**2. The mRNA transcription cycle**

Regulation of gene expression is essential for all single- and multi cellular organisms. This fundamental process is executed at the level of mRNA transcription by RNApII, typically in distinct transcription steps. The different stages of mRNA transcription, initiation, promoter clearance, elongation, mRNA processing and release of RNApII from DNA are tightly regulated by modifications of the CTD of RNApII (Sims *et al.*, 2004, Fig1). This characteristic domain is in mammalian cells composed of 52 repeats of the consensus sequence YS2PTS5PS. During mRNA transcription several posttranslational modifications of the CTD are occurring, most prominently phosphorylation of Serine 5 (Ser5) and Serine 2 (Ser2). These phosphorylation events are requisite for binding of proteins essential for RNA processing, splicing and polyadenylation. Using antibodies specifically detecting these phosphorylation events enables determination of global mRNA transcription activity in single cells in vivo.

Gene expression in mammalian cells has been comprehensively studied at the transcription initiation step that is controlled by cell-specific transcription factors. In fact, it has been thought for long time that assembly of the preinitiation complex and subsequent recruitment on RNApII is the rate-limiting step for gene transcription. However, early results indicated that RNApII is initiated, but paused at Drosophila heat shock genes (Boehm *et al.*, 2003, Ni *et al.*, 2004). More recently, it was observed using genome-wide association studies that initiated but stalled polymerase is not only present on immediateresponse or developmentally regulated genes, but also many non-expressed genes,

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

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

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

Regulation of gene expression is essential for all single- and multi cellular organisms. This fundamental process is executed at the level of mRNA transcription by RNApII, typically in distinct transcription steps. The different stages of mRNA transcription, initiation, promoter clearance, elongation, mRNA processing and release of RNApII from DNA are tightly regulated by modifications of the CTD of RNApII (Sims *et al.*, 2004, Fig1). This characteristic domain is in mammalian cells composed of 52 repeats of the consensus sequence YS2PTS5PS. During mRNA transcription several posttranslational modifications of the CTD are occurring, most prominently phosphorylation of Serine 5 (Ser5) and Serine 2 (Ser2). These phosphorylation events are requisite for binding of proteins essential for RNA processing, splicing and polyadenylation. Using antibodies specifically detecting these phosphorylation events enables determination of global mRNA transcription activity in single cells in vivo. Gene expression in mammalian cells has been comprehensively studied at the transcription initiation step that is controlled by cell-specific transcription factors. In fact, it has been thought for long time that assembly of the preinitiation complex and subsequent recruitment on RNApII is the rate-limiting step for gene transcription. However, early results indicated that RNApII is initiated, but paused at Drosophila heat shock genes (Boehm *et al.*, 2003, Ni *et al.*, 2004). More recently, it was observed using genome-wide association studies that initiated but stalled polymerase is not only present on immediateresponse or developmentally regulated genes, but also many non-expressed genes,

HSC and should lead to characterization of haematopoietic stem cell subpopulations.

niche, in order to expand immature stem cells in vitro.

HSC in vitro will be discussed.

**2. The mRNA transcription cycle** 

suggesting transcription elongation as the critical step in gene expression (Guenther *et al.*, 2007, Muse *et al.*, 2007, Zeitlinger *et al.*, 2007).

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

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.*, 2004).

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

Transcriptional Quiescence of Hematopoietic Stem Cells 65

Most cells, including proliferating, terminally differentiated and senescent cells, actively synthesize mRNA. In these cells, RNApII is phosphorylated on CTD Ser2 and Ser5 independent of the cell cycle (Garriga *et al.*, 2003, Marshall *et al.*, 2005). However, some cells do not display this active phosphorylation pattern. For example, deeply dormant cells, such as primary T and B lymphocytes, exhibit an almost complete absence of RNApII phosphorylation (Garriga *et al.*, 1998, Marshall *et al.*, 2005). Activation of these cells by antigen encounter results in upregulation of Ccnt1 both on mRNA and protein level and

On the other hand, RNApII in yeast cells in the stationary phase (Patturajan *et al.*, 1998, Radonjic *et al.*, 2005) or on Drosophila heat shock genes (Boehm *et al.*, 2003, Ni *et al.*, 2004) is phosphorylated on Ser5, but not on Ser2. Stimulation of these cells, such as the addition of nutrients or heat shock, ensures rapid activation of gene transcription. Thus, analysis of the specific phosphorylated sites in RNApII can distinguish cells featuring phases of productive

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,

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

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

subsequent phosphorylation of CTD Ser2 (Marshall *et al.*, 2005).

mRNA elongation or paused mRNA transcription initiation.

