A) Restriction map of the breakpoint region. Vertical arrow indicates the position of the breakpoint. Hatched box indicates position of MAR. 6) In vitro binding assay. The pP-end-labelled Xbal restriction fragments seen in A) above were incubated with nuclear matrices prepared from HeLa cells. Horizontal anows indite MAR-containing fragments. (1) input DNA, (A) matrix-associated DNA. The amount of unincubated 32P-end-labelled DNA (input DNA) loaded into each lane is indicated as a percentage of that used in the incubation. 43 The two restriction fragments showing stmng mahix binding activity in the in vitro assay were used as probes on Southem blots containing DNA prepared using the in vivo MAR isolation rnethod. However, in the resuiting hybridization patterns, individual restriction fragments cdd not be distinguished, due to the presence of a repeat sequence in these two probes. Thus the in vivo rnethod was not idormative in this case (data not show).

DNA sequence-based MAR predictions

Based on sequence characteristics commonly observed in known MARS, Singh et ai. (1997) designed an analytical model (MARFinder) to predict the locations of MARS using only DNA sequence. This model calculates a Matrix Association Potential for DNA sequence based on the presence of the following motifs: high AT-content, topoisornerase II consensus sites, curved DNA motifs, kinked DNA motifs, TG-rich spans and OR1 motifs (ATïA, ATITA, ATïTïA). The model's algorithm was used in this study to anaiyze 5 kb of intron 9 DNA sequence and approximately 20 kb of intron 7 sequence to determine the positions of pdcted MARS in these introns. The inmn 9 breakpoint region was sequenced by the Canadian Genetic Disease

Network Sequencing Centre (Accession nurnber AF276054). Intron 7 was sequenced by McNaughton et al. (1993; Accession number U60822). As seen in Figure 2-7, the Matrix Association Potentiai showed a single peak in intron 9 which coincided with the position of the MAR determineci in the functiond analysis describeci above and with the duplication breakpoint. MARFinder predicts a MAR in the intron 7 breakpoint region whose position occurs within the restriction fragments for which we demonstrated matrix attachment experimentaiiy, and occurs precisely at the location of the dupikation breakpoint (Figure 2-8). The experimental results, therefore, are consistent with the sequence-based predictive model. MAR

Figure 2-7. InWon 9 MAR: Exprimentally detemined position coincides wiai mqwn~analyasir Matrix association potential detemined for DNA sequence from intron 9 using the sequence-basecl mode1 deveîoped by Singh et al. (1997) is alignd with the restriction map of the intron 9 breakpoint region above in a collinear fashion. Vertical amms indicate the position of the bmakpoint. Hatched box indicates poslion of MAR. MAR

Figure 28. lntron 7 MAR: Exprimentally deteminrd position coinciâes with uqwnce-basmi analysia. Matrix association potential determined for DNA sequence from intmn 7 using the sequence-based mode1 developed by Singh et al. (1997) is aligned with the restriction map of the intron 7 breakpoint region above in a collinear fashon. Vertical amsindicate the position of the breakpoint. Hatched box indicates position of MAR. This study suggests tbaî the duplication in patient HSC689 occurred by non-homologous recombination between two MARS within the dystrophin gene. Using functional MAR assays, the existence of MARS at both the intron 7 and intron 9 breakpoints has been demonstrated in this patient. In addition, these MARS correspond to the positions of predicted peaks in Matrix Association Potential determine. using a sequence-based mode1 (Singh et al., 1997). Previously, the high A+T content of these breakpoint regions, a characteristic typical of MARS, and topoisomerase 1 consensus cleavage sites occwring at both breakpoints had been i&nified. Topoisornerse 1 cleaves only a single strand of DNA, suggesting that a breakage or second enzymaîic cleavage must occur to achieve a double-stranded break (Buliock et al., 1985). One topoisomerase 1 site occurs precisely at each of the intron 9 and 7 breakpoints, while second topoisomerase 1 sites exist one and two base pairs away, respectively, on the complemenw strands (Fig. 14B). It is possible, therefore, that this recombination involved up to four topoisomerase 1 cleavages. Together these results suggest a mechanism for the non- homologous recombination event which occurred in this patient. Although these brieakpoints are linearly distant from one another dong the dystrophin gene sequence, they may be in close physical proximity where they are anchored to the nuclear macrix (Fig. 2-9). Such proximity could provide the opportunity for recornbination to occw, perhaps initiated by topoisomerase 1 cleavage.

Earlier studies of this DMD patient indicated that the duplication originated in the gem-line of his matemal grandfather by unequal sister chromatid exchange (Hu et al., 1989). As spermatogenesis cm involve hundreds of rnitotic œll divisions prior to the fmal meiotic divisions (Hurst et al., 1998), it may be more likely that the recombination event leading to DMD in this patient occurred during a mitotic rather than meiotic œll division in the grandpaternal germ-line. Figure 2-9. Modd ot chromatin orginWon in th. dy.tiophin qem. The intron 7 and intron 9 dystrophin gene MARS on the sister chromatids of the X chromosome may be in close proximity where they are anchored to the nuclear rnatrix, allowing for a recornbination event to occur between them (as indicated by the X). Boxes inâicated exon numben. Shaded objects repn#rent proteins of the nuclear rnatrix. 48 The usud des of tbe dystrophin gene intron 7 and intron 9 MARS have not yet been determined. Tbe psence of topoisometase 1 consensus cleavage sites at both breakpoint regions suggests a role for &se MARs in controllhg DNA topology, especially important during DNA replication. The DNA region surroundhg the intron 9 breakpoint is rich in AITA, ATITA and ATTiTA sequences, a motif whkh has been noted at origins of nplication, indicating a possible rok for the intmn 9 MAR as an origin (Boulikas, 1992). The intron 7 and intron 9 MARS are not located near known promoters or enhancers in tbe dystrophin gene and, therefore, are not currently suspected of having a role in transcriptional control.

