The role of enhancers and transcription factors in the reorganisation of chromatin architecture

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In this proposal we are asking fundamental questions about the mechanisms employed to activate gene expression within the normal context of chromosomes. The mammalian genome encompasses about 1 metre of DNA which is assembled as chromatin and is compacted ~10,000 fold to fit it all into the cell nucleus as condensed chromatin. The genome is packaged as repeating units of structures termed nucleosomes which are made up of bundles of histone proteins surrounded by 147 base pairs (bp) of DNA which makes 1.7 turns around the outside of the nucleosome. Nucleosomes are themselves separated from each other by a short linker of about 40 bp, and are organised as a highly regular array with a repeat length of ~180 - 200 bp. In order to activate gene expression it is necessary for genes to undergo many distinct levels of chromatin modification and decondensation. The whole process of gene activation is tightly controlled by control modules on the DNA, namely proximal promoter elements where mRNA synthesis starts and distal enhancer elements. These elements function by first interacting with specific proteins named transcription factors that bind to DNA, which then recruit complexes that modify and remodel chromatin, and at the same time engage with the transcription apparatus that reads the genetic information required for the synthesis of gene products. Gene activation can involve the displacement of nucleosomes from regulatory elements and significant modification of the whole nucleosome array encompassing genes and their enhancers. Our laboratory is actively engaged in defining the transcription factors and chromatin modifying complexes that interact with and activate transcription of the human GM-CSF gene. This is a gene that functions during haemopoiesis and within the immune system to control the growth and function of specific classes of white blood cells termed granulocytes and macrophages. We have shown that this gene is very tightly regulated and is induced ~10,000 fold upon stimulation of pro-inflammatory pathways in cells such as T cells and mast cells, which also represent part of the immune system. In this context, GM-CSF functions to receive and send signals between different parts of the immune system, and it is essential that its expression is carefully controlled. In our studies we have identified an inducible transcriptional enhancer that is located 3000 bp upstream of the GM-CSF gene. We have shown that this enhancer responds to activation of the T cell antigen receptor, and undergoes extensive remodelling at the level of chromatin structure. We have shown that the recruitment of inducible transcription factors such as NFAT and AP-1, or developmentally regulated factors such as GATA-2, leads to the eviction of nucleosomes from the enhancer and profound remodelling of the flanking nucleosomes. We find that at least 4000 bp of DNA (~ 20 nucleosomes) is restructured such that (i) the regular array is disrupted and randomised, and (ii) the average nucleosome repeat length is reduced from the normal value of 180-190 bp to a much shorter value of 150-160 bp. It is not known how such relatively small enhancer and promoter elements can alter the structure of the chromatin fibre over such long distances. We have indications that this may be done by a combination of (i) transcription initiated not from the normal promoter element but from the enhancer, and (ii) chromatin remodelling complexes that are recruited by the enhancer and then spread across the locus. In this proposal we wish to test whether these mechanisms do indeed operate within the GM-CSF gene, and we want to work out the precise details of the remodelling process. We also aim to determine whether additional genetic elements exist that block the spread of chromatin remodelling from active genes into neighbouring genes.