**1. Introduction**

60 Advances in Hematopoietic Stem Cell Research

Wilson, A., Laurenti, E., & Trumpp, A. (2009) Balancing dormant and self-renewing

Wu, W.S., Heinrichs, S., Xu, D., Garrison, S.P., Zambetti, G.P., Adams, J.M., & Look, A.T.

Xiao, J., & Chen, H.S. (2004) Biological functions of melanoma-associated antigens. *World J* 

Yamazaki, S., Iwama, A., Takayanagi, S., Eto, K., Ema, H., & Nakauchi, H. (2009) TGF-beta

Yoshikawa, K. (2000) Cell cycle regulators in neural stem cells and postmitotic neurons.

Yuasa, H., Oike, Y., Iwama, A., Nishikata, I., Sugiyama, D., Perkins, A., Mucenski, M.L.,

Zeng, H., Yücel, R., Kosan, C., Klein-Hitpass, L., & Möröy, T. (2004) Transcription factor Gfi1

Zeng, S., Xu, Z., Lipkowitz, S., & Longley, J.B. (2005) Regulation of stem cell factor receptor signaling by Cbl family proteins (Cbl-b/c-Cbl). *Blood* 105(1):226-232. Zhong, J.F., Zhao, Y., Sutton, S., Su, A., Zhan, Y., Zhu, L., Yan, C., Gallaher, T., Johnston, P.B,

during homeostasis and repair. *Cell* 135(6):1118-1129.

repressing puma. *Cell* 23(4):641-653.

hibernation. *Blood* 113(6):1250-1256.

*Gastroenterol* 10(13):1849-1853.

*Neurosci Res* 37:1–14.

24(11):1976-1987.

*Sci U S A* 102(7):2448-2453.

4116-4125.

hematopoietic stem cells. *Curr Opin Genet Dev* 19(5):461-468.

(2008) Hematopoietic stem cells reversibly switch from dormancy to self-renewal

(2005) Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by

as a candidate bone marrow niche signal to induce hematopoietic stem cell

Suda, T., & Morishita, K. (2005) Oncogenic transcription factor Evi1 regulates hematopoietic stem cell proliferation through GATA-2 expression. *EMBO J* 

regulates self-renewal and engraftment of hematopoietic stem cells. *EMBO J* 23(20):

Anderson, W.F., & Cooke, M.P. (2005) Gene expression profile of murine long-term reconstituting vs. short-term reconstituting hematopoietic stem cells. *Proc Natl Acad*  Haematopoietic stem cells (HSC) have the exceptional capacity to undergo continuous selfrenewal and differentiation into multiple lineages, which is essential for haematopoietic homeostasis and response to injury. To achieve this life long function, these cells have to be protected from cytotoxic and genetic damage. On the other hand, rapid activation of haematopoietic stem cell proliferation in response to stimuli must be ensured. While cellular quiescence is thought to be the key mechanism underlying this paradoxical nature of HSC, the molecular basis of induction and maintenance of quiescence remains unresolved.

Quiescence is commonly defined as a reversible cell cycle exit. Induction and maintenance of stem cell quiescence has been studied at the level of cell cycle regulation (Orford & Scadden, 2008), cellular metabolism (Tothova & Gilliland, 2007) or interaction with the specific niche (Fuchs *et al.*, 2004). Genome-wide association studies have been performed on a variety of quiescent model systems, such as serum starvation of fibroblasts (Coller *et al.*, 2006), primary lymphocytes (Garriga *et al.*, 1998) or yeast in stationary phase (Patturajan *et al.*, 1998, Radonjic *et al.*, 2005). All of these studies revealed a significant decrease of productive mRNA transcription in these model systems. However, if quiescent adult stem cells share this down regulation of mRNA transcription has never been examined.

Due to their relative ease of isolation, cells of the haematopoietic lineage have been extensively studied. Importantly, several assays for hematopoietic stem cell function have been developed, such as colony forming ability and rescue of lethally irradiated mice. These functional tests are lacking in most other adult stem cell models, with the exception of spermatogonia and mammary gland stem cells (Brinster & Nagano, 1998, Shackleton *et al.*, 2006). Functional assays for HSC ability have provided us with the notion that most defined populations of long term repopulating HSC still contain progenitor cells, which can only transiently contribute to repopulation of the haematopoietic system. This heterogeneity is not only evident in defined cell populations, but also in the in vivo niche for HSC, the bone marrow. HSC in the bone marrow are interspersed with transient amplifying cells and differentiated cells, complicating stem cell identification by spatial organization of the tissue. Other stem cell systems, such as spermatogonia, keratinocyte or crypt stem cells have a clearly defined niche architecture, enabling stem cell identification by location only (Fuchs *et al.*, 2004). In this case, resting stem cells and activated progenitors can be separated by

