**4. Transcriptional quiescence of hematopoietic stem cells**

Hematopoietic stem cells (HSC) have been in the focus of basic and applied research since many decades. Definition of subsets of transplantable HSC and their in vitro culture have advanced considerably in recent years. However, so far no reliable marker for the isolation

induction of chromatin remodelling complexes result in rapid activation of gene transcription by release of active P-TEFb from adjacent loci (Jiang & Peterlin, 2008). In this sense, assembly of the transcriptional machinery on inactive promoters can be seen as a

Activation of hematopoietic gene transcription can also be achieved via binding of P-TEFb to actively acetylated chromatin. Studies on LPS-induced inflammatory gene expression in macrophages revealed that primary response genes have a stalled polymerase at their promoters, already phosphorylated at Ser5 of the CTD (Hargreaves *et al.*, 2009). In response to LPS stimulation, acetylation of H4K5/8/12 recruits Brd4, this in turn engages P-TEFb leading to CTD Ser2 phosphorylation and mRNA transcription (Hargreaves *et al.*, 2009).

Hematopoietic lineages are very susceptible for inhibition of P-TEFb activity and require specific co-factors for their respective differentiation. For example, differentiating macrophages and murine erythroleukemic cells down regulate the 42kD isoform, and up regulate the 55 kD isoform of CDK9 (Liu & Herrmann, 2005). Megakaryocyte differentiation depends on activation of P-TEFb and can be blocked by CDK9 inhibitors or dominant negative CDK9 (Elagib *et al.*, 2008). Erythroid differentiation depends on stabilization of a GATA-1/SCL/LMO2 complex on β-globin chromatin, subsequent association of P-TEFb and RNApII Ser2 phosphorylation in the locus by the ubiquitous enhancer facilitator/chromatin factor Ldb1 (Song *et al.*, 2010). Interestingly, deletion of Ldb1 in vivo results in defects in adult haematopoietic stem cell maintenance and diminished long-term reconstitution potential upon transplantation (Li *et al.*, 2011). However, the authors did not examine activity of P-TEFb in their knock-out mice, which may be reduced and thus result in defects in HSC specification. Certainly, many other genes and cells depend on P-TEFb activity during development and differentiation. Using the hematopoietic lineage as a model system for the basal mRNA transcription machinery will shed light onto many

Conversely, some leukemic cancers are characterized by dysregulation of P-TEFb activity. Several fusion genes of the histone methytransferase MLL1 involved in chromosomal rearrangements leading to myeloid and lymphoblastic leukaemia associate with mRNA transcription elongation factor encoded by ELL or P-TEFb (Benedikt *et al.*, 2011, Lin *et al.*, 2010), suggesting that one major mechanism for leukomogenesis is deregulated transcription elongation (Shilatifard *et al.*, 1996). Indeed, targeting P-TEFb with the specific CDK9 inhibitor Flavopiridol induces apoptosis in chronic lymphocytic leukemic cells by suppression of short-lived anti-apoptotic genes, such as Mcl-1 (Chen *et al.*, 2005). Dysregulation of P-TEFb activity is involved in several other cancer types. For example Hexim1, a negative regulator of P-TEFb activity, was found down regulated in invasive breast cancer samples compared to normal breast tissue (Wittmann *et al.*, 2003). Exploiting the susceptibility of the hematopoietic lineage for disturbance of their mRNA transcription

Hematopoietic stem cells (HSC) have been in the focus of basic and applied research since many decades. Definition of subsets of transplantable HSC and their in vitro culture have advanced considerably in recent years. However, so far no reliable marker for the isolation

transcription bookmark, to facilitate future expression.

aspects of eukaryotic mechanisms of transcription control.

may result in novel targets of cancer therapy.

