Alter DNA Topology Research Articles

Recent progress in the discovery and development of DNA gyrase B inhibitors.

New antibacterials that modulate less explored targets are needed to fight the emerging bacterial resistance. DNA gyrase and topoisomerase IV are attractive targets in this search. These are both type II topoisomerases that can cleave both DNA strands, and can thus alter DNA topology during replication or similar processes. Currently, there are no ATP-competitive inhibitors of these two enzymes on the market, as the only aminocoumarin representative, novobiocin, was withdrawn due to safety concerns. The search for novel ATP-competitive inhibitors is a focus of ongoing industrial and academical research. This review summarizes the recent efforts in the design, synthesis and evaluation of GyrB/ParE inhibitors. The various approaches to achieve improved antibacterial activities are described, with particular reference to Gram-negative bacteria.

Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase.

Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the in vivo interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in Mycobacterium tuberculosis taking advantage of minimal topoisomerase representation in the organism. The study unveils the first in vivo genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in Mtb, near the Ter domain of the Mtb chromosome validates its function as a decatenase.

All-trans retinoic acid suppresses topoisomerase IIα through the proteasomal pathway.

Topoisomerase IIα is a nuclear enzyme that alters DNA topology. It is a well-known anticancer target and related to cell differentiation status. All-trans retinoic acid (ATRA), an important active metabolite of vitamin A, is a promising anticancer agent in numerous malignancies. However, there are little data on the effect of retinoids on topoisomerase IIα regulation. In the present study, we investigated the relationship between ATRA and topoisomerase IIα, and the potential mechanisms of ATRA on topoisomerase IIα regulation. In several human carcinoma cell lines, ATRA was shown to suppress topoisomerase IIα protein, but not mRNA expression. ATRA induced the degradation of topoisomerase IIα through the proteasome pathway, but not the lysosome pathway. Ubiquitination was involved in this degradation. Western blot and immunocytochemistry proved that ATRA-induced topoisomerase IIα repression occurred only in the cell nuclei. ATRA not only influenced the cycle procession but also reduced the expression of cyclin D1. Cyclin D1, which is involved in cell differentiation, was regulated by topoisomerase IIα. Similar to cyclin D1, knockdown of topoisomerase IIα resulted in the increased differentiation of the cells, which was in contrast to the overexpression of topoisomerase IIα in the cells. Taken together, these data suggested that ATRA could target topoisomerase IIα and exert potential beneficial effects on cell differentiation.

Sound packing DNA: packing open circular DNA with low-intensity ultrasound.

Supercoiling DNA (folding DNA into a more compact molecule) from open circular forms requires significant bending energy. The double helix is coiled into a higher order helix form; thus it occupies a smaller footprint. Compact packing of DNA is essential to improve the efficiency of gene delivery, which has broad implications in biology and pharmaceutical research. Here we show that low-intensity pulsed ultrasound can pack open circular DNA into supercoil form. Plasmid DNA subjected to 5.4 mW/cm2 intensity ultrasound showed significant (p-values <0.001) supercoiling compared to DNA without exposure to ultrasound. Radiation force induced from ultrasound and dragging force from the fluid are believed to be the main factors that cause supercoiling. This study provides the first evidence to show that low-intensity ultrasound can directly alter DNA topology. We anticipate our results to be a starting point for improved non-viral gene delivery.

Proteolytic Degradation of Topoisomerase II (Top2) Enables the Processing of Top2·DNA and Top2·RNA Covalent Complexes by Tyrosyl-DNA-Phosphodiesterase 2 (TDP2)

Eukaryotic type II topoisomerases (Top2α and Top2β) are homodimeric enzymes; they are essential for altering DNA topology by the formation of normally transient double strand DNA cleavage. Anticancer drugs (etoposide, doxorubicin, and mitoxantrone) and also Top2 oxidation and DNA helical alterations cause potentially irreversible Top2·DNA cleavage complexes (Top2cc), leading to Top2-linked DNA breaks. Top2cc are the therapeutic mechanism for killing cancer cells. Yet Top2cc can also generate recombination, translocations, and apoptosis in normal cells. The Top2 protein-DNA covalent complexes are excised (in part) by tyrosyl-DNA-phosphodiesterase 2 (TDP2/TTRAP/EAP2/VPg unlinkase). In this study, we show that irreversible Top2cc induced in suicidal substrates are not processed by TDP2 unless they first undergo proteolytic processing or denaturation. We also demonstrate that TDP2 is most efficient when the DNA attached to the tyrosyl is in a single-stranded configuration and that TDP2 can efficiently remove a tyrosine linked to a single misincorporated ribonucleotide or to polyribonucleotides, which expands the TDP2 catalytic profile with RNA substrates. The 1.6-Å resolution crystal structure of TDP2 bound to a substrate bearing a 5'-ribonucleotide defines a mechanism through which RNA can be accommodated in the TDP2 active site, albeit in a strained conformation.

