Mechanistic insights into priming and early gene activation processes in the haemopoietic system

All blood cells originate from stem cells which are capable of staying stem cells, but also can develop into different blood cells. Blood stem cells are first formed in the embryo and then stay with the individual for the rest of his natural life. In the embryo, the blood cell system develops via different stages. In the early embryo, primitive cells (mesoderm cells) are formed that can develop into many different cell types, such as the blood cells system, the blood vessel system and the nervous system. When these cells develop towards the blood cell system they first become cells that can make blood vessels as well as blood stem cells and only after a while develop into blood stem cells. Each of these different developmental stages is characterized by the carefully orchestrated onset of expression of different genes. It is the combination of these genes that specifies each cell type. A very important topic of recent research addresses the question of how these different genes are controlled and why they are specifically switched on in one cell type, but not in another. It is now clear that genes are switched on in a very distinct order. On top of this hierarchy are genes that encode for proteins that bind to other genes and control their expression. We have shown that it is possible to study very early events in the activation of such genes by using an experimental system based on mouse embryonic stem cells that recapitulates embryonic development in vitro. We have also been able to show that we can use this system to investigate the earliest events happening when genes are switched on. Moreover, we have shown that such early events can be followed by studying how genes are organized into chromatin. Chromatin is the combined name of all proteins that package the meter-long DNA molecule so it fits into the nucleus. In order for genes to become active, this 'packaging' needs to be opened up. We have developed techniques that follow this 'opening-up' and we could show that this process starts much earlier than the actual onset of gene expression. In this proposal we want to understand the order of events how chromatin alteration occurs and which molecules are driving such alterations. We also want to identify target genes for important factors already known to drive chromatin alterations. These experiments are important for future experiments where we want to use embryonic stem cells to generate specific blood cell types.

The work described in this proposal asks the important question of how individual genes are primed for expression in development. We have previously shown that alterations in chromatin precede the onset of gene expression and also the stable formation of transcription factor complexes. We hypothesized that transcription factors transiently bind to their recognition sequences and leave a remodelled chromatin structure behind. In the work described here we wish to use an in vitro differentiation system based on mouse ES cells to gain insights into the earliest events of epigenetic priming of individual genes. We will initially concentrate on two genes, Pu.1 and c-fms which are part of a transcriptional hierarchy. We aim to identify the earliest factors binding to these genes. We will also manipulate the levels of a transcription factor known to regulate the timing of onset of expression of both genes (Runx1) and directly measure the effect of such manipulation on Pu.1 and c-fms chromatin structure. In addition, we plan to use a novel technique (Dam-fusion proteins) to map short-lived interactions of Runx1 with its target genes and correlate this with alterations in chromatin. Last, but not least, we plan to use this technique to identify new target genes for Runx1 and another important transcription factor, Smad1. These target loci themselves will be subject to further mechanistic studies in the future. We regard this joint proposal as a powerful alliance between two laboratories with highly relevant complementary expertise. Our combined expertise of ES cell engineering, sophisticated ES cell culture and chromatin fine structure analyses techniques provides us with a unique opportunity to probe deep into the molecular details of how haemopoietic development is initiated. This grant is joint with grant BB/E025129/1