**6. References**


It has been long known that adult HSC can be isolated by their low retention of Pyronin Y, an RNA binding dye (Shapiro, 1981), suggesting that global mRNA transcriptional quiescence is a hallmark of quiescent stem cells. This observation however, has never been addressed further in terms of the precise molecular mechanism underlying global transcriptional quiescence. Analysis of the distinct phosphorylation patterns of the CTD of RNApII during the different stages of mRNA transcription can reveal initiation, paused transcription or productive elongation. Our analysis of various adult stem cell systems showed that down regulation of productive mRNA transcription elongation is a conserved, specific and early feature of adult stem cells (Freter *et al.*, 2010). In line with this, the CTD kinase CDK9 was absent and its inhibition improved cell survival during cellular stresses, suggesting a beneficial function of transcriptional quiescence for stem cell maintenance and survival. However, we could not induce activation of mRNA transcription by overexpression of CDK9 in vivo. It is thus not clear what the in vivo function of this down regulation is. New animal models with a constitutively active RNApII have to be developed to elucidate if transcriptional quiescence is necessary for adult stem cell maintenance or just

Interestingly, we could observe some heterogeneity in terms of mRNA transcription elongation in the HSC pool, where only 27% of cells showed a clear absence of mRNA transcription elongation. Yet, identification and isolation of these transcriptionally quiescent cells depends on the availability of genetically encoded reporters of mRNA transcription. CDK9 activity reporters need to be specific for this kinase, and a large dynamic range has to be provided to clearly separate cell populations. A particular challenge will be to convert the negative observation of CTD Ser2 dephosphorylation into a signal-positive output, in order

Taken together, we defined a novel molecular mechanism for adult stem cell quiescence, which may lead to the identification of pure stem cell-like cell populations from various sources, including heterogeneous adult stem cell populations or cancerous tissue. Even though some technical questions and functional tests are still to be answered, transcriptional quiescence is a novel and exciting mechanism to detect, isolate and characterize adult stem

Aida, M., Chen, Y., Nakajima, K., Yamaguchi, Y., Wada, T. & Handa, H. (2006)

Bagella, L., Stiegler, P., De Luca, A., Siracusa, L.D. & Giordano, A. (2000) Genomic

Barboric, M., Lenasi, T., Chen, H., Johansen, E.B., Guo, S. & Peterlin, B.M. (2009) 7SK

early expression of the junB gene. *Mol Cell Biol,* 26, 6094-6104.

Transcriptional pausing caused by NELF plays a dual role in regulating immediate-

organization, promoter analysis, and chromosomal mapping of the mouse gene

snRNP/P-TEFb couples transcription elongation with alternative splicing and is essential for vertebrate development. *Proc Natl Acad Sci U S A,* 106, 7798-7803. Barboric, M., Nissen, R.M., Kanazawa, S., Jabrane-Ferrat, N. & Peterlin, B.M. (2001) NF-

kappaB binds P-TEFb to stimulate transcriptional elongation by RNA polymerase

a symptom of their quiescence.

**6. References** 

to avoid false-positive events by untransfected cells.

cells in an unprecedented purity from various sources.

encoding Cdk9. *J Cell Biochem,* 78, 170-178.

II. *Mol Cell,* 8, 327-337.


Transcriptional Quiescence of Hematopoietic Stem Cells 73

Ho, C.K. & Shuman, S. (1999) Distinct roles for CTD Ser-2 and Ser-5 phosphorylation in the

Huang, F., Wagner, M. & Siddiqui, M.A. (2004) Ablation of the CLP-1 gene leads to down-

Jang, M.K., Mochizuki, K., Zhou, M., Jeong, H.S., Brady, J.N. & Ozato, K. (2005) The

Jiang, H., Zhang, F., Kurosu, T. & Peterlin, B.M. (2005) Runx1 binds positive transcription

Kanazawa, S., Soucek, L., Evan, G., Okamoto, T. & Peterlin, B.M. (2003) c-Myc recruits P-

Kawai, Y., Sato, M. & Umezawa, Y. (2004) Single color fluorescent indicators of protein

Kippin, T.E., Martens, D.J. & van der Kooy, D. (2005) p21 loss compromises the relative

Kohoutek, J., Li, Q., Blazek, D., Luo, Z., Jiang, H. & Peterlin, B.M. (2009) Cyclin T2 is

