Calling cards for DNA-binding proteins

Wang, Haoyi; Johnston, Mark; Mitra, Robi D.

doi:10.1101/gr.6510207

Cited by 35 publications

(35 citation statements)

References 34 publications

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“…Probing chromatin is not a new role for Ty5, because changes in integration patterns have previously documented the chromatin dynamics that occur during aging, particularly the movement of Sir4 from the telomeres to the rDNA (32). In addition, the recently developed calling card approach cleverly uses Ty5's ability to mark chromosomal occupancy of proteins (28). Ty5 calling cards are created by fusing the domain of Sir4 that interacts with Ty5 to a transcription factor, and Ty5 insertions mark chromosomal sites where the transcription factor is bound.…”

Section: Discussionmentioning

confidence: 99%

“…These insertions where generated to establish baseline patterns of Ty5 integration for calling card experiments (27). A given transcription factor can be made into a Ty5 calling card by fusing it to the domain of Sir4 that interacts with Ty5 IN (28). Ty5 insertion sites in yeast strains expressing the calling cards identify chromosomal sites occupied by the transcription factor.…”

Section: Relationships Between Ty5 Insertions and Chromosomal Featuresmentioning

confidence: 99%

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Access to DNA establishes a secondary target site bias for the yeast retrotransposon Ty5

Baller

Gao

Voytas

2011

Proc. Natl. Acad. Sci. U.S.A.

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Integration sites for many retrotransposons and retroviruses are determined by interactions between retroelement-encoded integrases and specific DNA-bound proteins. The Saccharomyces retrotransposon Ty5 preferentially integrates into heterochromatin because of interactions between Ty5 integrase and the heterochromatin protein silent information regulator 4. We mapped over 14,000 Ty5 insertions onto the S. cerevisiae genome, 76% of which occurred in heterochromatin, which is consistent with the known target site bias of Ty5. Using logistic regression, associations were assessed between Ty5 insertions and various chromosomal features such as genome-wide distributions of nucleosomes and histone modifications. Sites of Ty5 insertion, regardless of whether they occurred in heterochromatin or euchromatin, were strongly associated with DNase hypersensitive, nucleosome-free regions flanking genes. Our data support a model wherein silent information regulator 4 tethers the Ty5 integration machinery to domains of heterochromatin, and then, specific target sites are selected based on DNA access, resulting in a secondary target site bias. For insertions in euchromatin, DNA access is the primary determinant of target site choice. One consequence of the secondary target site bias of Ty5 is that insertions in coding sequences occur infrequently, which may preserve genome integrity.T he insertion of mobile genetic elements into new chromosomal sites profoundly impacts genome structure and evolution. For many mobile elements, integration sites are not chosen randomly. Target site biases are particularly well-documented for the LTR retrotransposons and retroviruses (1-3). These retroelements replicate by reverse-transcribing mRNA into cDNA and then inserting the cDNA into their host's genome using an element-encoded integrase (IN). Retrotransposons are among the most abundant interspersed repeats in eukaryotic genomes, and retroviruses are often used as vectors for gene therapy. Understanding mechanisms of retroelement target site choice, therefore, has value for both basic and applied research.In the best studied cases, retroelement target site choice is dictated by interactions between IN and specific DNA-bound proteins. HIV IN, for example, interacts with the transcription coactivator lens epithelial-derived growth factor (4), and sites of HIV integration are influenced by sites of this protein's chromosomal occupancy (5). The role of chromatin in target site choice is also well-established for model yeast retrotransposons. The Schizosaccharomyces pombe Tf1 element inserts preferentially into regions upstream of some genes transcribed by RNA polymerase (pol) II (6). Tf1 IN interacts with the transcription factor Atf1p (7), and at the fbp1 promoter, Atf1p alone mediates target site choice (8). The Saccharomyces cerevisiae Ty1 and Ty3 retrotransposons prefer to integrate upstream of genes transcribed by RNA pol III, likely because of interactions between IN and components of the pol III machinery or associated chromatin (9, 10)....

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Section: Discussionmentioning

confidence: 99%

Section: Relationships Between Ty5 Insertions and Chromosomal Featuresmentioning

confidence: 99%

Access to DNA establishes a secondary target site bias for the yeast retrotransposon Ty5

Baller

Gao

Voytas

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Identification of the genomic set of cis-regulatory sequences is challenging in all organisms because regulatory sequences are generally small in comparison to entire protein-coding regions, and because neither their location nor their precise identity is conserved even between highly related organisms. Identification of the cis-elements in DNA, which control transcription, has been made easier and faster by the development of both computational (GuhaThakurta 2006;Das and Dai 2007;Brohee et al 2011) and experimental tools (Gertz et al 2009;Schlabach et al 2010) to find these elements, as well as by the development of methods to link DNA sequences and transcription factors (Harbison et al 2004;Barrera and Ren 2006;Walhout 2006;Wang et al 2007). In contrast, although cis-elements in RNA mediate many aspects of post-transcriptional regulation, including RNA splicing, folding, cellular location, degradation, and translation, the large-scale discovery of these elements has been complicated by at least three factors.…”

Section: Introductionmentioning

confidence: 99%

RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay

Dean

Grayhack

2012

RNA

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We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.

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“…We attach the transposase of a transposon to a TF, thereby endowing the TF with the ability to direct insertion of the transposon into the genome near where it binds (Wang et al 2007) (Figure 1). The transposon becomes a calling card that permanently marks the transcription factor's visit to a particular genomic location.…”

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confidence: 99%

“Calling Cards” for DNA-Binding Proteins in Mammalian Cells

et al. 2012

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The ability to chronicle transcription-factor binding events throughout the development of an organism would facilitate mapping of transcriptional networks that control cell-fate decisions. We describe a method for permanently recording protein-DNA interactions in mammalian cells. We endow transcription factors with the ability to deposit a transposon into the genome near to where they bind. The transposon becomes a "calling card" that the transcription factor leaves behind to record its visit to the genome. The locations of the calling cards can be determined by massively parallel DNA sequencing. We show that the transcription factor SP1 fused to the piggyBac transposase directs insertion of the piggyBac transposon near SP1 binding sites. The locations of transposon insertions are highly reproducible and agree with sites of SP1-binding determined by ChIP-seq. Genes bound by SP1 are more likely to be expressed in the HCT116 cell line we used, and SP1-bound CpG islands show a strong preference to be unmethylated. This method has the potential to trace transcription-factor binding throughout cellular and organismal development in a way that has heretofore not been possible.

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Calling cards for DNA-binding proteins

Cited by 35 publications

References 34 publications

Access to DNA establishes a secondary target site bias for the yeast retrotransposon Ty5

Access to DNA establishes a secondary target site bias for the yeast retrotransposon Ty5

RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay

“Calling Cards” for DNA-Binding Proteins in Mammalian Cells

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