**4. Transcriptional quiescence of hematopoietic stem cells** 

of pure HSC exists and our use of transplants is limited by the inability to expand these cells ex vivo. A sensitive marker of HSC quiescence and activation could be useful to isolate unadulterated long-term repopulating HSC and screen for factors that enable stem cell expansion while maintaining their undifferentiated state. It has been known for three decades that HSC down regulate productive mRNA transcription. Low retention of Pyronin Y, an RNA binding dye, can be used to isolate HSC (Shapiro, 1981), suggesting that global suppression of mRNA transcription is a feature of quiescent HSC. However, this observation has not been followed up by analysis of the global status of RNApII activity, which is responsible for all mRNA transcription.

We have previously observed that adult melanocyte stem cells (MelSC) down regulate many housekeeping genes, including ActB, ActG and GapDH, suggesting a global repression of mRNA synthesis in these cells (Osawa *et al.*, 2005). Melanocytes are pigmented cells in the hair follicle and skin, providing melanin granules to differentiating keratinocytes. The MelSC system in mouse hair follicles can serve as a model system for adult stem cell systems. It has the advantage of spatial separation of stem and differentiated cells and a non-lethal but obvious hair graying phenotype if this system is perturbed (Nishimura *et al.*, 2002). We observed that adult MelSC show a complete absence of RNApII CTD Ser2 phosphorylation, while Ser5 was phosphorylated (Freter *et al.*, 2010), suggesting a global down regulation of productive mRNA transcription. In line with this, CDK9 protein and mRNA was down regulated in MelSC as well. This suggests that RNApII is present at many genes in quiescent MelSC, but P-TEFb levels are not sufficient to induce active transcription elongation. Importantly, inhibition of CDK9 in vitro protected melanocyte precursors from stress-induced apoptosis and converted them to a stem cell-like state (Freter *et al.*, 2010).

We then expanded our observation to other stem cell systems, and found CTD Ser2-negative cells in all stem cell systems tested, including keratinocyte, muscle, spermatogonia and hematopoietic stem cells. This suggests that global suppression of mRNA transcription elongation is a conserved feature of adult stem cells. Interestingly, some stem cell systems showed heterogeneity of CTD Ser2 staining. For example, we observed that spermatogonia stem cells attached to the basal lamina are negative for CTD Ser2 phosphorylation, while those detaching up-regulate Ser2 phosphorylation, even though they are still positive for the spermatogonia stem cell marker CD9 (Freter *et al.*, 2010). Attachment to the basal lamina is often a requirement for stem cell function by directing planes of division or maintenance of the undifferentiated state. Thus, the CTD Ser2 negative population seems to be the more stem cell-like population in CD9 positive spermatogonia.

Similarly, murine CD34- c-Kit+Sca1+Lin- (KSL) long-term repopulating hematopoietic stem cells clearly showed two different populations. One population exhibited CTD Ser2 phosphorylation levels as high as short-term repopulating CD34+ KSL cells, while ~27% of all CD34- KSL cells were negative for CTD Ser2 phosphorylation (Freter *et al.*, 2010). Heterogeneity of the HSC pool has been described previously, with a transplantable fraction of 15-25% of CD34- KSL HSC population, also using additional markers (Ema *et al.*, 2005, Foudi *et al.*, 2009, Wilson *et al.*, 2008). Importantly, analysis of transcriptionally quiescent HSC requires isolation of pure subpopulations of cells. We found that adult MelSC exhibit up to 100fold lower levels of total RNA per cell (Osawa *et al.*, 2005), suggesting that one activated stem cell may be sufficient to mask the RNA signal of 100 quiescent stem cells.

Transcriptional Quiescence of Hematopoietic Stem Cells 69

recruitment of Ccnt1 and activation of hematopoietic gene transcription (Tian *et al.*, 2003). Interestingly, antagonists of the Aryl receptor prevent differentiation of HSC in vitro (Boitano *et al.*, 2010), suggesting that transcriptional quiescence may be beneficial for in vitro stem cell expansion. It would be very interesting to determine if inhibitors of P-TEFb activity

Transcriptional quiescence could also be used as a read-out of stem cell function in vitro. Screens of small molecular compounds in in vitro culture of primary HSC for maintenance of the human HSC markers CD34 and CD133 have led to some promising results (Boitano *et al.*, 2010). However, surface proteins may be unstable or unreliable, and may not be an immediate read-out of stem cell function. Transcriptional quiescence could serve as an alternative marker for stem cell identity. The development of fluorescent reporters for this screening is required to evaluate the impact of cytokines or small molecules on maintenance

