LSD1

Histone modifications are instrumental for the control of gene expression and chromatin architecture and a strict regulation of histone marks is critical for normal development. Defects in chromatin organization and gene expression due to aberrant activity of chromatin modifying enzymes have been implicated in tumorigenesis. Our current knowledge regarding histone methylation largely stems from the study of histone methyltransferases, and only recently the discovery of histone demethylases showed that histone methylation is reversible supporting a more dynamic role of histone methylation. The first histone lysine demethylase to be identified was LSD1/KDM1. Since its discovery, LSD1 is emerging as a key regulator of chromatin. Although its activity is potentially widespread, evidence suggests that LSD1 performs pathway-specific functions. In addition, several studies have implicated LSD1 in tumorigenesis and there is a growing interest in LSD1 as a drug target. However, despite the tremendous progress in understanding the dynamic regulation of histone methylation by LSD1, the biological function of LSD1 is just beginning to be uncovered. We still have a limited knowledge of LSD1 pathway-specific functions and a genome-wide study of LSD1 role in a whole organism is lacking. I have taken advantage of the streamlined network of proteins present in Drosophila, to demonstrate that dLsd1 activity is conserved in flies and to find novel functions of dLsd1 during development. Using a genetic approach, I found a surprising antagonism between the two histone demethylases dLsd1 and Lid. 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. In the proposed work (Task 1), I plan to take advantage of the vast array of tools available in Drosophila to identify novel pathways involving dLsd1. Specifically, I am carrying out an in vivo RNAi screen and I have already found several enhancers and suppressors of a dLsd1 loss of function phenotype. This approach has the key advantages of being unbiased and to take place in the context of a whole animal. This is crucial because it could unveil context dependent interaction important for development, which might be lost in cells grown tissue culture. The screen will be followed by a detailed functional characterization of key genetic interactions, with a special focus on transcription factors, chromatin regulators and proteins involved in signaling and RNA metabolism. In the second approach (Task 2), I will use ChIP-Seq technologies and transcriptome analysis to extend the characterization of dLsd1 mutant flies. The goal is to understand how dLsd1 and its interactors control gene transcription and chromatin homeostasis in specific developmental contexts. An important aspect of this strategy is the idea that the mechanisms that enhance or suppress dLsd1 mutant phenotypes are conserved between flies and mammals. In a first attempt to verify this, we already found that this is true for the interplay between dLsd1 and the Notch pathway. In Task 3, I plan to take a few new key interactions discovered in aim 1 and to test whether this newly acquired information is conserved in mammals and can be exploited to target tumor cells. I will start by investigating the role of the human orthologs of dLsd1 and Lid in heterochromatin formation in mammalian cells. Heterochromatin is deregulated in many human diseases, including cancer, but a detailed understanding of how this state is established and maintained at specific loci and how its misregulation contributes to tumorigenesis is still missing. The experiments proposed are aimed at gaining a better understanding of these processes and might provide key insights for the development of selective epigenetic strategies to kill cancer cells.