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Isolation and characterization of 5’ upstream regulatory region of survival motor neuron gene He, Ming 1999

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I S O L A T I O N A N D C H A R A C T E R I Z A T I O N OF 5' U P S T R E A M R E G U L A T O R Y R E G I O N OF S U R V I V A L M O T O R N E U R O N G E N E by M i n g He, M . D . The Shanghai No . 2 Medical University, 1989 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF S C I E N C E in T H E F A C U L T Y O F M E D I C I N E Department o f Surgery We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A Apr i l 1999 © M i n g He, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ./ <-> The University of British Columbia Vancouver, Canada Date Af " f /W DE-6 (2/88) A B S T R A C T Spinal muscular atrophy ( S M A ) is a common autosomal recessive disorder characterized by loss or degeneration of lower motor neurons in the spinal cord, leading to progressive symmetrical limb and trunk paralysis and muscular atrophy. The disease has been classified into three types and mapped to chromosome 5ql3 . The telomeric survival motor neuron (SMNt) gene has been identified to be the S M A determining gene. The centromeric S M N (SMNc) may be related to the S M A phenotype. Five nucleotides differences between S M N t and S M N c do not affect the encoded protein. S M N proteins are distributed widely in all tissues with highest concentration in C N S and liver. Little is known about the regulatory mechanism of the S M N gene in cells. To identify the regulatory sequence elements for the expression of the S M N gene in both neuronal and non-neuronal cells, we cloned a 3132 bp fragment of 5' upstream region of S M N gene isolated from the chromosome 5 library into the luciferase reporter gene system. Serial deletion constructs containing various lengths of 5-flanking region of S M N gene were transfected into both S Y 5 Y and Vero cells. The results of the promoter activity assay showed that 1) The reporter gene could be expressed in both neuronal and non-neuronal cells; 2) 48 potential binding sites for transcription factors were localized in the region from -1530 to +34; 3) The region from -157 to +114 displayed the highest promoter activity while the lowest activity was shown in the region from - 28 to +114; 4) The 5' upstream region beyond - 899 showed lower promoter activity than that of more approximal region (-306 to +114); 5) The promoter was more active in the neuronal cells in general than that in the non-neuronal cells, especially when the region contained more upstream sequences. The above results suggested that the S M N gene might be well regulated by many potential transcription factors. A negative regulatory element might be located in the region between -899 to -306 and the region between -157 to +114 might be critical for the S M N gene expression. i i T A B L E O F C O N T E N T A B S T R A C T i i L I S T OF F I G U R E S v L I S T OF T A B L E S v i A C K N O W L E D G E M E N T S v i i I N T R O D U C T I O N 1 Spinal Muscular Atrophy 1 1) Historical review 1 2) Clinical symptoms and classification of S M A disease 3 3) Genetics of S M A disease 4 Upstream Regulatory Region (Promoters) 14 Objectives of the Present Study 18 M A T E R I A L S A N D M E T H O D S 20 1) General chemicals and enzymes 20 2) Chromosome library screening 20 3) Subcloning 21 4) Sequencing 21 5) Search for transcription factor binding sites 22 6) Reporter gene constructs 22 7) Ce l l culture and D N A trasnfection 24 8) Ce l l extract and reporter gene assays 25 9) Immunocytochemistry 25 10) Data analysis 26 R E S U L T S 27 1) Sequence of 5' upstream region of S M N gene 27 2) Potential binding sites for transcription factors 27 3) Promoter activity of the 5' flanking region of S M N gene in transfection assays 28 4) Immunocytochemistry 30 i i i D I S C U S S I O N S U M M A R Y S I G N I F I C A N C E F U T U R E I N V E S T I G A T I O N S F I G U R E S 1-16 T A B L E S 1-3 R E F E R E N C E S A P P E N D I X 1 L I S T OF F I G U R E S Figure 1. Schematic illustration of 5ql3 region 44 Figure 2 Structure of S M N gene 45 Figure 3 Different scenarios of S M N gene arrangement 46 Figure 4 Diagram of subcloning procedure for sequencing 47 Figure 5 Cloning procedure of reporter gene constructs 48 Figure 6 The two basic reporter gene constructs 49 Figure 7 Diagram of 5' upstream region of S M N gene 50 Figure 8 A comparison between the sequences generated from the present study and in the Genbank 51 Figure 9 Distribution of potential binding sites for transcription factors in the 5' -upstream region of S M N gene 52 FigureTO Structure and activity of S M N promoter (-3132/ -306) analysis in S Y 5 Y neuroblastoma cells 53 Figure 11 Structure and activity of S M N promoter (-306/-28) analysis in S Y 5 Y neuroblastoma cells 54 Figure 12 Structure and activity of S M N promoter (-3132/ -306) analysis in Vero cells 55 Figure 13 Structure and activity of S M N promoter (-306/-28) analysis in Vero cells 56 Figure 14 Difference in expression levels of luciferase driven by S M N promoters containing various upstream regions in neuroblastoma cell line S Y 5 Y and kidney cell line Vero 57 Figure 15 Expression of S M N in S Y 5 Y cells 58 Figure 16 Expression of S M N in Vero cells v 59 L I S T O F T A B L E S Table 1, Oligonucleotides used to sequence the 5' flanking region of S M N gene 60 Table 2, Mismatched nucleotides 61 Table 3, Potential binding sites for transcription factors 62 \ v i A C K N O W L E D G M E N T I would like to acknowledge Drs. S. Kim and J.Francis, for providing the SY5Y cells and the antibody against S M N protein, respectively. I would like to thank Dr. Shiv Prasad for his advice in various aspects of this study. I also appreciate committee members for reviewing my thesis. Special thanks are given to my supervisor Dr. William Jia for his guide through the entire M.Sc. program. I would like to express my gratitude to Dr. Zheng Chen, Ms. Rena Chen and other colleagues in the lab for their assistance. I would also like to thank my husband, John Zhang, for his love and support in the past 2 years. vii I N T R O D U C T I O N S P I N A L M U S C U L A R A T R O P H Y 1) Historical review The spinal muscular atrophies (SMAs) are a genetically heterogenous group of inherited conditions characterized by degeneration of anterior horn cells or cranial nerve motor nuclei and resultant wasting and weakness of voluntary muscles (Dubowitz, 1995b). They are classified into two types: the childhood proximal autosomal recessive S M A , and the distal autosomal dominant S M A , or named X-l inked forms (see review in Thomas, 1994). This thesis w i l l focus on the childhood S M A , which represents the second most common fatal autosomal recessive disorder after cystic fibrosis (Pearn, 1978a). The history of the childhood S M A can be traced back to 1883, when Bennett discussed a chronic atrophic spinal paralysis in children (Bennett, 1883). One case was clearly the first infantile spinal muscular atrophy ( S M A type I), although not recognized as such. In 1891, Guido Werdnig, a neurologist in Graz, Austria, published his classic paper entitled " Two hereditary cases of progressive muscular atrophy in early infancy presenting as muscular dystrophy, but on a neural basis" (Werdnig, 1891). He described two brothers who developed a progressive proximal weakness affecting the legs and arms. One child died of pertussis aged 3 years; an extensive autopsy revealed bilateral symmetrical loss of anterior horn cells. In the following year, Johann Hoffmann of Heidelberg first used the term, in German, spinale Muskelatrophie, (spinal muscular atrophy), in his paper entitled "Ueber chronische spinale Muskelatrophie im Kindesalter, auf familarer Basis." (Hoffmann, 1892). In 1894, Guido Werdnig gave his further report about six children 1 who were commented on the variability of severity (Werdnig, 1894). In 1900, Hoffmann reported six cases in four families, and there were 21 other affected relatives in three families. The milder form of spinal muscular atrophy were first reported by Kugelberg and Welander in 1956, who described 12 cases with onset between 2 and 17 years and survival into adult life, with continued ambulation (Kugelberg & Welander, 1956). Since then, numerous papers reported many cases of S M A with various ages of onset and severity (see review in Thomas, 1994). Meanwhile, classification of the disease with different severity and localization of the S M A gene on human chromosomes has attracted much attention. In 1961, Byers and Banker first classified the S M A according to severity, and this classification was used to facilitate the prognostication of the disease (Byers & Banker, 1961). In 1964, Dubowitz described the co-relation between age of onset and severity (Dubowitz, 1964). In 1991, International S M A Collaboration, based on age of onset and clinical course, subdivided the childhood S M A into three clinical groups, including type I, Wernig-Hoffmann disease; type II, intermediate severity form and type III, Kugelberg-Welander disease (Munsat, 1991). In the 1970's, population studies had revealed that all types of S M A were likely to be inherited in an autosomal recessive manner (see review in Morrison, 1996). Therefore, hard research work on trying to locate the SMA-determining gene was carried out by a concerted international collaboration including researchers from the U . S . A , U . K , Finland and Germany. In 1990, it was concluded that all types of the S M A mapped to 2 chromosome 5q, which was also confirmed in French population by a French group working on a similar project (Munsat et al, 1990; Brzustowicz et al, 1990; Burghes et al, 1994; Daniels et al, 1992; Francis et al, 1993; Gi l l iam et al, 1990; MacKenzie et al, 1993; M e l k i et al, 1990; Simard et al, 1992; Wirth et al, 1994). In 1995, Suzie Lefebvre and his colleagues identified and characterized a spinal muscular determining gene named survival motor neuron (SMN) gene (Lefebvre et al, 1995). 