Torsionally Constrained DNA for Single-Molecule Assays: An Efficient, Ligation-Free Method

Controlled twisting of individual, double-stranded DNA molecules provides a unique method to investigate the enzymes that alter DNA topology. Such twisting requires a single DNA molecule to be torsionally constrained. This constraint is achieved by anchoring the opposite ends of the DNA to two separate surfaces via multiple bonds. The traditional protocol for making such DNA involves a three-way ligation followed by gel purification, a laborious process that often leads to low yield both in the amount of DNA and the fraction of molecules that is torsionally constrained. We developed a simple, ligation-free procedure for making torsionally constrained DNA via polymerase chain reaction (PCR). This PCR protocol used two “megaprimers,” 400-base-pair long double-stranded DNA that were labelled with either biotin or digoxigenin. We obtained a relatively high yield of gel-purified DNA (∼500 ng per 100-µl PCR reaction). The final construct in this PCR-based method contains only one labelled strand in contrast to the traditional construct in which both strands of the DNA are labelled. Nonetheless, we achieved a high yield (84%) of torsionally constrained DNA when measured using an optical-trap-based DNA-overstretching assay. This protocol significantly simplifies the application and adoption of torsionally constrained assays to a wide range of single-molecule systems.

Studies of DNA Gyrase at the Single Molecule Level

Topoisomerases are proteins that manipulate the topology of DNA. Unique among topoisomerases is DNA gyrase, a Type II bacterial topoisomerase, which is the only one capable of introducing negative supercoils. In addition, DNA gyrase can relax positive supercoils, an ability shared by other members of both Type I and II topoisomerases. To generate negative supercoils, DNA gyrase forms a double stranded break in DNA and passes a second strand of DNA through the resulting break, a process that is linked to ATP hydrolysis. To elucidate the conformational changes necessary for this action and to obtain a comprehensive picture of the mechanism of gyrase, we will utilize a novel single molecule technique, which combines both magnetic tweezers and TIRF microscopy to simultaneously observe protein and DNA movement during the DNA supercoiling process. Incorporating information from both dynamic single molecule and static structural studies promises to provide a more comprehensive picture of the mechanism used by this molecular machine to alter DNA topology.

On the structural basis and design guidelines for type II topoisomerase-targeting anticancer drugs.

Type II topoisomerases (Top2s) alter DNA topology via the formation of an enzyme–DNA adduct termed cleavage complex, which harbors a transient double-strand break in one DNA to allow the passage of another. Agents targeting human Top2s are clinically active anticancer drugs whose trapping of Top2-mediated DNA breakage effectively induces genome fragmentation and cell death. To understand the structural basis of this drug action, we previously determined the structure of human Top2 β-isoform forming a cleavage complex with the drug etoposide and DNA, and described the insertion of drug into DNA cleavage site and drug-induced decoupling of catalytic groups. By developing a post-crystallization drug replacement procedure that simplifies structural characterization of drug-stabilized cleavage complexes, we have extended the analysis toward other structurally distinct drugs, m-AMSA and mitoxantrone. Besides the expected drug intercalation, a switch in ribose puckering in the 3′-nucleotide of the cleavage site was robustly observed in the new structures, representing a new mechanism for trapping the Top2 cleavage complex. Analysis of drug-binding modes and the conformational landscapes of the drug-binding pockets provide rationalization of the drugs’ structural-activity relationships and explain why Top2 mutants exhibit differential effects toward each drug. Drug design guidelines were proposed to facilitate the development of isoform-specific Top2-targeting anticancer agents.

