Rapid activation of the bivalent gene Sox21 requires displacement of multiple layers of gene-silencing machinery

Abstract

The rapid formation of numerous tissues during development is highly dependent on the swift activation of key developmental regulators. Recent studies indicate that many key regulatory genes are repressed in embryonic stem cells (ESCs), yet poised for rapid activation due to the presence of both activating (H3K4 trimethylation) and repressive (H3K27 trimethylation) histone modifications (bivalent genes). However, little is known about bivalent gene regulation. In this study, we investigated the regulation of the bivalent gene Sox21, which is activated rapidly when ESCs differentiate in response to increases in Sox2. Chromatin immunoprecipitation demonstrated that prior to differentiation, the Sox21 gene is bound by a complex array of repressive and activating transcriptional machinery. Upon activation, all identified repressive machinery and histone modifications associated with the gene are lost, but the activating modifications and transcriptional machinery are retained. Notably, these changes do not occur when ESCs differentiate in response to retinoic acid. Moreover, ESCs lacking a functional PRC2 complex fail to activate this gene, apparently due to its association with other repressive complexes. Together, these findings suggest that bivalent genes, such as Sox21, are silenced by a complex set of redundant repressive machinery, which exit rapidly in response to appropriate differentiation signals.

Figure 1.

Expression of Sox21 in mouse ESCs undergoing differentiation in response to elevated levels of Sox2. qPCR was performed on cDNA prepared from RNA isolated from untreated i-Sox2-ESCs or i-Sox2-ESCs treated with 4 μg/ml Dox for 9, 24, or 72 h, as described in Materials and Methods. Results are presented as fold-enrichment relative to GAPDH in Dox-induced cells vs. uninduced cells.

Figure 2.

Sox2 and Oct4 are associated with a region of the Sox21 gene that contains a putative HMG/POU cassette of the Sox21 gene in i-Sox2-ESCs. A) Schematic diagram of the Sox21 gene, including a putative enhancer containing an HMG/POU cassette that is located ∼5 kb downstream of the Sox21 promoter. B, C) ChIP analysis of Sox2 association (B) or Oct4 association (C) with a region within the Sox21 gene containing the putative HMG/POU cassette. ChIP was carried out before and 24 h after induction of differentiation in i-Sox2-ESCs via treatment with 4 μg/ml Dox. ChIP analyses were repeated and similar results were obtained. *P < 0.05.

Figure 3.

Sox21 loses its inhibitory bivalent domain after i-Sox2-ESCs are induced to differentiate by elevating Sox2 levels. A) Schematic diagram of the Sox21 gene indicating approximate location of primers (triangle) used in ChIP analysis. B–D) ChIP analysis to determine enrichment of H3K27me3 and IgG (B), H3K4me3 (C), and the PRC2 component Suz12 (D) on the Sox21 promoter region in i-Sox2-ESCs before and 24 h after induction of differentiation via treatment with 4 μg/ml Dox. Primers flanking the regions located ∼1.2 kb upstream and ∼250 bp downstream relative to the Sox21 TSS, as well as the Sox21 TSS, were utilized to determine enrichment of H3K4me3 and Suz12. Additional primers amplifying regions ∼1.9 kb upstream and ∼1.46 kb downstream of the Sox21 TSS were utilized to measure enrichment of H3K27me3 on the Sox21 gene. Representative dataset for enrichment at the Sox21 gene using a control IgG antibody is displayed in panel B. All ChIP analyses were repeated, and similar results were obtained. *P < 0.05.

Figure 4.

Multiple repressive complexes are associated with the Sox21 gene in mouse ESCs. A) Schematic diagram of the Sox21 gene indicating approximate location of primers (triangle) used in ChIP analysis. B–E) ChIP analysis of H2Aub (B), HDAC1 (C), HDAC2 (D), and H3Ac (E) on the Sox21 gene in i-Sox2-ESCs before and 24 h after i-Sox2-ESCs were treated with 4 μg/ml Dox. Sox21 TSS, and regions ∼1.2 kb upstream and ∼250 bp downstream relative to the Sox21 TSS, were monitored for occupancy of H2Aub, HDAC2, and H3Ac. HDAC1 association was monitored at the Sox21 TSS, the region ∼250 bp downstream of the Sox21 TSS, and the region containing the putative Sox21 HMG/POU cassette. ChIP analyses were repeated with similar results. Values of P > 0.05 are indicated next to specific bar graphs in panel B. *P < 0.05.