MARS are frequently found in the flanking regions of genes, indicating that, generally, a gene exists entirely within a single chromatin loop or domain. Tbe finding of multiple intronic MARS in the dystrophin gene suggests that this enormous gene is organized into multiple chromatin domains, which may be condensed to varying levels. The degree of condensation of the various chmmatin domains in the dystrophin gene at any given time could be related to tbe pmmoter hmwhich the gene is king transcribed.

MARS have previously been irnpiicated in non-homologous recombination events. MARS have been mapped to bfeakpomts of chromosomai murangements in the mouse Ig K light chain (Sperry et al., 1989), a human ring chromosome 2 1 (Sperry et al., 1989), the human f3 globin gene (Anand et al., 1988) and the MIL gene (Strissel Broeker et al., 1996). However, this study of duplication breakpoints in the human dystrophin gene represents an example of the existence of MARs at both breakpoints of a recombination event. Preliminary resuits suggest that a third dystrophin gene MAR may be locaîed in intmn 19 at a duplication breakpoint found in DMD patient HSC627 (unpublished results). Patient HSC627 was previously shown to have DMD resulting hma non-homologous recombination event between introns 19 and 4 1 (Hu et al., 1991). Together these data support the possible deof MARS in non-homologous recombination events. The Role of Matrix Atkchment Regions in the Gene Expression from the Muscle-Specific Promoter in the Dystrophin Gene.

The experiments in this chapter were conducted by tbe author. Duchenne muscular dystrophy, a &generative muscle disorder, is caused by mutations in the gene encoding dystrophin. This 2.3 Mb gene is the largest known, and has seven promoters/fmt exons expressing different tissue-specific isoforms of the protein. The size and complexity of the dystrophh gene make it a unique mode1 for the study of the deof rnatrix attachment regions (MARS) in gene expression. Known inter-MAR distances are typidy 60 kb in size, indicating that the dystrophin gene should contain multiple MARS. In sorne genes, MARS are located at or near cis-regdatory sequences, while in others, MARS may serve a boundary function, suggesting des for MARS in transcriptional regdation. MARs may be expected to CO-localizewith regdatory regions in the dystrophin gene. To test this hypothesis, 1 have searcheci for MARS in a 24 kb DNA region surrounàing the intemal muscle-specinc promoter of the gene. 1 have found two MARS in tbis region: one upstream and one downstrearn of the muscle-specific promoter of the dystrophin gene. Evidence suggests that matrix attachment regions play a role in gene expression by defining chmatin domain boundaries and participaîing in aanscriptional regulation at cis-acting elements (Phi-Vanet al., 1988; Levy-Wilson et al., 1989; Stief et al., 1989; Phi-Van et al., 1990; Klehr et al., 199 1; Kalos et al., 1995; McKnight et al., 1992; Bonifer et al., 1994).

Frequently found in the flanking regions of genes, some MARS may serve a boundary fùnction, isolating a DNA region from cis-acting elements in a neighùouting region (Gasser et al., 1986; Phi-Van et al., 1988; Bode et al., 1992; Levy-Wilson et al., 1989). MARS from the chicken lysozyme, the human 0-interferon, and the human apolipoprotein-B genes, for example, have been shown to isolate reporter genes hm chmmosomal position effects in stably-transfected cells (Stief et al., 1989; Phi-Van et al., 1990; Klehr et al., 1991; Kalos et al., 1995). In addition, human apolipoprotein-B and alpha 1-trypsin gene M.showed position- independent effects of transgene expression in Drosophila (Namciu et al., 1998), while the chicken lysozyme gene MAR can suppress ectopic expiession of a transgene in mice (Bonifer et al., 1994).

It has been hypothesized that the nuclear rnatrix may act to localize or concentrate transcription factors within the nucleus (Stein et al., 1991). In fact, transcription factors and their binding activities have been found in the nuclear matrix (Bidwell et al., 1993; van Wijnen et al., 1993). Therefore, MARS whiçh co10calize with cis-regulatory elemenîs may influence transcriptional control through their association with transcription factors on the nuclear matrix. A nwnber of MARS are locaîed at or near promoters and enhancers, and interactions of such MARS with transcription factors of the nuclear matrix has been shown (Gasser et al., 1986; Cockeril1 et al., 1986; Jankelevich et al., 1992; Dworetzky et al., 1992; Jannan et al., 1988; Brotherton et al., 1991; Stein et al., 1991). 52 The nuclear xnatrix is known to demonstrate tissue-specific characteristics. Some nuclear matth proteins exhibit tissue-specifk expression (Dickinson et ai., 1992; Bidwell et al., 1993). In addition, the distribution of biidbg activities for some transcription factors between the matrix and non matrix compartments of the nucleus is tissue specific (van Wijnen et ai., 1993). As well, some MARS have been shown to bind to the nuclear matrix in a tissue-specific manner (Levy-Wilson et al., 1989; Brotherton et al., 1991).