We and others have shown that inhibition of P-TEFb activity can be favourable for cell survival during cellular stresses, such as serum or growth factor starvation (Freter *et al.*, 2010, Kanazawa *et al.*, 2003). Down regulation of P-TEFb activity could thus also be advantageous for cancer cell survival during metastasis or therapy. Flavopiridol, a very specific CDK9 inhibitor has been used in BLL with some success (Chen *et al.*, 2005), but failed in most cases as a single agent in cancer chemotherapy (Blagosklonny, 2004). If transcriptionally quiescent cancer cells are present in primary or metastatic tumours, further inhibition of CDK9 activity may not be required. Rather, activation of mRNA transcription in these cells may render them susceptible to therapy and prevent metastasis and relapse. The first step towards this goal would be to identify and isolate transcriptionally quiescent cancer cells from a given tumour using a kinase activity reporter or CDK9 promoter reporter. Next, it would be necessary to determine if transcriptionally quiescent tumour cells have an enhanced tumour forming capacity in vivo or survive treatment with chemotherapeutic agents better than transcriptionally activated cells. Finally, high throughput screens for small molecular compounds which activate transcriptional quiescent cancer cells using the same reporter systems will enable us to activate dormant cancer stem cell-like cells in vivo and improve treatment of metastasis and prevent relapse of cancer in

Adult stem cells have the unique capacity to self-renew and give rise to differentiated cells. To fulfil their lifelong function, these cells must be protected from cellular and genetic damage. Most adult stem cells are thought to enter a state of reversible cell cycle quiescence to preserve their role (Orford & Scadden, 2008). Indeed, activation of the cell cycle leads to premature stem cell exhaustion (Cheng *et al.*, 2000, Kippin *et al.*, 2005, Park *et al.*, 2003). However, most cells in the adult body have withdrawn from the cell cycle and can be induced to proliferate again, resulting in their eventual depletion (Bond *et al.*, 2004, Pajalunga *et al.*, 2007). Some somatic cells are able to resume proliferation and even selfrenew, for example differentiated T and B lymphocytes (Fearon *et al.*, 2001). It has thus been proposed that adult stem cells are distinguished by other mechanisms rather than cell cycle quiescence (Mikkers & Frisen, 2005). Yet, what kind of defining mechanisms or marker this property integrates, and if it is shared by various adult stem cells, is unclear at the moment.

have an effect on maintenance of undifferentiated HSC.

and expansion of HSC in vitro.

patients.

**5. Conclusion** 

In order to identify the transcriptionally quiescent subpopulation of CD34- KSL cells, we performed sorting of bone marrow cells and antibody staining of sorted populations. Unfortunately, this procedure always includes fixation of cells, so they can not be used for assessment of in vivo repopulation ability. In order to validate stem cell function in vivo, it is necessary to convert the negative CTD Ser2 phosphorylation event into a readout which can be measured in living cells. Kinase activity can be measured using fusion proteins of Cyan and Yellow fluorescent proteins (CFP and YFP respectively), separated by a kinase target, a flexible linker and a phosphoprotein binding domain. Phosphorylation events result in binding of the phosphoacceptor to the kinase target and folding of the fusion protein resulting in Foerster resonance energy transfer (FRET) between CFP and YFP (Sato *et al.*, 2007). However, autofluorescence of cellular organelles in low energetic wavelengths, such as CFP, results in low signal to noise ratios, which is particularly difficult for cell sorting of multiparametric cell suspensions, such as bone marrow cells. Furthermore, even a complete lack of FRET by spatial separation results in FRET signal due to high concentration of expressed fluorescent proteins (Nguyen & Daugherty, 2005). Thus, a FRET-based approach can be useful for single-cell based imaging approaches, such as time lapse imaging of individual cells in vitro, but rather not for FACS sorting of heterogenic cell populations.

