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1.
FIGURE 2

FIGURE 2. From: Two-step binding of transcription factors causes sequential chromatin structural changes at the activated Interleukin-2 promoter.

The removal kinetics of histone H3 and H1 from the Il2 promoter following T cell activation. A and B, In ChIP assays using anti-histone H3 (A) or anti-histone H1 (B), the Il2 promoter region (PCR position: −77 to +39) was precipitated from PR-T cells either unactivated or activated hourly for up to 4 hrs.

Satoru Ishihara, et al. J Immunol. ;187(6):3292-3299.
2.
FIGURE 4

FIGURE 4. From: Two-step binding of transcription factors causes sequential chromatin structural changes at the activated Interleukin-2 promoter.

Expression profiles of transcription factors at the protein level before and during T cell activation, and the effect of CHX on the chromatin remodeling at the Il2 promoter. A, Western blotting experiments were performed on total lysates from resting and activated PR-T cells. Fos proteins are marked with a circle (c-Fos) or a triangle (FosB/Fra-2 as indistinguishable bands), all of which were newly synthesized following activation. B, The prevention of induction of Fos proteins in CHX-treated PR-T cells was confirmed by Western blotting. C, The SEVENS assay on 1 hr-activated PR-T cells revealed that their pretreatment with CHX resulted in an even distribution in the gradient of the Il2 promoter (PCR position: −77 to +39, closed columns), which is different from that seen in cells not treated with CHX (open columns). As a control, the enrichment of the Actb promoter in the upper gradient fractions in the presence of CHX is shown in Supplementary Fig. S2A.

Satoru Ishihara, et al. J Immunol. ;187(6):3292-3299.
3.
FIGURE 6

FIGURE 6. From: Two-step binding of transcription factors causes sequential chromatin structural changes at the activated Interleukin-2 promoter.

ChIP assays to examine the recruitment of the “late-binding” transcription factors and chromatin-related factors to the Il2 promoter (PCR position: -220 to -111). A, The culture conditions are represented in this key by four different colors. Red is cells without activation and drugs; blue is cells activated without drugs for 3 hrs; yellow is cells activated for 3 hrs in the presence of 10 nM FK506; and green is cells activated for 3 hrs in the presence of 30μM SP600125. B-E, The recruitment of the “late-binding” factors Rel A (B), c-Rel (C), Oct2 (D) and NFAT2 (E), was blocked by SP600125 as well as FK506. F, CBP, a coactivator with HAT activity, bound to the Il2 promoter upon T cell activation. Treatment with either FK506 or SP600125 blocked this recruitment. G, Histone H3 was acetylated at lysine 27 in the activated PR-T cells. Similar to the pattern for CBP, the level of acetylation was not increased when either of the drugs was added to the culture. These values are normalized by total amount of histone H3. H and I, TCR activation did not enhance Brg1 binding (H) or enrich for the presence of H2A.Z (I), at the Il2 locus.

Satoru Ishihara, et al. J Immunol. ;187(6):3292-3299.
4.
FIGURE 5

FIGURE 5. From: Two-step binding of transcription factors causes sequential chromatin structural changes at the activated Interleukin-2 promoter.

The effect of FK506 or SP600125 on the nucleosome rearrangement and occupancy of core and linker histones at the Il2 promoter. A, The prevention of dephosphorylation of NFAT1 in FK506-treated PR-T cells was confirmed by Western blotting. The arrow or the arrowhead denotes the dephosphorylated or the phosphorylated form of NFAT1, respectively. B, The prevention of phosphorylation of c-Jun in SP600125-treated cells was confirmed by Western blotting using anti-phopho-c-Jun (serine 63). C, The SEVENS assays for 3 hr-activated PR-T cells revealed that the pre-treatment with FK506 (yellow columns), but not SP600125 (green columns), blocked the enrichment of the Il2 promoter (PCR position: −77 to +39) in the upper fractions. For comparison, the distribution of the Il2 promoter in unactivated (red columns) or 3 hr-activated drug-non-treated cells (blue columns) is shown. The distribution of the Actb promoter was not affected by either of the drugs (Supplementary Fig. S2B). D, ChIP assays using anti-histone H3 (left panel) or H1 (right panel) showing that FK506 and SP600125 prevented core/linker histones from being removed from the Il2 promoter following 3 hr-activation (arrows and arrowheads, respectively). As controls, the constant level of these histones binding to the Actb or Adad1 promoters was monitored. Because of different lots of the Abs used, the actual values in this figure are not the same as those shown in Fig. 2

Satoru Ishihara, et al. J Immunol. ;187(6):3292-3299.
5.
FIGURE 3

FIGURE 3. From: Two-step binding of transcription factors causes sequential chromatin structural changes at the activated Interleukin-2 promoter.

The recruitment kinetics of transcription factors to the Il2 promoter region. A, Many transcription factors bind to the Il2 promoter in the 300 bp region upstream from the TSS. The binding sites of the ones examined are shown as colored ovals. The black box and arrow represent the 1st exon and transcription direction, respectively. B-H, The recruitment kinetics of transcription factors at the Il2 promoter in PR-T cells were analyzed with ChIP assays using Abs against RelA (B), c-Rel (C), Oct2 (D), NFAT2 (E), NFAT1 (F), Jun proteins (G), or Fra-2 (H). The activation led these transcription factors to be gradually recruited to the Il2 promoter (PCR position: −220 to −111) (closed columns). In each experiment the level of the Adad1 promoter in the precipitates was used as a control for non-specific binding in the ChIP assay (open columns). Colored ovals in each chart correspond to their binding sites in the Il2 promoter shown in A. I, The normalized recruitment kinetics for all the factors are summarized. Since the value of the Il2 promoter precipitated by all Abs in unactivated PR-T cells was close to that of the Adad1 promoter, this value was used as the “0%” background for the ChIP assay. The maximal level was generally observed in PR-T cells activated for 3 hrs (except for the Fra-2 ChIP, where it was 4 hrs). The percentage of the binding at these time points was designated “100%”.

Satoru Ishihara, et al. J Immunol. ;187(6):3292-3299.
6.
FIGURE 1

FIGURE 1. From: Two-step binding of transcription factors causes sequential chromatin structural changes at the activated Interleukin-2 promoter.

Expression profiles of IL-2 mRNA production and protein secretion compared to the recruitment kinetics of a Pol II complex to the Il2 promoter following T cell activation. A, IL-2-expressing cells were counted in a secretion capture assay. Naïve T cells or PR-T cells, which were unactivated or activated for 4 hrs, were stained with anti-IL-2 and anti-CD4 Abs. The percentages of IL-2-expressing CD4+ T cells are indicated in the upper right quadrants. B, The relative values of mRNA for Il2 and 4 control genes were calculated by qRT-PCR compared to a standard curve obtained using genomic DNA (see Materials and Methods). The PCR reactions were performed using primer sets annealing to a region within a single exon, which is shown as an ordinal number (1st to 5th) in the key. The results are shown as relative values compared to the signal for β-actin mRNA encoded by the 3rd exon in resting PR-T cells, which is designated as 100%. Because there were no signals in the PCR reactions using the primers for BDNF or Adad1, the possibility of contamination with genomic DNA was ruled out. C and D, The recruitment kinetics of Pol II (C) and TBP (D) were evaluated with ChIP assays. The Il2 promoter (PCR position: −77 to +39, closed columns) and the Adad1 promoter (open columns) regions were examined in the immunoprecipitates.

Satoru Ishihara, et al. J Immunol. ;187(6):3292-3299.

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