Transcription termination and 3’end pre-mRNA processing

Most eukaryotic mRNA possess a poly(A) tail at their 3’ extremity, which is formed by endonucleolytic cleavage of the transcript followed by addition of a poly(A) tail (reviewed by Danckwardt et al., 2008 ; Mandel et al., 2008). These reactions are catalyzed by multiprotein complexes which bind specific elements at the 3’ end of the pre-mRNA. The polyadenylation signal, located 10-30 nucleotides upstream of the cleavage site, is bound by the cleavage/polyadenylation specific factor (CPSF), while the downstream sequence element (DSE) is recognized by the cleavage-stimulating factor (Cstf) (Fig. 4). Other RNA elements upstream of the cleavage site have been identified to anchor the 3’ end-processing machinery or to recruit other components such as the cleavage factor CFIm. After assembly of the multimeric complexes at their elements and endonucleolytic cleavage by CPSF, a nuclear poly(A) polymerase (PAP) synthesizes the 3’ end poly(A) tail.

Similarly to capping and splicing, transcription termination and 3’ end processing is regulated by multiple interactions with the elongating polymerase, and CTD truncation results in 3’ end processing defects (McCracken et al., 1997b ; Fong et al., 2003 ; Rosonina and Blencowe, 2004). Several components of both CPSF and Cstf complexes directly interact with the phosphorylated CTD (Barilla et al., 2001 ; Licatalosi et al., 2002 ; Proudfoot et al., 2002 ; Rosonina et al., 2006). Recently, it was demonstrated that Ser-2 phosphorylation is critically required for proper 3’ processing of the transcripts. The loss of Ser-2 phosphorylation caused by Cdk9 (Ctk1) mutation in yeast (Ahn et al., 2004) or following flavopiridol inhibition of P-TEFb in Drosophila (Ni et al., 2004) leads to inefficient mRNA processing. Intriguingly, the authors found that inactivation of the Cdk9 kinase activity had little effects on transcription elongation, while the recruitment of 3’ processing factors was impaired resulting in rapid degradation of the transcripts. Thus it was proposed that Ser-2

phosphorylation is necessary for accurate transcription termination and 3’ end processing, but appeared dispensable for transcription elongation of certain genes such as the intron-less Drosophila heat shock genes (Ni et al., 2004).

Transcription termination, which results in the dissociation of the elongation complex and the release of the mRNA, is intimately coupled with the 3’ processing machinery and requires additional factors that bind RNAPII and its CTD such as TTF2 (transcription termination factor 2) and Pcf11 (pre-mRNA cleavage complex 2 protein Pcf11) (Jiang et al., 2004 ; Zhang and Gilmour, 2006 ; West and Proudfoot, 2008). However, in contrast to the 3’

processing factors, the recruitment of these proteins does not depend on the phosphorylation status of the CTD.

As discussed above, transcription elongation is an integrated process that is tightly coupled to pre-mRNA processing, and the phosphorylation cycle of the RNAPII CTD occurs to coordinate these events. Extensive crosstalk between the pre-mRNA processing activities is also required for efficient transcription (Fig. 4). Indeed, some proteins that bind to the cap were shown to facilitate the recruitment of the splicing machinery at the cap-proximal splice site (Lewis et al., 1996 ; Colot et al., 1996). Similarly, many studies have reported a physical interaction of the cleavage/polyadenylation complexes with splicing factors, which stimulates both terminal intron splicing and 3’ end processing (Vagner et al., 2000 ; Li et al., 2001 ; McCracken et al., 2002 ; Awasthi and Alwine, 2003 ; Millevoi et al., 2006 ; Kyburz et al., 2006 ; Danckwardt et al., 2007). The cooperation between the different co-transcriptional mechanisms ensures the efficient production of mature mRNA, and the packaging of the pre-mRNA within the processing complexes is thought to protect the transcript from nuclease degradation and to prevent interactions with the DNA template during transcription (Li and Manley, 2006 ; Hicks et al., 2006).

