Hematopoietic Stem Cell Self-renewal, the Transcription Factor HoxB4, and the Polycomb and Trithorax Group Epigenetic Regulator Complexes

Lifelong hematopoiesis depends upon the sustained self-renewal activity of the long-term reconstituting hematopoietic stem cell (LTRC). A distinct multipotent downstream stage, the intermediate-term reconstituting cell (ITRC), also robustly self-renews during initial but not subsequent divisions. My work aimed at uncovering mechanisms that specify the longevity of self-renewal. Before I began my studies, the transcription factor HoxB4 had been extensively studied in the context of hematopoiesis and was believed to enhance LTRC self-renewal divisions, based on the dramatic expansion of reconstituting cell numbers following ectopic delivery to murine bone marrow. However, alternative mechanisms underlying this apparent expansion had not been explored, and details of where and when endogenous HoxB4 was expressed remained to be ascertained. I mapped expression of a panel of Hox cluster genes over the hematopoietic precursor hierarchy. Among this panel, expression of HoxB4 uniquely correlated with active cell cycling of permanently and transiently reconstituting cells, while expression was repressed in more advanced cycling progenitors. This expression pattern suggested that endogenous HoxB4 might specify self-renewal divisions, and that failure of more advanced precursors to sustain self-renewal might be explained by loss of HoxB4 expression. I tested this model by enforcing constitutive expression of exogenous HoxB4 in purified ITRC and later precursor stages normally capable of only transient self-renewal. Transduced ITRC and more advanced cells were endowed with permanent reconstituting capacity entirely comparable with that normally shown only by LTRC. The result implied that much of the stem cell amplification originally observed following HoxB4 transduction would have derived from the recruitment of downstream progenitors into the functional stem cell compartment. My observations have critical implications for proposed use of HoxB4 for clinical stem cell expansion. To explore the possibility that LTRC and ITRC may differ at the level of genetic regulation of Hox, I undertook a comprehensive transcript-level screen of the members of the Trithorax and Polycomb Group complexes, which epigenetically regulate the Hox loci. This survey revealed a small subset of Polycomb Group subunits that were preferentially expressed in LTRC versus ITRC. These differences merit future investigation of their possible involvement in prospective specification of sustained self-renewal.Ph