2) Clinical symptoms and classification of S M A disease The childhood spinal muscular atrophy, or proximal spinal muscular atrophy is a group of inherited neuromuscular disorders characterized by the degeneration of motor neurons in anterior horn of the spinal cord, leading to progressive symmetrical weakness and wasting in the proximal muscles (Dubowitz, 1995a). It represents the second common fatal autosomal recessive disease after cystic fibrosis in children, with an estimated incidence of 1 in 10,000 newborns and a carrier frequency of 1/40-1/60 (Pearn, 1978b). Clinically, it has been classified into three groups on the basis of the age of onset and clinical course (Munsat, 1991): - Type I (Wergnig-Hoffmann disease) is the most severe form, with onset at birth or before 6 months and 90% of them die within 2 years of age due to the respiratory failure. Type I patients are never able to sit or walk. - Type II (Intermediate severity form) S M A patients are able to sit but can not achieve the ability to stand or walk without any support. The age of onset is usually within 6-18 month. Survival of type II individuals depends on the degree of respiratory complications. Some of them can survive to 2nd or 3rd decade. 3 - Type III (Kugelberg-Welander disease) is the mildest form of all three childhood S M A types. The type III patients show their first symptom after 18 months, usually in late childhood or adolescence, and are characterized by the ability to walk unaided. Many type III patients can survive with a normal life expectancy. Whatever the variation in the clinical severity, all three forms of S M A patients are characterized by the following common features (see review in Thomas, 1994): - Muscular weakness is symmetrical and more proximal than distal. It affects legs more than arms. The trunk is also affected. - Deep reflexes are absent or markedly decreased. - Electromyographic analyses show muscle denervation with neither sign of sensory denervation nor marked decrease in conduction velocities of the motor nerves. - Histopathological examinations show atrophic fibres in all biopsies. 3) Genetics of S M A disease A l l three types of S M A have been mapped, by linkage analysis, to chromsome 5ql 1.2-13.3 (Munsat et al, 1990; Brzustowicz et al, 1990; Burghes et al, 1994; Daniels et al, 1992; Francis et al, 1993; Gi l l iam et al, 1990; MacKenzie et al, 1993; M e l k i et al, 1990; Simard et al, 1992; Wirth et al, 1994), a region of complex genomic organization that contains numerous repeated sequences, including polymorphic markers and genes (Burghes etal, 1994; Carpten etal, 1994; DiDonato etal, 1994; Francis etal, 1993; K leyn et al, 1993; Lefebvre et al, 1995; M e l k i et al, 1994; Roy et al, 1995; Thompson et al, 1995), suggesting that the S M A are allelic disorders. Further characterization of 4 S M A locus has revealed a choromsomal region containing a large inverted duplication of a 500kb-element and several classes of pseudogenes (Lefebvre et al., 1995). The element contains three genes (Figure 1): the Survival motor neuron (SMN) gene (Lefebvre et al., 1995), the neuronal apoptosis inhibitory protein (NAIP) gene (Roy et al, 1995; Thompson et al., 1995) and p44 gene, which encodes a subunit of the basal transcription factor TFI IH (Burglen et al., 1997; Carter et al., 1997). According to the position in the chromosome 5q, all these three genes have their corresponding telomeric locus and a centromeric locus. It is most likely that the two loci can exist in either orientation, depending on the particular chromosome (Burghes, 1997). Only the telomeric version is related to the large scale deletions in S M A patients (Burglen,et al., 1997; Carter et al., 1997; Lefebvre et al, 1995; Roy et al, 1995). The N A I P gene shows a similarity with baculoviral genes involved in inhibition of apoptosis in infected insect cells and is present in multiple copies. Only the copy of N A I P t (telomeric copy) containing exon5, named N A I P 5 , is associated with deletions in S M A patients (Liston et al, 1996; Roy et al, 1995). This copy is deleted in 45% of type I S M A patients and 18% of type II and III S M A patients (Burlet et al, 1996; Cobben et al, 1995; Hahnen et al, 1995; Roy et al, 1995; Simard et al, 1997; Velasco et al, 1996), but also in 2% o f unaffected carrier individuals (Roy et al, 1995; Thompson et al, 1995). Thus, loss of N A I P is not sufficient to cause the S M A disease. However, N A I P deletion may contribute to severity of the phenotype, by generating an effect additive to that of S M N t deletion (Simard et al, 1997; Wirth et al, 1995). Another gene p44 also exists as multiple copies, but only one copy, p44t (telomeric copy) is associated with S M A 5 deletions. Deletion or interruption of p44t gene have been observed in 73% of type I patients, but also in normal individuals (Burglen et al., 1997; Carter et al., 1997). In addition, the structure and function of the TFIIH protein appear normal in the patients homozygously deleted for p44t gene, which suggests that this copy may not play a role in any disease pathology of S M A (Burglen et al, 1997). Deletion of both N A I P 5 and P44t genes were found in 50% of type I S M A patients, which may represent the extent of deletion on severe S M A chromosomes (Roy et al, 1995; Thompson et al, 1995; Wirth et al, 1995). The S M N gene is about 20kb in length and consists of nine exons interrupted by eight introns (Burglen et al, 1996) (Figure 2). The two duplicated S M N gene copies are termed to the centromeric S M N (SMNc) gene and tolemeric S M N (SMNt) gene according to their relative location on chromosome 5q (Figure 1) (Lefebvre et al, 1995). The S M N t and S M N c gene can be distinguished by five nucleotides changes, which do not alter the encoded amino acids. These include one base change in exon 7 which has been the marker for distinguishing S M N t from S M N c , and one base change in exon 8, one base change in intron 6 and two base changes in intron 7 (Burglen et al, 1996; Lefebvre et al, 1995). Analysis of a control population shows that the S M N t gene is present in all individuals while S M N c gene is absent in 7.5% of individuals (Lefebvre et al, 1995; McAndrew et al, 1997). The copy number of the S M N t and S M N c gene varies in different individuals (McAndrew et al, 1997). A new multicopy marker A g - C A (C272), located at with the 6 upstream 5' end of the S M N c and S M N t genes (Figure 1), has a highly significant allelic association with S M A in both the American and French Canadian populations and can be used to estimate the copy number of S M N t and S M N c genes (Burglen et al, 1996; Simard et al, 1997). N A I P 5 gene copy which lies 3' to the S M N t gene can be used as a marker indicating the presence of the S M N t locus (Figure 1) (Roy et al., 1995). It has been shown that three copies of S M N t gene exist in nearly 6% of normal individuals, suggesting that one of the two chromosome 5 has two copies of the S M N t , while 94% of individuals have only one copy on each chromosome 5 (McAndrew et al., 1997). Analysis of the copy number of S M N c gene in normal population showed that 43.4% of individuals have two copies; 47.2% one copy; 7.5% no copies; and 1.9% three copies (McAndrew et al, 1997). It has been well demonstrated that the S M N t gene is the S M A determining gene (Cobben et al, 1995; DiDonato et al, 1997; Hahnen et al, 1995; Lefebvre et al, 1995; Rajcan et al, 1996; Rodrigues et al, 1995; van der Steege et al, 1996; Velasco et al, 1996) and the S M N c gene may be related to the S M A phenotype (Campbell et al, 1997; DiDonato et al, 1997; McAndrew et al, 1997). Using exon 7 as a marker, the S M N t gene is not detectable in > 90% of S M A patients, regardless of the severity. The S M N t gene that can be detected in S M A patients is always with some mutations (Brahe et al, 1996; Bussaglia et al, 1995; Hahnen et al, 1997; Lefebvre et al, 1995; McAndrew et al, 1997; Parsons et al, 1998; Parsons et al, 1996; Talbot et al, 1997). Two different mechanisms have been implicated for the lack of SMNt-specific exon 7 in S M A patients (Figure 3): gene deletion or gene conversion from S M N t to S M N c (DiDonato et al, 1997; Hahnen et al, 1 1995; Hahnen et al, 1996; Lefebvre et al, 1995; Rodrigues et al., 1995; van der Steege et al, 1996; Velasco et al, 1996). In most SMA type I patients, SMNt-specific exon 7 is absent and the number of loci detected by C272 is also reduced, which means that a deletion in SMNt gene exists in this group of patients (DiDonato et al, 1994; Melki et al, 1994; Lefebvre et al, 1995; Wirth et al, 1995). In SMA type II and type III patients, SMNt exon 7 is not detected, but the NAIP5 gene, which is the marker of the existence of the SMNt locus, is still present, and number of the marker C272 is not changed, indicating that the SMNt gene has converted to SMNc gene in type II and type III SMA patients, leading to the increased SMNc copy number (Figure 3) (Campbell et al, 1997; Burghes, 1997). Thus, it seems that SMNt gene deletions are related to the severe phenotype, and gene conversions from SMNt to SMNc may be associated with a milder disease phenotype. Variable mutations have been detected in 75 % of the SMA patients retaining the SMNt gene copy (<10%) by current methods (McAndrew et al, 1997). These mutations include disrupted splicing of exon 7 (Lefebvre et al, 1995), deletion of 4 base pairs or 5 base pairs in exon 3 (Bussaglia et al, 1995; Brahe et al, 1996), an 11 bp duplication in exon 6 (Parsons et al, 1996), and a five different missence mutations in exon 6 and exon 7 (Hahnen et al, 1997; Lefebvre et al, 1995; McAndrew al, 1997; Talbot al, 1997). Many of these mutations have been found in more than one SMA patient. It has been proposed that a highly conserved tyrosine-glycine (Y-G) dodecapeptide motif in the region of protein encoded by exon 6 and 7 are crucial for the correct functioning of the 8 protein and that mutations affecting this region result in S M A (Talbot et al, 1997). Other mutations may result in premature truncation of the S M N protein (Brahe & Bertini, 1996; Bussaglia et al., 1995; Parsons et al., 1996). The above findings have provided strong evidence indicating that the S M N t is the primary S M A - determining gene, and the regions encoded by exon 6 and 7 are important for normal functions of the S M N t gene product. The S M N c gene was found to be present in all patients and absent in 7.5% healthy individuals (Lefebvre et al, 1995), which indicates that S M N c gene is not the S M A causing gene. However, the correlation between the S M N c copy number