Transcriptional programming in cord blood-derived haematopoietic stem cells

Molecular regulators of HSC fate decisions have been identified through involvement in leukaemia or by post-genomic gene discovery approaches. Most candidate regulators have been studied in mouse, but the clinical potential of HSC fate modulation emphasises the need to study regulatory mechanisms in the human context. In order to identify regulators of human haematopoiesis, the global expression profile of cord blood (CB) CD133+Go cells was characterised. Less immature CD133+G1 cells served as comparison. CD133+Go cells had been shown to be highly enriched for LTC-IC, a surrogate in vitro assay of HSC function. Further functional characterisation of CD133+Go cells revealed enhanced mixed-lineage and erythroid progenitor activity. In line with the functional data, CD133+Go-enriched transcripts contained an erythroid expression signature. Moreover, global expression of myelo-Iymphoidaffiliated genes was reduced in CD133+Go cells, suggesting that myelo-Iymphoid programmes may be activated downstream of the HSC compartment. A significant proportion of transcripts enriched in CD133+Go cells were associated with transcription regulation. These included MLLT3, a common fusion partner of MLL in acute myeloid leukaemia. Analysis of MLLT3 expression in prospectively isolated progenitors from human CB revealed higher expression in HSC and megakaryocytic (Meg)/E progenitors (MEP). This suggested a role for MLLT3 in E/Meg fate decisions; it may also underline an association between E/Meg and HSC compartments. Lentiviral vectors were used to modulate MLLT3 expression in primitive human CB cells. Forced MLLT3 expression promoted E/Meg progenitor output and analysis of MLLT3 mutants suggested that MLLT3 functional effects depend on its transcription regulatory activity. Gene expression and cis-regulatory element analyses revealed cross-regulatory interactions between MLLT3 and E/Megaffiliated transcription factor GATA1. MLLT3 expression knockdown indicated a requirement for MLLT3 in the elaboration of E/Meg lineages. Gene expression knockdown also impaired multilineage engraftment of human HSC in immunodeficient NOD/SCID mice, with more significant effects on B-cell than on myelo-monocytic output. The latter observation may have implications for the lineage affiliation of MLUMLLT3 leukaemia. In summary, MLLT3 was identified as a novel regulator of human E/Meg lineage decisions and positioned in a regulatory circuit with GATA1. The data supports the notion that E/Meg commitment occurs hierarchically upstream of myelolymphoid lineage decisions and suggests that recently revised hierarchical models of murine haematopoiesis may also apply to the human system.EThOS - Electronic Theses Online ServiceGBUnited Kingdo