Histone Demethylases as Modulators of Hematopoietic Cell Fate.

Abstract 393

Epigenetic modifications influence cell fate decisions, such as stem cell self-renewal and differentiation, both in normal and leukemic stem cells (LSC). On this premise, an expression profile study of the emerging class of histone demethylating enzymes (HDE) in stem cell enriched populations was designed. Transcripts of all these enzymes (n=27) were detected in isolated HSC populations (frequency 1:2) from fetal liver and bone marrow. Similar results were obtained in HoxA9-Meis1 induced leukemia, with an inverse correlation between LSC content and the HDE Jarid1b expression level. As a functional follow-up, systematic knockdown of known or presumed HDE was undertaken in primary HSC using a RNA interference (RNAi) based approach. Five sh-RNA were designed and generated against every HDE and cloned into a retroviral vector, containing a GFP reporter gene. The screen was set as a loss-of-function assay, where sorted CD150+CD48-Lin- cells were co-cultured on retroviral producers for 5 days, in a 96-well setting, and a fraction of each well was then transplanted into 2 mice (day 0), along with competitor cells. The remaining cell fraction served to asses gene transfer by GFP epifluorescence measurements, and RNA isolated from sorted GFP+ cells was used to evaluate gene knockdown levels by Q-PCR analysis. Blood reconstitution was evaluated at regular intervals, tracking the contribution of the donor CD45.1+ transduced (GFP+) cells to recipient hematopoiesis over time. As baseline references, sh-RNA to luciferase (no effect) and the histone acetyl transferase myst3 (stem cell loss) were used, as well as hoxb4 over-expression (stem cell expansion). In the primary screen, RNAi against jarid1a, lsd1 and an arginine demethylating enzyme, resulted in a progressive decline in blood reconstitution levels from the transduced cells, ranging from a 50% to 80% reduction of the GFP content within the graft. On the other hand, knockdown of Jarid1b conferred an in vivo competitive advantage to the transduced cells. As a validation procedure, a 7 day culture step was introduced to the assay, hence increasing the signal to noise ratio, to better detect an increase in HSC activity, as recently reported. After a week in vitro (day 7), 1/8 equivalents of single well cultures were transplanted into 3 independent mice, and blood reconstitution levels serially assessed. Cells transduced with sh-RNA against jarid1b (3 constructs out of 6) contributed more significantly to host hematopoiesis than sh-RNA luciferase transduced cells (58±16% vs 26±3% GFP), or hoxb4 over-expressing cells (37±2% GFP), at comparable gene transfer rates, at the 14 week time point and beyond (3 independent experiments). In long-term recipients, differentiation potential of these cells was preserved, as evidenced by CD4+CD8+ thymic cells, B220+ splenic cells and CD11b+ bone marrow cells in the GFP positive contingent. Clonality studies on DNA isolated from these sorted populations are ongoing. There were no cases of leukemic transformation in all of the transplant recipients (n>30).

In conclusion, this RNAi based study performed on primary hematopoietic cells links histone demethylases to modulations in cell fate. An opposite role seems to emerge among the H3K4me3/me2 HDE JARID1a and JARID1b regarding HSC activity, yet these proteins are highly homologous. Sequence alignments pinpoint to differences within the C-terminal portion, containing a PHD domain, and interestingly, dysregulation of this chromatin-binding PHD finger (NUP98-JARID1a) has been recently shown to play a direct role in leukemogenesis