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

Lead Research Organisation: University of Manchester
Department Name: Medical and Human Sciences

Abstract

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.

Technical Summary

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
 
Description The most significant achievements from this grant are:
- Demonstration that Runx1 activate the Pu.1 locus by a "hit and run" mechanism (Blood. 2009 Jul 9;114(2):299-309.)
- Generation and validation of mouse ES lines expressing either Runx::Dam or Dam construct using the inducible Tet-on system. The Runx1::Dam construct was shown to be biologically functional as it can rescue the progression of Runx1-/- ES cells towards blood progenitors
- Multiple high-throughput RUNX1 binding datasets have been generated using DamID and ChIP-sequencing at different stages of haematopoietic development. They are currently integrated with microarray expression studies using the iRUNX1 system to identify genes that are transcriptionally activated after RUNX1 induction in a RUNX1 -/- background. A publication describing these results is in preparation.
Exploitation Route These findings allowed us to successfully apply for a BBSRC Lola funding
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Establishment of the haemopoietic transcriptional programme: From systems approaches to molecular mechanisms
Amount £1 (GBP)
Funding ID BB/I001794/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2011 
End 04/2016
 
Title RUNX1 binding sites in haemogenic endothelium 
Description We, therefore, developed and implemented a highly sensitive DNA adenine methyltransferase identification-based methodology, including a novel data analysis pipeline, to map early RUNX1 transcriptional targets in HE cells. This novel transcription factor binding site identification protocol should be widely applicable to other low abundance cell types and factors. Integration of the RUNX1 binding profile with gene expression data revealed an unexpected early role for RUNX1 as a positive regulator of cell adhesion- and migration-associated genes within the HE. 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact Integration of the RUNX1 binding profile with gene expression data revealed an unexpected early role for RUNX1 as a positive regulator of cell adhesion- and migration-associated genes within the HE.This suggests that RUNX1 orchestrates HE cell positioning and integration prior to the release of hematopoietic cells. Overall, our genome-wide analysis of the RUNX1 binding and transcriptional profile in the HE provides a novel comprehensive resource of target genes that will facilitate the precise dissection of the role of RUNX1 in early blood development. 
URL http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc5GSE55335
 
Description Collaboration with Dr Kouskoff/BBSRC grant 
Organisation University of Manchester
Department Cancer Research UK Manchester Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Scientific collaboration
Collaborator Contribution Scientific collaboration
Impact publications
Start Year 2006
 
Description Collaboration with Prof C. Bonifer/ BBSRC project 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Scientific collaboration
Collaborator Contribution Scientific collaboration
Impact Publications, datasets
Start Year 2010