Title
Polycomb-group complex 1 and Hoxa9/Hoxb4 sustain the hematopoietic stem and progenitor cell activities through the direct regulation of protein stability of Geminin, which determines cellular proliferation and differentiation

Shin’ichiro YASUNAGA
Associate Professor
Department of Stem Cell Biology
Research Center for Radiation Casualty Medicine
Research Institute for Radiation Biology and Medicine (RIRBM)
Hiroshima University

Abstract:
Polycomb-group (PcG) complex 1 (also designated as Polycomb repressive complex 1; PRC1) plays a pivotal role for sustaining the activities of hematopoietic stem cells (HSCs) and progenitors. PcG complex 1 is a multimetric nuclear protein complex consisting of Ring1A/B, Bmi1, Rae28 (Phc1) and Scmh1, which is known to maintain transcriptional repression through mono-ubiquitination of histone H2A. Although Bmi1 is known to regulate HSCs through the transcriptional repression of the Ink4a locus, which encode p16 INK4a and p19 ARF , the mice deficient in Rae28 displayed HSC defect without derepression of the Ink4a locus, indicating that the molecular role for PcG complex 1 has not been sufficiently understood. Curiously we found that the HSC defect in Rae28 -deficient mice was genetically compensated by transduction of Hoxb4, which enhances HSC activity ex vivo and in vivo and induces development of HSCs from ES and iPS cells. Based on these genetic findings we attempted to elucidate a molecular role for PcG complex 1 and Hoxb4 in the regulation of HSCs and progenitors.
PcG complex 1 maintain transcriptional repression of developmental regulators including Hox genes, while Hox genes determine the segment specificity during animal development. Hox genes are believed to act as a transcriptional regulator through the conserved DNA binding domain, ‘homeodomain’. We previously uncovered that either PcG complex 1 or a subset of Hox directly bind Geminin, a regulator of DNA replication licensing and cellular differentiation (Nature 2004). And we clarified that PcG complex 1 acts as an E3 ubiquitin ligase for Geminin to down-regulate Geminin protein level through the ubiquitin proteasome system and that accumulated Geminin-protein gave rise to HSC defect in Rae28 -deficient mice (PNAS 2008), and further demonstrated that Hoxb4 also composes an E3 ubiquitin ligase complex with a Roc1-Ddb1-Cul4a ubiquitin ligase core component to regulate the protein stability of Geminin, i.e. , Hoxb4 transduction reduced accumulated Geminin to recover the HSC activity in Rae28 -deficient mice. (PNAS 2010). We further revealed that Hoxa9, a key factor for supporting the activities of HSCs and leukemia stem cells (LSCs), also acts as an E3 ubiquitin ligase for Geminin that is similar to Hoxb4 (manuscript submitted). These findings suggest that Geminin plays a central role in sustaining the activity of HSCs and LSCs.
To further elucidate the molecular role for Geminin in HSCs, we have recently generated Geminin-EYFP knock-in mice to visualize Geminin expression and also devised retroviral gene transduction systems for up- and down-regulating Geminin expression. In this meeting, we argue for a role for dynamic expression profile of Geminin in hematopoiesis and leukemogenesis and further for a possibility of regulating the HSC and LSC activities through the manipulation of Geminin expression levels.

References:
1. Ohno, Y.*, Yasunaga, S .*, Ohtsubo, M., Mori, S., Tsumura, M., Okada, S., Ohta, T., Ohtani, K., Kobayashi, M. & Takihara Y. (* equal contribution)

2. Hoxb4 transduction down-regulates Geminin protein, providing hematopoietic stem and progenitor cells with proliferation potential.
Proc. Natl. Acad. Sci. USA , 107 (50 ), 21529-21534, 2010.

3. Ohtsubo, M.*, Yasunaga, S .*, Ohno, Y., Tsumura, M., Okada, S., Ishikawa, N., Shirao, K., Kikuchi, A., Nishitani, H., Kobayashi, M. & Takihara Y. (* equal contribution)

4. Polycomb-group complex 1 acts as an E3 ubiquitin ligase for Geminin to sustain hematopoietic stem cell activity.
Proc. Natl. Acad. Sci. USA , 105 (30) , 10396-10401, 2008.