Mapping and dissecting human HSC self-renewal networks


Hematopoietic stem cells (HSC) are found principally in the bone marrow. HSCs sustain lifelong production of all mature blood cells and self-renew.

Self-renewal is the key cellular process that underlies HSC expansion. It is therefore critical to understand its basis for the development of new HSC-expansion tools.

Our laboratory current research projects aim to map and dissect HSC self-renewal networks, using 3 complementary approaches:

1. Polycomb group genes (PcG) as key self-renewal determinants
2. Identification of non-coding element contributing to HSC activity
3. Chemo-genomic dissection of self-renewal determinants

1. Polycomb group genes (PcG) as key self-renewal determinants


Hox and Polycomb Group (PcG) genes are acknowledged key regulators of HSC self-renewal. PcG proteins form 2 distinct complexes: BMI1-RING1 (PRC1) and EZH1/2 (PRC2). We showed that PRC1 exert pro-proliferative, and PRC2 anti-proliferative effects on HSC1-2.

Through interactions with numerous co-factors identified in our laboratory such E4F13, UBAP2L4 and MAP1S, we found that BMI1 exerts several non-canonical functions to regulate HSC activity. Most intriguingly, we recently identified a BMI1-E4F1-CHK1 interaction complex that appears to control centrosome numbers and cell cycle checkpoints in HSCs (Figure).

We now aim to decipher the role of PcG genes to HSC self-renewal, by focusing on the non-chromatin functions of this complex. We will also continue to study the molecular bases underlying the opposing function of PRC1 and PRC2 in HSC activity using shRNA, gene targeting and gain of function approaches.

2. Identification of non-coding element contributing to HSC activity


The retroviral-based system developed in our lab5 creates nested chromosomal deletions in embryonic stem cells (ESC). This system has the unique potential to identify non-coding elements contributing to HSC activity.

For example, using this method, we showed that proteins located in the mRNA tunnel region of ribosomes are essential for ESC differentiation but dispensable for their self-renewal6-7.

We now focus our studies on the identification of structural elements that specify HSCs and validate these results in standard gain and loss of function approaches.

3. Chemo-genomic dissection of self-renewal determinants


After screening for proteins and small molecules that have the ability to expand human HSC, our lab identified the small molecule UM729 and its optimized version UM171. These molecules enhance self-renewal (expansion) of human HSCs8-9.

Using molecular probes, we will identify the target of these molecules, as currently performed for UM171. The identified targets will likely represent central proteins governing HSC self-renewal. In-depth elucidation of the relevant mechanisms will probably require more sophisticated approaches: transcriptome sequencing, phosphoproteomic analyses and RNA interference sensitized screens using shRNA libraries available at IRIC.



References

  1. Sauvageau M and Sauvageau G. (2010). Polycomb Group Proteins: Multi-Faceted Regulators of Somatic Stem Cells and Cancer. Cell Stem Cell, 7(3): 299-313.

  2. Sauvageau M, Sauvageau G. (2008). Polycomb group genes: keeping stem cell activity in balance. PLoS Biol., 6(4): e113.

  3. Chagraoui J, Niessen SL, Lessard J, Girard S, Coulombe P, Sauvageau M, Meloche S, Sauvageau G. (2006). E4F1: a novel candidate factor for mediating BMI1 function in primitive hematopoietic cells. Genes Dev. 20, 2110-20.

  4. Bordeleau ME, Aucagne R, Chagraoui J, Girard S, Mayotte N, Bonneil E, Thibault P, Pabst C, Bergeron A, Barabé F, Hébert J, Sauvageau M, Boutonnet C, Meloche S, Sauvageau G. (2014). UBAP2L is a novel BMI1-interacting protein essential for hematopoietic stem cell activity. Blood. 124(15):2362-9.

  5. Bilodeau M, Girard S, Hébert J, Sauvageau G. (2007). A retroviral strategy that efficiently creates chromosomal deletions in mammalian cells. Nat. Methods 4(3), 263-8.

  6. Fortier S, Bilodeau M, MacRae T, Laverdure JP, Azcoitia V, Girard S, Chagraoui J, Ringuette N, Hébert J, Krosl J, Mayotte N, Sauvageau G. (2010). Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions. PLoS Genetics 6(12): e1001241.

  7. Fortier S, MacRae T, Bilodeau M, Sargeant T, Sauvageau G (2015). A Haploinsufficiency Screen Highlights Two Distinct Groups Of Ribosomal Protein Genes Essential For Embryonic Stem Cell Fate. PNAS. 112(7): 2127-32.

  8. Fares I, Chagraoui J, Gareau Y, Gingras S, Ruel R, Mayotte N, Csaszar E, Knapp DJHF, Miller P, Ngom M, Imren S, Roy DC, Watts KL, Kiem HP, Herrington R, Iscove N, Humphries RK, Eaves C, Cohen S, Marinier A, Zandstra PW, Sauvageau G. (2014). Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal. Science 345(6203):1509-12.

  9. Pabst C, Krosl J, Fares I, Boucher G, Ruel R, Marinier A, Lemieux S, Hébert J, Sauvageau G. (2014). Identification of small molecules that support human leukemia stem cell activity ex vivo. Nature Methods. 11(4): 436-42.