"SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes",

Shutao Cai, Charles C Lee, and Terumi Kohwi-Shigematsu

Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720, USA.
Correspondence should be addressed to Terumi Kohwi-Shigematsu: terumiks@lbl.gov

SATB1 (special AT-rich sequence binding protein 1) organizes cell type–specific nuclear architecture by anchoring specialized DNA sequences and recruiting chromatin remodeling factors to control gene transcription. We studied the role of SATB1 in regulating the coordinated expression of Il5, Il4 and Il13, located in the 200-kb T-helper 2 (TH2) cytokine locus on mouse chromosome 11. We show that on TH2 cell activation, SATB1 expression is rapidly induced to form a unique transcriptionally active chromatin structure at the cytokine locus. In this structure, chromatin is folded into numerous small loops, all anchored to SATB1 at their base. In addition, histone H3 is acetylated at Lys9 and Lys14, and the TH2-specific factors GATA3, STAT6 and c-Maf, the chromatin-remodeling enzyme Brg1 and RNA polymerase II are all bound across the 200-kb region. Before activation, the TH2 cytokine locus is already associated with GATA3 and STAT6, showing some looping, but these are insufficient to induce cytokine gene expression. Using RNA interference, we show that on cell activation, SATB1 is required not only for compacting chromatin into dense loops at the 200-kb cytokine locus but also for inducing Il4, Il5, Il13 and c-Maf expression. Thus, SATB1 is a necessary determinant for the hitherto unidentified higher-order, transcriptionally active chromatin structure that forms on TH2 cell activation.

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.

Figure 2. ChIP analysis of the TH2 cytokine locus in D10.G4.1 (TH2) cells uncovers SATB1 binding sequences (SBSs).

(a) Schematic representation of the TH2 cytokine locus with the positions of SATB1-binding sequences (SBS-C1 to SBS-C9), DNase I–hypersensitive sites (RHS and HS) [31, 36] and the primer sets designed for ChIP analysis (bars indicated by 1–24).
(b) Semiquantitative PCR (top) and real-time PCR (bottom) analyses of Sau3AI-digested, crosslinked chromatin from resting and activated D10.G4.1 cells purified by urea-gradient centrifugation, digested with Sau3AI and precipitated with anti-SATB1. PCR amplification was performed using primer sets 1–24, indicated below each panel. For the semiquantitative analysis, 28-cycle PCR amplification was performed with Sau3AI-digested genomic DNA, with Sau3AI-digested chromatin from resting and activated D10.G4.1 cells immunoprecipitated (IP) with anti-SATB1, or with preimmune serum as a control. For real-time PCR analysis, we determined the quantity of target sequence in IP DNA from resting (blue diamonds) and activated (pink squares) D10.G4.1 cells as a multiple of target sequence in input DNA (see Methods). We did not detect any amplification signals beyond the threshold at >45 cycles for all primer sets using the IP DNA from resting D10.G4.1 cells (data not shown). Similarly, only primer sets that generated PCR products from activated D10.G4.1 IP DNA in semiquantitative analysis gave rise to detectable amplification signals in real-time PCR, indicating that SATB1 binding in vivo occurs at specific sites in the TH2 cytokine locus. Bars indicate s.e.m. from three experiments.

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 beta-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 beta-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 beta-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 <15% at all points.

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 <15% at all points.

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 <15% at all points.

