Simple repetitive DNA sequences, unusual DNA structures and transcriptional control

Abstract

With the progressive understanding of genome structure, organization, and sequence information, it has become evident that repetitive DNA sequences form a significant and substantial fraction of eukaryotic genomes. These sequences occur, although less predominantly, in the genomes of lower organisms. For a long time, repetitive sequences were considered to be superfluous and non-functional for normal cellular function and were termed as “Junk DNA.” In the last few years, our concept of these repetitive sequences has changed dramatically. Systematic genome sequence analysis has revealed that many such simple repetitive sequences are concentrated at biologically significant positions like telomeres and centromeres. Structural studies have established that many such repeat sequences can adopt non-B-DNA structures, leading to speculations on the possible role of such sequence motifs in modulation of DNA-protein interactions and thereby gene expression. Experimental evidence also suggests that many of these sequences show length polymorphism at certain chromosomal loci. This observation raises an important question: whether such sequences, when present within or around genes, influence gene expression as a function of the repeat length. Even though structural studies have established the sequence requirements for various unusual DNA structures, the functional significance of such sequence motifs still remains unclear. The identification of many proteins which specifically bind to various non-B-DNA structures as well as repeat motifs and the discovery of the existence of such structures in vivo has further invigorated the claim for a biological role of repetitive DNA sequences. Experimental evidence has shown that mutations or deletions of these apparently non-functional sequences lead to the loss of many vital regulation processes. These observations suggest that many control elements might be hidden within these monotonously repeated sequences. The nature of these control elements and the mechanism by which they establish the regulation process is still a subject of extensive study. Therefore, it is quite logical and interesting to speculate that repetitive DNA sequences, which exhibit such structural variability, can influence gene expression by modulating DNA-protein interactions. The length of the repeat unit can also differentially modulate gene expression, as it is speculated that these sequence motifs can adopt different structures depending on repeat length. To delineate the functional significance of the presence of such repetitive DNA sequences, we have developed a “structural cassette” approach wherein designed sequences with a potential to adopt various non-B-DNA structures can be introduced within and around genes, and the extent to which such sequence motifs influence gene expression in vivo can be studied. In this strategy, degeneracy of the genetic code has been used to design different structural motifs. Chapter 1 provides a brief account of the major advances made in our understanding of the distribution of simple repetitive DNA sequences in the genome, the unusual DNA structures some of the repeat units adopt, and the possible biological roles of such repeat sequences in the regulation process. The biological significance of various simple repetitive DNA sequences, the unusual structures they adopt, transcription, and DNA supercoiling have been discussed. An account of proteins which bind various repeat motifs and unusual DNA structures has also been given. The techniques which are commonly used to study various non-B-DNA structures have also been discussed. Chapter 2 describes the “Structural Cassette” approach which has been used to design intramolecular triplex potential polypurine/poly pyrimidine sequences within genes to study their effect on gene expression. Polypurine/polypyrimidine sequences have been shown to adopt intramolecular triplex structure under physiological superhelical density and/or low pH. We have introduced polypurine/polypyrimidine sequences (pSBT1 and pSBmT11) with or without mirror symmetry respectively in the N-terminal region of the ?-galactosidase gene in pBluescriptIISK+ to understand the role of such sequences adopting triplex structure in modulation of transcription elongation in vivo. We find that the designed sequences with and without mirror symmetry indeed adopt intramolecular triplex structure under physiological superhelical density. 