Exchange of GATA factors mediates transitions in looped chromatin organization at a developmentally regulated gene locus

Abstract

Enhancers can regulate designate promoters over long distances by forming chromatin loops. Whether chromatin loops are lost or reconfigured during gene repression is largely unexplored. We examined the chromosome conformation of the Kit gene that is expressed during early erythropoiesis but is downregulated upon cell maturation. Kit expression is controlled by sequential occupancy of two GATA family transcription factors. In immature cells, a distal enhancer bound by GATA-2 is in physical proximity with the active Kit promoter. Upon cell maturation, GATA-1 displaces GATA-2 and triggers a loss of the enhancer/promoter interaction. Moreover, GATA-1 reciprocally increases the proximity in nuclear space among distinct downstream GATA elements. GATA-1-induced transitions in chromatin conformation are not simply the consequence of transcription inhibition and require the cofactor FOG-1. This work shows that a GATA factor exchange reconfigures higher-order chromatin organization, and suggests that de novo chromatin loop formation is employed by nuclear factors to specify repressive outcomes.

Figure 1. Profile of GATA-1-ER and GATA-2 occupancy at the Kit locus

(A) Organization of the Kit locus. Numbers indicate distance in kb from the transcriptional start site. Shading delineates the transcribed region. Bars denote conserved GATA sites. Diamonds denote GATA elements within regions of high regulatory potential. Arrows denote sites of highest GATA factor occupancy. (B, C) ChIP analysis of GATA-1-ER in estradiol-treated G1E-ER4 cells (B) and G1E cells (C). (D, E) ChIP for GATA-2 in G1E cells (D), and in estradiol-treated G1E-ER4 cells (E). Results are averages of 3 independent experiments. Error bars represent standard deviation. IgG, isotype matched control.

Figure 2. ChIP assays of pol2

(A) and acetylated histone H3 (acH3, B) at the Kit locus in G1E cells (-GATA-1, striped bars) or estradiol-treated G1E-ER4 cells (+GATA-1, grey bars). Results are averages of 2 independent experiments.

Figure 3. The -114 kb region is a GATA-2 dependent enhancer

(A) Structure of the reporter constructs. Grey boxes represent regions that were placed upstream of the Kit core promoter (prom). (B) G1E and MEL cells were transfected with reporter constructs along with a plasmid expressing Renilla luciferase. The activity of the enhancer-less Kit promoter was set as 1. mt-114 represents a construct in which both GATA sites were destroyed. Results are averages of 3 independent experiments.

Figure 4. 3C analysis of the Kit locus

(A) Schematic of the Kit locus. Vertical bars demarcate sites bound by GATA-1 or GATA-2. Bgl II fragments used for 3C assay are indicated by Roman numerals. (B) 3C assay in G1E cells or estradiol-treated (21 hours) G1E-ER4 cells. Fragment V (vertical bar at position +5) served as “fixed” fragment. GATA-2⁺/GATA-1^- represents parental G1E cells. GATA-2^-/GATA-1⁺ represents estradiol-treated G1E-ER4 cells. Results are averages of 5 independent experiments. Error bars represent standard deviation. (C). Time course 3C analysis of the interaction between -114/+5 (left panel), +5/+58 (middle panel) and HS2/β-major (right panel) in G1E-ER4 cells treated with estradiol for 0, 4, 8, 16 and 24hrs. Results are averages of 4 (left panel), 3 (middle panel), and 3 (right panel) independent experiments.

Figure 5

A) 3C analysis of indicated fragments in NIH 3T3 cells. Results are averages of 3 independent experiments. B) 3C analysis of indicated BglII fragments in G1E cells in the presence or absence of 75μM DRB (6 hours). Results are averages of 2 independent experiments.

Figure 6. ChIP analysis of FOG-1 in G1E cells

(A) and in estradiol-treated G1E-ER4 cells (B). (C) anti-GATA-1 ChIP in estradiol-treated cells expressing GATA-1(V205M)-ER. (D) 3C analysis of -114/+5, +5/+58 in GATA-1(V205M)-ER expressing cells and G1E-ER4. Bars denote standard deviation and results are averages of 2 (GATA-1(V205M)-ER) and 5 (G1E-ER4) independent experiments.

Figure 7

Model of the chromosomal configuration of the Kit gene. In immature erythroid cells GATA-2 binds the -114 enhancer to form an activating chromatin loop. Upon maturation, GATA-1 replaces GATA-2 and increases the interaction among downstream elements at the expense of the enhancer/promoter interaction. Loss of the enhancer loop might contribute to repression of Kit. The effects of GATA-1 require FOG-1. The resolution limits of 3C preclude us from distinguishing whether the enhancer loops to the transcription start site or the +5 region. We favor the former possibility as enhancers commonly target promoter elements. The question mark represents possible intermediary protein complexes involved in loop formation.