Torsionally constrained DNA for single-molecule assays: an efficient, ligation-free method

Controlled twisting of individual, double-stranded DNA molecules provides a unique method to investigate the enzymes that alter DNA topology. Such twisting requires a single DNA molecule to be torsionally constrained. This constraint is achieved by anchoring the opposite ends of the DNA to two separate surfaces via multiple bonds. The traditional protocol for making such DNA involves a three-way ligation followed by gel purification, a laborious process that often leads to low yield both in the amount of DNA and the fraction of molecules that is torsionally constrained. We developed a simple ligation-free procedure for making torsionally constrained DNA via polymerase chain reaction (PCR). This PCR protocol used two ‘megaprimers’, 400-base-pair long double-stranded DNA that were labelled with either biotin or digoxigenin. We obtained a relatively high yield of gel-purified DNA (∼500 ng/100 µl of PCR reaction). The final construct in this PCR-based method contains only one labelled strand in contrast to the traditional construct in which both strands of the DNA are labelled. Nonetheless, we achieved a high yield (84%) of torsionally constrained DNA when measured using an optical-trap-based DNA-overstretching assay. This protocol significantly simplifies the application and adoption of torsionally constrained assays to a wide range of single-molecule systems.

Type IA topoisomerase inhibition by clamp closure

Bacterial DNA topoisomerase I (topoI) catalyzes relaxation of negatively supercoiled DNA. The enzyme alters DNA topology through protein-operated DNA gate, switching between open and closed conformations during its reaction. We describe the mechanism of inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis topoI by monoclonal antibodies (mAbs) that bind with high affinity and inhibit at 10-50 nM concentration. Unlike other inhibitors of topoisomerases, the mAbs inhibited several steps of relaxation reaction, namely DNA binding, cleavage, strand passage, and enzyme-DNA dissociation. The enhanced religation of the cleaved DNA in presence of the mAb indicated closing of the enzyme DNA gate. The formation of enzyme-DNA heterocatenane in the presence of the mAbs as a result of closing the gate could be inferred by the salt resistance of the complex, visualized by atomic force microscopy and confirmed by fluorescence measurements. Locking the enzyme-DNA complex as a closed clamp restricted the movements of the DNA gate, affecting all of the major steps of the relaxation reaction. Enzyme trapped on DNA in closed clamp conformation formed roadblock for the elongating DNA polymerase. The unusual multistep inhibition of mycobacterial topoisomerases may facilitate lead molecule development, and the mAbs would also serve as valuable tools to probe the enzyme mechanism.

Consistent mutations in Type II Topoismerases of Vibrio cholerae O1 and global reduction in fluoroquinolone susceptibility

Vibrio cholerae O1 the epidemic strains causing devastating cholera harbors two related type II topoisomerases, gyrase and topoisomerase IV which alter DNA topology through a DNA cleavage complex. Fluoroquinolones stabilizing the DNA breakage‐reunion complex are effective WHO recommended antibiotics used in the treatment of severe cholera cases. A total of 200 Vibrio cholerae O1 strains (clinical and environmental) collected from different parts of India were subjected for MIC calculations for ciprofloxacin according to the CLSI, 2010 guidelines. The strains showed reduced susceptibility to ciprofloxacin (MIC 0.1 to 1 μg/ml). Two mutation in quinolone resistance determining region, 1 mutation in gyrA (83S&gt;;I) and 1 mutation in parC (85S&gt;;L) were found consistent in all strains. These amino acid substitutions are previously known to be associated with quinolone resistance in V. cholerae. Molecular modelling studies of mutant gyrase and topoisomerase IV revealed change in binding pocket for ciprofloxacin and would lead to alter holding over DNA cleavage complex. Such type of strains experiences the inhibitory dose of ciprofloxacin as sub‐inhibitory concentration, and could be the reason of longer duration of diarrhoea. The reduced susceptibility to ciprofloxacin was also noticed in the hybrid V. cholerae O1 strains from recent large cholera epidemics in Western Africa and Haiti. The study suggested that the circulating epidemic strains are consistently spreading globally. It hampers the outbreak control policies which can be a serious concern in future. The use of alternate chemotherapeutic agents stabilizing the transition complex of typeII topoisomerases with DNA breakage‐reunion should be of pressing importance.

