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1.
Fig. 1.

Fig. 1. From: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements.

BRG1 binding, H3K4 trimethylation, and H3K27 trimethylation have distinct global distributions relative to transcriptional start sites. BRG1 binding regions and H3K4me3 and H3K27me3 were identified using ChIP-seq analyses and assigned to the nearest transcription start site (TSS) using CisGenome. The distance to the nearest TSS was calculated using Excel. The amount of binding and the number of modified regions over all genes were summed in each window using Excel. The transcription start site and direction are indicated by arrows. BRG1 binding data are from this study; H3K4me3 and H3K27me3 data were published previously (85). BRG1 binding is prominent at promoters and distal regions (10 to 100 kb), and H3K4 trimethylation is predominantly found from the TSS to kb +1, while H3K27 trimethylation is mostly distal from the TSS (3 to 300 kb). (A) BRG1 binding sites with respect to TSS. (B) H3K4 trimethylation with respect to TSS. (C) H3K27 trimethylation with respect to TSS.

Supriyo De, et al. Mol Cell Biol. 2011 April;31(7):1512-1527.
2.
Fig. 2.

Fig. 2. From: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements.

BRG1 binding positively correlates with gene activity in T helper cells. ChIP-seq and expression data were compared essentially as described previously (6). Genes were sorted based on gene expression values (Z scores) and binned into groups of 100 genes, and the average gene expression value for each bin was calculated. After BRG1 tags in binding regions were assigned to the nearest transcriptional start site, the BRG1 tags contained by the genes in each bin was counted, and the average value was calculated. BRG1 binding in naïve, Th1S, Th2S, and Th17S cells was graphed against gene expression for each bin. All graphs were scaled to the same tag frequency and Z score range to facilitate direct comparison. Gene expression data were published previously (85).

Supriyo De, et al. Mol Cell Biol. 2011 April;31(7):1512-1527.
3.
Fig. 11.

Fig. 11. From: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements.

Distal BRG1 binding sites in Gata3 are programmed by Stat6. (A) Standard ChIP was used to measure BRG1 binding in resting Th1, stimulated Th1, resting Th2, and stimulated Th2 cells. The samples were biological replicates of the ChIP-seq samples (but with different mice, with cells harvested on different days, etc.). x axis labels indicate distances in kilobases from promoter B: promoter A is at kb −10.2; CNS3 is at kb +1.7. Nfm is a negative-control locus for BRG1 in T cells (Nefm exon 1 in Fig. 8). The results of ChIP-seq for BRG1 from Fig. 7D are shown again to facilitate direct comparisons. (B) Standard ChIP was used to measure H3K4 trimethylation in the Gata3 locus in resting Th1, stimulated Th1, resting Th2, and stimulated Th2 cells. Chromatin from the same cells whose results are shown in panel A was analyzed at the same time, to facilitate direct comparisons. (C and D) Standard ChIP was used to measure BRG 1 binding (C) and H3K4 monomethylation (D) in the Gata3 locus in stimulated cells cultured under stimulated Th2 conditions and made from cells of wild-type (WT) mice (white bars) and STAT6-null mice (black bars). The same chromatin was analyzed in both panels to facilitate direct comparisons.

Supriyo De, et al. Mol Cell Biol. 2011 April;31(7):1512-1527.
4.
Fig. 10.

Fig. 10. From: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements.

BRG1 binding occurs at STAT binding sites. ChIP-seq profiles from Th2S cells for BRG1, STAT6, STAT5A, and STAT5B are shown. BRG1 data are from this study, while Stat6 data are from reference 86 and Stat5 data are from reference 42. Occupancy range values (y axis) are identical for all graphs to allow direct comparison (minimum tag frequency of 0, maximum tag frequency of 1.14 × 10−5). Asterisks indicate statistically significant binding regions identified using CisGenome. Each panel contains a scale bar for the x axis (genomic location). Gene exons are indicated as vertical bars with names on the side, and arrows indicate the direction of transcription. Gata3 promoter A is 10 kb upstream (rightward) of the indicated Gata3 gene. The BRG1 data from Fig. 6 are repeated here to facilitate direct comparison. The genomic coordinates represented (MM9 assembly) are as follows: (A) for Gata3, Chr2, nt 8700000 to 10000000; (B) for Il4, Il5, and Il13, Chr11, nt 53370000 to 53570000.

