Engineering an allosteric transcription factor to respond to new ligands
Ontology highlight
ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE75009 | GEO | 2015/12/20
SECONDARY ACCESSION(S): PRJNA302178
REPOSITORIES: GEO
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Project description:Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consist- ing of multiple zinc finger domains are currently be- ing developed for clinical applications. However, no genome-wide investigations into their binding speci- ficity have been performed. We have created six- finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB do- main (SKD) that interacts with a complex contain- ing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites ∼5- fold, with a majority of the new sites located out- side of promoters. De novo motif analyses suggest that the lack of binding specificity is due to sub- sets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger bind- ing sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications.
2014-08-01 | GSE59980 | GEO
Project description:Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consist- ing of multiple zinc finger domains are currently be- ing developed for clinical applications. However, no genome-wide investigations into their binding speci- ficity have been performed. We have created six- finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB do- main (SKD) that interacts with a complex contain- ing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites M-bM-^HM-<5- fold, with a majority of the new sites located out- side of promoters. De novo motif analyses suggest that the lack of binding specificity is due to sub- sets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger bind- ing sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications. ATF plasmids were designed and stably integrated into MCF7 cells as described in (Stolzenburg et al. 2012). Stable lines were grown at 30M-bM-^@M-^S80% confluency in DulbeccoM-bM-^@M-^Ys Modified EagleM-bM-^@M-^Ys Medium (Corning, Corning, NY) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Life Technologies, Grand Island, NY) and 1% penicillin/streptomycin; cells were selected using 5 M-BM-5g/ml puromycin (VWR, Radnor, PA) and 200 M-BM-5g/ml G418 (VWR, Radnor, PA). ATF expression was induced by treatment with media containing 1M-BM-5g/ml doxycycline (VWR, Radnor, PA) at 0 h, doxycycline media was refreshed at 48 h, and cells were harvested at 72 h. ATF expression was confirmed by hemagglutinin (HA) tag western blot prior to HA ChIP-seq, histone ChIP-seq and RNA-seq analysis. For HA-tag ChIP-seq, stable MCF7 cell lines were induced using 100 ng/ml doxycycline (Sigma) at 0 h, doxycycline media was refreshed at 48 h, and cells were harvested at 72 h by crosslinking in a final concentration of 1% formaldehyde. Crosslinking was stopped after 5 min by adding glycine to a final concentration of 125 mM. Crosslinked cells were washed in cold phosphate buffered saline, lysed using 1 mL low-salt IP buffer (150 mM NaCl, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, NP-40 (0.5%), Triton X-100 (1%) containing protease inhibitors) and aliquoted at 1 M-CM-^W 10M-bM-^HM-'7 cells/mL. Cells are then sonicated to a fragment size range of 500M-bM-^@M-^S800 bp. Samples were then diluted in 1 mL low-salt buffer and incubated with 3 M-BM-5L of anti-HA antibody (Covance, Princeton, NJ). Three-hundred microliter Sepharose A beads (GE Healthcare Life Science) were used for pull-down. Samples were sequenced at the UNC-CH Genome Analysis Facil- ity (Chapel Hill, NC) on an HiSeq (Illumina, San Diego, CA) to read counts of 4.1M-bM-^@M-^S67.3 M total reads. For histone ChIP-seq, antibodies to H3K4me3 (Cell Signaling Tech- nologies CST9751), H3K9Ac (Cell Signaling Technologies CST9649) and H3K9me3 (Diagenode pAb-056-050) were used; samples were prepared as previously described (OM-bM-^@M-^YGeen et al. 2011).
2014-08-01 | E-GEOD-59980 | biostudies-arrayexpress
Project description:We tested various architectures of artificial transcription factors (ATFs) in HEK293 cells to test the contribution of the interaction domain, nuclear localization domain, size of DNA-binding domain, and activation domain. Then we made a genome-scale ATF library and tested it in reprogramming mouse embryonic fibroblasts to induced pluripotent stem (iPS) cells. Three combinations of ATFs (C2, C3, and C4) could induce pluripotency when expressed with Sox2, Klf4, and c-Myc. We harvested polyadenylated RNAs from 11 cell types derived with different sets of factors. The transcriptional profiles of ATF-induced iPS cells are similar to that of iPS cells induced with Oct4, Sox2, Klf4, and c-Myc and mouse embryonic stem cells, exhibiting up-regulation of pluripotency markers and down-regulation of fibroblast markers. Comparisons of cells undergoing reprogramming at an intermediate stage before becoming fully reprogrammed suggest that ATFs activate a different set of genes than the set activated by Oct4. This study provides a proof-of-principle that a gene-activating ATF library can be used to identify cell fate-defining transcriptional networks in an unbiased manner.