Li, L., Jothi, R., Cui, K., Lee, J.Y., Cohen, T., Gorivodsky, M., Tzchori, I., Zhao, Y., Hayes,

Li, Q., Price, J.P., Byers, S.A., Cheng, D., Peng, J. & Price, D.H. (2005) Analysis of the large

Lin, C., Smith, E.R., Takahashi, H., Lai, K.C., Martin-Brown, S., Florens, L., Washburn, M.P.,

Liu, H. & Herrmann, C.H. (2005) Differential localization and expression of the Cdk9 42k

Liu, H. & Rice, A.P. (2000) Genomic organization and characterization of promoter function

essential for mouse embryogenesis. *Mol Cell Biol,* 29, 3280-3285.

possible mechanism of CD4 silencing. *Mol Cell Biol,* 25, 10675-10683. Kanazawa, S., Okamoto, T. & Peterlin, B.M. (2000) Tat competes with CIITA for the binding

*Cell,* 3, 405-411.

*Immunity,* 12, 61-70.

*Chem,* 76, 6144-6149.

and fetal death. *Mech Dev,* 121, 559-572.

immature thymocytes. *Mol Cell Biol,* 28, 907-912.

proliferation capacity. *Genes Dev,* 19, 756-767. Kohoutek, J. (2009) P-TEFb- the final frontier. *Cell Div,* 4, 19.

hematopoietic stem cells. *Nat Immunol,* 12, 129-136.

at threonine 186. *J Biol Chem,* 280, 28819-28826.

and 55k isoforms. *J Cell Physiol,* 203, 251-260.

of the human CDK9 gene. *Gene,* 252, 51-59.

transcription elongation to leukemia. *Mol Cell,* 37, 429-437.

recruitment and allosteric activation of mammalian mRNA capping enzyme. *Mol* 

regulation of the HAND1 gene and abnormality of the left ventricle of the heart

bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. *Mol Cell,* 19, 523-534. Jiang, H. & Peterlin, B.M. (2008) Differential chromatin looping regulates CD4 expression in

elongation factor b and represses transcriptional elongation by RNA polymerase II:

to P-TEFb and blocks the expression of MHC class II genes in HIV infection.

TEFb for transcription, cellular proliferation and apoptosis. *Oncogene,* 22, 5707-5711.

phosphorylation for multicolor imaging of intracellular signal flow dynamics. *Anal* 

quiescence of forebrain stem cell proliferation leading to exhaustion of their

S.M., Bresnick, E.H., Zhao, K., Westphal, H. & Love, P.E. (2011) Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of

inactive P-TEFb complex indicates that it contains one 7SK molecule, a dimer of HEXIM1 or HEXIM2, and two P-TEFb molecules containing Cdk9 phosphorylated

Conaway, J.W., Conaway, R.C. & Shilatifard, A. (2010) AFF4, a component of the ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link


Eissenberg, J.C., Shilatifard, A., Dorokhov, N. & Michener, D.E. (2007) Cdk9 is an essential

Ema, H., Sudo, K., Seita, J., Matsubara, A., Morita, Y., Osawa, M., Takatsu, K., Takaki, S. &

Fearon, D.T., Manders, P. & Wagner, S.D. (2001) Arrested differentiation, the self-renewing

Foudi, A., Hochedlinger, K., Van Buren, D., Schindler, J.W., Jaenisch, R., Carey, V. & Hock,

Freter, R., Osawa, M. & Nishikawa, S. (2010) Adult stem cells exhibit global suppression of RNA polymerase II serine-2 phosphorylation. *Stem Cells,* 28, 1571-1580. Fuchs, E., Tumbar, T. & Guasch, G. (2004) Socializing with the neighbors: stem cells and

Fujinaga, K., Irwin, D., Huang, Y., Taube, R., Kurosu, T. & Peterlin, B.M. (2004) Dynamics of

Fujinaga, K., Irwin, D., Taube, R., Zhang, F., Geyer, M. & Peterlin, B.M. (2002) A minimal

Gojo, I., Zhang, B. & Fenton, R.G. (2002) The cyclin-dependent kinase inhibitor flavopiridol

Grana, X., De Luca, A., Sang, N., Fu, Y., Claudio, P.P., Rosenblatt, J., Morgan, D.O. &

Guenther, M.G., Levine, S.S., Boyer, L.A., Jaenisch, R. & Young, R.A. (2007) A chromatin

Hanyu-Nakamura, K., Sonobe-Nojima, H., Tanigawa, A., Lasko, P. & Nakamura, A. (2008)

