Partridge, Janet Fay; (1993) The characterisation of a developmentally regulated transcription factor which complexes with the retinoblastoma gene product. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Murine embryonic development relies on the precise temporal and spatial expression of genes, the protein products of which dictate cellular phenotype. Proteins controlling the transcription of genes during early embryogenesis might be expected to be regulated on the differentiation of F9 embryonal carcinoma stem cells. One such regulated activity is DRTF1 (differentiation regulated transcription factor), a transcription factor which can associate with the product of the retinoblastoma tumour suppressor gene (pRb), pi07, and cyclin A. The embryonic expression pattern and biochemical characterisation of DRTF1 are the subject of this study. DRTF1 is a sequence specific DNA binding protein that consists of at least 3 DNA binding activities referred to as la, b, and c. Complexed DRTF1, referred to as DRTF1a, has similar DNA binding specificity and DNA binding polypeptides to DRTF1b/c, which lack pRb and cyclin A. DRTF1b/c are abundant in embryonal carcinoma (EC) and embryonic stem cells (ES), and down regulated on differentiation. In contrast, DRTF1a is not abundant in EC or ES cells, and is not down regulated as EC cells start to differentiate. The binding activity of DRTF1 was investigated during murine embryogenesis by preparing microextracts from tissues and whole embryos at various stages of development. DRTF1b was present in blastocyst stage embryos, and was abundant up to about 14 days of gestation. However, as embryogenesis progressed, the levels of DRTF1 b/c decreased whereas in contrast, the binding activity of DRTF1a increased. DRTF1 b/c were abundant in all tissues examined during early stages of embryogenesis, but became tissue restricted during later stages of development, and were frequently excluded from terminally differentiated tissues, for example liver and brain. In summary, DRTF1 is a group of DNA binding activities which share common DNA binding polypeptides, and which complex with other non-DNA binding polypeptides such as pRb and cyclin A in a developmentally regulated and tissue dependent fashion. The DNA binding polypeptides of DRTF1 were purified by sequence-specific affinity chromatography from F9 EC cells. The purification procedure was modified to incorporate a mutant site affinity column, which improved the purity of DRTF1. A unique 360 nucleotide cDNA was isolated by using degenerate oligonucleotides against peptide sequences derived from one purified polypeptide. The distribution of its mRNA was investigated by Northern blot and RNAse protection assays. These data, combined with a detailed examination of the expression during embryogenesis by in situ hybridisation, indicated that the mRNA expression was developmentally regulated, with high levels of expression at 6.5 to approximately 13 days post coitum, and lower levels of expression at later stages of development. This expression pattern was similar to the quantitative regulation of DRTF1 DNA binding activity, and the cDNA was therefore likely to encode a polypeptide involved in the DRTF1 activity. In conclusion, DRTF1 is a developmentally regulated and tissue-dependent transcription factor, that is subject to different levels of regulation. It is likely that DRTF1 plays an important role during embryonic development.

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