2D chloroquine gel electrophoresis, SI nuclease, DEPC probing, and antibody retardation assay have been used to probe the intramolecular triplex structure in vitro. SI nuclease and DEPC probing experiments clearly indicate the formation of H-y5 isomer in the case of pSBT1. E. coli cells containing pSBT1 and pSBmT11 plasmids showed 5-fold inhibition of ?-galactosidase expression compared to the transformants harboring the parent plasmid pBluescriptIISK+. Another construct pSBmT12 was made using less preferred codons and containing identical amino acid sequences but lacking contiguous polypurine/polypyrimidine sequence motifs. Cells harboring this plasmid showed the same level of ?-galactosidase activity as those harboring the control plasmid pBluescriptIISK+. Northern blot analysis of the total RNA showed that the triplex structure could partially block transcription elongation in vivo. These set of experiments suggest that the presence of polypurine/polypyrimidine sequences with or without mirror symmetry could adopt intramolecular triplex structure when present within a gene and can down-regulate gene expression in vivo. A schematic model has been suggested to show how a stretch of polypurine/polypyrimidine repeat can equilibrate between B-DNA and triplex DNA and downregulate gene expression by partially blocking the movement of RNA polymerase over the template DNA. Chapter 3 deals first with the construction of a suitable eukaryotic model system to extend the observation of chapter 2 as well as to study the effect of upstream polypurine/polypyrimidine sequences on the regulation of gene expression. The construction of plasmids pSC110, pSCDT72, and pSCUT61 carrying various structural cassettes has been described. Designed polypurine/polypyrimidine sequences were cloned both upstream and downstream of the promoter of the ?-galactosidase gene. As the ?-galactosidase gene is under the control of both prokaryotic (gpt) and eukaryotic (SV40) promoters, the effect of such structural motifs on gene expression was studied both in prokaryotes and eukaryotes. The downstream polypurine/polypyrimidine sequences were found to downregulate gene expression as observed earlier; however, the extent of downregulation in eukaryotes was found to be lesser than that in prokaryotes. The polypurine/polypyrimidine sequences, when present -300 bp upstream of the SV40 promoter, were found to downregulate gene expression drastically. Chapter 4 deals with the design of inverted repeat sequences with a potential to adopt a cruciform within the ?-galactosidase gene. The effect of such inverted repeat sequences on transcription elongation in vivo has been studied. Taking advantage of the degeneracy of the genetic code and employing the structural cassette approach as described earlier, we have designed two inverted repeat sequences, duplex Cl and duplex C2, having different stem lengths. Another sequence, duplex mCl, was designed as a control where the inverted repeat symmetry was abolished by shuffling codons. The plasmids pSBCl, pSBC2, and pSBmCl, carrying the designed duplex sequences, have identical amino acid sequences in their ?-galactosidase gene. Chemical probing experiments, 2D gel electrophoresis, and SI nuclease mapping show that the designed inverted repeat sequences indeed adopt cruciform both in vitro and in vivo under physiological superhelical density. The extruded cruciform acts in cis and influences gene expression in vivo by partially blocking the movement of RNA polymerase over the template DNA and thus downregulating gene expression. The biological significance of inverted repeat sequences present in the vicinity of many genes has been discussed. Chapter 5 describes the strategy to introduce a Z potential dinucleotide repeat sequence motif within the ?-galactosidase gene. The extent to which such sequences influence gene expression has been studied. Various in vitro studies as discussed earlier showed that the designed alternating purine/pyrimidine sequence in plasmid pSBZ1 indeed adopts left-handed Z-DNA structure under physiological superhelical density. The in vivo ?-galactosidase activity of the cells harboring the plasmid pSBZ1 did not show any change of ?