Supriyo De, et al. Mol Cell Biol. 2011 April;31(7):1512-1527.
5.
Fig. 5.

Fig. 5. From: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements.

Global activation-specific BRG1 binding. (A and B) Global redistribution of BRG1 binding. BRG1 binding regions were compared in Th2 cells (A) and Th1 cells (B). The numbers of BRG1 binding regions that are unique to stimulated cells, unique to resting cells, and shared are shown. The percentage for the regions unique to stimulated cells (Th2S and Th1S) represents the number of BRG1 binding regions unique for stimulated cells divided by the total number of BRG1 binding regions in stimulated cells. The percentage for regions unique to resting cells (uTh2 and uTh1) represents the number of BRG1 binding regions unique for resting cells divided by the total number of BRG1 binding regions in resting cells. (C) For each immediate-early gene, the ratio of BRG1 binding regions in stimulated versus resting Th2 cells was calculated; the horizontal line at 1 on the y axis indicates the position at which unchanged genes would be located. (D) Regulation of mRNA amounts by activation of Th2 cells. mRNA was prepared from resting and activated Th2 cells, synthesized with cDNA, and quantified using Q-RT-PCR. Each mRNA ratios represents the log2 amount of mRNA in activated cells divided by the amount in resting cells, so values greater than zero represent activation-induced genes (black bars) whereas genes expressed at higher levels in resting cells have values less than zero (light bars).

Supriyo De, et al. Mol Cell Biol. 2011 April;31(7):1512-1527.
6.
Fig. 4.

Fig. 4. From: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements.

Promoter CpG content and BRG1 binding in Th2S cells. Genes were grouped by promoter type as high CpG promoter (Hi CpG Pro), intermediate CpG promoter (Int CpG pro), or low CpG promoter (Low CpG pro) according to a previously published promoter CpG content list (47). Genes not found in that list are not shown as a separate type; “All pro” includes all 4 classes. Genes were also grouped by BRG1 binding class; “all” includes genes with at least 1 BRG1 binding region anywhere; “distal” includes genes that have at least one BRG1 binding region in the interval Mb −1 to kb −10 or kb +10 to Mb +1; “proximal” includes genes that have at least one BRG1 binding region in the interval kb −10 to kb −300 or kb +100 to kb 10; “promoter” includes genes that have at least one BRG1 binding region in the interval kb −300 to +100. For each BRG1 binding class, the height of a promoter class bar indicates the percentage of promoters of that type with BRG1 binding of that class. As shown in Table 1, BRG1 binding to genes as promoter sites and BRG1 binding to genes as distal sites are not mutually exclusive. High CpG promoter genes included those encoding Tbx21, Gata3, c-Maf, Ltβ, Rad50, Stat1, Stat3, Stat5a, Stat6, IL-12r-β2, and Ahr; intermediate CpG promoter genes included those encoding IFN-γ, IL-4, IL-5, IL-17a, IL-10, IL-2, FoxP3, TNF, Stat4, and Stat5b; low CpG promoter genes included Rorc, Il17f, Il13, Il21, Il3, Gmcsf, Ccr2, Ccr3, and Ccr5.

Supriyo De, et al. Mol Cell Biol. 2011 April;31(7):1512-1527.
7.
Fig. 9.

Fig. 9. From: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements.

BRG1 binding to miRNA loci is specific with respect to lineage and activation. BRG1 binding to miRNA loci was measured by ChIP-seq, and the results obtained for T helper subsets were compared. The BRG1 occupancy range values (y axis) are identical for all graphs to allow direct comparison (minimum tag frequency of 0, maximum tag frequency of 1.14 × 10−5). Asterisks indicate statistically significant BRG1 binding regions identified using CisGenome. Each panel contains a scale bar for the x axis (genomic location). Genes are indicated as boxes with names below, and arrows inside the boxes indicate the direction of transcription. Mature miRNA locations are marked as bars; the direction of transcription is indicated with arrows. Predicted transcriptional start sites are indicated with arrows (“T” represents T cell predictions [11], “C” represents cancer cell predictions [59]). CpG islands are marked as vertical bars. (A) For miR-146a, Chr11, nt 43157899 to 43217963. (B) For miR-155, Chr16, nt 84684385 to 84744449. (C) For miR-326, Chr7, nt 106670000 to 106730000. (D) For miRNA 181c, 181d, 23a, 27a, and 24-2 clusters, Chr8, nt 86690000 to 86750000.

