**3. mRNA transcription in the hematopoietic lineage**

Most of our knowledge of P-TEFb function derived from studies involving HIV replication (Barboric & Peterlin, 2005). Human Cyclin T1 is an essential co-factor of the immediate-early HIV gene product Tat, which recruits P-TEFb to the tar RNA located 5' on HIV genes to activate gene expression (Mancebo *et al.*, 1997, Zhu *et al.*, 1997). In resting lymphocytes, P-TEFb activity is low and thus HIV replication is blocked. Upon stimulation, upregulation of Cyclin T1 results in activation of P-TEFb and transcription of viral genes (Garriga *et al.*, 1998). Human Cyclin T1 (Ccnt1), the major Cyclin associated with CDK9, is characterized by a TAR recognition motif, which is essential for the formation of a ternary complex between tar RNA/Tat and P-TEFb to activate HIV gene transcription in cells (Wei *et al.*, 1998). This motif contains an essential Cysteine, which is required for complex formation with HIV Tat. Mutagenesis of mouse Ccnt1 at this position, which normally contains a Tyrosine in mouse, activates HIV transcription in murine cells (Fujinaga *et al.*, 2002).

P-TEFb is also required for normal hematopoietic development and function. Knock down of CDK9 in zebrafish embryos results in severe defects in definitive erythropoiesis, but no gross developmental defects despite a smaller body size (Meier *et al.*, 2006). Given the ubiquitous requirement of CDK9 for mRNA transcription this surprising observation may be explained by incomplete knockdown using morpholino DNA. Similarly, partial depletion of Ccnt1 in mice results in modest immunological phenotypes, such as appearance of autoimmunity due to impaired negative selection of autoreactive T cells in thymus (Oven *et al.*, 2007). Together, these results suggest that the hematopoietic lineage may be very susceptible for small changes in P-TEFb activity.

Recruitment of P-TEFb by transcription factors to heterochromatin converts this general elongation factor to a repressor of transcription. Runx1 binds the CD4 silencer in thymocytes and leads to active suppression of CD4 transcription during development. Interestingly, despite an engaged RNApII on the CD4 promoter and in the presence of an active CD4 enhancer in these cells, CD4 transcription is silenced (Jiang *et al.*, 2005). This is achieved by binding of Cyclin T1 to Runx1 and sequestering of P-TEFb into inactive chromatin loops (Jiang & Peterlin, 2008). Thus, inactive genes can be loaded with a poised polymerase and

Transcriptional Quiescence of Hematopoietic Stem Cells 67

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,

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

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

which is responsible for all mRNA transcription.

stem cell-like population in CD9 positive spermatogonia.

quiescent stem cells.

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 transcription bookmark, to facilitate future expression.

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 aspects of eukaryotic mechanisms of transcription control.

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 may result in novel targets of cancer therapy.