Studies of bacterial topoisomerases I and III at the single-molecule level

Topoisomerases are the enzymes responsible for maintaining the supercoiled state of DNA in the cell and also for many other DNA-topology-associated reactions. TypeIA enzymes alter DNA topology by breaking one DNA strand and passing another strand or strands through the break. Although all typeIA topoisomerases are related at the sequence, structure and mechanism levels, different typeIA enzymes do not participate in the same cellular processes. We have studied the mechanism of DNA relaxation by Escherichia coli topoisomerases I and III using single-molecule techniques to understand their dissimilarities. Our experiments show important differences at the single-molecule level, while also recovering the results from bulk experiments. Overall, topoisomerase III relaxes DNA using fast processive runs followed by long pauses, whereas topoisomerase I relaxes DNA through slow processive runs followed by short pauses. These two properties combined give rise to the overall relaxation rate, which is higher for topoisomerase I than for topoisomerase III, as expected from many biochemical observations. The results help us to understand better the role of these two topoisomerases in the cell and also serve to illustrate the power of single-molecule experiments to uncover new functional characteristics of biological molecules.

A novel nucleoid-associated protein specific to the actinobacteria

Effective chromosome organization is central to the functioning of any cell. In bacteria, this organization is achieved through the concerted activity of multiple nucleoid-associated proteins. These proteins are not, however, universally conserved, and different groups of bacteria have distinct subsets that contribute to chromosome architecture. Here, we describe the characterization of a novel actinobacterial-specific protein in Streptomyces coelicolor. We show that sIHF (SCO1480) associates with the nucleoid and makes important contributions to chromosome condensation and chromosome segregation during Streptomyces sporulation. It also affects antibiotic production, suggesting an additional role in gene regulation. In vitro, sIHF binds DNA in a length-dependent but sequence-independent manner, without any obvious structural preferences. It does, however, impact the activity of topoisomerase, significantly altering DNA topology. The sIHF–DNA co-crystal structure reveals sIHF to be composed of two domains: a long N-terminal helix and a C-terminal helix-two turns-helix domain with two separate DNA interaction sites, suggesting a potential role in bridging DNA molecules.

Studies of DNA Gyrase at the Single Molecule Level

Gyrase, a bacterial type II topoisomerase, is the only topoisomerase capable of introducing negative supercoils into DNA. Negative supercoiling results from the coordinated movement of double stranded DNA and protein domains linked to ATP hydrolysis. Thus, it is important to combine structural data with dynamic information of protein and/or DNA movements to obtain a comprehensive picture of the mechanism of gyrase. Although the structures of several individual domains of different type II enzymes have been elucidated, there are still no structures of the intact gyrase heterotetramer or an intact gyrase/DNA complex precluding the correlation of conformational changes in the protein with movements of DNA. To alleviate this shortcoming, we are combining structural and single molecule studies to investigate the conformational changes that occur in gyrase as it alters DNA topology. To facilitate our research, we have optimized a procedure for purifying stable gyrase/DNA complexes in the amounts required for biophysical studies. This method results in homogeneous complexes that are captured in well-defined states along the catalytic cycle. We are also developing a novel single molecule technique combining magnetic tweezers and fluorescence microscopy to study the movements of both protein and DNA during the DNA supercoiling process. The combination of dynamic single molecule and structural information promises to provide a more comprehensive picture of the mechanism used by this molecular machine to alter DNA topology.

Restoration of Topoisomerase 2 Function by Complementation of Defective Monomers in Drosophila

Type II topoisomerases are essential ATP-dependent homodimeric enzymes required for transcription, replication, and chromosome segregation. These proteins alter DNA topology by generating transient enzyme-linked double-strand breaks for passage of one DNA strand through another. The central role of type II topoisomerases in DNA metabolism has made these enzymes targets for anticancer drugs. Here, we describe a genetic screen that generated novel alleles of Drosophila Topoisomerase 2 (Top2). Fifteen alleles were obtained, resulting from nonsense and missense mutations. Among these, 14 demonstrated recessive lethality, with one displaying temperature-sensitive lethality. Several newly generated missense alleles carry amino acid substitutions in conserved residues within the ATPase, Topoisomerase/Primase, and Winged helix domains, including four that encode proteins with alterations in residues associated with resistance to cancer chemotherapeutics. Animals lacking zygotic Top2 function can survive to pupation and display reduced cell division and altered polytene chromosome structure. Inter se crosses between six strains carrying Top2 missense alleles generated morphologically normal trans-heterozygous adults, which showed delayed development and were female sterile. Complementation occurred between alleles encoding Top2 proteins with amino acid substitutions in the same functional domain and between alleles encoding proteins with substitutions in different functional domains. Two complementing alleles encode proteins with amino acid substitutions associated with drug resistance. These observations suggest that dimerization of mutant Top2 monomers can restore enzymatic function. Our studies establish the first series of Top2 alleles in a multicellular organism. Future analyses of these alleles will enhance our knowledge about the contributions made by type II topoisomerases to development.

