The aim of our research is to understand how chromatin factors regulate developmental programs. We are mainly interested to decipher how histone methylation control transcription during embryonic and postembryonic development.
POSTTRANSLATIONAL MODIFICATION OF HISTONES
In the nuclei of all eukaryotic cells, genomic DNA is tightly wrapped around histone complexes into a compacted structure called chromatin. Each histone complex is an octamer consisting of two copies of each of the histone core proteins H2A, H2B, H3 and H4. The N-terminal tails of these histones protrude from the octameric structure and are subject to a vast variety of post-translational modifications. These modifications remodel the structure of chromatin and represent a combinatorial "histone code" specifying the function of genomic regions.
METHYLATION OF HISTONE TAILS
The levels of lysine methylation are modulated by the action of histone demethylases and histone methyltransferases.
Trimethylated K4, K36 and K79 on histone H3 generally mark actively transcribed regions, whereas trimethylated K9 and K27 on histone H3 and K20 on histone H4 mark regions of transcriptionally silenced gene. Until recently, methylation was considered to constitute a permanent and irreversible histone modification. However, the recent discovery of histone demethylases has challenged this view. The largest group of histone demethylases contains a Jumonji (Jmj)C-domain, which catalyzes demethylation of specific methyl-lysine residues.
Since methylation state of histones is important during development, we use C.elegans as suitable model system to study the in vivo roles of enzymes regulating methylation, largely conserved in nematode.
OUR MODEL SYSTEM: C.ELEGANS
C.elegans is our favorite model system for several reasons. First, the organism has the advantage of a multicellular organism without the complexity of higher eukaryotes. The fact that C.elegans is a multicellular organism provides an opportunity to study tissue- or organ-specific proteins. Second, it is an appropriate system for genetic studies. Third, the C.elegans genome has been completely sequenced and other additional information such as the phenotypes of RNA interference (RNAi) of each gene (even with some limitation of the analysis) or that of some mutants is publicly available. Finally, in C. elegans gene families existing in higher eukaryotes are often represented by single genes, allowing a more suitable study in absence of functional redundancy.
C. Elegans growing in a bacteria plate
We are mainly interested in Neuronal development and Germline integrity after stresses and we are performing screens to identify enzymes regulating methylation states that are relevant for theseprocesses
Histone Lysine demethylases:
They are enzymes removing methyl marks from the histone tails and they often contain a JmjC domain. In the past few years, we have uncover the functions of a subset of histone demethylases (Christensen et al 2007, Agger et al, 2007, Vandamme et al, 2012, Kleine-Kohbrecher et al, 2010). We are further exploring the functions of these demethylases using classical genetic and biochemical approaches, including chromatin immunprecipitation followed by deep sequencing and transcriptome analysis with the aim to identify direct target genes. We are additionally expanding our analysis to other uncharacterized demethylases.
Histone methyl transferases:
A new project regarding histone methylatransferases, enzymes that add a methyl group to the histone tails, has been recently initiated in collaboration with the groups of Kristian Helin, Anja Groth, Klaus Hansen and Ole Nørregaard Jensen and supported by the Danish National Research Foundation. In this project we aim to uncover the role of histone methyltransferases during neuronal and germline development.
C.elegans is an excellent system to study many aspects of neuronal development, including the final steps of differentiation such as axon pathfinding and neuronal cell body migration.
Figure: neuron in C.elegans, identified using GFP reporters. From wormbook
In human aberrant histone lysine methylation has been linked to neurological disorders. We aim to identify modulators of histone lysine methylation that play relevant role in these aspects of neuronal development. To this end, screens have been performed and we are at the moment characterizing the mechanisms undelining the phenotypes.
Figure: A) axon pathfinding in wild-type animals and B) Example of aberrant axon pathfinding in a demethylase mutant allele.
GERMLINE INTEGRITY AFTER STRESSES
Histone lysine methylation is regulating DNA damage responses after different stresses. We aim to identify modulators of histone lysine methylation that play relevant roles germline integrity. To this end, screens have been performed using a variety of stressors, including IR, UV and agents interferring with DNA replication, and we are at the moment characterizing the mechanisms undelining the observed phenotypes.
Figure: A) C. elegans germline stained by DAPI (from wormbook) and B) resistance to HU observed in a demethylase mutant allele (Black bars)
Catalytic-independent roles of UTX-1 in C. elegans development. Vandamme J, Salcini AE (2012). Worm 2:2, 1-5; April/May/June 2013.
The C. elegans H3K27 Demethylase UTX-1 Is Essential for Normal Development, Independent of Its Enzymatic Activity. Vandamme J, Lettier G, Sidoli S, Di Schiavi E, Nørregaard Jensen O, Salcini AE (2012). PLoS Genet 8(5): e1002647.
A functional link between the histone demethylase PHF8 and the transcription factor ZNF711 in X-linked mental retardation. Kleine-Kohlbrecher D, Christensen J, Vandamme J, Abarrategui I, Bak M, Tommerup N, Shi X, Gozani O, Rappsilber J, Salcini AE, Helin K. Mol Cell. 2010 Apr 23;38(2):165-78. Epub 2010 Mar 25.
C.elegans intersectin: a synaptic protein regulating neurotransmission. Rose S, Malabarba MG, Krag C., Schultz A, Tsushima H, Di Fiore PP, Salcini AE. (2007). Mol Biol Cell 18(12):5091-9.
UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Agger K, Cloos PA, Christensen J, Pasini D, Rose S, Rappsilber J, Issaeva I, Canaani E, Salcini AE, Helin K. (2007). Nature. 449(7163):731-4.
RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3. Christensen J, Agger K, Cloos PA, Pasini D, Rose S, Sennels L, Rappsilber J, Hansen KH, Salcini AE, Helin K. (2007). Cell. 128(6):1063-76.
TTP specifically regulates the internalization of the transferrin receptor. Tosoni D, Puri C, Confalonieri S, Salcini AE, De Camilli P, Tacchetti C, Di Fiore PP. (2005). Cell 123:875-88.
EH and UIM: endocytosis and more. Polo S, Confalonieri S, Salcini AE, Di Fiore PP. (2003). Sci STKE 2003(213):re17.
The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling. Salcini AE, Hilliard MA, Croce A, Arbucci S, Luzzi P, Tacchetti C, Daniell L, De Camilli P, Pelicci PG, Di Fiore PP, Bazzicalupo P. (2001).Nat Cell Biol 3:755-60.
Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module. Salcini AE, Confalonieri S, Doria M, Santolini E, Tassi E, Minenkova O, Cesareni G, Pelicci PG, Di Fiore PP. (1997). Genes Dev 11:2239-49.