Journal: Nature genetics

Article Title: SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes.

doi: 10.1038/ng1913

Figure Lengend Snippet: Figure 1 Upon D10.G4.1 (TH2) cell activation, SATB1 expression is induced, and cytokine expression levels increase. (a) RNase protection assay on total RNA isolated from D10.G4.1 cells 0, 6 and 24 h after Con A activation. We compared expression of genes in the 200-kb TH2 cytokine locus before and after cell activation: levels of Il4, Il13 and Il5 transcripts increased markedly upon cell activation, whereas Rad50 and Kif3a expression levels remained low. (b) Immunofluorescence staining of D10.G4.1 cells using antibodies against SATB1 and GATA3. In resting cells, SATB1 expression is very low and cannot be detected by immunostaining, but it is detected in activated cells, where it shows a cage-like distribution. GATA3 is expressed in both resting cells and activated cells. After activation, SATB1 and GATA3 mostly colocalize.

Article Snippet: 1 28 6 VOLUME 38 [ NUMBER 11 [ NOVEMBER 2006 NATURE GENETICS 20 06 N at u re P u b lis h in g G ro u p h tt p :/ /w w w .n at u re .c o m /n at u re g en et ic s anti-SATB1 antibody (1583)18 and rabbit anti-acetylated H3 Lys9/14 (Upstate); mouse anti-RNA polymerase II (Abcam) and rabbit anti-c-Maf, mouse anti-GATA3, rabbit anti-STAT6 and rabbit anti-Brg1 (Santa Cruz Biotechnology), with pre-immune rabbit serum as a control.

Techniques: Activation Assay, Expressing, Rnase Protection Assay, Isolation, Staining, Immunostaining

Journal: Nature genetics

Article Title: SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes.

doi: 10.1038/ng1913

Figure Lengend Snippet: Figure 3 Chromatin loop analyses from SBS-C1 and SBS-C9 show dense SATB1-bound chromatin looping upon D10.G4.1 activation. SBS-C1 and SBS-C9 (red stars) interact with cytokine promoters, LCR and other regulatory elements in TH2 cytokine locus of D10.G4.1 cells (D10) depending on activation status. (a) Schematic representation of TH2 cytokine locus and positions of 20 Sau3AI DNA fragments used in ChIP assay are shown. SBS-C1 and SBS-C9 were used as fixed reference points (red stars). Forward and reverse primers were designed within these fragments. Black lines indicate DNA fragments located in spatially close proximity upon looping in resting cells. Pink lines indicate DNA fragments showing enhanced interactions upon cell activation. Red lines indicate positions brought to close proximity after activation. In activated cells, all DNA fragment interactions shown by black, pink and red lines are observed. (b) Relative crosslinking frequencies (indicated by blue bars in the histograms) between fragment 2 (SBS-C1) as a fixed reference point (indicated by red bar in each histogram) and the other fragments of the locus. Histograms are based on the ligation products by PCR (gels) produced by the 3C assay with resting and activated D10.G4.1 cells. Also shown are the results of the ChIP-loop assay using anti-SATB1 to immunoprecipitate Sau3AI-digested chromatin fragments from activated D10.G4.1 cells. (c) Similar to b, except that fragment 20 (SBS-C9) was used as a fixed reference point. In b and c, lane numbers correspond to DNA fragments numbered as shown in the map. A 100-bp ladder size marker (M) is shown in lane 2 (b) and lane 20 (c). Relative crosslinking frequency was calculated as described in Methods. Genomic DNA from cells subjected to 3C assay was amplified with primer pairs from different DNA fragments or S2 and S3 primers of the b-actin locus (Supplementary Fig. 2), and their PCR products are indicated as TH2 or actin, respectively. As a control used to correct for ligation and amplification efficiencies, a mixture of Sau3AI-digested and ligated BAC DNA covering the 200-kb region and the plasmid DNA containing the b-actin locus was subjected to PCR amplification with primers from DNA fragments in the 200-kb region or with S2 and S3 primers for actin, and their PCR products are indicated as TH2* or Actin*, respectively. For ChIP-loop assay, IP DNA was premixed with the plasmid DNA containing the b-actin locus to correct for the amount of DNA used in each PCR, and PCR products are indicated as Actin*. Experiments were repeated three times, and s.e.m. for relative crosslinking frequencies is o15% at all points.

Article Snippet: 1 28 6 VOLUME 38 [ NUMBER 11 [ NOVEMBER 2006 NATURE GENETICS 20 06 N at u re P u b lis h in g G ro u p h tt p :/ /w w w .n at u re .c o m /n at u re g en et ic s anti-SATB1 antibody (1583)18 and rabbit anti-acetylated H3 Lys9/14 (Upstate); mouse anti-RNA polymerase II (Abcam) and rabbit anti-c-Maf, mouse anti-GATA3, rabbit anti-STAT6 and rabbit anti-Brg1 (Santa Cruz Biotechnology), with pre-immune rabbit serum as a control.