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

activates HIV transcription in murine cells (Fujinaga *et al.*, 2002).

susceptible for small changes in P-TEFb activity.

TATA box upstream of the housekeeping-type promoter of the 42kD isoform (Shore *et al.*, 2003). The expression of both isoforms varies between developmental stages and organs (Shore *et al.*, 2005). For example, the expressed isoform shifts from the longer 55kD form to the shorter 42kD isoform during lymphocyte activation (Liu & Herrmann, 2005). However, the target gene specificity is very similar between the two isoforms (Liu & Herrmann, 2005).

P-TEFb is present in the cell as either a large or small multiprotein complex. The inactive, large complex consists of 7SK non-coding RNA, Hexim1, LARP7 and MEPCE (Li *et al.*, 2005), while the small, active complex is formed by binding of Brd4 to P-TEFb (Yang *et al.*, 2005). Brd4 binds to acetylated histones and may therefore target P-TEFb to actively transcribed genes, when no specific transcription factor is present (Jang *et al.*, 2005) but can also recruit P-TEFb to inducibly acetylated histones (Hargreaves *et al.*, 2009). However, even the large complex contains primarily active P-TEFb, which is sequestered away from the kinase targets. Cellular stresses, such as UV irradiation, cytokines or changes in the microenvironment result in release of active P-TEFb to support quick mRNA transcription to respond to stimuli. Several transcription factors have been shown to interact directly with P-TEFb to stimulate transcription elongation of RNApII. These include NF-kB (Barboric *et al.*, 2001), c-Myc (Eberhardy & Farnham, 2001), CIITA (Kanazawa *et al.*, 2000), GATA1 (Elagib *et al.*, 2008) and Runx1 (Jiang *et al.*, 2005), amongst others. Thus, P-TEFb can be either recruited directly to promoters by specific transcription factors or to acetylated histones by Brd4.

Phosphorylation of Ser2 in the CTD of RNApII and productive transcription elongation is the critical target for eukaryotic gene expression (Bentley, 1995, Chao & Price, 2001). Inhibition of the CTD Ser2 kinase CDK9 by 5,6-Dichloro-1-β-Dribofuranosylbenzimidazole (DRB) or Flavopiridol results in degradation of most mRNA (Chao & Price, 2001, Sehgal & Darnell, 1976) and induces apoptosis (Chen *et al.*, 2005, Gojo *et al.*, 2002). Similarly, knockdown of CDK9 in vivo results in complete absence of mRNA synthesis and embryonic lethality (Eissenberg *et al.*, 2007, Shim *et al.*, 2002). Cyclin T1 KO mice exhibit minor immunological defects (Oven *et al.*, 2007), while the Cyclin T2 KO mouse is embryonic lethal (Kohoutek *et al.*, 2009) with an extremely early phenotype before implantation of the blastocyst. This difference in phenotype of CDK9 Cyclin partners can partly be explained by a limited overlap in target genes of these isoforms (Ramakrishnan *et al.*, 2011).

On the other hand, ectopic activation of P-TEFb by ablation of Hexim1 results in embryonic lethality as well, due to hypertrophy of the heart (Huang *et al.*, 2004). Similarly, inactivation of the inhibitory large complex member LARP7 results in aberrant splicing and embryonic death in zebrafish, highlighting the essential role of P-TEFb for pre-mRNA splicing (Barboric *et al.*, 2009). However, overexpression of CDK9 from the endogenous Rosa26 promoter did not result in any phenotype in mice, mainly due to a low level of expression of CDK9 from this promoter (Freter *et al.*, 2010). Limiting P-TEFb activity is necessary for development of germ line cells in both D. melanogaster and C. elegance (Batchelder *et al.*, 1999, Hanyu-Nakamura *et al.*, 2008, Zhang *et al.*, 2003). Ectopic activation of P-TEFb by overexpression or knock down of inhibitors results in misexpression of somatic genes in germ line cells and their subsequent degeneration, resulting in sterile offspring. In summary, levels of P-TEFb and thus the global activity of RNApII need to be maintained within a certain limits, not exceeding and not below a basal threshold.