and S M A phenotype reveals that the S M N c gene may play an important role in modifying the phenotypes of the S M A (Campbell et al, 1997; McAndrew et al, 1997). It has been reported that 33% of unaffected individuals who only have one copy of S M N t gene (carriers) have three or four copies of the S M N c gene, as compared to only 1.9% of three copies (not four copies) in the rest of normal population (McAndrew et al, 1997). Using the C272 marker, a correlation between the number of copies of C272 and S M A phenotype has been revealed, which demonstrates that most of type II/III S M A patients who have milder phenotype have three copies of S M N c gene (Campbell et al, 1997; DiDonato et al, 1994; Hahnen et al, 1996; McAndrew et al, 1997; van der Steege et al, 1996; Velasco et al, 1996; Wirth et al, 1995). It is apparent that the increased copy number of S M N c gene may compensate for the lack of S M N t gene, suggesting that the S M N c gene is translated into an at least partially functional protein (Lefebvre et al, 1998), which also has been demonstrated at R N A and protein levels (Coovert et al, 9 1997; Lefebvre et al, 1997). Both S M N c gene and S M N t gene are expressed in all normal human tissue (Coovert et al, 1997; Lefebvre et al, 1995; Lefebvre et al, 1997; Novel l i et al, 1997). The nucleotide changes in exon 7 and exon 8 between the S M N c and the S M N t gene do not influence the encoded amino acid, which means both S M N c and S M N t encode an identical protein (Lefebvre et al, 1995). However, analysis of the S M N gene transcripts in lymphoblastoid cell line, human muscle and central nervous system (CNS) tissues shows alternative splicing occurs in both S M N c and S M N t gene copies. The majority of transcripts (approximately 90%) from the S M N t gene are full-length (1.7kb), thereby encoding a fully functional protein, while the remainder (approximately 10%) is missing exon 5 (Gennarelli et al, 1995; Lefebvre et al, 1995; Parsons et al, 1996). S M N c gene can produce all kinds of isoforms of S M N , but only 20-30% of these are the full-length transcripts. The spliced transcripts from S M N c copy include transcripts without exon 5, or 7 or both. A l l these non-full-length transcripts generate truncated proteins (Gennarelli et al, 1995; Lefebvre et al, 1995; Parsons et al, 1996). The translated protein from the full-length transcript is a novel protein of 294 amino acids with a molecular weight of 38 kDa (Lefebvre et al, 1995). Light and electron-microscopy studies have demonstrated that S M N protein is localized in the cytoplasm and the nucleus (L iu & Dreyfuss, 1996). Nuclear S M N protein is detected by an t i -SMN antibody in prominent new sub-nuclear bodies called "gems" for 'Gemini of the coiled bodies' nucleus (Liu & Dreyfuss, 1996). The S M N protein is ubiquitously expressed in 10 humans; is conserved throughout mammalian species and shows no resemblance to any known protein in database (Lefebvre et al, 1995). Quantitative western blot analysis has shown that the S M N protein is abundantly expressed in human brain and spinal cord although it is detected at similar levels in non-neural tissues such as kidney, liver as well (Coovert et al., 1997). In human C N S , cell specific expression has been observed by in situ hybridization. The expression of the S M N gene is mainly located in specific neuronal populations, including motor neurons, which are the target cells in S M A , central canal, dorsol root ganglia, cerebral pyramidal cells and cerebellum Pukinje cells (Tizzano et al, 1998). Immunohistochemical analysis of S M N protein shows that motor neurons of the spinal cord from normal fetuses have a large amount of cytoplasmic S M N protein and large gems as compared with other cells and tissues (Lefebvre et al., 1997). In situ hybridization analysis also shows the large motor neurons of the spinal cord are the main cells that express S M N (Tizzano et al, 1998). The function of the S M N protein still remains unknown. However, many research results have supported the notion that it may be related to the R N A metabolism. First, the "gems", which are labeled by ant i -SMN antibodies, appear to be associated with the nuclear coiled bodies, which play a role in pre-RNA metabolism (L iu & Dreyfuss, 1996). The "gems" appear to interact directly with the coiled bodies and undergo similar changes in response to enviromrrental and metabolic conditions of the cell (L iu & Dreyfuss, 1996). Secondly, the S M N protein was shown to form a complex with a novel protein named SIP1 ( S M N interacting proteinl) (Fischer et al, 1997; L i u et al, 1997) and also interact with small nuclear ribonucleprotein U l and U5 of spliceosome, the catalytic core 11 of the splicing reaction. Finally, the region of amino acids 262 to 279 of the S M N contains a tyrosine/glycine-rich motif that is present in various R N A binding proteins (Talbot et al., 1997). Interestingly, mutations in this region of S M N t gene have been frequently found in S M A patients, which further emphasizes.the functional importance of this region and suggests that the loss of function in interacting with R N A s may contribute to the S M A pathology. A recent study also suggested that the ability of S M N self-association through the portion of the S M N protein encoded by exon 6 and 7 may be important (Lorson et al, 1998). In addition, it has also been reported that S M N protein may have a synergistic anti-apoptotic activity with Bcl-2 protein (Iwahashi et al., 1997). Since the majority (90%) of the full-lengh S M N m R N A , which can be translated into a functional protein, are transcribed from S M N t gene (Gennarelli et al., 1995; Lefebvre et al., 1995; Parsons et al, 1996), loss of S M N t gene results in a decrease in the functional level of S M N protein and the severity of S M A phenotype may directly correlate with the levels of S M N protein in cells. However, the amount of full-length transcripts could vary due to the variable copy numbers of S M N c gene in different individuals (McAndrew et al., 1997). In human fetal tissues (liver and spinal cord), the relative amount of the S M N protein is dramatically reduced in all S M A type I, but not in type III, especially in spinal cord (Lefebvre et al., 1997). For type II patients, the protein level is either decreased or normal (Lefebvre et al., 1997). Similar protein levels are observed in S M A type I patients carrying large-scale deletions (involving S M N t and telomeric versions of N A I P and P44 on both mutant chromosomes), small deletions or intragenic mutations of the S M N gene (Coovert et al., 1997). A l l these results suggest that the remarkable reduction of the S M N 12 protein is related to the type I S M A patients and milder decrease usually occurs in type II/ type III patients. A s mentioned before, evidence from cytogenetics suggest that the loss of S M N t gene by deletion is related to the severe S M A phenotype while loss of S M N t gene by gene conversions, resulting in increased S M N c gene copy number, may be associated with a milder phenotype. The coincidence among the copy number of S M N c , the levels of S M N protein and the severity of S M A disease phenotype supports a hypothesis that, despite the low efficiency in the production of the full-length m R N A by the S M N c , the presence of higher number of S M N c copies results in an increased levels of fully functional protein, leading to a less severe disease phenotype. However, efforts to demonstrate a correlation between S M N c copy number and disease severity have produced conflicting results. Rare cases o f severe forms are also associated to an increased number of S M N c , suggesting that other factors may affect the severity of disease, and a new factor named H4f5 (human 4f5) was reported recently to be a S M A -modifying gene (Scharf et al., 1998). It has also been proposed that the S M A phenotype could be modified by different kinds of conversion extending to a different part of the S M N t gene (for review see Burghes, 1997). On the other hand, immunohistochemical analysis of gems in S M A fibroblats shows the number of gems in the type II patients is clearly more than that in type I patients even though the copy number of S M N c genes are identical in both patients, suggesting that not all S M N c genes in S M A patients are functionally equivalent (Coovert et al., 1997). The most likely explanation is that the S M N c gene copy converted from the S M N t gene is different from the original S M N c gene. The converted S M N c gene copy in type II patients may produce more functional 13 protein sufficient for the gem formation, even though the amount is reduced, whereas the remaining S M N c gene in type I patients is not capable of expressing the appropriate amounts of S M N protein for the formation of gems (Coovert et al., 1997). According to this point, the increased S M N c gene copies that can modify the phenotype of S M A (modifying S M N c ) should be distinguishable from the original S M N c gene. A new hypothesis has been proposed that no copies of modified S M N c genes exist in type I S M A , one modified copy exists in type II and two of those copies in type III (Burghes, 1997; Coovert et al, 1997). The amount of S M N proteins produced from S M N c copy also appears to be cell-type specific. In S M A patients lacking the S M N t (therefore all of S M N protein are produced by S M N c copies), the most significant reduction in the levels of S M N protein was found in the spinal cord (100-fold) while the modest decrease were observed in fibroblasts and skeletal muscle (Coovert et al, 1997). The apparent unequivalence between the original S M N c and converted S M N c and cellTtype specific levels of expression of S M N from the S M N t copy suggest that regulatory factors may play a role at the level of transcription, which may also contribute to the phenotype of S M A disease. U P S T R E A M R E G U L A T O R Y R E G I O N f P R O M O T E R S I The fundamental dogma of molecular biology is that D N A carrying the genetic information variable in each individual produces R N A , which in turn produces proteins that presents the corresponding characteristics of the individual (the phenotype). The process of transcription, whereby an R N A product is produced from D N A , therefore 14 plays