Specificity quantification of Glucocorticoid receptor binding using high-throughput sequencing
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE69386 | GEO | 2015/07/01
SECONDARY ACCESSION(S): PRJNA285340
REPOSITORIES: GEO
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Project description:In this work, we used our recently developed method Spec-seq to characterize the binding specificity of Glucocorticoid receptor in vitro. This GR Spec-seq experiment has been run twice separately (Dec 2014 and Mar 2015) with different library compositions. The basic workflow is the same as our previous work for lac repressor published in Genetics 198.3 (2014): 1329-1343. Recombinant human GR protein was used to facilitate in vitro DNA-binding and separation experiments. Bound and Unbound DNA fragments were separated in EMSA gels, purified, barcoded for further Illumina sequencing. An initial experiment was performed using the putative consensus sequence: AGAACA GGG TGTTCT; randomized library 1: AGAACN NSN NGTTCT [diversity =512]; randomized library 2: AGAANN GGG NNNTCT [diversity = 1024]; randomized library 3: AGAACA GGG TGNNNN [diversity =256]; randomized library 4: AGAACA GGGC NNNTCT [diversity = 64]. The initial total library diversity was ~1853 with a library composition of 10% positive control sequence + randomized libraries 1-4 + 5% negative control sequence. A 5th library containing 2304 sequences based on DDNACW KKN KGTTCT, where D="not C", N="any base", W="A or T" and K="G or T" was subsequently prepared and analyzed using similar ratios of control sequences. Binding conditions for EMSA were 100ng FAM-labelled dsDNA+ 0/0.5/1/2/4uM GR DBD protein for each lane, 1X NEB buffer 4. GR DBD was prepared as previously described. The EMSA was performed using a 9% 33:1 acrylamide gel and TB buffer, and was run at 200V for 30mins @ 0 degrees C. The 2uM protein lane used for final sequencing. Bound/unbound fractions resulting from EMSA of these libraries and conditions were used to generate PWMs as described. The GR PWM that was generated through this analysis was used to define relative binding energies using the patser program (35), which can be accessed online at (http://stormo.wustl.edu/consensus/cgi-bin/Server/Interface/patser.cgi ). Derived binding affinities are proportional to the inverse of the natural log of the calculated energies.
2015-07-01 | E-GEOD-69386 | biostudies-arrayexpress
Project description:The glucocorticoid receptor (GR), is a hormone-activated transcription factor which binds to GR binding sequences (GBS) encoded in the genome. Interestingly, of the many genomic sequences matching the GR consensus sequence only a small subset is actually bound. This indicates that the presence of a GBS alone is insufficient to specify where in the genome GR binds. We identified sequences that can locally prevent GR binding and mass-spec analysis was used to identify proteins that bind to these sequences.
2016-03-30 | PXD002252 | Pride
Project description:BACKGROUND: Protein quantification is an essential step in many proteomics experiments. A number of labeling approaches have been proposed and adopted in mass spectrometry (MS) based relative quantification. The mTRAQ, one of the stable isotope labeling methods, is amine-specific and available in triplex format, so that the sample throughput could be doubled when compared with duplex reagents. METHODS AND RESULTS: Here we propose a novel data analysis algorithm for peptide quantification in triplex mTRAQ experiments. It improved the accuracy of quantification in two features. First, it identified and separated triplex isotopic clusters of a peptide in each full MS scan. We designed a schematic model of triplex overlapping isotopic clusters, and separated triplex isotopic clusters by solving cubic equations, which are deduced from the schematic model. Second, it automatically determined the elution areas of peptides. Some peptides have similar atomic masses and elution times, so their elution areas can have overlaps. Our algorithm successfully identified the overlaps and found accurate elution areas. We validated our algorithm using standard protein mixture experiments. CONCLUSIONS: We showed that our algorithm was able to accurately quantify peptides in triplex mTRAQ experiments. Its software implementation is compatible with Trans-Proteomic Pipeline (TPP), and thus enables high-throughput analysis of proteomics data.
| S-EPMC3044303 | biostudies-other
Project description:Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we show using selective isolation of chromatin-associated proteins that the protein network around chromatin-bound GR is affected by SUMOylation, with several nuclear receptor coregulators and chromatin modifiers being more avidly associated with SUMOylation-deficient than SUMOylation competent GR. This difference is reflected in our chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in opening chromatin at glucocorticoid-regulated enhancers and inducing expression of their target loci. Our results thus show that SUMOylation determines GR specificity by regulating the chromatin protein network and accessibility at GR-driven enhancers. We speculate that a similar mechanism is utilized by many other SUMOylated TFs.