-galactosidase activity compared to the control plasmid. This indicated that although the designed sequence adopted left-handed structure under physiological superhelical density, the movement of RNA polymerase was not inhibited by such sequences in vivo. This observation also rules out the possibility of stabilization of Z-DNA structure in vivo by Z-DNA binding proteins in E. coli. Chapter 6 deals with the construction of a model system for triplet repeat-related genetic disorders. The triplet repeat sequences have attracted much attention from the scientific community because of their involvement with many human genetic diseases. Recently, trinucleotide repeat expansion mutations have been identified in disorders such as Fragile-X, Myotonic Dystrophy, Huntington’s disease, etc. In these cases, the expansion of certain triplet repeat sequences within and around genes by an unknown mechanism leads to the disruption of gene function and, eventually, the onset of the disease process. To understand the mechanism of triplet repeat expansion and the effect of expansions on gene expression, we have created an E. coli model system where a stepwise increase of CTG and CAG repeat length is made in frame with the ?-galactosidase gene in pBluescriptIISK+ and pBluescriptIIKS+ respectively. We find that in vivo expression of ?-galactosidase decreases with the increase in the repeat length due to the reduced level of transcript formation. We also find that the DNA fragments containing the triplet repeat show anomalous mobility on polyacrylamide gel with increasing repeat length, indicating the formation of a compact structure. On the basis of the above observation, we propose that the expanded triplet repeat can inhibit transcription elongation by RNA polymerase and these truncated transcripts might eventually be degraded as they may not be polyadenylated. This may lead to the onset of the disease process. We also propose the probable structure which the elongated triplet repeats can adopt. A certain length of the repeat unit can form a slipped structure, which can then fold back into a compact structure. Various structural and genetic studies on these clones are likely to provide a molecular understanding of the disease process. The appendix of the thesis presents an account of the dynamic mutations that we observed when triplet repeat-containing clones were grown in E. coli cells. We found that in many cases, the length of the CTG triplet repeat decreases, and this reduction in length always takes place in frame with lacZ, while the flanking sequences remain unaltered. The clone containing (CTG)60 was transformed in E. coli JM109 and DH10B cells and plated LB agar plates containing X-gal and IPTG. All the colonies showed the appearance of blue color. When a number of such colonies were randomly picked up, grown in 10 ml of LB, and sequenced, a range of triplet repeat-containing clones were observed. The length of the triplet repeat in most cases was found in the range of 13-35. Some of the clones also showed the presence of a mixture of CTG repeat lengths, as confirmed by denaturing polyacrylamide gel electrophoresis. The clones with reduced length of triplet repeat also showed higher levels of ?-galactosidase activity. In summary, our experimental evidence suggests that it is not only the two-dimensional disposition of nucleotide sequences but also the three-dimensional nature of the DNA double helix that is important for specific DNA-protein interaction and quantitative gene expression. Regulation of gene expression is a well-orchestrated and perfectly timed process. This finely tuned transmission of messages is primarily established by highly specific DNA-protein interactions. Various regulatory proteins bind specifically to their cognate DNA sequences and establish tissue- and cell-specific gene expression. A crucial and fundamental question arises: whether double-helical DNA merely provides nucleotide sequences to the cognate proteins, thus acting as a passive participant in its own utilization, or if its three-dimensional structure is also important for modulating DNA-protein interactions and thus gene expression. In conclusion, we can say that simple repetitive sequences, which are present in the close proximity of various genes, indeed can modulate gene expression by modulating DNA-protein interactions, by virtue of their structural variability, and the level of gene expression is also dependent on the length of the repeat unit. • A novel approach to design cis-acting DNA structural elements for regulation of gene expression in vivo. Partha S. Sarkar, R. Bagga, P. Balagurumoorthy & Samir K. Brahmachari, Current Science (1991), 61, 586-591. • In vivo regulation of transcription elongation by a designed potential cruciform structure. R. Bagga, Partha S. Sarkar, and Samir K. Brahmachari, 15th International Congress of Biochemistry, Israel, 1991. • Synthetic gene design to investigate the role of cis-acting DNA structural elements in regulation of gene expression in vivo. Samir K. Brahmachari, Partha