Dystrophin gene isoforms am expressed hmat least 7 different promoters/fmt exons in a tissue-specific manner (Fig. 1-11. Thtee of these fmt exons, transcfibed hmthe brain, muscle and Purkinje promoters, are spliced to exon 2 (Boyce et al., 199 1; Klamut et al., 1990; Gorecki et al., 1992). Four others, the retinal, central nervous systenn, peripheral nerve and non- muscle promoters, are located in introns 29, 44, 55 and 62, respectively, and, in tum, are spiiced to exons 30, 45, 56 and 63, respectively @'Souza et al., 1995; Lidov et al., 1995; B yers et al., 1993; Hugnot et al., 1992).

Transcription hmthe muscle pmmoter produces a 14 kb transcript, which is spliced fkom 79 exons (Roberts et al., 1993). Due to the extraordinary size of the dystrophin gene, the tim required for its transcription is 16 hours (Tennyson et al., 1995). The 427 kD full-length muscle isoform of dystrophin is expressed in skeletal and cardiac muscle, and at lower levels in smooth muscle and brain (Chelly et al., 1988). Klarnut et al. (1990) identifieci an 850 bp promoter which can direct muscle-specific expression of a reporter gene. This promoter contains binding site motifs known to be involved in the ~legulatedexpression of other muscle- specific genes, including a muscle-CAAT consensus sequence, a CArG box and three MW-1 (myocyte-specific enhancer-binding nuclear factor 1) consensus sequence motifs. A muscle- specific enhancer has also been idenified approximately 6 kb downstream hmthe muscle exon 1 in intron 1 (Klamut et al., 1996). This core enhancer, 195 bp in length, contains three MEF- 1 and two MEF-2 consensus binding sites (Klamut et al., 1997). 53 The extraordinary size of the dystrophin gene far exceeds that of known inter-MAR distances (typicaliy 90 kb), and presents a unique challenge in tenns of its chr0amti.n domain organization during transcription. Domain organization in this gene may involve interactions with the nuclear matrix, and may dBer hmsmaller genes, which have domain boundaries only in the flanking regions. Li addition, MARS may coinci& with cis-acting regdatory sequences in any or aii of the promoter regions and may play a role in regulating transcription.

To cletennine if MARS are involved in the gene expression of dystrophin, 1 have examined a 24 kb DNA region surmunding the muscle-specific promoter in the dystrophin gene. Utilizing two standard assays for MAR identification, 1have conflTmed thaî MARS are located both upstream and downstream of this promoter.

DNA clones

A cosmid clone conaining the normai DNA region smunding muscle-specifk promoter.exon1 region was previously isolated from a 49,XXXXY genomic Lorist B cosmid library (Klamut et al., 1990). Restriction fragments hm the cosmid clone were further subcloned into the plasmid vector ptkGH (Klarnut et al., 1996).

Plasmid clone p3'MAR (containing the 3' MAR hmthe apolipoprotein B (ApoB) gene) was used as a control in both the in vitro DNA-binding assay and the in vivo MAR isolation method (a generous gift hmDr. B. Levy-Wilson). The plasmid clone 1.8 WH containing the known MAR hmintron 9 of the dystrophin gene was dso used as a control in these experiments. This clone was previously isolated in our laboratory (Chapter 2, Hu et al., 1990). HeLa cells were grown on plastic culture dishes in aMEM coataining 16 mM glucose, 10% fetal bovine serum (FBS)and 10 pg/ml gentamycin.

Clonal human fetal myoblast cultures were prepared from primary fetal muscle cultures (Klamut et al., 1990). Rirnary fetai muscle cultures were dilution-plated (10 celldml) in cloning medium (F12 media (BRL) containing 20% fetal bovine semm (FBS) and a Skeletal Muscle Builet Kit (Clontech, USA)) ont0 96-weU plates at 0.1 Wwell. Clonal colonies were frozen in cloning medium with 10% dimethyl sulfoxide. The determination of myogenicity was based on each clona1 colony's ability to differentiate into mdtinucleated myotubes in fusion medium (aMEM, 16 mM glucose, 2% fetai bovine serum (FBS) and 10 pg/d gentamycin).

For use in the in vivo MAR Isolation Method experiments, primary human fetal myogenic clones were grown in cloning media. Myogenic differentiation was induced by transfemng cells plated at high density to fusion media. Cells were allowed to differentiate into fused myotubes for 3 to 5 days.

in vitro DNA-Binding Assay

The method of Cockeril1 and Garrard (1986) was used in the identification of MAR-containing restriction fragments using nuclear matrices isolated hmHeLa cells. See Chapter 2. in vivo MAR isolation Method

The method of Mirkovitch, et al. (1984) was used in the isolation of MARS from HeLa ceUs and undifferentiated myogenic cells. See Chapter 2. 55 The isolation of nuclei hmdifferentiated myotubes was adapted hmthe rnethod of Meilon and Bhorjee (1982). The fused myotubes were harvested in 0.25% trypsin and washed twice with PBS. The foliowing steps were carried out at 4OC. Cells were suspended in TECK

solution (l0mM Tris-HC1 pH7.8, 1mM EDTA, 3 mM CaCl*, lûmM KCl) and allowed to swell

for 10 min. The suspension was homogenized with 30 strokes of a tight-fitting Dounce

homogenizer. Nuclei were collected by centrifiigation for 3 min at 1000 x g, and resuspended in TECK solution. The suspension was Dounce homogenized once again with 2 strokes and cenrrifuged. The nuclei hmthis procedure were then used in the in vivo method of Mirkovitch et al. (1984).

