Generation and analysis of a conditional mutation of the HirA gene

Lay Summary The chromosomes within our cells contain the genetic instructions for life on the strands of DNA. In order to form the chromosomes the DNA is wrapped very tightly around proteins known as histones. The DNA and histone together is known as chromatin. However, the chromatin is not merely an ingenious way of getting a long length of DNA into a small space. Chromatin is dynamic, i.e. it is constantly changing, and alters its structure during important events such as fertilization of the egg by sperm, division of the cells, aging of cells and the switching on/off the genes themselves. HirA is a key protein for certain aspects of making and changing chromatin. We already know it is vital for life, without HirA DNA from the sperm cannot correctly join up with the egg at fertilization (at least in flies), and mouse embryos without HirA die in the middle of the pregnancy. However, since embryos lacking HirA die so early making it is very difficult to do certain experiments to work out how HirA is involved in development and regulating genes and cells. This project will allow us to make the mutation of HirA occur at different times and in different cells at will, providing an exquisite probe of HirA function. We hope to shed light on developmental pathways and cellular functions of HirA and will collaborate actively with other groups interested in these phenomena.

HirA is a WD40 domain protein we first identified while searching for the gene haploinsufficient in DiGeorge syndrome. While not a major player in that condition we have gone on to report HirA protein interactions with bicoid homeodomain transcription factors and histone H3.3, clone the Fugu and Drosophila homologues, and created a targeted mutation of HirA in mouse. With Tom Misteli we recently showed altered chromatin dynamics and differentiation of murine embryonic stem cells. Other groups have shown, in a variety of organisms, that Hira is involved in cellular senescence, chromatin remodelling immediately post fertilization, and epigenetic control of gene expression. HirA is required for embryonic viability with embryos dying shortly after gastrulation. This makes it exceeding difficult to define the role of Hira is specific developmental processes, senescence at the level of the whole organism, and even examine the cell cycle due to inability to culture mouse embryonic fibroblasts successfully. We request support to create a conditional mutation of the HirA gene in order to identify the timing requirements for HirA during development using a ubiquitously expressed, tamoxifen inducible, Cre. The targeting strategy will borrow heavily from our successful creation of a constitutive null allele and single loxP knock in (for chromosome engineering). We will create MEFs and analyse the cell cycle and cellular senescence in the absence of HirA. Following on from our observations concerning Hira expression and the phenotype of the constitutive null, we will examine the requirements for Hira specifically in the developing heart and neural crest. Given the recent interest in Hira from various quarters, we will immediately make mice available to a number of groups who have expertise in specific areas and where it would be unrealistic for us to propose equivalent experiments in a three year time frame.