LSD1 IMPACT · Dissecting the interaction network of the histone demethylase LSD1 in vivo

Dissecting the interaction network of the histone demethylase LSD1 in vivo

LSD1 IMPACT The casual relationships between histone modification and transcription are complex and incompletely understood. Decoding the rules that govern the dynamic regulation of chromatin organization in living cells, how chromatin contributes to gene expression during normal development and how its misregulation is implicated in cancer, are major biological questions. Our path to responding involves studying chromatin modulation by histone demethylation. Specifically, we focus on the histone lysine demethylase LSD1/KDM1a, which has in the past few years emerged as a key chromatin regulator. LSD1/KDM1A catalyzes the demethylation of mono and dimethyl marks at lysine (K) 4 and 9 of histone H3. K4 methyl marks are normally associated with actively transcribed genes and enhancers while K9 methylation is a repressive mark. Therefore, LSD1 dual activity towards these residues enables it to regulate both gene repression and activation. It has been hypothesized that association with specific co-factors can determine LSD1 substrate specificity. In support of this hypothesis, LSD1 has been found to promote gene silencing by removing activating methyl marks from H3K4 in association with the REST corepressor (coREST). In contrast, when LSD1 interacts with the androgen or estrogen receptor, it promotes transcriptional activation by demethylating the repressive H3K9me2 mark. How these complexes are assembled in response to specific signals, the tissue and temporal specificity of their targeting and the maintenance mechanisms deployed in vivo remain to be elucidated. Understanding the mechanisms underlying the tissue-specific roles of LSD1 is especially important given its crucial role in development and in cancer. Deletion of mouse LSD1 results in embryonic lethality and conditional knockdown of its activity in specific tissues or in embryonic stem cells causes differentiation defects. In addition, LSD1 is overexpressed in a wide variety of cancers. Further, its depletion or chemical inhibition can block the proliferation of tumor cell lines. However, evidence has been provided that LSD1 can also suppress metastasis. LSD1 role in development and cancer appears to be highly context dependent, therefore, a careful systematic analysis of the different biological effects of LSD1 inhibition is needed, especially considering its enormous potential for being used as a drug target. Despite the considerable progress of the past few years, our knowledge of the biological role(s) of LSD1, of the different LSD1 containing complexes in vivo, of their mechanisms of action at target genes and the signals modulating their activity, remain limited. To address these questions, we take advantage of a simple and yet very powerful model organism, Drosophila. We then examine to what extent the mechanisms identified in Drosophila are conserved in mammals, and deregulated in cancer. We have shown that mutation of the Drosophila LSD1 ortholog, dLsd1, results in oogenesis and wings defects and we have unveiled a novel interplay between dLsd1 and the histone demethylase Lid in the control of gene transcription and heterochromatin homeostasis. These studies revealed the crucial role of dLsd1 in the control of chromatin and transcription during Drosophila development and opened the road to more detailed analysis of dLsd1 specific role in these processes. We are therefore addressing with our studies three primary questions: 1) How is LSD1 activity controlled and in which context? 2) Where does LSD1 bind in the genome and how is LSD1 targeted to specific genomic regions? 3) How does LSD1 misregulation affect normal development and contribute to tumorigenesis? To answer these questions, we couple genetic screens, high-throughput genomic technologies, biochemical and in vivo assays. Specifically, we have performed a genetic screen in Drosophila to find novel modulators of LSD1 activity in vivo (WP1). This strategy has allowed us to identify around 200 candidates. Among the hits of the screen, we found two different subunits of the Pumilio translational regulator complex, Pumilio and Brat. The Pumilio complex is a multi-subunit complex containing a PUF RNA-binding protein, Pumilio; a Trim-NHL protein, Brat and a zinc-finger RNA binding protein, Nanos. We were able to discover a new post-transcriptional mechanism regulating LSD1 activity and to suggest the existence of feedback loop between LSD1 family and the PUM complex which may be functionally important during development and in human malignancies (Miles et al, MCB 2015). These results encouraged us to continue the analysis of the genetic interaction between dLsd1 and other candidates from the screen, with a special interest in chromatin associated proteins and components of signaling pathways. In addition, we have decided to combine this genetic approach with high throughput proteomic and genomic approaches to identify the proteins directing LSD1 targeting in the genome or modulating its activity in vivo in specific developmental contexts (WP2). Specifically, we are focusing on the role of dLsd1 in oogenesis and in wing development, two processes strongly affected by dLsd1 mutation. Combining ChIP-Seq and transcriptomics studies with genetic experiments, we found that: 1) dLsd1 affects oogenesis by controlling the expression of developmental genes and transposons. In addition, we found an interplay between dLsd1 and a GATA factor. 2) We recently discovered that dLsd1 controls wing growth. Our data show that dLsd1 depletion in the wing results in increased DNA damage and apoptosis and altered expression of growth genes and transposons. 3) Through genetic screens and mass spectrometry analysis, we have generated data on the dLsd1 interactome, which direct a series of questions that we plan to address in the next years. Collectively our results provide novel insights into the role of the histone demethylase dLsd1 in oogenesis and in wing development and provide the basis for future studies directed at understanding the molecular mechanisms by which dLsd1 complexes control the transcription of target genes and transposons in response to upstream signals. Ultimately, the scope of our analysis in Drosophila is to identify novel mechanisms that could be relevant to mammalian cells and to verify their potential implication in diseases, such as cancer. We have already shown that the interplay between dLsd1 and the Notch pathway and between dLsd1 and the Pumilio complex, both discovered in Drosophila, are also present in mammals (WP3). These results encouraged us to continue in this direction. The ultimate goal is to shed light on the most evolutionary conserved and therefore fundamental function of LSD1 complexes and on their interplay with signaling pathways. Luisa.di-stefano@univ-tlse3.fr http://cbi-toulouse.fr/eng/equipe-di-stefano

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