2021-09-08 | PXD015183 | Pride
Project description:In this work we introduce a new high-throughput method, Spec-seq, that directly measures specificity by sequencing. It has several advantages over existing methods to quantify large collections (thousands) of binding site energies in one experiment. Using lac repressor as an example, we show that this method has excellent reproducibility giving energy measurements generally consistent within about 0.1kT. While not measuring in parallel as many different sequences as some of the higher throughput methods, we obtain high accuracies because we measure exactly what is necessary, the relative affinity to a large collection of sequences, without any mathematical fitting procedures or approximations required. It is similar to MITOMI in the number of different sites that can be analyzed in parallel, but it is much simpler to perform, requiring only a means to separate bound and unbound fractions which are then sequenced using standard high throughput, short read sequencing machines that are now readily available. When applied to the lac repressor we obtain, in a single experiment, data covering a large fraction of all the previous studies, plus thousands of additional variants, allowing us to compile a much more comprehensive profile of its specificity. We learn that the lac repressor can bind to sites of different lengths but that the preferred sequence, and the mode of binding, depends on the length. We also apply the method to two other members of the LacI/GalR protein family, PurR and YcjW, to obtain extensive models of their specificity and test the generality of the lac repressor's ability to bind to operators of variable length. We find that the lac repressor is apparently unique in its ability to bind with high affinity to sites of different lengths and with different modes of binding. 4 independent experiments.
2014-10-01 | E-GEOD-61223 | biostudies-arrayexpress
Project description:Purpose of these experiments was to assess the effect of glucocorticoid receptor SUMOylation on its protein-protein interactions using BioID. Experiments were performed with SUMOylation deficient GR3KR (K277,293,703R) fused to the BirA* biotin ligase tag.
2021-01-11 | PXD017713 | Pride
Project description:Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and glucocorticoid resistance in leukemia cells confers a poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 newly diagnosed ALL patients and found significantly higher expression of caspase 1 (CASP1) and its activator NLRP3 in glucocorticoid resistant leukemia cells, due to significantly lower somatic methylation of CASP1 and NLRP3 promoters. Over-expression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1 overexpressing ALL. Our findings establish a new mechanism by which the NLRP3/CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on glucocorticoid transcriptional response suggests this mechanism could also modify glucocorticoid effects in other diseases.
2015-03-24 | GSE67044 | GEO
Project description:The specificity of STAT1 transcription factor was determined quantitatively by measuring the relative binding affinity to hundreds of variants of the gonsensus site. The binding specities of eight mutants of STAT1 were also investigated. The mutants showed either overall reduced binding specificity or altered specificity accross the binding site. The effect of CpG methylaton within the binding site was also investigated, which demonstrated verry little effect on binding. A previously described method, Spec-Seq, was used to determine the binding specificititys of STAT1 and the mutants. Libreries of DNAs were enzymatically methylated and the same method was used for studying their binding affinities in comparison with the non-methylated counterparts. The proteins and DNAs were incubated for 30 min in in suitable buffer and the bound and unbound bands were resolved in 12% polyacrylamide gels. the DNAs were purified from the bound and unbound bands and sequenced.
2017-04-01 | GSE95526 | GEO
Project description:Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and glucocorticoid resistance in leukemia cells confers a poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 newly diagnosed ALL patients and found significantly higher expression of caspase 1 (CASP1) and its activator NLRP3 in glucocorticoid resistant leukemia cells, due to significantly lower somatic methylation of CASP1 and NLRP3 promoters. Over-expression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1 overexpressing ALL. Our findings establish a new mechanism by which the NLRP3/CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on glucocorticoid transcriptional response suggests this mechanism could also modify glucocorticoid effects in other diseases.
2015-03-24 | GSE67043 | GEO
Project description:Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and glucocorticoid resistance in leukemia cells confers a poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 newly diagnosed ALL patients and found significantly higher expression of caspase 1 (CASP1) and its activator NLRP3 in glucocorticoid resistant leukemia cells, due to significantly lower somatic methylation of CASP1 and NLRP3 promoters. Over-expression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1 overexpressing ALL. Our findings establish a new mechanism by which the NLRP3/CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on glucocorticoid transcriptional response suggests this mechanism could also modify glucocorticoid effects in other diseases.
2015-03-24 | GSE67042 | GEO