Recently, the development of circular permuted green fluorescent proteins (cpGFP) has enabled researchers to measure phosphorylation events in living cells using a single wavelength (Kawai *et al.*, 2004). However, the increase in fluorescence was only around 10- 15%, which would be too little for separation by FACS. Novel mutations of circular GFP and ratiometric measurement of absorbance at different wavelengths increased the dynamic range up to 16fold between free and saturated forms of cpGFP, at least for measurement of pH or Ca2+ in living cells (Bizzarri *et al.*, 2006, Souslova *et al.*, 2007). Yet, if a kinase reporter can be constructed using these advanced cpGFP mutants has still to be shown.

We could observe a specific down regulation of CDK9, the RNApII CTD Ser2 kinase, in adult melanocyte stem cells both on the mRNA and protein levels (Freter *et al.*, 2010). The CDK9 promoter has many features of a housekeeping gene promoter (Bagella *et al.*, 2000, Liu & Rice, 2000), thus down regulation of this promoter may be due to a similar mechanism as other down regulated housekeeping genes in MelSC. Reporters for promoter function, for example GFP, Luciferase or LacZ have been used extensively to isolate or trace specific cells in vivo. However, isolating CDK9 promoter negative cells as transcriptionally quiescent stem cells could be biased by secondary effects on the reporter, such as silencing of reporter constructs or heterogeneity of expression between cells. One solution would be to label all cells with a constitutive reporter, and isolate constitutive promoter positive, CDK9 promoter reporter negative cells. The constitutive promoter has to be carefully chosen, as for example expression from the CMV promoter highly depends on CDK9 (Peng *et al.*, 1998). Given our observation of low activity of CDK9 in HSC, it is not surprising that HSC show limited CMV promoter activity (Salmon *et al.*, 2000). Constitutive promoters, but also promoters used for overexpression of genes in quiescent stem cells thus need to be validated for activity in vivo.

In vitro culture and expansion of immature hematopoietic stem cells could help to achieve better transplantation response in patients, but has not been achieved yet. So far culture of immature HSC leads to almost immediate differentiation and loss of multi-lineage repopulation ability. It has been shown recently that the xenobiotic Aryl receptor is present on HSC (Singh *et al.*, 2009). Activation by ligands results in nuclear translocation,

In order to identify the transcriptionally quiescent subpopulation of CD34- KSL cells, we performed sorting of bone marrow cells and antibody staining of sorted populations. Unfortunately, this procedure always includes fixation of cells, so they can not be used for assessment of in vivo repopulation ability. In order to validate stem cell function in vivo, it is necessary to convert the negative CTD Ser2 phosphorylation event into a readout which can be measured in living cells. Kinase activity can be measured using fusion proteins of Cyan and Yellow fluorescent proteins (CFP and YFP respectively), separated by a kinase target, a flexible linker and a phosphoprotein binding domain. Phosphorylation events result in binding of the phosphoacceptor to the kinase target and folding of the fusion protein resulting in Foerster resonance energy transfer (FRET) between CFP and YFP (Sato *et al.*, 2007). However, autofluorescence of cellular organelles in low energetic wavelengths, such as CFP, results in low signal to noise ratios, which is particularly difficult for cell sorting of multiparametric cell suspensions, such as bone marrow cells. Furthermore, even a complete lack of FRET by spatial separation results in FRET signal due to high concentration of expressed fluorescent proteins (Nguyen & Daugherty, 2005). Thus, a FRET-based approach can be useful for single-cell based imaging approaches, such as time lapse imaging of individual cells in vitro, but rather not for FACS sorting of heterogenic cell populations. Recently, the development of circular permuted green fluorescent proteins (cpGFP) has enabled researchers to measure phosphorylation events in living cells using a single wavelength (Kawai *et al.*, 2004). However, the increase in fluorescence was only around 10- 15%, which would be too little for separation by FACS. Novel mutations of circular GFP and ratiometric measurement of absorbance at different wavelengths increased the dynamic range up to 16fold between free and saturated forms of cpGFP, at least for measurement of pH or Ca2+ in living cells (Bizzarri *et al.*, 2006, Souslova *et al.*, 2007). Yet, if a kinase reporter

can be constructed using these advanced cpGFP mutants has still to be shown.