Isolation of Total Genomic DNA

HeLa celis or undifferentiated myogenic cells were removed from plates in the presence of 0.25% trypsin and centrifuged at 900 x g. The pellet was then washed in ice-cold PBS and resuspended in 0.1 M NaCi, 10 mM Tris-HCl (pH 8). 25 mM EDTA, 0.5% SDS. Pmteinase K was added to a fmal concentration of 75 pg/mi with an overnight incubation at 37OC. Total genornic DNA sample was extracted with phenoYchlorofom and precipitated with ethanol.

Ident@cation of MARS using the in vitro method

In vitro binding assays were conducteâ according to the experimental conditions established by CakeriJi and Garrard (1986). Since large amounts of ce11 extracts were required to conduct these experiments, it was not pmtical to use primary myogenic cells with ïimited growth potential for this assay. Instead HeLa cells were selected for these experiments. Cloned restriction fragments spanning 24 kb across the muscle promoterlexon 1 region (Fig. 3-1A) lntron 9

Figure 3-1. In vitro binding 8ssay of the muscle piomotwlexonl region. A) Restriction map of the muscle promoter/exonl region indicating the positions of Hindlll (H) and Xbal (X) sites, with sires of restriction fragments indicated in kilobases. (P) promoter, (Ml) muscle exonl , (E) enhancer. B) and C)in vitro binding assay. The mP-end-labelled restriction fragments in part A) were incubated with nuclear matrices prepared from HeLa cells in two incubations (B and C). The PP-end-labelled restriction fragments containing the apdipoprotein-6 (Apo-B) MAR or the dystrophin gene intron 9 MAR were included in incubations B and C, respectively, as poslive contrds. The vectors in which these bomiMARS were contained wen, included as negative controls. Horizontal anows indicate the MAR-containing fragments. (1) input DNA, (A) math-essociated DNA, (V) vector DNA. The amount of unincubated sP-end-labelleci DNA (input DNA) loaded into each lane is indicated as a percentage of that used in the incubation. 57 were analyzed for matrix-binding activity. In vitro binding assays using these ftagments indicated binding to the nuclea. maûix across an 11.9 kb region, including the 6.6 kb Hindm fragment immediately upstFeam of the muscle promoter, the 3.4 kb HindIII fiagrnent containhg the muscle pmmoter and exon 1, and the neighbouring 1.7 kb HindIIIIXba.1 fiagrnent from intron 1 suggesting that one or more MARS span the region. The other restriction fragments from this 24 kb region, including the 3.3 kb XbaI/HinàIiI fragment that contained an enhancer, did not show ma*-binding activity (Fig. 3-lB,C). Known MARS (eitber the ApoB gene 3' MAR or the dystrophin gene intron 9 MAR) were used as positive controls in this experiment, while the plasmid vectors in which tl~ywere cloned were used as negative controls. A second in vitro binding assay using the same restriction fragments hm the muscle promotedexon 1 region confirmed matru-binding activity for the 3.4 kb HindIII and 1.7 kb Hindm/XbaaI fragments, but not the 6.6 kb HindIII fiagrnent (data not shown). The other restriction fragments hmthe region again did not show rnatrix-binding activity, confyming that they do not contain MARS.

Refinement of MARs using the in vivo methai

The 6.6 kb HindIïI fragment upstream of the muscle-specific promoter was used as a probe on a Soutbem blot prepared using the in vivo MAR isolation methoâ from HinàIWBamHI-digested undifferentiated primary myogenic cells (Fig. 3-2A). This probe hybridized to a 5.4 kb HindLWBamHi fragment in the pellet fraction and to a srnalier 1.2 kb HindIIVBamHI fragment in the supernatant fisbction (Fig. 3-2B). These two restriction fragments can be distinguished despite the existence of a DNA riepeat sequence in this probe. A 2.0 kb HindIII/XbaI probe, a subclone of the 6.6 kb fragment, hybndized only to the 5.4 kb HincüIl/BamHI fragment in the

pellet fraction (Fig. 3-2C). These nsults CO- that a MAR exists within the upstream 6.6 kb Hindm fragment but define a 5' limit to the MAR at 5.4 kb upstream of the promoter. As a control, a probe containhg the Apo-B MAR (Fig. 3-3A) was Figum 3-2. In VIVOMAR iwlation midy of the upamm6.6 W Hindlll fragment. A) Restriction map of the muscle promoter region. Hatched boxes indicate the restriction fragments used as probes. (P) promoter, (Ml) muscle exonl . B) Southem blot containing DNA frorn HindlllBamHI-digested nuclear haloes, prepared from undifferentiated myogenic cell nuclei hybridized with probe 1 (6.6 kb Hindlll fragment). C) Southem blot from part B) hyôridizeâ with probe 2 (2.0 kb HindllIMbal fragment). Horizontal anom, indicate the MAR-containing fragments. (S) supernatant or unbound DNA, (P) pellet or matrix-bound DNA, (T) total genomic DNA. Figure 3-3. Apdipoprotein-0 3' MAR - in vivo stuây A) Restriction map of Apo-8 MAR. B) In vivo study - Supernatant (S) and pellet (P) DNA was prepaied from human fetal fibroblast nuclei digested with HindlllBamHI. Total (T) human genomic DNA was digested with the same enzymes. Sizes of restriction fragments are indicated. 60 hybricüzed to the blot used in Fig. 3-2. The 1.4 kb HindXIüBamHI testriction fiagment containing the MAR can be seen in the pellet lane, indicating that it contains a MAR (Fig. 3-3B). This restriction fragment appears as a doublet in both the total and pellet lanes because this MAR occurs within a known hypew&able region, giving rise to multiple alleles. (The MAR DNA and the total genomic DNA were isolated hmtwo different iridividuals, which explains the difference in the size of this fragment.) The neigh-g 0.4 kb restriction fragment which does not contain a MAR appears primady in the supematant fraction.

