Regulation of replication and genome stability by B-Myb

The properties of all cells are governed by the patterns of genes that are turned on to give rise to the proteins that perform various functions. Specific proteins (transcription factors) act like molecular switches to control those genes that should be turned on. This project aims to investigate one of these switches called B-Myb, which belongs to a small group of related proteins that control cell growth and differentiation. B-Myb is the ancestral Myb protein and appears to be essential for fundamental processes in all dividing cells, whereas the related c-Myb protein has evolved more specialised functions, most notably in the regulation of blood cell development. All cells that require c-Myb also contain B-Myb, raising the issue of whether the common origin of these proteins is reflected in a degree of overlap in their function. A large body of evidence indicates that B-Myb is involved in the control of cell division through an influence on replication of the genome. Interestingly, recent studies have suggested that at least part of this influence of B-Myb on replication is crucial to prevent damage to chromosomes that could lead, for example, to the development of cancer. Although cellular division is a fundamental process shared by most cells, some cells have an altered process that is adapted to their particular function. An example is the cell type that constitutes the inner ball of cells in the early embryo. These cells, termed embryonic stem cells (ES cells), are individually capable of giving rise to all tissues of the adult and can be isolated and grown in the laboratory almost indefinitely without becoming changed in any way. ES cells express very high levels of B-Myb and cannot be cultured in its absence. This project aims to define precisely how B-Myb influences the replication and stability of the genome, and to determine whether its role is distinct in cells that also contain the related c-Myb protein. Comparison will also be made between normal cells and ES cells to determine if B-Myb performs additional roles in specialised cellular division. A secondary major aim is to identify the molecular mechanisms through which B-Myb acts, including identification of the genes that it controls. The main approach underlying the project will be to reduce the levels of B-Myb protein in cells and then to analyse a range of parameters that reflect the efficiency of replication and integrity of the genome. Reduction of B-Myb levels will be achieved chemically ('RNA interference knockdown') or using cells derived from mice that have been genetically modified so that it is possible to remove the B-Myb protein when desired. This study will increase our basic knowledge of the control of cellular division and genome stability. The findings may be of practical significance in terms of understanding mechanisms underlying diseases that result from acquired chromosomal defects (eg cancer). An understanding of the function of B-Myb in ES cells will be crucial in their future application in translational medicine, especially in relation to its importance in maintaining the capacity for indefinite division without loss of genomic integrity.

Myb transcription factors are implicated in the regulation of cell growth and differentiation. B-Myb is ubiquitously expressed in proliferating cells. Although cyclin B1 has been identified as a target of B-Myb action, little else is known about how B-Myb controls cell division and to what extent this may vary in cell types that co-express other Myb family proteins or have specifically adapted cell cycles, such as embryonic stem (ES) cells. Recent studies suggest that B-Myb prevents damage to chromosomes, and that at least in some cell types this may be independent of direct actions on the replication process. We aim to define precisely how B-Myb influences the replication and stability of the genome. A range of parameters will be analysed following RNA interference knockdown or conditional deletion of B-myb. Embryonic fibroblasts (MEFs) will be the main focus for these studies, subsequent comparisons to erythroblasts (c-Myb expressing cells) and ES cells (specialised cell cycle) being made in light of the initial results. Effects of B-Myb on replication dynamics will be studied through observation of incorporation of halogenated deoxynucleosides into DNA in whole nuclei and at individual replication forks. Replication timing will be investigated by sorting of cells at distinct stages of S phase and immunoprecipitation of BrdU labelled DNA to use as probes against specific gene sequences. We will also investigate to what extent B-Myb influences checkpoint responses to potentially genotoxic insults that act in S phase. We aim to identify the mechanisms underlying the effects of reduced B-Myb expression. Immuno-FISH will be used to examine whether proteins that are suspected to be partners in multiprotein complexes co-localise with B-Myb. We will utilise conditional deletion of B-myb in MEFs combined with microarray screening to identify direct target genes that underlie the effects of B-Myb on replication and genome stability.