We could observe a specific down regulation of CDK9, the RNApII CTD Ser2 kinase, in adult melanocyte stem cells both on the mRNA and protein levels (Freter *et al.*, 2010). The CDK9 promoter has many features of a housekeeping gene promoter (Bagella *et al.*, 2000, Liu & Rice, 2000), thus down regulation of this promoter may be due to a similar mechanism as other down regulated housekeeping genes in MelSC. Reporters for promoter function, for example GFP, Luciferase or LacZ have been used extensively to isolate or trace specific cells in vivo. However, isolating CDK9 promoter negative cells as transcriptionally quiescent stem cells could be biased by secondary effects on the reporter, such as silencing of reporter constructs or heterogeneity of expression between cells. One solution would be to label all cells with a constitutive reporter, and isolate constitutive promoter positive, CDK9 promoter reporter negative cells. The constitutive promoter has to be carefully chosen, as for example expression from the CMV promoter highly depends on CDK9 (Peng *et al.*, 1998). Given our observation of low activity of CDK9 in HSC, it is not surprising that HSC show limited CMV promoter activity (Salmon *et al.*, 2000). Constitutive promoters, but also promoters used for overexpression of genes in quiescent stem cells thus need to be validated for activity in vivo. In vitro culture and expansion of immature hematopoietic stem cells could help to achieve better transplantation response in patients, but has not been achieved yet. So far culture of immature HSC leads to almost immediate differentiation and loss of multi-lineage repopulation ability. It has been shown recently that the xenobiotic Aryl receptor is present on HSC (Singh *et al.*, 2009). Activation by ligands results in nuclear translocation, recruitment of Ccnt1 and activation of hematopoietic gene transcription (Tian *et al.*, 2003). Interestingly, antagonists of the Aryl receptor prevent differentiation of HSC in vitro (Boitano *et al.*, 2010), suggesting that transcriptional quiescence may be beneficial for in vitro stem cell expansion. It would be very interesting to determine if inhibitors of P-TEFb activity have an effect on maintenance of undifferentiated HSC.

Transcriptional quiescence could also be used as a read-out of stem cell function in vitro. Screens of small molecular compounds in in vitro culture of primary HSC for maintenance of the human HSC markers CD34 and CD133 have led to some promising results (Boitano *et al.*, 2010). However, surface proteins may be unstable or unreliable, and may not be an immediate read-out of stem cell function. Transcriptional quiescence could serve as an alternative marker for stem cell identity. The development of fluorescent reporters for this screening is required to evaluate the impact of cytokines or small molecules on maintenance and expansion of HSC in vitro.

We and others have shown that inhibition of P-TEFb activity can be favourable for cell survival during cellular stresses, such as serum or growth factor starvation (Freter *et al.*, 2010, Kanazawa *et al.*, 2003). Down regulation of P-TEFb activity could thus also be advantageous for cancer cell survival during metastasis or therapy. Flavopiridol, a very specific CDK9 inhibitor has been used in BLL with some success (Chen *et al.*, 2005), but failed in most cases as a single agent in cancer chemotherapy (Blagosklonny, 2004). If transcriptionally quiescent cancer cells are present in primary or metastatic tumours, further inhibition of CDK9 activity may not be required. Rather, activation of mRNA transcription in these cells may render them susceptible to therapy and prevent metastasis and relapse. The first step towards this goal would be to identify and isolate transcriptionally quiescent cancer cells from a given tumour using a kinase activity reporter or CDK9 promoter reporter. Next, it would be necessary to determine if transcriptionally quiescent tumour cells have an enhanced tumour forming capacity in vivo or survive treatment with chemotherapeutic agents better than transcriptionally activated cells. Finally, high throughput screens for small molecular compounds which activate transcriptional quiescent cancer cells using the same reporter systems will enable us to activate dormant cancer stem cell-like cells in vivo and improve treatment of metastasis and prevent relapse of cancer in patients.