an essential role in the gene expression. In addition, the expression of genes in particular cell types is regulated by a number of processes, including synthesis of the primary R N A transcripts, posttranscriptional processing of m R N A , m R N A degradation, protein synthesis (translation), posttranslational modification of proteins, and protein degradation. However, much of this regulation occurs at the level of transcription and is mediated by regulatory proteins that either inhibit (repressors) or activate (enhancers) transcription from specific region of a gene. The promoter region is a D N A specific sequence that the R N A polymerase can bind to. The promoter region is normally located at the 5' end, upstream to a coding region of the gene. This 5'-upstream regulatory region is essential for either basal or regulated gene expression. The regulation of transcription initiation is therefore the regulation of interaction of R N A polymerase with its promoter. In prokaryotes such sequences are found immediately upstream of the start site of transcription, the sequences are not identical for all promoters, but certain nucleotides are found much more often than others at each position. These sequences are called consensus sequence which can be used for distinguishing a promoter region from a non-promoter region on D N A template. For most promoter in prokaryotes, comparative analysis of promoter sequence has identified two consensus sequence elements located upstream around the -10 and -35 region respectively, which are A T rich sequence. A variety of proteins called transcription factors bind to a sequence in and around a promoter and either activate transcription by facilitating R N A polymerase binding or repress transcription by blocking the binding activity. 15 In eukaryotic cells, the promoter region is more complex. The promoter elements include two parts: the core elements and the upstream promoter elements. The core elements include initiator element ( Y A Y T C Y Y Y ) and an A T rich sequence ( T A T A A A A ) called T A T A box, supporting basal transcription (Breathnach & Chambon, 1981). The T A T A boxes are commonly found about 25 to 30 bp before the transcription initiation site, used to assemble initiation complex composed by TFIID (Transcription Factor IID), T F I I A (Transcription Factor IIA), R N A polymerase II and other transcription factors. It plays an essential role in accurately positioning the start site of transcription (Breathnach & Chambon, 1981). Although T A T A boxes are relatively common, many genes have been found to be expressed without T A T A boxes (Weis & Reinberg, 1992). In these promoters, a sequence known as the initiator element, which is located over the start site of transcription itself appears to play a critical role in determining the initiation point and acts as a minimal promoter capable of producing basal levels of transcription (Weis & Reinberg, 1992). A variety of other short sequence elements that function in regulation of a given promoter are often found within hundred base pairs from the transcription start site, usually called upstream promoter elements (UPE). Those elements dramatically increase the low activity of the promoter itself. Two common elements are found between -110 and -40 region: the C C A A T ( G C C A A T ) boxes and G C boxes ( G G G C G G ) , bound by the C T F ( C C A A T transcription factor) and SP1 transcription factor respectively (Dynan & Tjian, 1985). 16 Additional regulatory sequence elements with more complex sequence structure lie in thousands of base pairs away from the core promoter elements. These elements are cis-acting, orientation independent and position independent. Although they lack promoter activity themselves, these elements act by increasing the activity of a given promoter and hence referred to as upstream activator sequence or enhancers (Muller et al., 1988). These sequence elements act preferentially on the nearest promoter, and are variable in sequence. Each sequence is recognized by different transcription factors and therefore believed to be responsible to tissue specific expression. The transcription factors that bind to the upstream promoter elements interact with and facilitate assembly of the R N A polymerase II initiation complex. A t least three types of transcription factors regulate transcription initiation by R N A polymerase: 1) specific factors which alter the specificity of R N A polymerase for a given promoter or set of promoters; 2) repressors which bind to a promoter, blocking access of R N A polymerase to the promoter; and 3) activators which bind near promoter, enhancing the RNA-promoter interaction. Regulation by means of a repressor protein that binds to D N A and blocks transcription is referred to as negative regulation. In contrast, regulation mediated by an activator is called positive regulation. Most eukaryotic promoters are positively regulated and initiation of transcription is almost always dependent on the action of one or, more often, several activator proteins. This may be due to the large size of the eukaryotic genome. Negative regulation appears to be less common, although many eukaryotic regulatory proteins can be either activators or repressors under some circumstances. To date, although a few transcription factors function have been studied, 17 the detailed molecular function of most transcription factors remains unknown. O B J E C T I V E S OF T H E P R E S E N T S T U D Y Since the S M A determining gene - S M N gene was identified and characterized in 1995, S M A research on revealing the genetic basis of this devastating neuromuscular disorder has made substantial progress. One of the areas that have not been well studied is the promoter region of S M N gene. We virtually know nothing about how the gene is controlled by its promoter and cis-acting elements in the upstream region. A s mentioned before, about 95% of S M A patients have lost their S M N t copy therefore rely on the S M N c copy. Only 30% of transcripts from the S M N c gene are full length, the patients therefore need more active transcription to compensate. One way is to have more copies of the S M N c gene, which has been implied by the correlation between copy number of S M N c and phenotype. The other way that could compensate for the loss of S M N t gene is to have a stronger promoter for the remaining S M N c gene, which has not been demonstrated in S M A patients. However, the fact that type II/III S M A patients with a converted S M N c at the original S M N t locus have milder phenotype compared to type I patients who may have the same copy number of S M N c but do not have the conversion implies possible differences in promoter activity between the two genes. Understanding the promoter structure of the S M N gene wi l l help to investigate this possibility and may further benefit to explore new avenues for the treatment of this disease as new drugs may be developed to enhance the promoter activity for S M N c gene expression. A s a first step towards this goal, the present study was aimed at cloning and partially sequencing the 5' upstream regulatory region of normal human S M N gene and further characterizing the 18 region using a luciferase reporter gene assay in both neuronal and non-neuronal cells. 19 MATERIALS AND METHODS 1) General chemicals and enzymes P 3 3 labeled ddNTP were purchased from Amersham (Oakville, Ont. Canada), and D N A -modifying enzymes including restriction endonucleases were from Gibco B R L (Burlington, Ont., Canada), New England (Mississauga, Ont. Canada), and Pharmicia (Baie d'Urfe, Que. Canada). Fine chemicals were purchased from Sigma (Mississauge, Ont. Canada). Ce l l culture mediums and transfection reagents were also purchased from Gibco B R L (Burlington, Ont., Canada). 2) Chromosome library screening Two primers (upstream: 5' G G G C G A G G C T C T G T C T C A A A 3 ' ; downstream: 5 ' C A G C A C C C T T C T T C C G G C C C 3 ' ) were designed according to the published sequence of S M N gene (Gurglen, et al. 1996) and used to generate a fragment of 405 bp (from -267 to +138) from human genomic D N A by Polymerase Chain Reaction (PCR). This 32 fragment was subsequently used as the template to generate P labeled probes with a random primer labeling kit (Gibco B R L Burlington, Ont., Canada). E.col i (LE392) were infected with the phage Charon 21A library (10,000 pfu, American Type Culture Collection, A T C C , Rockvil le, M D , U S A ) containing the human chromosome 5 EcoR I fragments and were plated on 150 mm dishes for screening with the above labeled probes. A positive clone harboring the 5' upstream region of the S M N gene (4370 bp, from -3132 to +1238) was identified and purified more to obtain 100% positives by subsequent screening with the same probes. 20 3) Subcloning The clone obtained from the screening procedure was digested with the restriction endonuclease EcoRI. A 4.4kb S M N upstream fragment was subcloned into the plasmid vector pZErO - 1 . 1 (Invitrogen and Corporation, U S A ) named p S M N (Figure 4) for sequence characterization, specifically the region from -1210 to -565 (counting from the A T G translation start codon) using the primers S M N 1 and S M N 2 (Table 1). The primers were designed corresponding to different regions of the promoter in an anti-sense orientation (Table 1). The 4370 bp fragment was excised and subcloned from the p S M N using B a m H I and EcoR I restriction enzymes to generate two constructs, S M N a and S M N b (Figure 4). These constructs were eventually found to contain the S M N promoter regions from -3132 to -1580 and from -1580 to +1238, respectively, as these were used for further sequencing with the p Z E r O - l . l / M 1 3 universal reverse primer (Invitrogen Corporation,USA). Q I A G E N mini-preparation K i t (Mississauga, Ont. Canada) was used to purify the above plasmid D N A . 4) Sequencing The sequencing was performed with the Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham Life Science, Inc., Oakville, Ont. Canada) as per manufacturer's instructions. Briefly, a set of four termination reactions, each with a different specific labeled dideoxynucleotids (ddNTP) was produced by the thermal cycling program with perkin Elmer Thermal cycler 9600. The.sequencing reaction products were denatured at 95°C for 5 minutes and electrophoresed on a denaturing 6% acrylamide gel containing 8 M urea. The gels were subsequently transferred onto 21 Whatman blotting paper, dried for 30 min., and exposed to Scientific Imaging F i l m (Kodak, Rochester, New York U S A ) at room temperature for 12-24 hours. 