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. 2009 Feb 17;106(7):2259-64.
doi: 10.1073/pnas.0806420106. Epub 2009 Jan 26.

BRG1 requirement for long-range interaction of a locus control region with a downstream promoter

Shin-Il Kim  1 , Scott J Bultman, Christine M Kiefer, Ann Dean, Emery H Bresnick
Affiliations

BRG1 requirement for long-range interaction of a locus control region with a downstream promoter

Shin-Il Kim et al. Proc Natl Acad Sci U S A. .

Abstract

The dynamic packaging of DNA into chromatin is a fundamental step in the control of diverse nuclear processes. Whereas certain transcription factors and chromosomal components promote the formation of higher-order chromatin loops, the co-regulator machinery mediating loop assembly and disassembly is unknown. Using mice bearing a hypomorphic allele of the BRG1 chromatin remodeler, we demonstrate that the Brg1 mutation abrogated a cell type-specific loop between the beta-globin locus control region and the downstream beta major promoter, despite trans-acting factor occupancy at both sites. By contrast, distinct loops were insensitive to the Brg1 mutation. Molecular analysis with a conditional allele of GATA-1, a key regulator of hematopoiesis, in a novel cell-based system provided additional evidence that BRG1 functions early in chromatin domain activation to mediate looping. Although the paradigm in which chromatin remodelers induce nucleosome structural transitions is well established, our results demonstrating an essential role of BRG1 in the genesis of specific chromatin loops expands the repertoire of their functions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Progressive assembly of a cell-specific chromatin loop. (A) Models of GATA-1-mediated chromatin loop assembly. Model 1, GATA-1 occupancy at the LCR induces looping; Model 2, simultaneous GATA-1 occupancy at the LCR and the βmajor promoter induces looping. Model 3, GATA-1 occupancy at the LCR precedes occupancy at the βmajor promoter and concomitant looping. (B) G1E-ER-GATA-1 cells were treated with β-estradiol at 25 °C for 48 h, and the culture temperature was then increased to 37 °C. At various times thereafter (in h), cells were harvested and analyzed. (C) ER-GATA-1-mediated activation of βmajor transcription. Real-time RT-PCR was used to analyze βmajor primary transcripts and mRNA in G1E-ER-GATA-1 cells under conditions indicated in B. Values were normalized by Gapdh mRNA (mean ± SE, three independent experiments). (D) Quantitative ChIP analysis of ER-GATA-1 occupancy at HS2 and the βmajor promoter in G1E-ER-GATA-1 cells under conditions indicated in B (mean ± SE, four independent experiments). (E) Murine β-globin locus organization. HSs are depicted as filled circles, and embryonic (Ey and βH1) and adult (βmaj and βmin) globin genes are depicted as boxes. The diagram depicts the 3C strategy. BglII fragments and primers are depicted as shaded rectangles and triangles, respectively. (F) Quantitation of BglII cleavage efficiencies at the indicated sites using real-time PCR. (G) 3C analysis of the proximity of a BglII fragment containing the LCR (HS2) relative to fragments containing the −84 and −45 kb regions lacking known regulatory elements or the βmajor promoter in G1E-ER-GATA-1 cells under conditions indicated in B (mean ± SE, three independent experiments). (H) Linear regression analyses of GATA-1 occupancy at the LCR versus looping (Top) and GATA-1 occupancy at the βmajor promoter versus looping (Bottom).
Fig. 2.
Fig. 2.
GATA-1 rapidly mobilizes BRG1 at the promoter. (A–D) Co-regulator-LCR interactions. Quantitative ChIP was used to measure FOG-1 (A), CBP (B), MED1 (C), and BRG1 (D) occupancy at the LCR (HS2) in G1E-ER-GATA-1 cells under conditions indicated in Fig. 1B. (E–K) Factor-promoter interactions. Quantitative ChIP was used to measure FOG-1 (E), CBP (F), MED1 (G), BRG1 (H), Pol II and Ser-5-Pol II (J), and EKLF (K) occupancy at the βmajor promoter in G1E-ER-GATA-1 cells under conditions indicated in Fig. 1B. Quantitative ChIP analysis (in relative units) of BRG1 occupancy at the LCR (HS2), βmajor promoter, and necdin promoter (negative control) in G1E-ER-GATA-1 cells under conditions of Fig. 1B (I) (mean ± SE, three or four independent experiments). The shaded area indicates the range of times in which maximal factor occupancy is achieved. (L) Linear regression analysis of EKLF versus BRG1 occupancy at the βmajor promoter.
Fig. 3.
Fig. 3.
BRG1 requirement for chromatin looping. (A) 3C strategy: BglII fragments and primers are depicted as shaded rectangles and triangles, respectively. (B–D) 3C analysis of higher-order structure. The proximity of a BglII fragment containing the LCR (HS2) was measured relative to fragments lacking known regulatory regions (−84 kb, −45 kb, and 3′ of βminor), as well as the βh1 and βmajor promoters, using the following samples: deproteinized BAC DNA (B), untreated or β-estradiol-treated G1E-ER-GATA-1 cells (C), and E12.5 fetal liver cells from WT or BRG1 mutant (MT) mice (D) (mean ± SE, three independent experiments). The vertical dotted line denotes HS2. (E) Real-time PCR quantification of BglII cleavage efficiencies. Untreated or β-estradiol-treated G1E-ER-GATA-1 cells (Top); WT or BRG1 MT fetal liver cells (Bottom).
Fig. 4.
Fig. 4.
Mechanism underlying BRG1-dependent looping. (A) Impaired looping in Brg1-mutant cells is not associated with down-regulation of GATA-1, FOG-1, LDB1, and EKLF. Real-time RT-PCR analysis of mRNA levels in WT or Brg1-mutant fetal liver cells at embryonic day 12.5. mRNA levels were normalized to 18S rRNA. Each graph depicts the relative expression of a given gene in WT versus mutant samples [mean ± SE, two to five (Eklf mRNA) independent experiments]. (B) Quantitative ChIP analysis of EKLF occupancy at the LCR (HS2), βmajor promoter, and Ey promoter in WT or BRG1 mutant (MT) fetal liver cells at embryonic day 12.5 (mean ± SE, two independent experiments). IgG, mouse IgG. (C) Co-immunoprecipitation of ER-GATA-1 (arrow) and endogenous BRG1 in untreated and β-estradiol-treated (24 h) G1E-ER-GATA-1 cells. (D) BRG1-independent chromatin loop at c-Kit. The diagram depicts the 3C strategy (Top). BglII fragments and primers are depicted as shaded rectangles and triangles, respectively. The graph depicts 3C results measuring the proximities of a BglII fragment containing the +5 kb region and a fragment containing the +58 kb region in fetal liver cells from WT and Brg1-mutant mice at embryonic day 12.5 (mean ± SE, three independent experiments). (E) BRG1-independent chromatin loop at Gata2. The diagram depicts the 3C strategy (Top). HindIII fragments and primers are depicted as shaded rectangles and triangles, respectively. The graph depicts 3C results measuring the proximities of a HindIII fragment containing the −77 kb region and fragments containing either the 1S promoter or the +9.5 kb region in fetal liver cells from WT and Brg1-mutant mice at embryonic day 12.5 (mean ± SE, two independent experiments). (F) Model depicting BRG1 as a mediator of GATA-1-dependent looping. I, The LCR complex assembles before the promoter complex and looping. II and III, GATA-1 rapidly induces BRG1 occupancy at the promoter. IV, BRG1 is required for looping, and looping occurs concomitantly with promoter complex assembly.
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