TATA box upstream of the housekeeping-type promoter of the 42kD isoform (Shore *et al.*, 2003). The expression of both isoforms varies between developmental stages and organs (Shore *et al.*, 2005). For example, the expressed isoform shifts from the longer 55kD form to the shorter 42kD isoform during lymphocyte activation (Liu & Herrmann, 2005). However, the target gene specificity is very similar between the two isoforms (Liu & Herrmann, 2005). P-TEFb is present in the cell as either a large or small multiprotein complex. The inactive, large complex consists of 7SK non-coding RNA, Hexim1, LARP7 and MEPCE (Li *et al.*, 2005), while the small, active complex is formed by binding of Brd4 to P-TEFb (Yang *et al.*, 2005). Brd4 binds to acetylated histones and may therefore target P-TEFb to actively transcribed genes, when no specific transcription factor is present (Jang *et al.*, 2005) but can also recruit P-TEFb to inducibly acetylated histones (Hargreaves *et al.*, 2009). However, even the large complex contains primarily active P-TEFb, which is sequestered away from the kinase targets. Cellular stresses, such as UV irradiation, cytokines or changes in the microenvironment result in release of active P-TEFb to support quick mRNA transcription to respond to stimuli. Several transcription factors have been shown to interact directly with P-TEFb to stimulate transcription elongation of RNApII. These include NF-kB (Barboric *et al.*, 2001), c-Myc (Eberhardy & Farnham, 2001), CIITA (Kanazawa *et al.*, 2000), GATA1 (Elagib *et al.*, 2008) and Runx1 (Jiang *et al.*, 2005), amongst others. Thus, P-TEFb can be either recruited directly to promoters by specific transcription factors or to acetylated histones by

Phosphorylation of Ser2 in the CTD of RNApII and productive transcription elongation is the critical target for eukaryotic gene expression (Bentley, 1995, Chao & Price, 2001). Inhibition of the CTD Ser2 kinase CDK9 by 5,6-Dichloro-1-β-Dribofuranosylbenzimidazole (DRB) or Flavopiridol results in degradation of most mRNA (Chao & Price, 2001, Sehgal & Darnell, 1976) and induces apoptosis (Chen *et al.*, 2005, Gojo *et al.*, 2002). Similarly, knockdown of CDK9 in vivo results in complete absence of mRNA synthesis and embryonic lethality (Eissenberg *et al.*, 2007, Shim *et al.*, 2002). Cyclin T1 KO mice exhibit minor immunological defects (Oven *et al.*, 2007), while the Cyclin T2 KO mouse is embryonic lethal (Kohoutek *et al.*, 2009) with an extremely early phenotype before implantation of the blastocyst. This difference in phenotype of CDK9 Cyclin partners can partly be explained by a limited overlap in target genes of these isoforms

On the other hand, ectopic activation of P-TEFb by ablation of Hexim1 results in embryonic lethality as well, due to hypertrophy of the heart (Huang *et al.*, 2004). Similarly, inactivation of the inhibitory large complex member LARP7 results in aberrant splicing and embryonic death in zebrafish, highlighting the essential role of P-TEFb for pre-mRNA splicing (Barboric *et al.*, 2009). However, overexpression of CDK9 from the endogenous Rosa26 promoter did not result in any phenotype in mice, mainly due to a low level of expression of CDK9 from this promoter (Freter *et al.*, 2010). Limiting P-TEFb activity is necessary for development of germ line cells in both D. melanogaster and C. elegance (Batchelder *et al.*, 1999, Hanyu-Nakamura *et al.*, 2008, Zhang *et al.*, 2003). Ectopic activation of P-TEFb by overexpression or knock down of inhibitors results in misexpression of somatic genes in germ line cells and their subsequent degeneration, resulting in sterile offspring. In summary, levels of P-TEFb and thus the global activity of RNApII need to be maintained

within a certain limits, not exceeding and not below a basal threshold.

Brd4.

(Ramakrishnan *et al.*, 2011).

Most cells, including proliferating, terminally differentiated and senescent cells, actively synthesize mRNA. In these cells, RNApII is phosphorylated on CTD Ser2 and Ser5 independent of the cell cycle (Garriga *et al.*, 2003, Marshall *et al.*, 2005). However, some cells do not display this active phosphorylation pattern. For example, deeply dormant cells, such as primary T and B lymphocytes, exhibit an almost complete absence of RNApII phosphorylation (Garriga *et al.*, 1998, Marshall *et al.*, 2005). Activation of these cells by antigen encounter results in upregulation of Ccnt1 both on mRNA and protein level and subsequent phosphorylation of CTD Ser2 (Marshall *et al.*, 2005).

On the other hand, RNApII in yeast cells in the stationary phase (Patturajan *et al.*, 1998, Radonjic *et al.*, 2005) or on Drosophila heat shock genes (Boehm *et al.*, 2003, Ni *et al.*, 2004) is phosphorylated on Ser5, but not on Ser2. Stimulation of these cells, such as the addition of nutrients or heat shock, ensures rapid activation of gene transcription. Thus, analysis of the specific phosphorylated sites in RNApII can distinguish cells featuring phases of productive mRNA elongation or paused mRNA transcription initiation.
