Development of a chromatin immunoprecipitation protocol applicable to small cell populations and its application to embryo research.

The different types of cells that make up our bodies all contain the same set of genes, half inherited from our mothers and half from our fathers. This simple fact raises the interesting question of why different types of cell are so very different; a muscle cells, skin cells and white blood cells, for example, not only look different, but do very different tasks in our bodies. The answer to this question is that although all cells have the same set of genes, they use them in different ways. A small number of genes are 'switched on', or 'expressed', in all cell types. These genes encode crucial RNAs and proteins that are essential for basic cell functions, such as generating energy or making essential cell components. They are called 'housekeeping' genes. Other genes are required only in specific cell types. Genes responsible for making the oxygen carrying protein haemoglobin are expressed only in those blood cells that become red blood cells. In all the different cell types in our bodies a different set of key 'tissue-specific' genes are expressed, making proteins and RNAs that allow that cell to adopt its own particular shape and carry out its own specific function. If we can understand the control mechanisms by which genes are switched on and off, then we can begin to intervene to turn one cell type into another. For example, cancers often occur due to abnormal gene expression so understanding how to alter the expression of key genes opens up the possibility of halting, or reversing the growth of tumour cells. Genes can be regulated in several ways, but a key factor applying to all, or the great majority of genes, is the packaging of DNA into a DNA-protein complex called chromatin, primarily by a small group of proteins, the histones. If the DNA containing specific genes is tightly bundled up by histones, then those genes will be inaccessible to the enzymes and other factors required to make RNA and will switched off. Conversely, if the DNA is unwrapped, opened up and made accessible, then the gene can be expressed. The early embryo represents a stage in life at which changing patterns of gene expression are particularly crucial. The fertilised egg is capable of making all the different cell types in our bodies. It is said to be 'totipotent'. However, as the embryo divides, possibly as soon as the four cell stage, then individual cells begin to change their patterns of gene expression and become committed to turning into a particular cell type. As cell numbers increase during the first few days and weeks of embryonic life, cells continue to change their patterns of gene expression as they become more specialised, and more limited in what sort of cell they can become. Understanding the mechanisms that control these early changes in gene expression is crucial in understanding how environmental factors can alter embryonic development at this vulnerable stage of life. Until now it has been impossible to study the packaging of DNA by histones in early embryos because the number of cells available is so small. Even by combining multiple embryos, the numbers are measured in hundreds, ten thousand times less than what is needed for current experimental techniques. We have developed a modified 'chromatin immunoprecipitation' protocol in which we use antibodies to isolate genes packaged by particular modified histones from as few as 50-100 cells.This new technique allows us, for the first time, to study mechanisms of DNA packaging and regulation of key genes in cells of the early embryo. In order to fully exploit the enormous potential of this new approach, we wish to determine, firstly how it can be adapted to study a variety of non-histone proteins, some of which, the 'transcription factors, play key roles in gene expression and secondly whether it can be applied to the very earliest embryos (2 -16 cell stages) in order to study environmental effects (toxins, dietary components etc) on gene regulation and embryonic development

We have recently developed a new procedure, CChIP, that uses Drosophila cells to provide 'carrier' chromatin and that allows ChIP assays on 1000 mammalian cells or fewer. Successful assays with such small cell numbers are made possible by the high efficiency of ChIP with native, unfixed chromatin (NChIP). We wish to determine whether the CChIP protocol can be applied to formaldehyde cross-linked chromatin. Such a cross-linking step is necessary to allow ChIP to be used to explore the distribution across specific genes of transcription factors and other DNA-binding proteins, the majority of which become detached from DNA when chromatin is prepared by nuclease digestion (as in NChIP). Precipitation of cross-linked chromatin (XChIP) is widely used, but recoveries can be low. As a model system to study the possible application of CChIP to cross-linked chromatin, we will use decreasing numbers of cultured mouse embryonic stem cells as target cells and will, initially, explore binding of the transcription factor Oct4 to well-defined target genes. By comparing undifferentiated and differentiated ES cells, in which Oct4 is either present or absent, we can ask whether the expected patterns of binding are detected. Our own specific interest has been to use CChIP to assay the levels of specific histone modifications at the promoters of key regulator genes in Inner Cell Mass and trophectoderm derived from the blastocyst stage embryo. We now wish to ask whether the same approach can be used to study even earlier embryonic stages, from 4 / 16 cells. We can reach the number of cells required (at least 100) by combining embryos, but need to establish efficient procedures for chromatin preparation in the presence of Drosophila carrier cells. This will then allow us to study the effects of environmental agents (toxins) on the histone modifications associated with key regulator genes, possible changes in expression, either immediately or at some later developmental stage.