Results hmthe in vivo method also conf"um the existence of a MAR in the 3.4 kb Hindm fiagrnent using nuclear haloes fiom primary human fetal myogenic œils digested with Hindm and BarnHI (Fig. 3-4A). This fragment hybridized to itself in the pellet fraction @ig. 3-4B). (The 3.4 kb HindiII fragment contains no BamHI restriction site.) Sirnilar results were obtained with HindIIVEcoRI-digested HeLa nuclei using the same 3.4 kb fhgment as a probe (Fig. 34C). The 2.7 kb EcoRVHindm restriction fragment was seen in the pellet lane, indicating that it is ma&-bound. The sxnailer 0.32 kb I-iindIWEcoRI and 0.38 kb EcoRI fragments fkom the promoter were detected primarily in the supematant, indicating that they a~p not part of the MAR or that they anz too small to remain bound to the matrix in the absence of neighbouring sequences. To determine whether the promoter restriction fragments were too small to mmain bound to the matrix, this expriment was repeated using HindIWBgiII and HindlIVPstI digests of HeLa nuclei (Fig. 3-SA), both of which preserve the muscle promoter within a larger restriction fragment. The lack of matrix binding across the muscle-specific promoter was confinned. Using a Hindm/BglII digest, the 3.4 kb HinâIII probe hybridized to the 0.9 kb promoter-containing HindIWBglII hgment, which was found predominantly in the supematant fraction. The BglII site occurs in the coding region of exon 1. The detection of 0.9 kb pmmoter-containing fragment in the supernatant fraction suggests that the MAR downs~ of the promoter likely does not extend into the exon 1 coding region. The 2.5 kb HindIIIlBglIl exonl-containing frPgment was seen primarily in the pellet fraction (Fig. 3-SB). Similarly, ihe Myoblasts Hela MIS HindllWamHl HindllVEcoRl SPT

Figure 34 In vivo MAR isolation study ot the promoter-containing 3.4 W Himnil fragment. A) Restriction map of the musde promoter region. Hatched box indicates the restriction fragment used as a probe. (P) promoter, (Ml) muscle exonl . 6) Southern blot containing DNA f rom Hindll VBamHI-digested nuclear haloes prepared from undifferentiated myogenic cell nuclei. C) Southem blot containing DNA from HindllVEcoRl digested nuclear haloes prepared from HeLa cells. Horizontal awsindicate the MAR-containing fragments. (+) incompletely digested fragment. (') restriction fragment still detectable from previous hybridization. (S) supernatant or unbound DNA, (P) pellet or mat&-bound DNA, (T) total genomic DNA. HeLa Cella HindllWgHI HindllllP.11 SPTSPT

Figure 3-5. In vivo MAR isolation stuây of the promoterontaining 3.4 kb Hindlll fragment. A) Restriction map of the muscle promoter region. Hatched box indicetes the restriction fragment used as a probe. (P) promoter, (Ml) musde exonl . B) Southem blot cantaining DNA from HindllllBgll t- and HindllIPstl-digested nudeai haloes piepared from HeLa ceIl nuclei. Horizontal amsindicate the MAR-containing fragments. (S) supernatant or unbound DNA. (P) pellet or mat&-bound DNA, (T) total genomic DNA. 63 1.4 kb HindIII/PstI fragment containing the muscle-specific promoter/exonf was observed primarily in the supernatant fraction, while the downstream 2.0 kb fhgnient was in the matrix- bound pellet DNA (Fig. 3-SB). The 3.4 kb Hindm fkagment seen in the pellet fiaction of this

HincüïI/PstI digest indicates incoqlete digestion at the Pst1 site possibly because this site is embedded in the nuclear matrix.

Confimation of matrix-binding was also observed using the downstream 1.7 kb HïndIII/XbaI restriction fragrnent as a probe on Southern blots prepared using the in vivo rnethod (Fig. 3- 6A). This probe hybridized to a ma&-bound 5 kb Hindm fragment in undifferentiated myogenic cells (Fig. 3-6B)and to a 4 kb HindWEcoRI fragment in HindIII/EcoRI-digested HeLa cells (Fig. 3-6C). These results suggest that the downstream MAR extends from the 3.4 kb Hindm pmmoter-containhg fragment across tbe intervening Hindm site into tbe neighbouring 1.7 kb Hindm;/xbaI restriction fragment.

These &ta demonstrate the existence of two MARS, both upstream and downstream of the muscle-specific promoter of the dystrophin gene. The positions of these MARS near the promoter suggest that they rnay play a role in facilitating transcriptional regdation fiom this promoter.