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Project description:Allosteric communication between distant sites in proteins is central to nearly all biological regulation but still poorly characterised for most proteins, limiting conceptual understanding, biological engineering and allosteric drug development. Typically only a few allosteric sites are known in model proteins, but theoretical, evolutionary and some experimental studies suggest they may be much more widely distributed. An important reason why allostery remains poorly characterised is the lack of methods to systematically quantify long-range communication in diverse proteins. Here we address this shortcoming by developing a method that uses deep mutational scanning to comprehensively map the allosteric landscapes of protein interaction domains. The key concept of the approach is the use of ‘multidimensional mutagenesis’: mutational effects are quantified for multiple molecular phenotypes—here binding and protein abundance—and in multiple genetic backgrounds. This is an efficient experimental design that allows the underlying causal biophysical effects of mutations to be accurately inferred en masse by fitting thermodynamic models using neural networks. We apply the approach to two of the most common human protein interaction domains, an SH3 domain and a PDZ domain, to produce the first global atlases of allosteric mutations for any proteins. Allosteric mutations are widely dispersed with extensive long-range tuning of binding affinity and a large mutational target space of network-altering ‘edgetic’ variants. Mutations are more likely to be allosteric closer to binding interfaces, at Glycines in secondary structure elements and at particular sites including a chain of residues connecting to an opposite surface in the PDZ domain. This general approach of quantifying mutational effects for multiple molecular phenotypes and in multiple genetic backgrounds should allow the energetic and allosteric landscapes of many proteins to be rapidly and comprehensively mapped.
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Project description:Ovarian cancer is the leading cause of death among gynecological malignancies. It is usually detected at late stages when the disease is spread through the abdominal cavity in form of ascitic fluids. Thus, there is an urgent need to develop novel therapeutic interventions to target advanced stages of ovarian cancer, particularly metastatic disease. Mammary serine protease inhibitor (Maspin) represents an important metastasis suppressor initially identified in breast cancer. In this work we have generated a sequence-specific zinc finger artificial transcription factor (ATF) to up-regulate the Maspin promoter in aggressive ovarian cancer cell lines, to interrogate the therapeutic potential of Maspin in ovarian cancer. We found that while Maspin was expressed in some primary ovarian tumors, the promoter was epigenetically silenced in cell lines derived from ascites. Transduction of the ATF in MOVCAR 5009 cells derived from ascitic cultures of a TgMISIIR-Tag mouse model of ovarian cancer resulted in tumor cell growth, impaired cell invasion, and severe disruption of actin cytoskeleton. Systemic delivery of lipid-protamine-RNA (LPR) nanoparticles encapsulating a chemically modified ATF mRNA resulted in inhibition of ovarian cancer cell growth in nude mice, accompanied with Maspin re-expression in the treated tumors. Finally, gene expression microarrays of ATF-transduced cells revealed an exceptional potency and specificity for the Maspin promoter. These analyses revealed targets co-regulated with Maspin in human short-term cultures derived from ascitic fluids from ovarian cancer patients, such as TSPAN12, which could mediate the anti-metastatic phenotype of the ATF. Our work outlined the first targeted, non-viral delivery of designer ATFs into tumors with potential application in clinical trials for metastatic ovarian cancers. reference X sample with 6 samples
2012-09-29 | E-GEOD-36396 | biostudies-arrayexpress
Project description:We tested a genome-scale artificial transcription factor (ATF) library in reprogramming mouse embryonic fibroblasts to induced pluripotent stem (iPS) cells. Three combinations of ATFs (C2, C3, and C4) could induce pluripotency when expressed with Sox2, Klf4, and c-Myc. The transcriptional profiles of ATF-induced iPS cells are similar to that of iPS cells induced with Oct4, Sox2, Klf4, and c-Myc and mouse embryonic stem cells, exhibiting up-regulation of pluripotency markers and down-regulation of fibroblast markers. We performed ChIP-seq on the active histone mark H3K27ac and the repressive histone mark H3K9me3 in C2+SKM iPS cells, C3+SKM iPS cells, in Oct4+SKM to validate the total transcriptome results. In addition, the ATFs from C2 were chromatin immunoprecipitated to determine where they are bound in the genome at an intermediate stage before being fully reprogrammed to iPS cells. This study provides a proof-of-principle that a gene-activating ATF library can be used to identify cell fate-defining transcriptional networks in an unbiased manner.
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