Mapping and dissecting human HSC self-renewal networks


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

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's 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 elements 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 key regulators of HSC self-renewal. PcG proteins form 2 distinct complexes: BMI1-RING1 (PRC1) and EZH1/2 (PRC2). Our lab has shown that PRC1 exert pro-proliferative, and PRC2 anti-proliferative effects on HSCs1-2.

Through interactions with numerous co-factors identified in our laboratory such as 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 basis underlying the opposing function of PRC1 and PRC2 in HSC activity using CRISPR, shRNA, gene targeting and gain of function approaches.

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


DelES, a 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 with 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 HSCs, 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. 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 sgRNA or 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.