Figure 6. Association of TH2-specific factors, RNA polymerase II, Brg1 and acetylated histone H3 with the cytokine region.
(a) Map of locus and position of 24 primer sets used for real-time PCR.
(b–g) Real-time PCR results from the Sau3AI-digested crosslinked chromatin, purified through urea-gradient centrifugation and immunoprecipitated from either resting (blue) or activated (red) D10.G4.1 cells with the use of antibodies against GATA3 (b), STAT6 (c), c-Maf (d), RNA polymerase II (e), Brg1 (f) and acetylated histone H3 at K9/14 (g). Resting cell data from the upper panel of g are shown at a finer scale in the lower panel of g. TH2-specific GATA3 and STAT6 are essentially preassembled with the 200-kb TH2 cytokine locus in resting cells. Upon activation, their binding to the 200-kb locus is increased at the indicated specific sites (b,c). c-Maf, which was induced upon activation, became strongly associated across the 200-kb TH2 cytokine locus, not limited to the Il4 promoter region. RNA polymerase II also predominantly bound the 200-kb locus after activation. Brg1 binding across the 200-kb locus was increased on activation. The level of histone H3 acetylation at K9/14 was markedly increased on activation. However, before activation, histone H3 in the TH2 cytokine locus was already acetylated, albeit to a much lesser degree. We used D10.G4.1 cells freshly purchased from ATCC for our experiments and avoided many cell passages. To obtain resting D10.G4.1 cells, we harvested cells 2 weeks after stimulation with APC. Our results contrast with results from others, who harvested cells 5 d after stimulation and obtained higher histone acetylation levels for resting cells [34]. Data indicate s.e.m. from three experiments.

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.

Figure 8. Summary of activation-dependent looping events and a model of transcriptionally active chromatin.
(a) Summary of 3C and ChIP-loop assays of the cytokine regions in resting and activated D10.G4.1 cells. Black lines connecting two positions indicate juxtaposition of these sites in resting cells. Pink lines represent two positions that show greatly increased crosslinking frequencies after activation. Red lines represent two positions that are brought into close proximity upon activation (based on both 3C and ChIP-loop assays). Black vertical arrowheads show direct SATB1-binding sites. The crosslinking frequency for each ligation product generated between any two positions is represented by the peak of the parabola connecting the positions.
(b) A schematic diagram based on the looping events shown in a, assuming that all looping events can occur in a single cell. In this model, all small loops converge on a common core base bound to SATB1 (blue spheres). As a consequence, the total physical volume of the active transcriptional complex is reduced, enhancing the accessibility of factors to genomic sites. Based on the urea-ChIP data (Fig. 2), which demonstrated that SATB1 binds predominantly to BURs in the 200-kb region and not in additional sites such as promoters, it is possible that at a given time point when chromatin was crosslinked, a large number of cells have SATB1 bound to BURs but that only a small subfraction of cells form the densely looped structure, giving rise to difference in the DNA template levels for PCR amplification. An alternative explanation is that indirect binding by SATB1 is much weaker than its direct binding and cannot be captured efficiently, thus requiring increased PCR amplification for detection.

Supplementary Fig. 1 (pdf 200K)
Cloning of genomic sequences that bind to SATB1 in vitro.

Supplementary Fig. 2 (pdf 1M)
ChIP loop assay and 3C assay to determine chromatin structure of the TH2 cytokine locus.

Supplementary Fig. 3 (pdf 3K)
Primer sequences and DNA fragment sizes.

Supplementary Fig. 4 (pdf 768K)
Immunolocalization of RNA polymerase II, c-Maf, STAT6, Brg1 and SATB1 in resting and activated D10.G4.1 cells.

Supplementary Fig. 5 (pdf 236K)
FISH analysis for the 200-kb cytokine region.
 

Nature Genetics - vol. 38, no. 11, pp. 1229 - 1230 (November, 2006)
doi:10.1038/ng1106-1229
http://www.nature.com/ng/journal/v38/n11/abs/ng1106-1229.html

News and Views

"Transcription in the loop",

Anita Göndör & Rolf Ohlsson

Anita Göndör and Rolf Ohlsson are at the Department of Development and Genetics, Evolution Biology Centre, Norbyvägan 18A, Uppsala University, SE-752 36 Uppsala, Sweden.
Anita.Gondor@ebc.uu.se

A new study shows that during transcription, the TH2 interleukin gene cluster is organized into several small chromatin loops connected at their base by the protein SATB1. This first detailed glimpse of chromatin folding provides a new perspective on the coordination of cell type–specific gene expression.

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4. Frenster JH, and Hovsepian JA, "Kissing Chromosomes and Paired Sense-Antisense RNA Synthesis", Cold Spring Harbor Symposium on Quantitative Biology, vol. 71, 62 (May, 2006).

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