Hargreaves, D.C., Horng, T. & Medzhitov, R. (2009) Control of inducible gene expression by

signal-dependent transcriptional elongation. *Cell,* 138, 129-145.

state expression is independent of SKP2. *Mol Cell Biol,* 23, 5165-5173. Garriga, J., Peng, J., Parreno, M., Price, D.H., Henderson, E.E. & Grana, X. (1998)

and down-regulation of Mcl-1. *Clin Cancer Res,* 8, 3527-3538.

stem cells from normal and Lnk-deficient mice. *Dev Cell,* 8, 907-914.

memory lymphocyte, and vaccination. *Science,* 293, 248-250.

hematopoietic stem cells. *Nat Biotechnol,* 27, 84-90.

differentiation. *Blood,* 112, 4884-4894.

their niche. *Cell,* 116, 769-778.

*Biol,* 24, 787-795.

3093-3102.

3834-3838.

germ cells. *Nature,* 451, 730-733.

77-88.

kinase in Drosophila that is required for heat shock gene expression, histone methylation and elongation factor recruitment. *Mol Genet Genomics,* 277, 101-114. Elagib, K.E., Mihaylov, I.S., Delehanty, L.L., Bullock, G.C., Ouma, K.D., Caronia, J.F., Gonias,

S.L. & Goldfarb, A.N. (2008) Cross-talk of GATA-1 and P-TEFb in megakaryocyte

Nakauchi, H. (2005) Quantification of self-renewal capacity in single hematopoietic

H. (2009) Analysis of histone 2B-GFP retention reveals slowly cycling

human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element. *Mol Cell* 

chimera of human cyclin T1 and tat binds TAR and activates human immunodeficiency virus transcription in murine cells. *J Virol,* 76, 12934-12939. Garriga, J., Bhattacharya, S., Calbo, J., Marshall, R.M., Truongcao, M., Haines, D.S. & Grana,

X. (2003) CDK9 is constitutively expressed throughout the cell cycle, and its steady-

Upregulation of cyclin T1/CDK9 complexes during T cell activation. *Oncogene,* 17,

induces apoptosis in multiple myeloma cells through transcriptional repression

Giordano, A. (1994) PITALRE, a nuclear CDC2-related protein kinase that phosphorylates the retinoblastoma protein in vitro. *Proc Natl Acad Sci U S A,* 91,

landmark and transcription initiation at most promoters in human cells. *Cell,* 130,

Drosophila Pgc protein inhibits P-TEFb recruitment to chromatin in primordial


Transcriptional Quiescence of Hematopoietic Stem Cells 75

Rahl, P.B., Lin, C.Y., Seila, A.C., Flynn, R.A., McCuine, S., Burge, C.B., Sharp, P.A. & Young, R.A. (2010) c-Myc regulates transcriptional pause release. *Cell,* 141, 432-445. Ramakrishnan, R., Yu, W. & Rice, A.P. (2011) Limited redundancy in genes regulated by

Salmon, P., Kindler, V., Ducrey, O., Chapuis, B., Zubler, R.H. & Trono, D. (2000) High-level

Sehgal, P.B. & Darnell, T. (1976) The inhibition by DRB (5,6-dichloro-1-beta-D-

Shackleton, M., Vaillant, F., Simpson, K.J., Stingl, J., Smyth, G.K., Asselin-Labat, M.L., Wu,

Shapiro, H.M. (1981) Flow cytometric estimation of DNA and RNA content in intact cells

Shilatifard, A., Lane, W.S., Jackson, K.W., Conaway, R.C. & Conaway, J.W. (1996) An RNA

Shim, E.Y., Walker, A.K., Shi, Y. & Blackwell, T.K. (2002) CDK-9/cyclin T (P-TEFb) is

Shore, S.M., Byers, S.A., Dent, P. & Price, D.H. (2005) Characterization of Cdk9(55) and

Shore, S.M., Byers, S.A., Maury, W. & Price, D.H. (2003) Identification of a novel isoform of

Sims, R.J., 3rd, Belotserkovskaya, R. & Reinberg, D. (2004) Elongation by RNA polymerase

Singh, K.P., Casado, F.L., Opanashuk, L.A. & Gasiewicz, T.A. (2009) The aryl hydrocarbon

Song, S.H., Kim, A., Ragoczy, T., Bender, M.A., Groudine, M. & Dean, A. (2010) Multiple