5) Search for transcription factor binding sites Sequences of the 5'-flanking region obtained from the above procedure and from the Genbank were analyzed for the presence of the potential transcription factor binding sites using the Matlnspector Release 2.0 (http://www.gsf.de/cgi-bin/matsearch.pl) with a minimum core similarity of 0.80 and a minimum matrix similarity of 0.85. Other databases, such as T E S S , v.3.3 (http://www.cbil.upenn.edu/cgi-bin/tess/tess33) and T F S E R C H (http://www.pdapl.trc.rwcp.or.jp/research/db/TFSEARCH.html), were also searched for putative binding sites. Since the results varied between the different database searches, Matlnspector was used as the base of analysis for the reason that it has been cited by published literature (Berger et al., 1998). 6) Reporter gene constructs A plasmid named p P C D N A 3 (Figure 5) containing the 4370 bp S M N gene fragment (-3132 to +1238) was constructed using the pHSV3 plasmid (5.4kb) construct which was generated previously in the lab. The latter construct was derived from the p c D N A 3 plasmid (Invitrogen Corporation, U S A ) with the following modifications: 1) Insertion of HSV-1 packaging sequence ("a" sequence) and a HSV-1 replication origin (OriS); 2) Removal of cytomegalovirus ( C M V ) promoter in the p c D N A / H S V 3 . Taking the advantage of the Eco47 III restriction site at position +114 downstream from the translation start site and a Not I site in the poly linker of the p P C D N A 3 , 3' to the S M N 22 gene (Figure 5), the +114 to +1238 region of S M N gene was deleted from the p P C D N A 3 . The Luciferase reporter gene (2.8kb) isolated from the p G L 2 - Basic vector (Promega, Madison, WI , U S A ) was then inserted into the p P C D N A 3 downstream to +114. This construct (11.4kb) containing the - 3132 to +114bp fragment of S M N gene was named LSH3132. The first stage of the isolation and identification of the upstream of S M N gene was focused on the segment from - 3132 to -306. Three constructs containing fragments of - 410 to +114 (LSH1410), -899 to +114 (LSH899), or -306 to +114 (LSH306) (Figure 5) were made by sequential deletions in the upstream region using the restriction sites H i n d i , Fok l , and PstI, respectively (Figure 6A). To further analyze the sequence elements between the - 306 position and the translation start site, more deletion constructs were made. The - 306 to +114 fragment was transferred into the KpnI/XhoI sites of the luciferase vector pGL2-basic vector (Promega, Madison, WI , U S A ) (LS306). Four additional constructs were generated by sequential deletions in this region between the Xho I and BstXI , Smal, A M I , and A p a l sites, respectively (Figure 6B). The resulting constructs contain the 5' upstream region o f -157 to +114 (LS157), -67 to +114 (LS67), -46 to +114 (LS46) and -28 to +114 (LS28), respectively, upstream of luciferase reporter gene (Figure 5). A construct LS74 was created by inserting a 7 oligonucleotides to LS67 to recover the potential binding site of neural specific transcription factor A P 2 (activator protein-2) (Figure 5). 23 LacZ plasmid A similar construct H S V / L a c Z containing a LacZ reporter gene under the control of a human cytomegalovirus ( C M V ) immediate early gene promoter was utilized in the present study as an internal control to normalize the variations among transfections. 7) Cel l Culture and D N A transfection African green monkey kidney (Vero) cells were cultured at 37°C in a humidified atmosphere of 95% air/5% CO2 in Dulbecco's Min imal Essential Medium ( D M E M , Gibco, B R L , Burlington, Ont., Canada), containing 10% fetal bovine serum, 2 m M L -glutamine (Gibco B R L , Burlington, Ont., Canada), and 100 units /ml of penicillin/streptomycin (Gibco B R L , Burlington, Ont., Canada) (complete medium). Human S H - S Y 5 Y neuroblastoma cells were kindly provided by Dr. Seong K i m (Department of Medicine, U B C ) . The cell culture dishes (Corning, N Y , U S A ) were pre-incubated with ploy-L-lysine (O.lmg/ml, Sigma, Mississauga, Ont. Canada) for 30 min at room temperature followed by 2x15 min washing in distilled water and air dried. The S H - S Y 5 Y cells were then cultured in the poly-L-lysine coated dishes under the same condition as the Vero cells. The day before transfection, the cell cultures were split and grown in a penicillin/streptomycin-free D M E M medium in 60 mm culture dishes to reach 80%) confluent for Vero cells and 60% for the S Y 5 Y on the next day. Both cells were transfected with 3 ug D N A purified with Q I A G E N maxi-preparation kit (Mississauga, Ont. Canada), using Lipofec tAMINE P L U S T M Reagent (Gibco B R L , Burlington, Ont., Canada) in a serum- and antibiotics-free medium according to the manufacturer's instruction. Transfected cells were incubated at 37°C for 3 hours before the medium was 24 replaced by a fresh completed D M E M containing 1 0 % serum and 100 units/ml of penicillin/streptomycin. Two days after the transfection, the cells were harvested for reporter gene expression assays (see below). A n equal amount of the H S V / L a c Z plasmid D N A was co-transfected with the test promoter constructs in all experiments. 8. Cell Extract and Reporter Gene Assays The cells were washed twice with phosphate-buffered saline (PBS) and lysed in 4 0 0 LLI of I X Reporter Lysis buffer (Promega, Madison, WI, U S A ) . Five microliters cell extract was mixed with 100 pi luciferase assay reagent (Promega, Madison, WI, U S A ) and the luciferase activity was measured with a Lumat luminometer (Berthold, Bad Wildbad, Germany). (3- Galactosidase activity was measured using the LacZ Assay System (Promega, Madison, WI, U S A ) according to the manufacturer's instructions with a spectrophotometer (U -2000, Hitachi, Japan) at visible light absorbance of 4 2 0 nm. Standard curves for both the Luciferase and LacZ enzyme assays were established according to the manufactures' instructions to determine the linear range. A series of sample dilutions were performed to ensure that the results of measurements were in the linear range. 9. Immunocytochemistry For immunocytochemical studies, cells were fixed with 4 % paraformaldehyde for 10 min. at room temperature followed by a rinse in PBS, and then were incubated with an anti-SMN rabbit serum, a gift from Dr. J. Francis's Lab, at Harvard University, at 1:1000 25 dilution overnight at 4°C. Following 3 x 5 min wash, the cultures were subsequently incubated with a biotin-labeled anti-rabbit antibody (Vector, Burlingame, C A , U S A , 1:1000) for 1 hour at room temperature followed by 3 x 5min wash in P B S . The cultures were processed with the horseradish peroxidase-based avidin-biotin complex system (Vector, Burlingame, C A , U S A ) for 1 hour at room temperature, and the immunoreaction was detected with 0.01% 3',3'-diaminobenzidine and 0.01% H 2 0 2 with 3 minutes reaction. 10. Data analysis Purity of all plasmids used for transfection was ensured by gel electrophresis and spectrophotometry with an A260/280 ratio over 1.8. Levels of luciferase activity were normalized by measuring the expression levels of LacZ gene and the data were expressed as ratios o f luciferase activity to beta-galactosidase activity for each sample. Each construct was trasfected on four individual culture dishes, and average ratios of luciferase activity to LacZ activity were calculated based on a minimum of two independent experiments. Statistical analysis of all the data in the present study was performed using student t-test and A N O V A with Statview 4.0 (Abacus Concepts Inc., Berkeley, C A , U S A ) on a Macintosh computer. 26 RESULTS 1) Sequence of 5' upstream region of S M N gene. A 4370 bp D N A fragment containing the 5' flanking region and a part of coding region of S M N gene was isolated from chromosome 5 library and subcloned into pZErO plasmid vector (pSMN) by Dr. Shiv Prasad (Dept. of ophthalmology, U B C ) . The primers S M N 1 , S M N 2 , and a universal reverse primer (RP) for pZErO-1.1 vector (see Table 1, p 18) were used to determine a total of 1385 bp sequence (Appendix I) in the 5' upstream region of S M N gene (Figure 7). B L A S T (http://www.ncbi.nlm.nih.gov/BLAST/) similarity search in the Genbank showed that a fragment of 2550 nucleotides was lacking at the position of -1530 in the cloned 5'flanking region of S M N gene in the p S M N construct. In addition, there were 17 nucleotides mismatches in the sequence from -565 to -1950 as shown in Table 2. 2) Potential binding sites for transcription factors (Table 3) The search of the potential transcription binding sites for sequence from -1530 to A T G showed that proximal sequence of the 5' flanking region of the S M N did not contain T A T A box or G C boxes that are usually required for transcription initiation. However, a putative C A A T box (gataaCCACtcg) was found at -146, which may be an important element for the initiation of transcription. The 5'flanking region contained numerous putative response elements. Possible binding domains with complete sequence matches were activator protein-1 (AP-1) at -57 (agTGACgactt), at -990 (ggTGACagagc), at -1420 (gtTGACcaagt); activator protein-2 (AP-2) at -16 (caCCCGcgggtt), at -67 27 (cCCCGggc) ; activator protein-4 (AP-4) at -400 (ctCAGCtatt), at -1024 (ctCAGCtcac); C R E B a t -5 8 (agTGACgacttc), at (-690 tcTGACgacaga); basic helix-loop-helix (bHLH) transcription factors MyoD/E47 at -927 (caCACCtgta, -1225 caCACCtgta); and NF-1 at -1164 (cctTGGCttcatatagta). In addition, there were several putative binding sites for P O U domain factors, Oct-1 at -858 (cacaatATGCtccaa), at -910 (cattttgggATGCc), and B R N - 2 (Brain-2) at -206 (gaaatgaaAAATatac), at-1267 (tagatgctTAATaaag). The distribution of putative binding sites in the promoter of S M N gene appeared clustered within a number of sequenced regions (Fig.9), including from —40 to -80 , -140 to -150, -200 to -250, -450 to -510, -570 to -590, -910 to -930, -1230 to -1240, and -1386 to -1420. 