The complex roles of histone demethylases in vivo

Eukaryotic genomes are organized into complex chromatin structures. The dynamic regulation of these structures is essential for development and their alterations have been implicated in diseases such as cancer. The combination of post-translational modifications in histones tails determines chromatin structure and influences the transcriptional status of genes.¹ Despite the tremendous progress made towards understanding the biochemical and cellular function of the enzymes responsible for adding these marks, the more difficult and important challenge to understand how their enzymatic activities are integrated in vivo to control biological processes is still largely unmet. In mammals, the analysis of the function of histone modifying enzymes is complicated by the presence of multiple orthologs of each protein. With its streamlined genome and the availability of powerful genetic tools, Drosophila provides an ideal model system for the study of chromatin dynamics in vivo. The large collections of mutants, deficiencies and inducible transgenic RNAi lines makes it possible to target the function of genes alone or in combination and to dissect the networks of chromatin regulation in the context of a whole organism. To this end, we have recently examined the effects of combining mutations in the histone H3K4 demethylases Lid and dLsd1.² Surprisingly, despite causing an increase in H3K4 methylation levels, Lid mutations strongly suppress dLsd1 mutant phenotypes. Investigation of the basis for this antagonism revealed that Lid opposes the functions of dLsd1 in promoting heterochromatin spreading. Moreover, these data unveiled a role for dLsd1 in Notch signaling in Drosophila and a network of interactions between dLsd1, Lid and Notch at euchromatic genes.²

These results, together with previous findings, support a model for the creation and maintenance of heterochromatin boundaries, in which dLsd1 demethylates H3K4me1,² promoting deacetylation of H3K9 by RPD3 ³ and subsequent methylation of H3K9 by Su(var)3–9, thereby facilitating spreading of heterochromatin. Interestingly, our study suggests that Lid opposes the spreading of heterochromatin by preventing dLsd1/Su(var)3–9/Rpd3 complex-dependent H3K9 methylation,² possibly by antagonizing Rpd3 histone deacetylase function on the H3K9 residues (Fig. 1).^4,5 In this context, Lid function appears to be independent of its histone H3K4 demethylase activity. Intriguingly, although Lid is essential for development, its histone demethylase activity is not.⁶

Figure 1.

A hypothetical model for the interplay between Lid and dLsd1 at euchromatin-heterochromatin boundaries and at Notch target genes.

Furthermore, the interplay between Lid and dLsd1 is not limited to chromatin boundaries but also extends to euchromatic genes, where H3K4 methylation is prevalent. Previous studies had implicated Lid as a crucial factor in the silencing of Notch target genes.⁷ Our study shows that dLsd1 can also repress Notch target gene expression and suggests that Lid and dLsd1 cooperatively contribute to repression by maintaining low levels of H3K4 methylation.² Repression of Notch target genes is essential for the establishment of Notch-inhibited cell fates,⁸ suggesting that Lid and dLsd1 could play a role in proper cell fate specification during Drosophila development. Interestingly, analysis of compound mutant flies suggests that, in a context in which the Notch signaling pathway is active, dLsd1 switches from a repressor to an activator role (Fig. 1).² Such a switch could be dependent on the availability of activating complexes triggered by Notch activation. In support of this hypothesis, a study in mammalian cells has shown that changes in composition of the complexes present at the Gh promoter modulate LSD1 activity and, depending on the cell type, LSD1 can act either as a co-repressor or as a coactivator.⁹ Alternatively, dLsd1 mutation could promote de-repression of negative regulators of Notch activity or could directly modulate Notch activity by demethylating components of the Notch activating complex. Further studies are required to distinguish between these possibilities and to determine if the dual role of dLsd1 in modulating Notch signaling is conserved in mammals. In mice, LSD1 has been shown to repress the Notch target Hey1 in late stages of pituitary development.⁹ Furthermore, a very recent study shows the presence of a SIRT1-LSD1 corepressor complex at Notch target genes in human cell lines,¹⁰ suggesting that LSD1 ability to regulate Notch target genes is conserved.

Our study underscores the importance of the chromatin context of demethylase targets as well as the status of key signaling pathways. It also highlights how an in vivo approach can reveal the complex network of interactions between histone demethylases, which would otherwise be difficult to predict by considering only their biochemical properties. Future steps will be to test whether the crosstalk between Lid and dLsd1 in the control of higher order chromatin structure at euchromatin-heterochromatin boundaries is conserved in mammals and to explore the possible involvement of these interactions in diseases. Deregulation of heterochromatin can increase cancer susceptibility by the aberrant silencing of tumor-suppressor genes and by increasing genomic instability;¹¹ we speculate that the human orthologs of Lid and dLsd1 may play a role in tumorigenesis by controlling heterochromatin homeostasis. Additionally, the modulating effects of LSD1 on the Notch signaling pathway could potentially be exploited to create novel strategies to manipulate Notchmediated carcinogenesis.

In conclusion, Drosophila research continues to provide invaluable tools to dissect the molecular pathways that contribute to chromatin plasticity in vivo, paving the way for the study of parallel pathways in mammalian systems.

Comment on: Di Stefano L, et al. Genes Dev. 2011;25:17–28. doi: 10.1101/gad.1983711.