Tissue specificity of MARS

Dystrophin gene MARS may bind to the nuclear 11151th in a tissue-specific manner, as noted for some known MARS (Levy-Wilson et al., 1989; Brothe~onet al., 1991). The experiments presented above were conducted using nuclei isolated hmHeLa celis and undifferientiated myogenic cells, neither of which express the muscle isofom of dystrophin. In order to determine the tissue specificity of dystrophin gene MARS, in vivo MAR identification experiments were lcpeated using fiised myotubes which express the muscle isofonn of C) HOU CeIts HindllUEcoRl SPT

Rgun 36. In vivo MAR kdation study of the downstream 1.7 kb HindllüXbal fragment A) Restriction map of the muscle promoter region. Hatched box indicates the restriction fragment used as a probe. (P) promoter, (M1 ) muscle exonl , (E) enhancer. B) Southem blot containing DNA from HindWBamHI- digested nuclear haloes prepared fmm undifferentiated myogenic cell nuclei. C) Southem blot containing DNA from HindllVEcoRl digested nuclear haloes prepared from HeLa cells. Horizontal amsindicate the MAR-containing fragments. (+) incompletely digested fragment, (S) supernatant or unbound ONA (P)pellet or matrix-bound DNA, (T) total genomic DNA. 65 dystrophin. Clone- myoblasts hm human fetai siceletal muscle were used in these experiments, and were induced to fke into myotubes. (Celi clones hma male fetus were used to avoid possible differences in matrix attachent between the active and inactive X chromosomes.) The 3.4 kb Hindm restriction fragment hybridized to the 2.7 kb HindIIUEcoRI fragment which was e~chedin the pellet fraction DNA in myotube nuclei (Fig. 3-7). (The smaîler 0.32 kb HindIU/EcoRI and 0.38 kb EcoRI promoter fragments were not detectable on this blot.) Therefore, the same nuclear rnatrix attachment previously demonstrated in the ceils which do not express the muscle isoform of dystrophin is observed in myotubes which do express this isofonn, indicating that this downstream MAR is constitutive.

Sequence analysis of the muscle promoter/exon 1 region

A 12 kb region across the promoter has been sequenced by the Canadian Genetic Disease Network Sequencing Centre (Accession Nurnber AF276053). My analysis has shown regions of high ATcontent and a number of topoisomerase II consensus cleavage sites, characteristics of MARS (Boulikas 1993). The 5.4 kb MAR-containing region upstream of the muscle-specific promoter has an ATcontent of 66%, while the 4.4 kb region containing the downstream MAR has an ATcontent of 70%. 1 found 204 strong vertebrate topoisomerase IJ consensus cleavage sites (RNYNNCNNGYNGKTNYNY., Spitzner and Muller, 1988) across the 12 kb region, occurring at higher frequency upstream of the promoter. The topoisomerase II consensus cleavage sites occw at an average frequency of 21 sites/kb in the 5.4 kb region containing the upstream MAR, at 14 sitedkb in the 4.4 kb mgion containing the downstream MAR, at 17 sitedkb in the upstream 1.2 kb HindIWBamHI restriction fragment (Fig. 3-2A), and at 11 siteskb across the 0.7 kb promoter region (0.32 kb HindrmEcoRI and 0.38 kb EcoRI restriction fragments; Fig. 3-4A). 1 have also found other protein binding consensus sites in this sequence, including consensus sites for myocyte-specific enhancer-binding nuclear factor 2 (MEF-2; Gossett et al., 1989), four of which fa11 within the downstream MAR (Fig. 3-8). Figure 3-7. In vivo MAR isolation study of the ti.rw specificity ot the muscle piornater MAR A) Restriction map of the musde promoter region. Hatched box indicates the restriction fragment used as a probe. (P) promoter, (Ml) muscle exonl . B) Souaiem blot containing DNA from HindllVEcoRl digested nuclear haloes prepared from differentiated myotubes. Horizontal amws indicate the MAR- containing fragments. (S) supematant or unbound DNA, (P)pellet or mat& bound DNA. (T) total genomic DNA. Figura 38. Romoter Region MARS: Expimentcrlly detemined positions cotncide Wh sequmcbba..d analy8is. Matrix association potential determined for DNA sequence from the muscle promoter region using the sequence-based model developed by Singh et al. (1997) is aligned with the restriction map of the muscle promoter region above in a collinear fashion. Hatched boxes indicate positions of MARS. Asterisks indicate positions of MEF-2 consensus sites. 68 In addition, I used the sequence-based mode1 desi@ by Singh et al. (1997) to dyzethe 12 kb pmmoter region DNA sequence for possible MARS. The model pdicfed two pealrs in Matrix Association Potential which a coincident with the functional MARS which 1 detennined, indicating the presence of sequence characteristics common to known MARS (Fig. 3-8).

Discussion

1have shown experirnentally the existence of two MARS: one upstream of the muscle-specific pmmoter and one downstream in intron 1 which is at least 2 kb upstream of the intmnic muscle- specific enhancer. These MARs have sequence characteristics typicai of MARS, such as high AT-content and topoisornerase 11 consensus cleavage sites (Boulikas, 1993). In addition, a DNA sequence-based model predicts two peaks in MAR Association Potential, one within each of the two experimentaliy determineci MARS (Singh et al., 1997). These results indicate that the

MARS at the dystrophin gene muscle-specific pmmoter show DNA sequence and topological simiiarities to other known MARS ,and, therefore, may also serve similar functions.

