Transcriptional programmes of haemopoietic stem cells: regulation of the gene encoding the murine stem cell antigen Cd34
Haemopoietic stem cells are capable of self-replicating and differentiating along eight distinct lineages; these properties allow them to reconstitute the entire blood system of irradiated recipients. How these cells choose whether to self-renew or commit to a specific lineage remains unclear, but transcription factors are likely to play an important role in this decision. To gain insight into the transcriptional programmes of haemopoietic stem cells, the regulation of the gene encoding the murine stem cell antigen Cd34 was analysed. Within haemopoiesis, Cd34 expression is restricted to early progenitor cells, but outside haemopoiesis, Cd34 is expressed in vascular endothelium, fibroblasts, brain and testis. Three key regulatory elements were identified: (a) a3 Kb upstream region which functions as a stem-cell specific, chomatin-dependent enhancer in cell-lines, (b) a6 Kb downstream region which seems to have the ability to buffer against position effects, (c) a 1.2 Kb promoter which does not exhibit any stem-cell specificity. These three regions were linked to a human CD34 cDNA reporter and were able to direct high-level expression of the transgene in a Cd34-expressing progenitor cell-line; this construct was then used to generate transgenic mice. RNA and protein analysis were performed on haemopoietic cells derived from bone marrow, AGM, and fetal liver: transgene RNA was detected in Cd34 expressing cells. However, expression was also seen in non-Cd34-expressing cells, as well as in other tissues. To improve the level and the specificity of the targetting, four more constructs were made using combinations of the regulatory elements described previously with different reporters and heterologous promoters to generate transgenic mice. In all these mice, Cd34-expressing cells of the bone marrow were analysed for transgene expression. To assess the extent to which these constructs were subject to position effect variegation, analysis of gene expression by single cell RT-PCR was conducted in stably-transfected clones. The work presented here on the regulation of the murine stem cell antigen Cd34 gene shows that although the elements identified have the potential to target transcription of a reporter to the stem cell compartment, further regions are probably necessary for specific and high-level expression.