S. Sarkar, P. Balagurumoorthy, P.K. Burma, and R. Bagga. Proceedings of the Symposium on “Synthetic Oligonucleotide: Frontiers and Applications.” Nucleic Acids Research (1991), 24, 163-166. • A novel approach to design cis-acting DNA structural elements to study regulation of gene expression in vivo. Samir K. Brahmachari, Partha S. Sarkar, P. Balagurumoorthy, R. Bagga, J. Biomol. Struc. Dyn. (1991), 8(6), A022. • Intramolecular triplex potential sequences within a gene downregulate its expression in vivo. Partha S. Sarkar and Samir K. Brahmachari, Nucleic Acids Research (1992), 20, 5713-5718. • Intramolecular DNA triple helix and regulation of gene expression. Samir K. Brahmachari and Partha S. Sarkar. Structural Biology: The State of the Art (1994), Adenine Press, NY. Editor: R.H. Sarma and M.H. Sarma. • DNA triple helix and regulation of gene expression. Samir K. Brahmachari and Partha S. Sarkar, J. Biomol. Struc. Dyn., 10, A108 (1993). • Unusual DNA structure and regulation of gene expression. Samir K. Brahmachari, Partha S. Sarkar, U. S. Shaligram, and A.K. Maiti, 16th IUBMB Congress, New Delhi (1994), Abs, Vol I, Page 47. • Involvement of poly purine/poly pyrimidine sequences in the control of gene expression in vivo. Samir K. Brahmachari, Partha S. Sarkar, U.S. Shaligram, and A.K. Maiti, Asian Transcription Congress, ACTIII (1994), Abs., Page 28. • Triplet Repeat Expansion: Structural Approach to Molecular Understanding of Myotonic Dystrophy. Partha S. Sarkar, G.S. Rajesh, U.S. Shaligram, and Samir K. Brahmachari, 16th IUBMB Congress, New Delhi (1994), Abs., Vol II, 183. • Synthetic Gene Design for Modulation of Gene Expression in Vivo. Samir K. Brahmachari, Partha S. Sarkar, P.K. Burma, U.S. Shaligram, and S.S. Pataskar. Proceedings of the Ranbaxy Symposium on Molecular Genetics and Gene Therapy (1995, In press). • Functional Significance of Simple Repetitive Sequences in the Genome. Samir K. Brahmachari, G. Meera, Partha S. Sarkar, P. Balagurumoorthy, J. Tripathi, S. Raghavan, U.S. Shaligram, and S.S. Pataskar. Proceedings of the 3rd International Conference on DNA Fingerprinting, Electrophoresis (1995, In press). • Inverted Repeat Sequences Adopting Cruciform Structures in Vivo Block Transcription Elongation. Partha S. Sarkar, R. Bagga, P.K. Burma, and Samir K. Brahmachari, (Submitted for publication). • Intragenic Amplification of Myotonic Dystrophy (CTG)n within Gene Inhibits Gene Expression. Partha S. Sarkar, U.S. Shaligram, G.S. Rajesh, and Samir K. Brahmachari, (Submitted for publication). • Alternative Pu/Py Sequences Adopting Left-Handed Z-DNA Structure Does Not Influence Gene Expression in Vivo. Partha S. Sarkar and Samir K. Brahmachari, (Manuscript in preparation). • Base Composition of the Polypu/Polypy Sequences Dictates the Isomerization in Intramolecular Triplex. Partha S. Sarkar, U.S. Shaligram, and Samir K. Brahmachari, (Manuscript in preparation). • Triplet Repeat Sequences Show Extensive Inframe Deletion Mutation in E. coli, and Gene Expression Is Modulated by the Length of Such Repeat Motifs. Partha S. Sarkar, Malathy N., and Samir K. Brahmachari, (Manuscript in preparation) Even though the designed sequence adopted a left-handed structure under physiological superhelical density, the movement of RNA polymerase was not inhibited by such sequences in vivo. This observation also rules out the possibility of stabilization of Z-DNA structure in vivo by Z-DNA binding proteins in E. coli. Chapter 6 deals with the construction of a model system for triplet repeat-related genetic disorders. The triplet repeat sequences have attracted much attention in the scientific community due to their involvement in many human genetic diseases. Recently, trinucleotide repeat expansion mutations have been identified in disorders such as Fragile-X, Myotonic Dystrophy, Huntington’s disease, etc. In these cases, the expansion of certain triplet repeat sequences within and around genes, by an unknown mechanism, leads to the disruption of gene function and the eventual onset of the disease process. To understand the mechanism of triplet repeat expansion and its effect on gene expression, we have created an E. coli model system where a stepwise increase in the length of CTG and CAG repeats is introduced in frame with the ?-galactosidase gene in pBluescriptIISK+ and pBluescriptIIKS+ plasmids, respectively. We find that in vivo expression of ?