The demonstration of MARS in the region of the muscle-specific pmmoter of îhe dystrophin gene suggests an important rde for these MARS in transcriptional regulation hm this promoter. MARS have previously been identified at or near promoters and enhancers in other genes (Gasser et al., 1986; Cockeril1 et al., 1986; Jankelevich et al., 1992; Dwotetzky et al., 1992; Jarman et al., 1988; Brotherton et al., 199 1). The pximity of the two MARS i&ntified in this study to the muscle-specific pmmoter and enhancer of the dystrophin gene suggests that their association with the nuclear matrix may involve interactions with transcription factors that may be localized or concentrated on the nuclear marrix. Consensus binding sites of the transcription factor MEF-2, identifid in the downstream MAR, may be Unportant in transcriptional control from tht muscle-specific pmmoter. Previously, Klamut et al. (1997) 69 identifieci MEF-2 biiding sites within the muscle-specific intron 1 enhancer of the dystrophin gene, and showed that mutation of one of these sites led to a 9-fold decrease in reporter gene activity relative to the wild-type construct in H9C2 myotubes.

In a gene as complex as the dystrophin gene, MARS may play a role in controlling hmwhich of the multiple dystmphin gene promoters transcription is initiated. It is possible that the upstream MAR could be acting as a chromatin domain boundary in celis which express tbe muscle isoform of dystrophin. DNA hmthis region could be examined for DNaseI-sensitivity to determine the possible coincidence of a DNaseI-sensitive border with the upstreaxn MAR. In addition, this MAR could be tested with a reporter gene for boundary fwictions in both stably- msfected cells and transgeic animals. A comprehensive study of matrix-binding activity at the other dystmphin promoters could yield further information about transcription across this complex gene.

The tissue-specific binding of MARS has previously been observed in other genes (bvy-

Wilson et al., 1989; Brotherton et al., 1991). In these genes, the facultative MARS were observed to bind to nuclear matrices in cells where the genes were expressed but not in cells where the gene was not expressed. In the present study, the existence of MARS at the muscle- specific promoter region has been investigated in HeLa and undifferentiated myogenic ceüs, neither of which express the muscle isoform of dystrophin, and in differentiated myotubes, which do express this protein. AIthough the inherent dificulties of isolating nuclei hm myotubes prevented an extensive investigation of tissue-specifkity of the MARS, it appears that the downstream MAR is matrix-bound in both dystrophin-expressing (myotubes) and non- expressing cells (HeLa cells, undifferentiated myogenic ceils).

It also appears thaî interaction of the muscle-specific promoter region of the dystrophin gene with the nuclear matrix is consistent on both the xtive and inactive X chromosomes. The two 70 nonexpressing ce11 types used in this study (HcLa ceils and undifferentiated myogenic cells) are both of female origin. The clear distinction between matrix-bound and unbound fragments in the above experiments would indicate that b'inding to nuclear matrix proteins is the same for this DNA region on both X chromosomes.

In summary, the identification of two MARS flanking the muscle-specific pmmoter of the dystrophin gene, suggests that these DNA sequences might play an importaut role in expression fiom the muscle-specific pmmoter of the dystrophin gene, perhaps through theîr impact on the activities of the muscle-specific pmmoter and nearby enhancer. CHAPTER 4

Discussion and Future Directions. DISCUSSION

The dystrophin gene is unique with respect to its extraordinary size, complexity and high rate of mutation. At a length of over 2300 kb, it is the largest known gene, spanning 79 exons. The expression of distinct dystrophin isofonns occurs from a least seven promoters within tbe dystophin gene in a tissue-specifk manner. Mutations in this gene lead to Duchenne muscular dystrophy, a lethal degenerative muscle disorder, and the milder, allelic Becker muscular dystrophy, and are mostly a result of chromosomal rearrangements.

The organization of chromath with the nucleus involves the attachment of DNA to the nuclear matrix. .DNA sequences involved in these interactions are called maÉrix aüachment regions. The association of MARS with the proteins of the nuclear maûix is thought to play roles in such nuclear metabolic events as iliegitimate recombiiation and regdation of transcription.

1 have addressed the question of the involvement of chmatin organization on iliegitllnate recombination in the dystrophin gene by studying a duplication event. A duplication within this gene, resulting from a non-homologous reçombination event, was identified in a patient with Duchenne muscular dystrophy. The breakpoint regions of this chmmosomal rearrangement contained DNA sequence characteristics observed in known matrix attachment regions. Tb experiments presented in this thesis show the existence of functional maüix attachmernt regions at both these breakpoints. In addition, topoisomerase 1 consensus sites occur at both breakpoints. A mode1 of the mechanism of this recombination event has been proposed whereby the two breakpoint regions, although distant hmone another on the DNA sequence, may have been anchored in close proximity to one anoher on the nuclear matrix. This positionhg on the nuclear rnaûix may have provided the opportunity for illegiimate recombination to occur, perhaps involving topoisomerase 1cleavage. 73 1 also investigated the role of chromath organization on transcriptional ngulation in the dystrophin gene. The extraordinary size and complexity of this gene present a unique problem with respect to the organization of its chromatin for transcription. 1 examined the muscle-

specific pmmoter region for the existence of MARS at tbis cis-acting eIement and the nearby intronic enhancer. MARs were found to flank the muscle-specific promer, suggesting a role for these MARS in transcriptional regdation. DNA sequence analysis of the pmmoter region showed high ATcontent and topoisornerase II consensus sites, typical characteristics of MARS

These results suggest important roles for the dystrophin gene MARS in both the transcriptional regdation of dystmphin and in an illegitimate recombination event resulting in Duchenne muscular dystrophy. To characterize fiuthet the roles of dystrophin gene MARS, I propose the study of a nwnber of aspects of the MARS identiaed in this study.