3) Promoter activity of the 5' flanking region of S M N gene in transfection assays To characterize the promoter region and identify the important regulatory elements of S M N gene, a serial deletion strategy was applied by constructing recombinant plasmids containing different 5'-deletions of upstream region fused to luciferase reporter gene. Sequences derived from the 5'-flanking region were tested for their effects on promoter activity by transient expression in neuroblastoma ( S Y 5 Y ) cells and African green monkey kidney (Vero) cells. To normalize the differences in transfection efficiency, a H S V / L a c Z reporter plasmid was co-transfected, and the relative promoter activity was calibrated as luciferase activity per unit of lacZ activity. Analysis of the progressive expression indicated that the plasmid LSH3132, which contained the S M N promoter sequence from —3132 to +114 bp, had a strong luciferase 28 activity in both S Y 5 Y and Vero cells (2568±159.9 fold for S Y 5 Y and 126.7±25.3 fold for Vero, respectively, over background expression levels). Figures 10 and 11 summarize the effects of deletions of 5' flanking region from -3132 to -28 luciferase activity in S Y 5 Y cells. The data showed that there was no significant difference in the luciferase activity comparing the constructs containing 5' upstream regions of-3132 to +114 bp, to that o f -1410 to +114 bp (LSH1410, p=0.389), or to that o f -899 to +114 bp (LSH899, p=0.241). The luciferase activity was increased by 30% when a deletion was made from -3132 to -306 bp (LSH306, p=0.0181). The highest luciferase activity was observed in the construct LS157 with upstream region of -157 to +114 (38485±2022 fold over the background), which was slightly higher than that in the LS306 construct (46.8%, PO.008) . Further deletion from -157 to -67 bp (LS67) resulted in a 76-fold decrease of the luciferase activity (pO.OOOl). Another 40-fold decrease (pO.OOOl) was found when the deletion was extended from -67 to -46 bp, which meant a deletion of 111 base pairs from -157 to -46 caused a total of 2750-fold decrease in the promoter activity (pO.OOOl). The construct LS46 expressed luciferase activity that was 13-fold higher than the promoterless construct (p= 0.02). Further deletion from —46 to -28 bp (LS28) increased the levels of expression of luciferase reporter gene by 84% at the statistical significant level of p= 0.02. The pattern of the luciferase activity affected by serial deletions in Vero cells was similar to that in S Y 5 Y cells (Figure 12 and 13). However, the expression levels were different between S Y 5 Y and Vero cells with an elongation of the promoter region towards 5' upstream (Figure 14). The data showed that there was no significant difference in the 29 expression levels of luciferase reporter gene between the two types of cells transfected with the construct containing the 5' flanking region from -28 to + 114 bp. Interestingly, the significant difference in the promoter activity between the two cell types appeared when the 5'-flanking region further extended to more upstream regions. The luciferase activity of construct containing -157 to +114 was 5-fold higher in S Y 5 Y cells than that in Vero cells (pO.OOOl), and it was 23-fold higher when extended to - 1410 bp (pO.OOOl). A putative A P 2 binding site was located between -71 and -62 , which was partially deleted in construct LS67. To test the function of this neural specific transcription factor (Mitchell et al., 1991; Schorle et al., 1996) in the expression of luciferase gene in both S Y 5 Y and Vero cells, a construct (LS74) based on LS67 but with extra 7 bp ( A A G C C C C ) to recover the putative domain for A P - 2 site was generated and transfected into both cell lines. Comparing to LS67, in which the putative A P - 2 site was lost, this construct resulted in a 50% increase (p<0.000l) in luciferase activity for S Y 5 Y cells and a 60% increase (p=0.002) for Vero cells (Figure 11 and 13). 4) Immunocytochemistry To confirm that the S Y 5 Y cells and Vero cells contain adequate levels of trans-acting factors to support the activation of the chimeric S M N promoter-reporter gene constructs, we examined the endogenous S M N expression in those two cell lines. Immunocytochemistry labeling using a polyclonal antibody for S M N protein showed high levels of expression of S M N in S Y 5 Y cells (Figure 15). The S M N immunoreaction products appeared granulous and distributed in both cytoplasm and nuclei for most S Y 5 Y cells (Figure 15a). The Vero cells expressed moderate levels of endogenous S M N (Figure 30 16a and 16b), but the distribution of S M N protein varied from cell to cell. In most Vero cells, the S M N appeared in a few highly localized regions of cytoplasm and the nuclei were negative to the labeling (Figure 16a), while in cells that seemed undergoing mitosis, the S M N proteins were concentrated in the nuclei and the cytoplasm was not labeled (Figure 16b). 31 DISCUSSION Survival motor neuron ( S M N ) gene has two duplicated copies named S M N t and S M N c , which can be distinguished only by the differences of 5 nucleotides (Chen et al, 1998; Lefebvre et al, 1995). S M N t is SMA-determining gene (Cobben et al, 1995; DiDonato et al, 1997; Hahnen et al, 1995; Lefebvre et al, 1995; Rajcan et al, 1996; Rodrigues et al, 1995; van der Steeg et al, 1996; Velasco et al, 1996) and S M N c may be related to the S M A phenotype (Campbell et al, 1997; DiDonato et al, 1997; McAndrew et al, 1997). The 5' flanking regions of both S M N t and S M N c gene are almost identical except 13 nucleotides differences (personal communication, A . Burghes). To identify the functional domains in the regulatory region of the S M N gene, a 4370 bp EcoRI D N A fragment containing the 5' flanking region of S M N gene was isolated from human chromosome 5 library and 3132 bp of upstream region was subcloned into the p H S V 3 vector upstream of the luciferase reporter gene. Due to the extreme homology between the S M N t and S M N c genes in the sequences of their promoter region and due to the approach by which the 4370 bp fragment was cloned, it was not possible to infer whether the 3132 upstream region in the pHSV3 was derived from the S M N t gene or the S M N c gene. A total of 1385 bp sequence (Figure 7) of the 3132 bp fragment was determined in the present study and compared with the published 5' flanking region sequences of S M N t and S M N c in the Genbank. The sequence showed better identity to the promoter sequence of S M N c than that o f S M N t gene (Table 2). Furthermore, based on the same sequence in the Genbank, a fragment of 2550 nucleotides at -1530 bp from the translation initiation site 32 was found to be absent in the cloned E c o R l / E c o R l D N A fragment isolated from the Chromosome 5 library (Figure 8). Absence of the 2550 bp sequence in this construct was confirmed by digestions o f two restriction enzymes that are unique for that deleted region. Although the possibility that the upstream region of S M N gene cloned from the chromosome 5 library indeed did not contain the 2550 bp fragment could not be excluded, it is still likely that this fragment of D N A was deleted by E.col i during the cloning procedure as similar deletions were not uncommon in this type of procedure. P C R screening of the human genomic D N A from different sources based on the sequences around the missing region w i l l be helpful in confirming the above explanation. The mismatches of 17 base pairs in the rest of 5'- flanking region were likely due to polymorphism. In order to determine the basic promoter region and putative regulatory elements, serial deletion constructs containing various lengths of 5'-fianking region of the S M N gene were constructed and transfected into S Y 5 Y cells. The deletions from -3132 to -1410, and to -899, had little effect on luciferase activity in S Y 5 Y cells, suggesting that this region did not have either positive or negative regulatory function on the expression of S M N gene. However, because of the missing 2550 bp sequence at the -1530 bp, the promoter activity tested with the construct LHS3132 (-3132 to +114) might not truly reflect the entire upstream regulatory region. It remains to be studied whether the region from -1530 to -4080 in the S M N promoter contains any regulatory elements. The promoter activity of further deletion from -899 to - 306 bp resulted in a significant 33 increase in promoter activity, suggesting a possibility of the presence of the negative regulator elements in this region. A putative binding domain for transcription repressor Delta E F I was found at -351. Delta E F I (Sekido et al, 1997; Sekido et al, 1996; Takagi et al, 1998) has been found to counteract basic helix-loop-helix (bHLH) activators through binding site competition and fulfill the conditions of the E2 box repressor (Sekido et al, 1994). Furthermore, sequence domain for M Z F 1 , a transcription factor which may repress transcription in non-hematopoietic cells (Hui et al, 1995; Morris et al, 1994; Morris et al, 1995; Thiele et al, 1998) was found at four position between -899 to -306. The construct L S I 5 7 with a further deletion from -306 to -157 displayed the highest promoter activity. This region thus might be important for the induction of a high transcription rate in response to the binding by various transcription factors. A further deletion of 90 bp from -157 to - 67bp, significantly reduced the promoter activity by 76-fold, indicating that a positive regulator element may be present in this region. Coincidentally, putative binding sites of various transcription factors such as G A T A 1 , L M 0 2 C O M , IK-2 , C E B P B , G A T A c , TH1E47, N Y F , C A A T , L Y F 1 , A P 2 , B A R B I E box were clustered in this region (Figure 9 and Table 3). Another 21 bp deletion (from -67 to -46bp) further reduced the promoter activity by 40-fold, suggesting that this region might also positively regulate the S M N gene transcription. A number of potential transcription binding sites, such as A P - 1 , C R E B , A T F , N R F 2 , CETSIP54, T C F 1 1 , are clustered in this region of 21 bp, suggesting the importance of this D N A fragment in the transcriptional activity of the S M N gene. 34 Transcription of a given gene depends upon the integrity of the upstream regulatory region and the initiator element near the proximal region including some part of the 5' untranslated region. The upstream transcription factors