A double take on bivalent promoters

Abstract

Histone modifications and chromatin-associated protein complexes are crucially involved in the control of gene expression, supervising cell fate decisions and differentiation. Many promoters in embryonic stem (ES) cells harbor a distinctive histone modification signature that combines the activating histone H3 Lys 4 trimethylation (H3K4me3) mark and the repressive H3K27me3 mark. These bivalent domains are considered to poise expression of developmental genes, allowing timely activation while maintaining repression in the absence of differentiation signals. Recent advances shed light on the establishment and function of bivalent domains; however, their role in development remains controversial, not least because suitable genetic models to probe their function in developing organisms are missing. Here, we explore avenues to and from bivalency and propose that bivalent domains and associated chromatin-modifying complexes safeguard proper and robust differentiation.

Keywords: Polycomb; Trithorax; bivalent domains; chromatin; embryonic stem cells.

Figure 1.

Bivalent domains mark CpG-rich promoters of developmental genes in ES cells. Whereas promoters of housekeeping genes such as Polm are marked solely with H3K4me3, bivalent promoters carry both H3K4me3 and H3K27me3. The latter genes either are expressed at low levels or are silent in ES cells but may become activated upon differentiation to neural precursor cells (NPCs) or be expressed in cells of reduced differentiation potential, such as MEFs. Conversely, other genes may become silent upon differentiation. Promoters with low CpG content may be activated during differentiation by other means, as they lack H3K4me3 and H3K27me3 in ES cells, and their regulation is likely distinct from CpG-rich promoters. The ChIP-seq traces are based on data from Mikkelsen et al. (2007).

Figure 2.

Bivalent domains and heterogeneity. Two scenarios could potentially explain the co-occurrence of H3K4me3 and H3K27me3 observed by ChIP-seq on bivalent promoters. As ChIP-seq cannot establish physical co-occurrence of two marks on the same allele, admixture of cells that either express (green) or do not express (red) the gene in focus could explain the occurrence of both marks as well as the low expression level in the overall population. In contrast, in the case of “true” bivalency, virtually all cells in the population carry both marks simultaneously at the promoter in question, leading to low, if any, expression for that gene in all cells.

Figure 3.

Potential conformations of bivalent domains. At bivalent promoters, H3K4me3 and H3K27me3 marks may be present in different conformations. As illustrated in A, H3K4me3 and H3K27me3 may occupy neighboring nucleosomes in the vicinity of the TSS. Alternatively, both modifications may co-occupy the same nucleosome in either an asymmetric fashion featuring differentially modified copies of H3 within a single nucleosome (B) or a symmetric conformation involving H3 molecules that carry both modifications simultaneously (C). Recent data indicate that the latter conformation is unlikely to be present in cells, whereas an asymmetric conformation can be observed. See the text for details.

Figure 4.

Modes of H3K4 methyltransferase and PRC recruitment to CpG-rich promoters. (A) SET1A/B and MLL complexes are recruited in both transcription-independent and transcription-dependent ways. Mechanisms involve recognition of unmethylated CpG islands by CXXC domain proteins, interactions with the OGT/TET system, TFs, H3K4me3 itself, and the transcription machinery. (B) Recruitment of PRC2 likely relies on interactions with DNA, histones, histone modifications, auxiliary proteins, and ncRNAs. PRC1 is targeted by recognition of unmethylated CpG islands through CXXC proteins such as KDM2B (by binding to H3K27me3) and through interaction with TFs. The box denotes the histone and DNA modifications present in this figure as well as in Figures 5 and 6.

Figure 5.

A step-wise model for the generation of bivalent domains. MLL and SET1A/B complexes “prime” CpG-rich promoters with basal levels of H3K4me3. For recruitment mechanisms, see Figure 4A. Loci where suitable TFs and activators are present will experience reinforcement of H3K4me3 deposition through cotranscriptional means, leading to sustained activation (active loci). In the absence of activating TFs, H3K4 demethylases can counterbalance the activity of MLL/SET1 complexes, allowing PRC2 to methylate one copy of H3 at the promoter to generate a bivalent set of marks. At some bivalent loci, PRC2 is joined by PRC1, reinforcing gene repression as well as PRC recruitment in a feed-forward loop. See Figure 4 for a key of the histone modifications present in this figure.

Figure 6.

Histone mark-binding proteins at bivalent promoters. Several binding proteins recognizing H3K4me3 and H3K27me3 may be present at bivalent genes. TFIID, SAGA, and CHD1 complexes, among others, are recruited at least in part by H3K4me3. Cfp1 binds to H3K4me3 as a subunit of SET1A/B complexes, supporting further H3K4me3 methylation. H3K4me3, together with other factors, prevents DNMT3-mediated DNA methylation. On the other hand, H3K27me3 recruits certain PRC1 complexes but also reinforces binding of PRC2 itself through interaction with its EED subunit. PRC1 complexes compact chromatin and interfere with preinitiation complex (PIC) assembly. Some PRC1 complexes further catalyze H2.Aub1, which may impair FACT recruitment, among other effects. See Figure 4 for a key of the histone modifications present in this figure.

Figure 7.

Bivalency represents a dynamic equilibrium between activation and repression that keeps genes in a plastic, inducible state and at the same time increases robustness. (A) In the bivalent state, activating stimuli and repressive complexes counterbalance each other in a metastable equilibrium. Activating TFs in concert with H3K27 demethylases and H2.A-deubiquitinating enzymes (DUBs) shift the equilibrium toward activation, ultimately displacing repressive factors and converting bivalent loci to active ones. In contrast, removal of the activating stimuli that are present at low levels at bivalent domains shifts genes to a repressed state, a process that requires robust H3K4 demethylase activity. H3K9 methylation and DNA methylation may buttress repression. (B) For proper differentiation, genes need to be activated once a developmental signal reaches a certain threshold, while ensuring that significant expression of these genes does not occur prior to reaching that signal threshold. Only H3K4me3-marked genes such as housekeeping genes are constitutively active, regardless of signal levels. In contrast, fully repressed genes may require extremely high levels of signal for sufficient activation. Genes carrying neither active nor repressive marks may be induced, but the absence of repressive signals renders them vulnerable to noise and may lead to subthreshold activation. Bivalent genes exhibit the correct activation thresholds because higher levels of signal are required to displace the counterbalancing repressive factors.