Techniques: Activation Assay, Ligation, Produced, Marker, Control, Plasmid Preparation

Journal: Nature genetics

Article Title: SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes.

doi: 10.1038/ng1913

Figure Lengend Snippet: Figure 4 Rad50 intronic SBSs contribute to chromatin loop organization of TH2 cytokine locus in D10.G4.1 cells. (a) Schematic representation of TH2 cytokine locus and positions of 20 Sau3AI DNA fragments used in the ChIP assay. Positions used as fixed reference points are indicated by red stars. (b–f) Relative crosslinking frequencies between fixed fragments SBS-C3 (b), SBS-C4 (c), SBS-C5 (d), SBS-C6 (e) and the 3¢ end of the LCR (f) and other fragments of the locus, calculated from 3C assay on resting D10.G4.1 cells, 3C assay on activated D10.G4.1 cells and SATB1 ChIP-loop assay on activated D10.G4.1 cells. The fixed reference points are shown by red bars in the histograms. Lane numbers correspond to DNA fragments as shown in the map. All Rad50 intronic SBSs are involved in chromatin looping induced by activation, but interaction involving the position at the 3¢ end of the LCR remains unchanged after activation. Experiments were repeated three times, and s.e.m. for relative crosslinking frequencies is o15% at all points.

Article Snippet: 1 28 6 VOLUME 38 [ NUMBER 11 [ NOVEMBER 2006 NATURE GENETICS 20 06 N at u re P u b lis h in g G ro u p h tt p :/ /w w w .n at u re .c o m /n at u re g en et ic s anti-SATB1 antibody (1583)18 and rabbit anti-acetylated H3 Lys9/14 (Upstate); mouse anti-RNA polymerase II (Abcam) and rabbit anti-c-Maf, mouse anti-GATA3, rabbit anti-STAT6 and rabbit anti-Brg1 (Santa Cruz Biotechnology), with pre-immune rabbit serum as a control.

Techniques: Activation Assay

Journal: Nature genetics

Article Title: SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes.

doi: 10.1038/ng1913

Figure Lengend Snippet: Figure 5 TH2 cytokine gene promoters are juxtaposed on D10.G4.1 activation, but the Rad50 promoter is not involved. (a) Schematic representation of TH2 cytokine locus and positions of 20 Sau3AI DNA fragments used in ChIP assay. Positions used as fixed reference points are indicated by red stars. (b–e) Relative crosslinking frequencies (histograms) between fixed fragments of the Il5 promoter (b), Rad50 promoter (c), Il13 promoter (d) and Il4 promoter (e) (red stars) and other fragments of the locus, as determined by 3C assay on resting D10.G4.1 cells, 3C assay on activated D10.G4.1 cells and SATB1 ChIP-loop assay on activated D10.G4.1 cells. Lane numbers correspond to DNA fragments as shown in the map. Experiments were repeated three times, and s.e.m. for relative crosslinking frequencies is o15% at all points.

Article Snippet: 1 28 6 VOLUME 38 [ NUMBER 11 [ NOVEMBER 2006 NATURE GENETICS 20 06 N at u re P u b lis h in g G ro u p h tt p :/ /w w w .n at u re .c o m /n at u re g en et ic s anti-SATB1 antibody (1583)18 and rabbit anti-acetylated H3 Lys9/14 (Upstate); mouse anti-RNA polymerase II (Abcam) and rabbit anti-c-Maf, mouse anti-GATA3, rabbit anti-STAT6 and rabbit anti-Brg1 (Santa Cruz Biotechnology), with pre-immune rabbit serum as a control.

Techniques: Activation Assay

Journal: Nature genetics

Article Title: SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes.

doi: 10.1038/ng1913

Figure Lengend Snippet: Figure 7 SATB1 is required for Il4, Il5 and Il13 expression, induction of c-Maf and dense looping of active chromatin upon TH2 cell activation. (a) Protein blot analysis of parental D10.G4.1 cells, stably transfected with a control construct expressing nonspecific shRNA (nonsilencing shRNA) or an shRNA against Satb1 at 0 and 24 h after ConA treatment. (b) RNase protection analysis of cells described under a before and after ConA treatment at 6 and 24 h. (c) Immunostaining of parental D10.G4.1 cells and D10.G4.1 cells stably transfected with shRNA against Satb1 at 0 and 24 h after Con A activation. The cells were stained with DAPI (DNA; blue) and anti–c-Maf (c-Maf; green). (d) The 3C assay for D10.G4.1 cells stably transfected with the shRNA against Satb1 24 h after ConA activation. SBS-C1 (DNA fragment 2) and SBS-C9 (DNA fragment 20) (red stars) were used as fixed reference points in this 3C assay. The 3C assay was performed in the identical manner as described in Figure 3, except that here we used D10.G4.1-Satb1-shRNA cells, whereas in Figure 3, we used parental D10.G4.1 cells.

Article Snippet: 1 28 6 VOLUME 38 [ NUMBER 11 [ NOVEMBER 2006 NATURE GENETICS 20 06 N at u re P u b lis h in g G ro u p h tt p :/ /w w w .n at u re .c o m /n at u re g en et ic s anti-SATB1 antibody (1583)18 and rabbit anti-acetylated H3 Lys9/14 (Upstate); mouse anti-RNA polymerase II (Abcam) and rabbit anti-c-Maf, mouse anti-GATA3, rabbit anti-STAT6 and rabbit anti-Brg1 (Santa Cruz Biotechnology), with pre-immune rabbit serum as a control.

Techniques: Expressing, Activation Assay, Stable Transfection, Transfection, Control, Construct, shRNA, Immunostaining, Staining