Work Package 9

Epigenetic transcriptional control in stem cells by the master developmental regulators PcG and trxG.

Introduction
A central question in developmental biology and disease is how cell fate decisions are regulated. A major level of regulation is at the level of gene transcription- turning on or off in a well controlled manner sets of master regulators that drive development and regulate in stem cells the decision to proliferate (self-renewal) or enter into differentiation pathways. Understanding stem cell fate regulation is of high importance as diseases such as many forms of cancer are ‘driven’ by a limited number of ‘cancer stem cells’, which have retained or acquired inappropriately a self-renewal ‘stem’ cell-like fate. Trithorax group gene activators (trxG) and Polycomb group (PcG) gene repressors are such master regulators, which form an epigenetic memory system of developmental fate by ensuring stability and inheritance of crucial target genes such as Hox gene clusters. PcG and trxG proteins do so by acting in large complexes to fix the local chromatin structure in either an active or repressed configuration.

Objective
Here we propose to investigate in detail the role of PcG and TrxG in controlling the balance between proliferation and differentiation in stem cells (with particular emphasis on Embryonic mouse and human stem cells (ES cells) and hematopoietic stem cells). We aim to characterise in detail the gene and protein expression profile changes when stem cells are undergoing specific differentiation pathways and to characterise in detail the mechanism of transcriptional control by master developmental regulators such as PcG and TrxG protein complexes. Furthermore, we will develop (stem)cell based assays to be used in screens with small molecule libraries (RNAi) to interfere with and control specific differentiation pathways. A major spin-off will be increased knowledge of proliferation/differentiation switches, which will hold promise for new targets for selected intervention in multifactorial diseases such as cancer.

Approach
Mouse ES cells or hematopoietic stem cells carrying conditional alleles of essential PcG genes will be used to eliminate their function in a time-controlled manner. In addition, stable inducible RNA-interference against PcG and trxG genes will be used to study loss-of-function phenotypes and effects on cell differentiation programs in mouse and human ES cells. These reagents are available at the start of the project. Essential target genes governing stem cell and differentiation programs will be identified using expression profiling on mouse and human cDNA and oligo arrays, and will be validated using specific RNA-interference. Changes in PcG and trxG protein complexes upon induction of specific differentiation pathways will be assayed using native purification with special epitope tags, followed by mass spectrometry. Using specific lineage-reporter constructs, we will use high complexity RNAi libraries generated at the NKI, to screen for differentiation controlling genes in an unbiased fashion.

Links with other parts of the program: The above-mentioned aims are strongly embedded within the unique and complementary research programs of the participants. Direct links exist with WP5: Study of transcription complexes by Biotin tagging/mass spec; WP10: control of cell proliferation in development/hemapoiesis; WP8: signal mechanisms for complete differentiation of ES cells to desired lineages.

Keywords: Polycomb, trithorax, Stem cell fate, master regulators.

Participants: van Lohuizen, Verrijzer, Mummery