Supriyo De, et al. Mol Cell Biol. 2011 April;31(7):1512-1527.
8.
Fig. 8.

Fig. 8. From: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements.

Tissue-specific BRG1 binding. (A) ChIP-seq profiles for Th2S cells are shown. BRG1 occupancy (y axis) is again plotted from the minimum tag frequency of 0 to a maximum tag frequency of 1.14 × 10−5. A scale bar indicates 20 kb along the x axis. No statistically significant BRG1 binding regions were identified using CisGenome in this interval for any T helper subset. Genes are indicated as arrows on the profiles, indicating the direction of transcription; Chr18, nt 35014000 to 35025000 (MM9) is shown. (B) BRG1 binding in mouse T cells was measured by standard ChIP and normalized to the input samples. The value representing 2 times the average control IgG IP result is indicated by the dashed line; Nfm is a negative-control locus in T cells (Nefm exon 1). The locus data represent details of features shown in panel A. This experiment was a biological replicate of the ChIP-seq whose results are shown in panel A (but with different mice, with cells harvested on different days, etc.). (C) BRG1 binding in mouse brain tissue was measured by standard ChIP and normalized to the input samples. The value representing 2 times the average control IgG IP result is indicated by the dashed line. RHS7 is a positive-control locus in Th2 cells. The locus data represent details of features shown in panel A.

Supriyo De, et al. Mol Cell Biol. 2011 April;31(7):1512-1527.
9.
Fig. 7.

Fig. 7. From: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements.

Lineage-specific BRG1 binding. Signature cytokines (A, C, and E) and transcription factors (B, D, and F) are shown. Th1-specific (A and B), Th2-specific (C and D), and Th17-specific (E and F) genes were compared. ChIP-seq profiles for naïve, Th1S, Th2S, and Th17S cells are shown for each gene; the uTh1 profile is shown for Th1 genes; and the uTh2 profile is shown for Th2 genes. BRG1 occupancy range values (y axis) are identical for all graphs to allow direct comparisons (minimum tag frequency of 0, maximum tag frequency of 1.14 × 10−5). Vertical arrows indicate lineage-specific BRG1 binding identified using CisGenome. Asterisks indicate statistically significant BRG1 binding regions identified using CisGenome. Each panel contains a scale bar for the x axis (genomic location). Genes are indicated as boxes with names below, and arrows inside the boxes indicate the direction of transcription. Note that the Rorc gene produces the ROR-γ mRNA; the ROR-γT (T-cell specific) transcript initiates from an internal, downstream promoter within the Rorc gene. Gata3 promoter A is 10 kb upstream of the indicated Gata3 gene. The genomic coordinates represented (MM9 assembly) are as follows: (A) for Ifnγ, Chr10, nt 117820000 to 117960000; (B) for Tbx21, Chr11, nt 96930000 to 97020000; (C) for Il4, Il5, and Il13, Chr11, nt 53370000 to 53570000; (D) for Gata3, Chr2, nt 8700000 to 10000000; (E) for Il17a and Il17f, Chr1, nt 20680000 to 20820000; (F) for Rorc, Chr3, nt 94140000 to 94230000.

Supriyo De, et al. Mol Cell Biol. 2011 April;31(7):1512-1527.
10.
Fig. 6.

Fig. 6. From: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements.

Activation-specific BRG1 binding at specific genes. (A and D) ChIP-seq profiles for resting (uTh1 and uTh2) and activated (Th1S and Th2S) cells are shown. The BRG1 occupancy values (y axis) are identical for all graphs to allow direct comparison (minimum tag frequency of 0, maximum tag frequency of 1.14 × 10−5). Asterisks indicate statistically significant BRG1 binding regions identified using CisGenome. Genes are indicated as boxes below the profiles, and the arrows inside the boxes indicate the direction of transcription. (A) For the Egr1 locus, Chr18, nt 35010000 to 35030000 (MM9) are shown. A scale bar indicates 2 kb along the x axis. Downward pointing arrows indicate regions analyzed by standard ChIP as shown in panels B and C. (D) For the Pdcd4 locus, Chr19, nt 53950000 to 54020000 (MM9) are shown. A scale bar indicates 10 kb along the x axis. (B) BRG1 binding was measured by standard ChIP, and the results were normalized to the input samples. Control IgG ChIP results were similar to those obtained with Nfm, a negative-control locus. The y axis labels indicate distances from the Egr1 TSS in kilobases. This experiment was a biological replicate of the ChIP-seq experiment whose results are depicted in Fig. 6A (but performed with different mice, with cells harvested on different days, etc.). (C) H3S10pK14ac levels were measured by standard ChIP and normalized to the input samples. Control IgG IP results were similar to those obtained with Nfm (Nefm exon 1), a negative-control locus. The locus position is indicated with respect to distance from the Egr1 TSS in kilobases. This experiment was performed using the same chromatin used for the BRG1 ChIP whose results are shown in panel B.