Transcriptional Quiescence of Hematopoietic Stem Cells 63

suggesting transcription elongation as the critical step in gene expression (Guenther *et al.*,

Transcription initiation requires phosphorylation of Ser5 of the CTD by TFIIH, a heterodimeric kinase consisting of CDK7 and Cyclin H. These phosphorylation events enable binding of the mRNA capping machinery (Ho & Shuman, 1999) and promoter clearance. Typically, a short (~40nt) nascent RNA is then produced by RNApII. However, mRNA transcription is paused at many genes due to the action of negative elongation factor (NELF) and DRB-sensitivity inducing factor (DSIF) (Wu *et al.*, 2003, Yamaguchi *et al.*, 2002).

Fig. 1. The mRNA transcription cycle is characterized by phosphorylation of the RNA polymerase (RNApII) CTD. Phosphorylation of Ser5 (5) by CDK7/Cyclin H induces

Ser2 (2) leads to productive elongation and release of mature mRNA.

2004).

promoter clearance and pre-mRNA capping. RNApII is then halted (STOP), until activation of CDK9/Cyclin T. Phosphorylation of the inhibitory complex and RNA polymerase II CTD

Recruitment of positive transcription elongation factor b (P-TEFb), a heterodimeric protein consisting of the kinase CDK9 and one of the regulatory subunits Cyclin T1, T2 or K, to stalled polymerases is required for alleviation of the transcriptional block by NELF and DSIF (Peterlin & Price, 2006, Rahl *et al.*, 2010). P-TEFb phosphorylates RNA recognition motif-containing protein RD, a component of NELF, and Spt5, a subunit of DSIF (Aida *et al.*, 2006, Fujinaga *et al.*, 2004). NELF then dissociates from RNApII, while DSIF remains associated with RNApII and becomes a positive transcription elongation factor (Chen *et al.*, 2009). Importantly, phosphorylation of Ser2 of the CTD by P-TEFb triggers transcription elongation, mRNA processing, and release of mature mRNA (Kohoutek, 2009, Ni *et al.*,

CDK9 has first been identified as a CDC2-related kinase with a PITALRE motif (Grana *et al.*, 1994). The cyclin partner of CDK9 is Cyclin T1, T2 or K. Unlike other CDK/Cyclin heterodimers, neither P-TEFb levels nor kinase activity is fluctuating during the cell cycle (Garriga *et al.*, 2003, Grana *et al.*, 1994). CDK9 exists in two isoforms, a major 42kD sized peptide and an N-terminal extended peptide, whose transcription starts from an alternative

2007, Muse *et al.*, 2007, Zeitlinger *et al.*, 2007).

location and molecular markers can be easily identified. If all adult stem cells share a repertoire of molecular markers, findings from other adult stem cells can be transferred to HSC and should lead to characterization of haematopoietic stem cell subpopulations.

In our previous work, we found that adult melanocyte stem cells exhibit a 10 to 100fold lower level of housekeeping gene mRNA compared to differentiated cells, suggesting a global repression of mRNA transcription (Osawa *et al.*, 2005). We could then show that the largest subunit of RNA polymerase II (RNApII), which is responsible for all mRNA transcription, exhibits a partly phosphorylated C-terminal domain (CTD), characteristic of initiated, but paused mRNA transcription (Freter *et al.*, 2010). In line with this, we found the RNApII kinase CDK9 absent in adult melanocyte stem cells. Inhibition of CDK9 resulted in cellular resistance to withdrawal of essential growth factors, conferring a stem cell-like phenotype to progenitor cells. Interestingly, various other adult stem cells, including keratinocyte, muscle, spermatogonia and also HSC exhibited a similar partial phosphorylation of RNApII (Freter *et al.*, 2010). We concluded that transcriptional quiescence is an early, specific and conserved marker for adult stem cells. This feature can be used to isolate and characterize pure populations of stem cell-like cells from any tissue, enabling a deeper understanding of stem cell biology and recapitulation of the stem cell niche, in order to expand immature stem cells in vitro.

In this chapter I would like to summarize our findings that HSC exhibit a reduction in productive mRNA transcription. I would like to elaborate on the implications arising from transcriptional quiescence of a subset of HSC, both in development and disease. Technical challenges and resulting applications of identifying and isolating transcriptionally quiescent HSC in vitro will be discussed.