Figure 5.

Sox21 loses its bivalent domains 9 h after i-Sox2-ESCs are induced to differentiate by elevating Sox2. A) Schematic diagram of the Sox21 gene indicating approximate location of primers (triangle) used in ChIP analysis. B–G). ChIP analysis of H3K27me3 (B), Suz12 (C), H3K4me3 (D), H2Aub (E), HDAC1 (F), and HDAC2 (G) on the Sox21 gene in i-Sox2-ESCs before and 9 h after i-Sox2-ESCs were treated with 4 μg/ml Dox by ChIP analyses. H3K4me3, Suz12, H2Aub, and HDAC2 association was monitored at the Sox21 TSS and at regions 1.2 kb upstream and ∼250 bp downstream relative to the Sox21 TSS; H3K27me3 association was determined at 2 additional regions, ∼1.9 kb upstream and ∼1.4 kb downstream relative to the Sox21 TSS. HDAC1 association was determined utilizing primers flanking the region containing the putative Sox21 HMG/POU cassette. ChIP analyses were repeated with similar results. Values of P > 0.05 are indicated next to specific bar graphs in panel E. *P < 0.05.

Figure 6.

Sox21 gene remains bivalent when i-Sox2-ESCs are induced to differentiate using retinoic acid. A) Schematic diagram of the Sox21 gene indicating approximate location of primers (triangle) used in the ChIP analysis. B–E) ChIP analyses of H3K27me3 (B), PRC2 component Suz12 (C), HDAC1 (D), and H3K4me3 (E) were performed in i-Sox2-ESCs before and 24 h after treatment with 10 μM RA. Primers amplifying the Sox21 TSS and the regions ∼1.2 kb upstream and ∼250 bp downstream relative to the Sox21 TSS were utilized to determine the association of Suz12 and H3K4me3 with the Sox21 promoter region. Additional primers amplifying regions ∼1.9 kb upstream and 1.4 kb downstream relative to the Sox21 TSS were utilized to monitor enrichment of H3K27me3. HDAC1 enrichment was monitored at the putative Sox21 HMG/POU cassette region, the Sox21 TSS, and 250 bp downstream of the Sox21 TSS. ChIP analyses were repeated with similar results.

Figure 7.

Sox21 gene remains silent in Eed-null ESCs due to the presence of redundantly functioning repressive machinery. A) qPCR was performed on cDNA prepared from RNA isolated from Eed-null ESCs or untreated i-Sox2-ESCs to measure Sox21 expression. Results are presented as fold-enrichment relative to GAPDH in Eed-null ESCs and compared to i-Sox2-ESCs, which was arbitrarily set to 1. B) Schematic diagram of the Sox21 gene indicating approximate location of primers (triangle) used in ChIP analysis. C–G) ChIP analysis of HDAC1 (C), HDAC2 (D), H2Aub (E), H3K4me3 (F), and Sox2 and Oct4 (G) was performed in Eed-null ESCs. Primers amplifying the region containing the putative HMG/POU cassette of Sox21 were utilized to determine the association of HDAC1, Sox2, and Oct4 with the Sox21 gene. Enrichment of HDAC2, H2Aub, and H3K4me3 at the Sox21 promoter was monitored at the Sox21 TSS and the regions ∼1.2 kb upstream and ∼250 bp downstream relative to the Sox21 TSS.

Figure 8.

Model for bivalent gene regulation in ESCs: Bivalent, lineage-specific developmental regulatory genes remain silent in ESCs due to the presence of multiple, redundantly functioning repressive complexes, which likely collaborate to maintain a repressive environment to prevent gene activation. However, the coexistence of positive regulatory features at these genes allows for rapid activation during differentiation when appropriate signals for specific bivalent genes appear. This is facilitated by rapid exit of the repressive complexes from bivalent genes undergoing activation (A). The bivalent features remain associated with the gene when ESCs are induced to differentiate along a different pathway, and the gene remains inactive under these circumstances, which argues that the spurious activation of poised genes is prevented due to the presence of bivalent features which continue to allow the gene to remain in the poised state while preventing its inappropriate activation (B). Finally, crucial lineage-specific developmental regulators are kept under tight control via the existence of multiple layers of redundantly functioning mechanisms, and, in this way, loss of a single repressive complex is insufficient to allow gene activation due to the presence of other repressive mechanisms (C).