Supriyo De, et al. Mol Cell Biol. 2011 April;31(7):1512-1527.
11.
Fig. 3.

Fig. 3. From: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements.

Distal and proximal BRG1 binding sites positively correlate with gene activity. (A) Highly expressed genes have more BRG1 binding regions. The number of BRG1 binding regions was counted for each gene. The percentage of genes that have the indicated number of binding regions was graphed for all genes (19,050 genes), “high” genes (the 2,032 genes with the most abundant mRNA), and “low” genes (the 1,208 genes with the least abundant mRNA). A total of 3.9% of the high genes had 7 or more BRG1 binding regions versus 0.08% of the low genes. (B) Genes with the lowest level of expression are more likely to lack BRG1 binding regions. A total of 80% of the low genes had no BRG1 binding versus 28% of the high genes. (C) Distal BRG1 binding and proximal BRG1 binding positively correlate with gene activity. Genes were sorted based on gene expression values (Z scores) and binned into groups of 100 genes, and the average gene expression level for each bin was calculated. BRG1 tags in binding regions were assigned to the nearest transcriptional start site, except for the proximal sites, for which only BRG1 binding from kb −10 to kb +10 with respect to the TSS was counted, while for distal sites only BRG1 binding from kb −100 to kb −10 and kb +10 to kb +100 kb was counted. BRG1 binding was graphed against gene expression for each bin. All graphs were scaled to the same tag frequency and Z score range to facilitate direct comparison. Gene expression data were published previously (85). (D) BRG1 binding is enriched in highly expressed genes at all distances relative to the TSS. The percentages of genes in each category with a BRG1 binding region at the indicated interval from the TSS are graphed. At every interval, the high genes are overrepresented and the low genes are underrepresented relative to all genes.

Supriyo De, et al. Mol Cell Biol. 2011 April;31(7):1512-1527.
12.
Fig. 12.

Fig. 12. From: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements.

Novel BRG1 binding sites in the Gata3 locus are in open chromatin and possess enhancer-like activity. (A, B, and C) Distal BRG1 binding sites have an open chromatin conformation. Nuclei from stimulated Th1 and Th2 cells were digested with DNase I; DNA was quantified using Q-PCR primers detecting the indicated regions. For each region, a representative dose response is shown; similar results were obtained by averaging three biological replicates digested with 8 μg/ml DNase I (data not shown). (A) Distal BRG1 binding regions are DHS in Th2 cells but not in Th1 cells. (B) Control regions lacking BRG1 binding are not DHS. (C) A control region with BRG1 binding in Th1 and Th2 cells is DHS in both. (D and E) Distal BRG1 binding sites possess enhancer-like activity. The indicated elements were placed immediately upstream (D) or 2.2 kb downstream (E) of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). (F) Enhancer-like activity is BRG1 dependent. The indicated elements were placed upstream of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. Cells were treated with BRG1 shRNA or a control, as indicated. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). (G) Distal BRG1 binding sites lack enhancer-like activity in a nonepisomal vector. The indicated elements were placed upstream of the SV40 promoter in the pGL3 promoter vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. The horizontal line at 1 on the y axis indicates the value obtained with the promoter alone. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). These same elements exhibited increased expression using episomal reporters (D and E). None of the five tested regions were positive for enhancer-like activity in this assay. (H) Distal regions without BRG1 binding in Th2 cells lack enhancer-like activity. The indicated elements were placed upstream of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with stimulation. The horizontal line at 1 on the y axis indicates the value obtained with the promoter alone. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values. All four tested regions lacking BRG1 binding were negative for enhancer-like activity in this assay.

Supriyo De, et al. Mol Cell Biol. 2011 April;31(7):1512-1527.

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