which can moderate the cell specific expression have been shown to interact with the TATA-bind ing protein TFIID (Sawadogo & Roeder, 1985a; Sawadogo & Roeder, 1985b) and with the initiator binding factor TFII-I (Roy et al., 1991). Sequence analysis showed that there was no conventional T A T A box element near the A T G start codon, but its 5' untranslated sequence contained a pyrimidine-rich region (-28 to -10), which might be the initiator element for the transcription of the S M N gene (Roy et al, 1991). The fact that the -28 to +114 5' flanking region did have a basic promoter activity seemed to support the view that an initiator element is present in the untranslated region of S M N gene, and this element is required for transcription initiation. It is worthwhile pointing out that the m R N A of the S M N gene starts at least 33 bp upstream (-33) to the A T G translation start codon (Burglen et al, 1996; Chen et al, 1998). Interestingly, the -28 to +114 region alone appeared to have some promoter activity. Thus, the basic promoter activity seemed to be present in the region after the messenger R N A transcription initiation site, suggesting that a 5' untranslated region of S M N gene might be important for promoter activity, and might play a role in the initiation of transcription of the S M N gene. To further confirm it, comparison of the promoter activity of the -306 to +114 region with and without the -28 to + 114 region should be helpful. On the other hand, it is also necessary to exclude the possibility that the 34 bp of introns in the -28/+114 fragment may play a role for the promoter activity. 35 It has been well known that both S M N t and S M N c gene copies are expressed widely in human tissues, and the expression level is highest in spinal cord and liver (Battaglia et al, 1997; Lefebvre et al, 1997). To investigate the expression of the S M N gene in both neuronal and non-neuronal cells, the African green monkey kidney cells (Vero) were also used for reporter gene expression assay with serial deletion constructs of S M N 5' flanking region. The results showed that the luciferase reporter gene was also expressed at high levels in Vero cells, and the pattern of the promoter activities resulted from the serial deletions was similar to that in S Y 5 Y cells. These results suggested that there might be no major difference in the regulatory mechanism for the promoter activity of S M N gene in neuronal and non-neuronal cells in the present experiment settings. However, despite the similarity in the regulatory pattern of the sequence elements, the promoter activity of S M N was not necessarily the same between neuronal cells and non-neuronal cells. Expression levels of luciferase were almost the same in S Y 5 Y and Vero cells for the constructs of LS28. But it was 5-fold higher in S Y 5 Y cells than that in Vero cells when the 5' flanking region extended to -157 bp. Further extension to -1410 showed greater difference between neuronal cells and non-neuronal cells. These results implied that the basic promoter regulatory mechanism for transcription initiation might be same for neuronal and non-neuronal cells. However, some additional neural specific enhancing factor binding sites might be present in the more upstream region, resulting in higher levels of promoter activity in neuronal cells. Sequence analysis for potential transcription factor binding sites revealed that some domains for tissue-specific transcription factors in -1410 to -28 region. A P O U domain transcription factor Brain-2 (BRN-2) at -206 and -1267 (Dawson et al, 1996; Fujii & Hamada, 1993; Hagino et al, 1997; Hagino et al, 36 1998; Josephson et al, 1998; Shimazaki et al, 1999), which is mainly expressed in the output neurons may play a role in the development of these output neurons (Hagino et al., 1999); The M y o D binding domain at -927 and -1225, is a muscle-specific regulatoy factor (Dias et al., 1994). The transcription factor A P - 2 has been shown to play an important role in the expression of neuronal genes (Mitchell et al., 1991; Schorle et al., 1996). A potential A P - 2 binding site was located between -71 to -62 . To investigate the possible role of this domain in the S M N gene, promoter activity between the LS74 and L S 67 was compared, since only the former contained the promoter region that was 7 bp longer than the latter to retain the A P - 2 site. Although the luciferase activity of LS74 was slightly higher than that of LS67 by 50-60% in both S Y 5 Y and Vero cells, the sequence domain might not have the neural cell-specific function in S M N gene since the effects were similar in both neuronal and non-neuronal cells. 95% of S M A patients who lack their S M N t copy therefore rely on the S M N c (Cobben et al, 1995; DiDonato et al, 1997; Hahnen et al, 1995; Lefebvre et al, 1995; Rajcan et al, 1996; Rodrigues et al, 1995; van der Steeg et al, 1996; Velasco et al, 1996). Increased S M N c copy number as a result of gene conversion from S M N t to S M N c are related to the milder phenotype (Campbell et al, 1997; DiDonato et al, 1997; McAndrew et al, 1997). It has been reported that the converted S M N c at the original S M N t locus is different from the original S M N c copy, and the former can produce more functional S M N products, leading to the milder phenotypes (type II/III) (Coovert et al, 1997). This fact implies possible difference in promoter activity between the S M N t and S M N c gene copies. However, no significant difference between the S M N t and S M N c copies has been found 37 in D N A sequence at the 5' flanking upstream regions as far as 6000 bp upstream. Expression of the reporter gene driven by the promoter from S M N t or S M N c also showed no difference in COS-7 cell line (personal communication, A . Burghes). Thus, difference in the expression levels of S M N from the two copies in S M A patients may not be simply due to the sequence difference in their promoter regions. However, the role of promoter activity in the above pathological phenomena of S M A still can not be ruled out. This is based on some of the basic findings from the present study: 1) A small change in length of the sequence of the proximal 5' flanking region of S M N gene could result in a dramatic difference in the promoter activity; and 2) promoter activity of S M N gene varied in different cell types. The first finding suggested that the proximal region was critical for the expression of the S M N gene and is therefore potentially vulnerable for any kinds of alteration, such as polymorphism, which result in a few base pair changes randomly along the genome, including the promoter regions. Polymorphism affecting the promoter activity of S M N gene has been noticed previously (Parsons et al, 1998). While polymorphism that reduces the promoter activity may not cause any problem in normal individuals due to multiple copies of S M N gene and high levels of expression from S M N t . It may be critical for patients who lack one S M N t copy, since the functional full length m R N A only composes 30% of total transcripts generated from the S M N c copy (Gennarelli et al., 1995; Lefebvre et al, 1995; Parsons et al, 1996). Given the fact that the S M N c copy might be evolutionarily new comparing to S M N t locus, higher frequency o f polymorphism may occur in this region, which means that the promoter activity of S M N c may be more variable than that of S M N t among individuals. To verify whether this is true, sequence analysis among both normal population and patients should be 38 conducted and the effect of sequence alteration due to polymorphism should be studied. The second finding suggested that the promoter activity was higher in neuronal cells than Vero cells. This was likely due to binding of certain transcription factors that only exist or highly expressed in neuronal cells. If the above polymorphism alters the binding affinity of the D N A sequence to these potential neuron specific transcription factors, lower expression o f S M N gene may be specifically apparent in neuronal cells. O n the other hand, lacking the neuronal specific tanscription factor in motor neurons secondary to the lack of S M N t gene in S M A patients can also reduce the promoter activity in neurons. To verify this hypothesis, it is necessary to identify the transcription factors that regulate the S M N promoter activity and their binding domains in the upstream regulatory region in both neuronal cells and non-neuronal cells. 39 SUMMARY In conclusion, the 5'-upstream regulatory region of the S M N c gene from human chromosome 5q library was functionally characterized. This study examined the functional domains of a 3246bp genomic D N A fragment, which contains 3132 5' flanking region, first exon and 34bp first intron of S M N gene. To identify the gene promoter sequence and the expression level in neural and non-neural cells, transfection of the S Y 5 Y cells and Vero cells were performed with a series of 5' deletion reporter gene constructs, that extended upstream to - 3132bp relative to +1(ATG). The luciferase activity analysis demonstrated that the -157/+114 construct had the strongest promoter activity in both cell lines, the -67/+114 and -74/+114 constructs showed a moderate activity; whereas -28/+114 and -46/+114 constructs showed a basic promoter activity. The upstream 5' flanking region (-899/-306) displayed a negative regulatory effect on promoter activity. Within the proximal region of S M N gene promoter, two blocks of the sequence, 83bp from -157 to -74 and 21bp from -67 to -46 acted as positive regulators for promoter activity. The region of -28 to +114 possessed minimal essential promoter activity. Given the fact that the transcription initiation site lies at least -34 bp upstream of the A T G , the basic promoter activity therefore was possibly located in the 5' untranslated region. A l l these data suggested that the transcription of S M N gene is tightly regulated in either neural cells or non-neural cells. Although the 5' untranslated region constituted the basic promoter activity, S M N gene transcription may be greatly influenced by regulatory factors such as C A A T , A P I , A P 2 , C R E B . A n y mutations in the -157/+114 region may dramatically affect the transcription of S M N gene. Comparison of the promoter activity of S M N between neural and non-neural cell lines revealed the higher expression of 40 luciferase reporter gene in neural cells when the promoter region extended to -74 suggesting the presence of possible domains for the neuronal specific enhancers in 3132 region. 