Souslova, E.A., Belousov, V.V., Lock, J.G., Stromblad, S., Kasparov, S., Bolshakov, A.P.,

Tian, Y., Ke, S., Chen, M. & Sheng, T. (2003) Interactions between the aryl hydrocarbon

Tothova, Z. & Gilliland, D.G. (2007) FoxO transcription factors and stem cell homeostasis:

insights from the hematopoietic system. *Cell Stem Cell,* 1, 140-152.

receptor has a normal function in the regulation of hematopoietic and other

functions of Ldb1 required for beta-globin activation during erythroid

Pinelis, V.G., Labas, Y.A., Lukyanov, S., Mayr, L.M. & Chudakov, D.M. (2007) Single fluorescent protein-based Ca2+ sensors with increased dynamic range. *BMC* 

receptor and P-TEFb. Sequential recruitment of transcription factors and differential phosphorylation of C-terminal domain of RNA polymerase II at cyp1a1

stained with Hoechst 33342 and pyronin Y. *Cytometry,* 2, 143-150.

differential regulation of two Cdk9 isoforms. *Gene,* 350, 51-58.

stem/progenitor cell populations. *Biochem Pharmacol,* 77, 577-587.

II: the short and long of it. *Genes Dev,* 18, 2437-2468.

transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors. *Blood,* 96, 3392-3398. Sato, M., Kawai, Y. & Umezawa, Y. (2007) Genetically encoded fluorescent indicators to

visualize protein phosphorylation by extracellular signal-regulated kinase in single

ribofuranosylbenzimidazole) of hnRNA and mRNA production in HeLa cells. *Cell,*

L., Lindeman, G.J. & Visvader, J.E. (2006) Generation of a functional mammary

polymerase II elongation factor encoded by the human ELL gene. *Science,* 271, 1873-

required in two postinitiation pathways for transcription in the C. elegans embryo.

Cyclin T2 and Cyclin T1. *BMC Res Notes,* 4, 260.

gland from a single stem cell. *Nature,* 439, 84-88.

living cells. *Anal Chem,* 79, 2570-2575.

9, 473-480.

1876.

*Genes Dev,* 16, 2135-2146.

Cdk9. *Gene,* 307, 175-182.

*Biotechnol,* 7, 37.

differentiation. *Blood,* 116, 2356-2364.

promoter. *J Biol Chem,* 278, 44041-44048.


Mancebo, H.S., Lee, G., Flygare, J., Tomassini, J., Luu, P., Zhu, Y., Peng, J., Blau, C., Hazuda,

Meier, N., Krpic, S., Rodriguez, P., Strouboulis, J., Monti, M., Krijgsveld, J., Gering, M.,

Muse, G.W., Gilchrist, D.A., Nechaev, S., Shah, R., Parker, J.S., Grissom, S.F., Zeitlinger, J. &

Nguyen, A.W. & Daugherty, P.S. (2005) Evolutionary optimization of fluorescent proteins

Ni, Z., Schwartz, B.E., Werner, J., Suarez, J.R. & Lis, J.T. (2004) Coordination of transcription,

Nishimura, E.K., Jordan, S.A., Oshima, H., Yoshida, H., Osawa, M., Moriyama, M., Jackson,

Orford, K.W. & Scadden, D.T. (2008) Deconstructing stem cell self-renewal: genetic insights

Osawa, M., Egawa, G., Mak, S.S., Moriyama, M., Freter, R., Yonetani, S., Beermann, F. &

Oven, I., Brdickova, N., Kohoutek, J., Vaupotic, T., Narat, M. & Peterlin, B.M. (2007) AIRE

Pajalunga, D., Mazzola, A., Salzano, A.M., Biferi, M.G., De Luca, G. & Crescenzi, M. (2007)

Park, I.K., Qian, D., Kiel, M., Becker, M.W., Pihalja, M., Weissman, I.L., Morrison, S.J. &

Patturajan, M., Schulte, R.J., Sefton, B.M., Berezney, R., Vincent, M., Bensaude, O., Warren,

Peng, J., Zhu, Y., Milton, J.T. & Price, D.H. (1998) Identification of multiple cyclin subunits of

Peterlin, B.M. & Price, D.H. (2006) Controlling the elongation phase of transcription with P-

Radonjic, M., Andrau, J.C., Lijnzaad, P., Kemmeren, P., Kockelkorn, T.T., van Leenen, D.,

required for haematopoietic development. *Development,* 133, 4913-4923.

transcriptional activation in vivo and in vitro. *Genes Dev,* 11, 2633-2644. Marshall, R.M., Salerno, D., Garriga, J. & Graña, X. (2005) Cyclin T1 expression is

human peripheral blood lymphocytes. *J Immunol,* 175, 6402-6411.

