Context and number of non‐canonical repeat variable diresidues (RVDs) impede the design of TALE proteins with improved DNA targeting
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PROVIDER: PRJNA530517 | ENA |
REPOSITORIES: ENA
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Project description:A particularly promising approach for engineering more precise TALE proteins is to better understand non-canonical RVDs. The RVD repertoire employed by Xanthomonas extends well beyond just the four canonical RVD types, and natural TALE repeat arrays often utilize serial combinations of both canonical and non-canonical RVDs in a manner which is not fully understood. Attempts to characterize these non-canonical RVDs have utilized various approaches, in terms of both TALE design and experimental method. Some studies have considered just TALEs of particular biological relevance, others have looked at large numbers of different RVDs within a fixed repeat-array-context, and still others have designed custom TALEs to address hypotheses about optimal RVD positioning within the repeat array. Methods for inferring the DNA binding activity of these TALEs have included sequencing of cleaved DNA fragments from TALE-nuclease fusions; ELISA assays for TALE-oligonucleotide binding; reporter assays for TALEs’ transcriptional-activating effects; and in silico modeling. In this study we employ a novel “wholesale swap-out” approach to characterize 3 non-canonical RVDs for thymine and 4 non-canonical RVDs for guanine.
2019-12-19 | GSE129193 | GEO
Project description: Not available
| S-EPMC6954735 | biostudies-literature
Project description:Transcription Activator Like Effector (TALE) proteins have been described as recognizing DNA using a simple DNA-binding code, which has allowed for their use in many techniques requiring precise targeting of genomic loci. The TALE DNA binding domain is composed of an array of short repeats that differ in two key DNA-contacting positions, the repeat variable diresidues (RVD); the identities of the amino acids at these positions determine which base the repeat binds. While this simple code has been used successfully to design TALEs to target specific sequences, off-target binding has also been observed, indicating the need for a more rigorous understanding of the factors that determine TALE specificity. In this study, we assayed the DNA binding specificities of 21 TALE proteins of different lengths and RVD compositions using custom-designed Protein Binding Microarrays (PBMs). Position weight matrices derived from these data were used to develop a model to predict the specificity of any TALE protein. This modeling shows the large influence of protein length, RVD position, and neighboring RVD identity on binding specificity, indicating that the simple cipher-like code does not fully capture the complexity of TALE-DNA binding.
2015-06-11 | GSE56978 | GEO
Project description:ChIP-Sequencing on Shox2-HA E12.5 and E13.5 Limb and Palate, as well as Pbx on E12.5 limb . Abstract: Vertebrate appendage patterning is programmed by Hox-TALE factors-bound regulatory elements. However, it remains enigmatic which cell lineages are commissioned by Hox-TALE factors to generate regional specific pattern and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis, and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.
2016-07-05 | GSE81897 | GEO
Project description:ChIP-Sequencing on Shox2-HA E12.5 and E13.5 Limb and Palate, as well as Pbx on E12.5 limb . Abstract: Vertebrate appendage patterning is programmed by Hox-TALE factors-bound regulatory elements. However, it remains enigmatic which cell lineages are commissioned by Hox-TALE factors to generate regional specific pattern and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis, and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis. Shox2/TALE For ChIP-Seq, the list of libraries below, including controls, were generated [listed in the format of (antibody)-target-tissue-stage]: (α-HA)-Shox2-Limb-E12.5, (α-HA)-Shox2-Limb-E13.5, (α-HA)-Shox2-Palate-E12.5, (α-HA)-Shox2-Limb/Palate-E12.5, (α-Pbx)-Pbx-Limb-E12.5, Input (control), (α-HA)-Mixed Limb/Palate from Shox2+/+ mice-E12.5 (control). *The attached signal tracks(*.bigwig) were generated by âbdgcmp (MACS2) to filter out background signal(by filtering against the signal track obtained from (α-HA)-Mixed Limb/Palate from Shox2+/+ mice-E12.5 (control)) and subsequently convert to bigwig for analysis and visualization.
2016-07-05 | E-GEOD-81897 | biostudies-arrayexpress
Project description:We present a strategy to investigate regulatory elements that leverages programmable reagents to selectively inactivate their endogenous chromatin state. The reagents, which comprise fusions between transcription activator- like effector (TALE) repeat domains and the LSD1 histone demethylase, efficiently remove enhancer-associated chromatin modifications from target loci, without affecting control regions. We find that inactivation of enhancer chromatin by these fusions frequently causes down- regulation of proximal genes. Our study demonstrates the potential of 'epigenome editing' tools to characterize a critical class of functional genomic elements. ChIP-seq analysis of TALE-Fusion Proteins
2013-07-26 | E-GEOD-48866 | biostudies-arrayexpress
Project description:Meis, Prep and Pbx1 TALE homeoproteins interactions with Hox proteins are essential for development and disease. Although Meis and Prep behave similarly in vitro, their in vivo activities remain largely unexplored. We show that Prep and Meis interact with largely independent sets of genomic sites and select different DNA binding sequences, with Prep associating mostly with promoters and house-keeping genes and Meis with promoter-remote regions and developmental genes. Hox target sequences associate strongly with Meis but not with Prep binding sites, while Pbx1 cooperates with both Prep and Meis. Accordingly, Meis1 shows strong genetic interaction with Pbx1 but not with Prep1. Meis1 and Prep1 nonetheless co-regulate a subset of genes, predominantly through opposing effects. Notably, the TALE homeoprotein binding profile subdivides Hox clusters into two domains differentially regulated by Meis1 and Prep1. After duplication of the ancestral gene, Meis and Prep thus specialized their interactions but maintained significant regulatory coordination. ChIP-seq of 3 TALE proteins in E11.5 C57BL/6 embryonic mice
2013-03-31 | E-GEOD-39608 | biostudies-arrayexpress
Project description:We use NGS to assess the ability of TALE-guided DNA methyltranferases to make targeted changes to DNA methylation Targeted bisulfite sequencing of cells infected with wild-type or mutant TALE-DNMT constructs directed to the CDKN2A (p16) locus
2015-05-01 | E-GEOD-66456 | biostudies-arrayexpress