41 S I G N I F I C A N C E Promoter function of S M N gene has been recognized as an important aspect for understanding the pathology of the disease beside the function of its proteins. Given the fact that little is known about how the genes is controlled by its promoter and cis-acting elements in the upstream region, our results may provide the information that is potentially important regarding the pathogenesis of S M A disease. Furthermore, understanding the regulatory mechanisms of the S M N gene expression may lead to developing new strategies for the treatment of S M A , such as through up-regulating the S M N promoter activity to increase the expression levels of the gene in patients who are otherwise lacking the efficient transcription of S M N gene. 42 F U T U R E I N V E S T I G A T I O N S Based on the preliminary results, the future studies should be focused on following aims: 1) To verify the missing region of -4080 to -1530 In order to exclude the possibility that the missing region in cloned construct was not an artifact, P C R and Southern blotting wi l l be utilized to screen human genomic D N A from deferent sources. 2) To investigate the function of the missing fragment (-4080 to -1530) The above fragment w i l l be inserted back to the promoter-reporter gene construct and the promoter activity w i l l be compared to the one with the deletion in both neuronal and non-neuronal cells. 3) To identify the essential promoter region The preliminary results of primer extension on the S M N gene showed that the transcription initiate site may located in the region more than hundred bases upstream to the translation start site. It is therefore necessary to verify that the region between -157 to +114, which displayed the highest expression levels of reporter gene is essential for the promoter activity. 4) To confirm the subcellular distribution of S M N protein in human non-neuronal cells The immunocytochemistry results for S M N . protein showed difference in subcellular distribution of the S M N protein between the Vero cells and S Y 5 Y neuroblastoma cells. To further confirm this observation, human non-neuronal cells such as glioma cells and fibroblastoma cells w i l l be used. 43 Fig 1. Schematic illustration of 5ql3 region The two reverse duplicates of 500kb elements in 5ql3 region are shown. C e n t r o m e r i c T e l o m e r i c p44c N A I P S M N C C 2 7 2 C272 S M N t N A I P ( 5 ) p44t 44 Fig.2, Structure of S M N gene The S M N gene contains 9 exons (black boxes) and 8 introns. 5' i 2a 2b 3 4 5 6 7 8 3' 1 1 1 1 1 1 1 1 I 45 Fig.3, Different scenarios of S M N gene arrangement in normal individuals and S M A patients. Note that the 3'-end of the S M N t has been replaced by the 3' region of the S M N c gene in the case of convertion. S M N c ' , converted S M N c . SMNC C272 C272 SMNt N A I P 5 Normal 5' 5' 3' C272 SMNt N A I P 5 5' 3' Deletion SMNc C272 V/-3' 5' C272 SMNc' N A I P 5 Convertion 5' 3' SMNc C272 C272 SMNc' N A I P 5 3' 5' 5' 3' 46 Fig . 4, Diagram of subcloning procedure for sequencing. Plasmid p S M N contained the EcoR 1(E) /EcoR 1(E) fragment o f the S M N gene from -3132 to +1238. Nucleotides -1210 to -990 and -990 to -565 were sequenced in the p S M N using primers S M N 1 and S M N 2 , respectively. The E c o R l fragment was digested with B a m H l ( B ) and subcloned into two plasmids (pSMNa and p S M N b ) for sequencing nucleotides from -1950 to -1580 in p S M N a and from -1580 to -1210 in p S M N b using the universal reverse primer. The arrows point to sequencing orientations. 47 Fig.5 Cloning procedure of reporter gene constructs. Cloned fragment (-3132 to +1238) was subcloned to plasmid p P C D N A 3 and the region from +115 to +1238 was replaced with a luciferase gene subsequently to generate LSH3132 followed by a series of deletions. Note that the L S H series and L S series are in different plasmid backbones (see Fig.6). 48 Fig.6 The two basic reporter gene constructs. A , the construct containing the 5' flanking region of -3132 to +114; B , the construct containing the region of -306 to +114. B y digestion with the restriction enzymes as marked, the constructs LSH1410, LSH899 and LSH306 were deducted from LSH3132, and the constructs L S I 5 7 , LS67, LS46, and LS28 were deducted from LS306 (see Fig.5). The L S H series are based on plasmid p C D N A 3 with a H S V - 1 packaging sequence and OriS; the L S series are based on plasmid p G L - 2 basic. 49 Fig.7, Diagram of 5' upstream region of S M N gene. Fragments that were investigated in the present study are showed. The fragments of -3132 to +1238 was.cloned, -3132 to +114 was tested for promoter activity and -1950 to -565 was sequenced. -3132 5' 5' upstream region of SMN gene -1950 -1580 i I -990 I -565 RP SMN2 1385 bp sequenced ATG+114 +1238 '3' SMN1 Tested region Cloned region 50 Fig.8, A comparison between the sequences generated from the present study and in the Genbank. SEQUENCE IN DATABASE 5' -4500 -4080 -1530 _565 3' SEQUENCE OF pSMN 5 -1950 -1531 -1530 -565 3' 51 Fig . 9, Distribution of potential binding sites for transcription factors in the 5'-upstream region of S M N gene. Distribution of putative binding sites for transcription factors in the 5'flanking region of SMN gene 27,43,26,7,8, 1.5.9.14,15,16. 28,29,30 33,34,35 l^ifl, A n X 3 8 ' 9 6.7.8 "U -.5.-4.1516 „ 21 V * * 4 * ? * T W ^ \ V L / 2,3,45 \ A 19.10.11 1213 I 9 717 i 19 910 t V » V ' 3 1 25 I 9,10 \ 4,5,10% I 32,34,36 3 7 2 1 1,9,10 '134.5,42, „ \ M 1 2 5 / 11 j ,111111 l l l l l Ml III I I II j l 1  1111, HI I I 1 , PHI l - ^ l l l II -1500 -1400 -1300 -1200 -1100 -1000 -900 -800 -700 -600 -500 -400 -300 -200 -100 0 Note: The numbers represent different transcription factors, see Table 3. 52 Fig . 10 Structure and activity of S M N promoter (-3132/ -306) analyzed in S Y 5 Y neuroblastoma cells. Promoter activity is expressed in ratios of a test construct over the background level after being normalized with levels of beta-galactosidase (see text p.28-30). Note that expression of reporter LSH306 is significantly higher than LSH3132. -1,410 -3,132 0 1000 2000 3000 4000 Promoter activity 53 F i g . l 1, Structure and activity of S M N promoter (-306/-28) analyzed in S Y 5 Y neuroblastoma cells. The promoter activity is expressed in logarithm of the ratios of a test construct over the background level after being normalized with levels of beta-galactosidase (see text p.28-30). -306 I ' 1 ' 1 > 1 • 1 1 1 0 1 2 3 4 5 Promoter activity (Log) 5 4 Fig. 12, Structure and activity of S M N promoter (-3132/ -306) analyzed in Vero cells. Promoter activity is expressed in ratios of a test construct over the background level after being normalized with levels of beta-galactosidase (see text p29-30). Note that expression of reporter LSH306 is significantly higher than LSH3132. -1,410 -3,132 I L S H 3 0 6 0 100 2 0 0 3 0 0 Promoter activity 55 Fig . 13 Structure and activity of S M N promoter (-306/-28) analyzed in Vero cells. The promoter activity is expressed in logarithm of the ratios of a test construct over the background level after being normalized with levels of beta-galactosidase (see text p.29-30). 56 Fig . 14 Difference in expression levels of luciferase driven by S M N promoters containing various upstream regions in neuroblastoma cell line S Y 5 Y and kidney cell line Vero. The data are expressed as ratios o f average promoter activity in S Y 5 Y to that in Vero cells (the activity in Vero cells is set as 1 for any given construct). It is evident that the ratio increases with the extension of promoter sequences towards the 5' upstream regions, suggesting the promoter becomes more neuron specific. 25 0 4 - J I • SY5Y CELLS W VERO CELLS -1410 -157 -67 -28 57 Fig. 15 Expression of SMN in SY5Y cells. a) Specific staining of the antibody for S M N is present in both the cytoplasm and the nuclei; b) The control sample shows negative immunoreaction when the primary antibody was omitted in the procedure. 58 Fig. 16 Expression of SMN protein in Vero cells. a) Immunoreactive products for the antibody against SMN were concentrated in certain regions of cytoplasm in most of cells while the nuclei were not stained; b) SMN immunoreactivity was concentrated in the nuclear region of the cells that were under mitosis, c) Negative control sample as 15b). a) b) c) 59 T A B L E 1, Oligonucleotides primers used to sequence the 5'-flanking region of S M N c gene. O L I G O N U C L E O T I D E POSITION S E Q U E N C E S M N 1 -565 5' A C A A A C A A G G A A G A C A A A C S M N 2 -990 5' - C T G G A A T G C A G T G G C G T G A T R P (Reverse pZEro-1.1 primer) 60 TABLE 2. Mismatched nucleotides. Numbers in the bracket are sequence positions of S M N c gene in the Genbank. The discrepancy in positions was due to the lacking region in the cloned fragment in p S M N (see Fig.8). Pos i t ion Sequence of S M N c in G e n b a n k p S M N sequence -1091 G T -1099 / G -1131 / C -1157 C G -1158 C G -1196 A -1358 A -1535 T (-4085) C -1545 C (-4095) T -1552 A (-4102) G -1553 / (-4103) G -1647 T(-4197) A -1652 T (-4202) -1695 T (-4245) C -1850 C (-4400) G -1915 T(-4415) C -1932 A (-4432) 61 TABLE 3, Potential binding sites for transcription factors. Transcription factors -157 +34 - 1530 -157 1 DELTAEF1 -1506,-1226,-938,-928,-351 2 XFD2 -1492 3 HFH1 -1491 4 GATA1 -151,-150 -1486,-1387,-1386,-887,-635,-496 5 LM02COM -148 -1484,-1384,-1226,-928,-885,-633,-494 6 NKX25 -1421 7 AP1FJ -57 -1420,-1181,-990 8 API -57 -1420,-990 9 IK2 -83,-106,-46 -1409,-1346,-1218,1084,-1043,-920, -908,-768,-736,-504,-348,-230, 10 MZF1 -1397,-1346,-1070,-643,-510,-504,-361 11 STAT -1346 12 CEBPB -121 -1281 13 BRN2 -1267,-206 14 E47 -1228,-930 15 MYOD -1225,-927 16 GFI1 -1224,-1090,-926 17 RORA1 -1184 18 NF1 > -1164 19 S8 -1120 20 PADS -1101,-479 21 AP4 -1024,-400 22 TCF11MAFG -994 23 OCT1 -910,-858 24 LYF1 -77 -907,-767 25 AH H A R M -29 -798 26 ATF -59 -691 27 CREB -58,-57, -690 28 HFH8 -663,-250,-246 29 HNF3B -663,-250,-248,-223 30 HFH3 -663,-591,-577,-250,-246 31 HFH2 -662,-249,-245 32 NFAT -583,-518,-229,-224 33 TATA -574 34 FREAC7 -572,-501 35 CDPCR3HD -562 36. 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