A Fundamental Regulatory Mechanism Operating through OmpR and DNA Topology Controls Expression of Salmonella Pathogenicity Islands SPI-1 and SPI-2

DNA topology has fundamental control over the ability of transcription factors to access their target DNA sites at gene promoters. However, the influence of DNA topology on protein–DNA and protein–protein interactions is poorly understood. For example, relaxation of DNA supercoiling strongly induces the well-studied pathogenicity gene ssrA (also called spiR) in Salmonella enterica, but neither the mechanism nor the proteins involved are known. We have found that relaxation of DNA supercoiling induces expression of the Salmonella pathogenicity island (SPI)-2 regulator ssrA as well as the SPI-1 regulator hilC through a mechanism that requires the two-component regulator OmpR-EnvZ. Additionally, the ompR promoter is autoregulated in the same fashion. Conversely, the SPI-1 regulator hilD is induced by DNA relaxation but is repressed by OmpR. Relaxation of DNA supercoiling caused an increase in OmpR binding to DNA and a concomitant decrease in binding by the nucleoid-associated protein FIS. The reciprocal occupancy of DNA by OmpR and FIS was not due to antagonism between these transcription factors, but was instead a more intrinsic response to altered DNA topology. Surprisingly, DNA relaxation had no detectable effect on the binding of the global repressor H-NS. These results reveal the underlying molecular mechanism that primes SPI genes for rapid induction at the onset of host invasion. Additionally, our results reveal novel features of the archetypal two-component regulator OmpR. OmpR binding to relaxed DNA appears to generate a locally supercoiled state, which may assist promoter activation by relocating supercoiling stress-induced destabilization of DNA strands. Much has been made of the mechanisms that have evolved to regulate horizontally-acquired genes such as SPIs, but parallels among the ssrA, hilC, and ompR promoters illustrate that a fundamental form of regulation based on DNA topology coordinates the expression of these genes regardless of their origins.

Structural Basis of Type II Topoisomerase Inhibition by the Anticancer Drug Etoposide

Type II topoisomerases (TOP2s) resolve the topological problems of DNA by transiently cleaving both strands of a DNA duplex to form a cleavage complex through which another DNA segment can be transported. Several widely prescribed anticancer drugs increase the population of TOP2 cleavage complex, which leads to TOP2-mediated chromosome DNA breakage and death of cancer cells. We present the crystal structure of a large fragment of human TOP2β complexed to DNA and to the anticancer drug etoposide to reveal structural details of drug-induced stabilization of a cleavage complex. The interplay between the protein, the DNA, and the drug explains the structure-activity relations of etoposide derivatives and the molecular basis of drug-resistant mutations. The analysis of protein-drug interactions provides information applicable for developing an isoform-specific TOP2-targeting strategy.

OsSpo11-4, a Rice Homologue of the Archaeal TopVIA Protein, Mediates Double-Strand DNA Cleavage and Interacts with OsTopVIB

DNA topoisomerase VI from Archaea, a heterotetrameric complex composed of two TopVIA and two TopVIB subunits, is involved in altering DNA topology during replication, transcription and chromosome segregation by catalyzing DNA strand transfer through transient double-strand breaks. The sequenced yeast and animal genomes encode only one homologue of the archaeal TopVIA subunit, namely Spo11, and no homologue of the archaeal TopVIB subunit. In yeast, Spo11 is essential for initiating meiotic recombination and this function appears conserved among other eukaryotes. In contrast to yeast and animals, studies in Arabidopsis and rice have identified three Spo11/TopVIA homologues and one TopVIB homologue in plants. Here, we further identified two novel Spo11/TopVIA homologues (named OsSpo11-4 and OsSpo11-5, respectively) that exist just in the monocot model plant Oryza sativa, indicating that at least five Spo11/TopVIA homologues are present in the rice genome. To reveal the biochemical function of the two novel Spo11/TopVIA homologues, we first examined the interactions among OsSpo11-1, OsSpo11-4, OsSpo11-5, and OsTopVIB by yeast two-hybrid assay. The results showed that OsSpo11-4 and OsTopVIB can self-interact strongly and among the 3 examined OsSpo11 proteins, only OsSpo11-4 interacted with OsTopVIB. Pull-down assay confirmed the interaction between OsSpo11-4 and OsTopVIB, which indicates that OsSpo11-4 may interact with OsTopVIB in vivo. Further in vitro enzymatic analysis revealed that among the above 4 proteins, only OsSpo11-4 exhibited double-strand DNA cleavage activity and its enzymatic activity appears dependent on Mg2+ and independent of OsTopVIB, despite its interaction with OsTopVIB. We further analyzed the biological function of OsSpo11-4 by RNA interference and found that down-regulated expression of OsSpo11-4 led to defects in male meiosis, indicating OsSpo11-4 is required for meiosis.