Mikkers, H. & Frisen, J. (2005) Deconstructing stemness. *EMBO J,* 24, 2715-2719.

melanocyte stem-cell fate determination. *Nature,* 416, 854-860.

for intracellular FRET. *Nat Biotechnol,* 23, 355-360.

into cell-cycle regulation. *Nat Rev Genet,* 9, 115-128.

niche. *Development,* 132, 5589-5599.

*Cell Biol,* 176, 807-818.

epithelial cells. *Mol Cell Biol,* 27, 8815-8823.

haematopoietic stem cells. *Nature,* 423, 302-305.

cerevisiae stationary phase exit. *Mol Cell,* 18, 171-183.

polymerase II. *J Biol Chem,* 273, 4689-4694.

human P-TEFb. *Genes Dev,* 12, 755-762.

TEFb. *Mol Cell,* 23, 297-305.

*Nat Genet,* 39, 1507-1511.

13, 55-65.

D., Price, D. & Flores, O. (1997) P-TEFb kinase is required for HIV Tat

regulated by multiple signaling pathways and mechanisms during activation of

Patient, R., Hostert, A. & Grosveld, F. (2006) Novel binding partners of Ldb1 are

Adelman, K. (2007) RNA polymerase is poised for activation across the genome.

RNA processing, and surveillance by P-TEFb kinase on heat shock genes. *Mol Cell,*

I.J., Barrandon, Y., Miyachi, Y. & Nishikawa, S. (2002) Dominant role of the niche in

Nishikawa, S. (2005) Molecular characterization of melanocyte stem cells in their

recruits P-TEFb for transcriptional elongation of target genes in medullary thymic

Critical requirement for cell cycle inhibitors in sustaining nonproliferative states. *J* 

Clarke, M.F. (2003) Bmi-1 is required for maintenance of adult self-renewing

S.L. & Corden, J.L. (1998) Growth-related changes in phosphorylation of yeast RNA

van Berkum, N.L. & Holstege, F.C. (2005) Genome-wide analyses reveal RNA polymerase II located upstream of genes poised for rapid response upon S.


**4** 

*1Japan 2Germany* 

**Markers for Hematopoietic Stem Cells:** 

Stephan Ewers2, Dietmar Vestweber2 and Yuzuru Kanakura1

Hematopoietic stem cells (HSC) are characterized with the capacity for self-renewal as well as multi-lineage differentiation, maintaining the immune system and blood cell formation throughout life. Although studies for the HSC biology have been in the forefront of the stem cell research field, many questions still remain with regard to the origin, development, and aging of HSC. Furthermore, needless to say, HSC are very useful for clinical medicine, particularly in the transplantation and/or regeneration therapy for hematological malignancies. Success of those therapies depends on how effectively HSC are purified and transplanted to the patients. In order to address those important issues in both basic and clinical science, information of cell surface molecules

Since the frequency of HSC in bone marrow or peripheral blood is extremely low, many studies have attempted to identify unique markers associated with those rare cells. As a result, it is now possible to purify long-term reconstituting HSC from mouse bone marrow with very high efficiency. However, many of these parameters change dramatically during ontogeny or inflammation, and what is worse still, they differ between mouse and man. Efficient HSC-based therapies and the emerging field of tissueregenerative medicine will benefit from more precise information about what defines

In this chapter, we summarize a large body of information with respect to the HSCrelated markers and introduce Endothelial cell-selective adhesion molecule (ESAM) as a novel marker for HSC (Yokota et al., 2009). Indeed, ESAM is expressed throughout the ontogeny in mouse and can be used as a gating parameter for sorting long-term repopulating HSC. In addition, the marker appears to be useful for the purification of

**1. Introduction** 

HSC.

human HSC.

that selectively mark HSC is essential.

**Histories and Recent Achievements** 

Takafumi Yokota1, Kenji Oritani1, Stefan Butz2,

*Osaka University Graduate School of Medicine, Suita* 

*Max-Planck-Institute for Molecular Biomedicine, Münster,* 

*1Department of Hematology and Oncology,* 

*2Department of Vascular Cell Biology,* 