FUTURE DIRECTIONS

Binding of Proteins to Dystropbin Gene MARS

MARS have now been identified in the dystrophin gene at the intron 7 and intron 9 duplication breakpoints and at the muscle-specific promoter region. These DNA sequences were show to interact with the proteins of the nuclear mauix. The precise positions of protein binding sites within these MARS can be investigated using in vitro DNAse footprinting assays.

Specific regions within each MAR can be studied for protein binding. For example, the binding of nuclear proteins to consensus sites identified in the DNA sequences, such as the topoisornerase 1 consensus sites at the bceakpoint MARS and MEF-2 consensus sites in the muscle-specific pmmoter region MAR, can be examined by this method. End-labelled restriction fragments containing such regions can be incubated with nuclear extracts, and then 74 treated briefly with DNase. Following gel electrophoresis of the purined DNA, the resulting DNase footp~t,oôserved by autoradiography, reflects the sequeme position at which a protein binds. The binding of nuclear protein at identifiai consensus sites would provide connnnation for the suspecteci roles of these MARS. A topoisornerase I footprint at the intron 7 and 9 duplication breakpoints wouid support the mode1 of recombinaîion involving cleavage by topoisornerase 1. Simüarly, protein binding at transcription factor binding sites, such as MEF- 2, in the muscle-specific pmmoter MAR would support its proposeci role in transcriptional regulation. In addition, suspecteci binding of tissue-specific factors, iike MEF-2, can be supported if tissue-specific binding can be shown, by using nuclear extracts hmcells that express the transcription factor under study and those which do not.

Transcriptional Regulation by Muscle-Specific Promoter Region MARS

The function of the dystmphin gene muscle-specific promoter region MARS in transcriptional regulation from the muscle-specific promoter can be examined by testing for enhancer properties. Enhancers act to increase gene expression independent of their position and orientation with respect to a test promoter in a tissue- or development-specific manner. Both the upstream and downstrearn muscle-specific promoter region MARS can be tested for classical enhatlcer fùnction in transient transfection assays. For such studies, the DNA regions containing the MARS (and subclones thereof) would be inserted into a vector constnict containing a core promoter and reporter gene, and assayed for gene expression in the myogenic cell lines C2C 12 and H9C2. The mouse skeletal C2C 12 and rat cardiac H9C2 muscle cell hes have previously been used in muscle-specific gene expression studies (Klamut et al., 1990). An increase in reporter gene expression of MAR-containing constmcts over MAR-less constructs, independent of position or orientation with respect to the promoter, would indicate the enhancer properties of the MAR. 75 Previously, Hindm fragments from tbe muscle-promoter rgion, including the MAR regions, were tested for enhancer iictivity in H9C2 cells (Klamut et al., 1996). The 6.6 kb Hindm restriction fragment, imiriPAiately upstream of the muscle-specific promoter (see Fig. 3-lA), showed low enhancer activity. However, many cis-acting elements may reside in this large fragment. Any cis-acting element in this fia&mentwhich may have a positive effect on transcription may be masked by another whose effects on transcription are negative. Testing subclones of this fiagment in the 5.4 kb MAR-cont-g region might yield more &tailed results of enbaacing activity in this region. The 4.4 kb downstream MAR appears to span a Hindm site and wouid not necessarily have demonstrated enhancer activity if such existed. The choice of restriction fragment containhg this downstream MAR region in its entirety would yield accurate results on enhancing activity of the whole MAR. An EcoRI restriction fragment would contain the entue MAR, extending hmthe promoter, across the Hindm site, through the neighbouring 1.7 kb HindIIyXbaI region, but excluding the previously identified muscle- specific enhancer (see Fig. 3- LA).

MARS at Other Breakpoints in the Dystrophin Gene

In addition to the duplication stuclied in this thesis, other breakpoints of chromosoma1 rearrangement in the dystrophin gene have DNA sequences characteristic of MARS. These rearrangernents, found in patients with DMD, may also have occurred as a result of their interactions with the nuclear matrix.

One of these patients has a duplication of exons 20 to 41, involving breakpo~ntsin introns 19 and 41 (Hu et al., 1991). Like the patient in this study, the duplication arose by non- homologous recombination of sister chrornatids in the materna1 grandfather. Both breakpoints ate AT-nch (about 70%). Topoisornerase 1 consensus sites have been identified in the breakpoint regions of both intmn f 9 and 4 1. Intron 4 1 contains a topoisornerase 1 site precisely 76 at the breakpoint, while intmn 19 contains a topoisomerase II site precisely at the breakpoint. Preliminary work, using the in vivo MAR isolation methoâ, has shown the existence of a MAR at the intron 19 breakpoint (Xiuyuan Hu, personal communication).

The bnakpoints of a translocation involving the dystropiiin gene, identined in a nue femaie DMD patient, also have MAR sequence characteristics (Bodmg et al., 1991). This translocation occuned by non-hodogous recombination and involved breakpoints on chromosome 4 and in intron 5 1 of the dystrophin gene. Non-random inactivation of the normal X chromosome led to disease in this patient. The origin of the recombination was detennined to be paternal, and probably resulted hma pst-meiotic rarrangement in spermiogenesis, since it is unlikelly that an X-autosome translocation can be transmitted through male meiosis. The breakpint regions have a high AT-content and contain topoisomerase II consensus sites at the breakpoints. A topoisomerase 1consensus site was found at the chromosome 4 breakpoint.

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