Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA.

In traditional electrophoresis mobility shift assay (EMSA) a single (32)P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions. An extension of this method uses a population of DNA restriction fragments derived from long genomic regions for the identification of fragments containing protein binding regions. Although the method allows simultaneous analysis of large fragments, it is relatively laborious and can be used to detect only fragments containing high affinity protein binding sites. Here we describe an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. With this strategy restriction fragments, derived from genomic DNA (<10 kb), containing high as well as low affinity protein binding regions may be easily identified.

Retroelements in human disease.

Retroelements are an abundant class of noncoding DNAs present in about half of the human genome. Among them, L1, Alu and SVA are currently active. They "jump" by retrotransposition, shuffle genomic regions by 5' and 3' transduction, and promote or inhibit gene transcription by providing alternative promoters or generating antisense and/or regulatory noncoding RNAs. Recent data also suggest that retroelement insertions into exons and introns of genes induce different types of genetic disease, including cancer. Retroelements interfere with the expression of genes by inducing alternative splicing via exon skipping and exonization using cryptic splice sites, and by providing polyadenylation signals. Here we summarize our current understanding of the molecular mechanisms of retroelement-induced mutagenesis which causes fifty different types of human disease. We categorize these mutagenic effects according to eleven different mechanisms and show that most of them may be explained either by traditional exon definition or transcriptional interference, a previously unrecognized molecular mechanism. In summary, this review gives an overview of retroelement insertions in genes that cause significant changes in their transcription and cotranscriptional splicing and show a remarkable level of complexity.

Intronic retroelements: Not just "speed bumps" for RNA polymerase II.

Two well-known retroelements, L1 and Alu, comprise about one third of the human genome and are nearly equally distributed between the intergenic and intragenic regions. They carry different regulatory elements and contribute structurally and functionally to the expression of our genes. Recent data also suggest that hundreds of intronic L1s and Alus interfere with the transcription of human genes by inducing intron retention, forcing exonization and cryptic polyadenylation. These novel features can be explained with the RNA polymerase kinetic model and suggest that intronic L1s and Alus are not just "speed bumps" in regulation of RNA polymerase traffic. Here we discuss the complexity of the regulation of gene transcription imposed by intronic retroelements and predict that in addition to transcriptional activity, transcription factor binding and nucleosomal occupancy play a significant role in the transcriptional interference effects of the host genes.

Intronic L1 retrotransposons and nested genes cause transcriptional interference by inducing intron retention, exonization and cryptic polyadenylation.

BACKGROUND: Transcriptional interference has been recently recognized as an unexpectedly complex and mostly negative regulation of genes. Despite a relatively few studies that emerged in recent years, it has been demonstrated that a readthrough transcription derived from one gene can influence the transcription of another overlapping or nested gene. However, the molecular effects resulting from this interaction are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using in silico chromosome walking, we searched for prematurely terminated transcripts bearing signatures of intron retention or exonization of intronic sequence at their 3' ends upstream to human L1 retrotransposons, protein-coding and noncoding nested genes. We demonstrate that transcriptional interference induced by intronic L1s (or other repeated DNAs) and nested genes could be characterized by intron retention, forced exonization and cryptic polyadenylation. These molecular effects were revealed from the analysis of endogenous transcripts derived from different cell lines and tissues and confirmed by the expression of three minigenes in cell culture. While intron retention and exonization were comparably observed in introns upstream to L1s, forced exonization was preferentially detected in nested genes. Transcriptional interference induced by L1 or nested genes was dependent on the presence or absence of cryptic splice sites, affected the inclusion or exclusion of the upstream exon and the use of cryptic polyadenylation signals. CONCLUSIONS/SIGNIFICANCE: Our results suggest that transcriptional interference induced by intronic L1s and nested genes could influence the transcription of the large number of genes in normal as well as in tumor tissues. Therefore, this type of interference could have a major impact on the regulation of the host gene expression.

Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA.

BACKGROUND: In a traditional electrophoresis mobility shift assay (EMSA) a 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb) for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE. Shifted DNA fragments containing protein binding sites are identified by using additional procedures, i. e. gel elution, PCR amplification, cloning and sequencing. Although the method allows simultaneous analysis of a large population of fragments, it is relatively laborious and can be used to detect only high affinity protein binding sites. Here we propose an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. This strategy allows the identification of DNA fragments containing low as well as high affinity protein binding regions, derived from genomic DNA (<10 kb) of known sequence. RESULTS: We have combined an EMSA-based selection step with subsequent denaturing PAGE for the localization of protein binding regions in long (up to 10 kb) fragments of genomic DNA. Our strategy consists of the following steps: digestion of genomic DNA with a 4-cutter restriction enzyme (AluI, BsuRI, TruI, etc), separation of low and high molecular weight fractions of resultant DNA fragments, 32P-labeling with Klenow polymerase, traditional EMSA, gel elution and identification of the shifted bands (or smear) by denaturing PAGE. The identification of DNA fragments containing protein binding sites is carried out by running the gel-eluted fragments alongside with the full "spectrum" of initial restriction fragments of known size. Here the strategy is used for the identification of protein-binding regions in the 5' region of the rat p75 neurotrophin receptor (p75NTR) gene. CONCLUSION: The developed strategy is based on a combination of traditional EMSA and denaturing PAGE for the identification of protein binding regions in long fragments of genomic DNA. The identification is straightforward and can be applied to shifted bands corresponding to stable DNA-protein complexes as well as unstable complexes, which undergo dissociation during electrophoresis.