The Telomere Binding Protein TRF2 Induces Chromatin Compaction

Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

Decreased Rate of β-GlobinL1 Allele Transcription Due to Intronic LINE-1 Insertion In the β-Globin gene Is Associated with β-globinL1 Promoter and Enhancer Hypermethylation Which Is Not Reverted by Decitabine

AbstractAbstract 2073We have previously described a unique etiology of β-thalassemia due retrotransposon LINE-1 (L1) insertion into the intron-2 of the β-globin gene in a mother and daughter of Ukrainian descent who exhibited typical laboratory features of β-thalassemia trait (1, 2). The affected β-globinL1 gene generated roughly 10–15% of the total β−globin message (1). In order to unravel how the intronic insertion of transposable element attenuates the β-globin expression, interspecific hybrids of the propositus′ lymphocytes and mouse erythroleukemia (MEL) cells were generated (laboratory of Dr. T. Papayannopoulou, Seattle, WA) and used for expression studies. The total RNA from differently treated (emetine, 5-aza-2′-deoxycytidine (Sigma)) in vitro cultured cell hybrids were isolated and β-globin transcripts were analyzed by Real-Time qPCR with gene-specific primers. Nuclei from hybrid MEL cells were isolated to performed nuclear run-on assay. Bisulfite modification was done on genomic DNA from MEL hybrids and the promoter and enhancer regions of β-globin gene were amplified, PCR products were subcloned and sequenced. We demonstrated that the observed reduction in steady-state level of β-globin mRNA is partially caused by aberrant splicing followed by activation of nonsense-mediated decay (NMD) pathway, leading to increased degradation of aberrant β-globin mRNA variants. Reduction in expression of β-globin mRNA from β-globinL1 allele comes also from altered rate of transcription. We performed PCR-based nuclear run-on assay and forty minutes of in vitro transcription revealed 30% decrease in β-globinL1 allele transcription rate compared to wild-type β-globin allele. It is known that L1 regulatory regions are highly methylated (3) but there is no evidence so far demonstrating retrotransposon-mediated epigenetic control of neighboring genes. Therefore we determined the methylation status of 6 CpGs between −415 bp and +110 bp (bases upstream and downstream of transcription start) on the promoter region and 3 CpGs between +442 bp to +592 bp on the enhancer region (bases downstream of the poly(A) signal) of the mutated and normal β−globin alleles. Enhancer region of the mutated allele was 100% methylated (100% Me-CpG versus 9% Me-CpG in control), and promoter region was partially methylated (≈ 65% Me−CpG versus 35% Me-CpG in control). Surprisingly, treatment of induced hybrid MEL cells with demethylation agent (5-aza-2′-deoxycytidine, decitabine) did not change the methylation profile and consequently did not increase the expression of β-globinL1 mRNA whereas the expression of γ-globin gene, which was used as an internal control, was increased. We therefore hypothesize that decreased rate of transcription from β-globinL1 allele is associated with altered DNA topology caused by the L1 insertion and β-globinL1 promoter-enhancer displacement. Resulting chromatin modifications lead to permanent β-globinL1 gene silencing and hypermethylation of regulatory sequence refractory to demethylating agent.Divoky V et al: Blood. 1996;88 (suppl1):148a.Kimberland ML, Divoky V et al: Hum Mol Genet. 1999;8:1557-1560.Hata K, Sakaki Y: Gene. 1997;189:227-234. Grant support:NS10281 −3/2009 Ministry of Health, Czech Republic and Internal grant agency of Palacky University grant No. LF_2010_013. Disclosures:No relevant conflicts of interest to declare.