-galactosidase decreases as the repeat length increases due to the reduced level of transcript formation. We also find that the DNA fragments containing the triplet repeat show anomalous mobility on polyacrylamide gel with increasing repeat length, indicating the formation of a compact structure. Based on these observations, we propose that the expanded triplet repeat can inhibit transcription elongation by RNA polymerase, and these truncated transcripts might eventually be degraded since they may not be polyadenylated. This may lead to the onset of the disease process. We also propose the probable structure that the elongated triplet repeats can adopt. A certain length of the repeat unit can form a slipped structure, which can then fold back into a compact structure. Various structural and genetic studies on these clones are likely to provide a molecular understanding of the disease process. The appendix of the thesis presents an account of the dynamic mutations that we observed during the experiment. ________________________________________ List of Publications: 1. A novel approach to design cis-acting DNA structural elements for regulation of gene expression in vivo. Partha S. Sarkar, R. Bagga, P. Balagurumoorthy & Samir K. Brahmachari, Current Science (1991), 61, 586-591. 2. In vivo regulation of transcription elongation by a designed potential cruciform structure. R. Bagga, Partha S. Sarkar, and Samir K. Brahmachari, 15th International Congress of Biochemistry, Israel, 1991. 3. Synthetic gene design to investigate the role of cis-acting DNA structural elements in regulation of gene expression in vivo. Samir K. Brahmachari, Partha S. Sarkar, P. Balagurumoorthy, P.K. Burma, and R. Bagga. Proceedings of the Symposium on “Synthetic Oligonucleotide: Frontiers and Applications.” Nucleic Acids Research (1991), 24, 163-166. 4. A novel approach to design cis-acting DNA structural elements to study regulation of gene expression in vivo. Samir K. Brahmachari, Partha S. Sarkar, P. Balagurumoorthy, R. Bagga, J. Biomol. Struc. Dyn. (1991), 8(6), A022. 5. Intramolecular triplex potential sequences within a gene downregulate its expression in vivo. Partha S. Sarkar and Samir K. Brahmachari, Nucleic Acids Research (1992), 20, 5713-5718. 6. Intramolecular DNA triple helix and regulation of gene expression. Samir K. Brahmachari and Partha S. Sarkar. Structural Biology: The State of the Art (1994), Adenine Press, NY. Editor: R.H. Sarma and M.H. Sarma. 7. DNA triple helix and regulation of gene expression. Samir K. Brahmachari and Partha S. Sarkar, J. Biomol. Struc. Dyn., 10, A108 (1993). 8. Unusual DNA structure and regulation of gene expression. Samir K. Brahmachari, Partha S. Sarkar, U. S. Shaligram, and A.K. Maiti, 16th IUBMB Congress, New Delhi (1994), Abs, Vol I, Page 47. 9. Involvement of poly purine/poly pyrimidine sequences in the control of gene expression in vivo. Samir K. Brahmachari, Partha S. Sarkar, U.S. Shaligram, and A.K. Maiti, Asian Transcription Congress, ACTIII (1994), Abs., Page 28. 10. Triplet Repeat Expansion: Structural Approach to Molecular Understanding of Myotonic Dystrophy. Partha S. Sarkar, G.S. Rajesh, U.S. Shaligram, and Samir K. Brahmachari, 16th IUBMB Congress, New Delhi (1994), Abs., Vol II, 183. 11. Synthetic Gene Design for Modulation of Gene Expression in Vivo. Samir K. Brahmachari, Partha S. Sarkar, P.K. Burma, U.S. Shaligram, and S.S. Pataskar. Proceedings of the Ranbaxy Symposium on Molecular Genetics and Gene Therapy (1995, In press). 12. Functional Significance of Simple Repetitive Sequences in the Genome. Samir K. Brahmachari, G. Meera, Partha S. Sarkar, P. Balagurumoorthy, J. Tripathi, S. Raghavan, U.S. Shaligram, and S.S. Pataskar. Proceedings of the 3rd International Conference on DNA Fingerprinting, Electrophoresis (1995, In press). 13. Inverted Repeat Sequences Adopting Cruciform Structures in Vivo Block Transcription Elongation. Partha S. Sarkar, R. Bagga, P.K. Burma, and Samir K. Brahmachari, (Submitted for publication). 14. Intragenic Amplification of Myotonic Dystrophy (CTG)n within Gene Inhibits Gene Expression. Partha S. Sarkar, U.S. Shaligram, G.S. Rajesh, and Samir K. Brahmachari, (Submitted for publication). 15. Alternative Pu/Py Sequences Adopting Left-Handed Z-DNA Structure Does Not Influence Gene Expression in Vivo. Partha S. Sarkar and Samir K. Brahmachari, (Manuscript in preparation). 16. Base Composition of the Polypu/Polypy Sequences Dictates the Isomerization in Intramolecular Triplex. Partha S. Sarkar, U.S. Shaligram, and Samir K. Brahmachari, (Manuscript in preparation). 17. Triplet Repeat Sequences Show Extensive Inframe Deletion Mutation in E. coli, and Gene Expression Is Modulated by the Length of Such Repeat Motifs. Partha S. Sarkar, Malathy N., and Samir K. Brahmachari, (Manuscript in preparation)