1.3.4. Transcription elongation and chromatin remodeling.

The packaging of the DNA template into chromatin represents an important block to transcription elongation. The basic repeating unit of chromatin, the nucleosome, contains two copies of the four core histones H2A, H2B, H3 and H4 wrapped around by a 147 base pairs DNA. During transcription, RNAPII associates with multiple factors that promote changes in the chromatin structure. These factors include ATP-dependent chromatin remodelers, histone-modifying enzymes and histone chaperones (reviewed by Kulaeva et al., 2007 ; Li et al.,

Figure 4. Schematic drawing of the 3’ end processing machinery in mammals (Danckwardt et al., 2008). The cleavage/polyadenylation complexes CPSF and CstF are recruited to the elongation complex through interaction with the Ser-2-phosphorylated CTD, and binds distinct RNA elements. CPSF recognizes the polyadenylation signal (AAUAAA) while CstF associates with the downstream sequence element (DSE). CPSF 73 catalyzes the endonucleolytic cleavage of the transcript at the cleavage site (CA) allowing the subsequent addition of the poly(A) tail by the poly(A) polymerase (PAP). Multiple stimulatory interactions between the pre-mRNA processes (capping, splicing and cleavage/polyadenylation) occur to coordinate their activities during transcription, and are indicated by an arrow (+).

2007). Many of these factors are recruited to the TEC through direct or indirect interaction with the phosphorylated RNAPII CTD, which thus coordinates transcriptional events with chromatin remodeling.

1.3.4.1. Histone eviction by chromatin-remodeling factors.

Transcription through chromatin implies the displacement of the nucleosomes, or a transient and partial removal of the histone octamers depending on the rate of gene expression. These mechanisms involve the concerted action of two classes of chromatin modifiers. One consists of chromatin-remodeling complexes, which utilize ATP hydrolysis to alter the histone-DNA contacts, and results in transient unwrapping of the DNA from the histone octamer, or in transient displacement of the nucleosome along the DNA template (nucleosome sliding) (reviewed by Bouazoune and Brehm, 2006 ; Saha et al., 2006). Histone eviction and reassembly require the activity of another set of chromatin modifiers, the histone chaperones, which selectively remove some histone protein from the octamer, and thus allow RNAPII to traverse the nucleosome (reviewed by Sims et al., 2004 ; Kulaeva et al., 2007).

Particularly, H2A/H2B histone dimers were shown to be evicted/exchanged at higher rate compared to H3 and H4 during transcription (Kimura and Cook, 2001 ; Schwabish and Struhl, 2004 ; Thiriet and Hayes, 2005 ; Workman, 2006), although H3 replacement by the H3.3 histone variant also occurs at transcriptionally active genes in Drosophila (Ahmad and Henikoff, 2002 ; Mito et al., 2005 ; Schwartz and Ahmad, 2005). Many of the remodelers associates with the polymerase after transcription initiation, including the conserved histone chaperones FACT (facilitates chromatin transcription) and Spt6 (suppressor of Ty 6), as well as Chd1 (chromodomain helicase DNA-binding protein 1). The ATP-dependent chromatin remodeler SWI/SNF (switch/sucrose nonfermentable) is known to function in early events of transcription such as PIC assembly, and may be well required during elongation. The current data suggest a model in which FACT, in cooperation with RNAPII and the chromatin remodelers, destabilizes the nucleosomes by selectively removing one H2A/H2B dimer. After passage of the polymerase, the chaperone activities of FACT, Spt6 and Chd1 may facilitate the reassembly of nucleosomes (Reinberg and Sims, 2006) (Fig. 5A). Other proteins that associate with the TEC were shown to influence this process in yeast, including the transcription factor TFIIS, the chromatin factor Spt2, and the histone chaperone Hir (histone regulatory protein) (Kulaeva et al., 2007). It is not known however how these factors coordinate their activities with the other chromatin factors FACT, Spt6 and Chd1.

Figure 5A. Putative role of FACT, Chd1, and Spt6 in nucleosome eviction/reassembly. Step 1, the removal of one H2A/H2B dimer by FACT (Spt16/SSRP1) facilitates transcript elongation through the nucleosome. Step 2, the chaperone activities of FACT, Chd1, and Spt6 may facilitate the reassembly of nucleosomes following the wake of transcript elongation (Reinberg and Sims, 2006).

Figure 5B. Distribution profiles of histone proteins and their modifications across genes.

Histone modifications associated with transcriptionally active genes are indicated by (+) on the right panel (Li et al., 2007).

1.3.4.2. Histone modifications and transcription.

The nucleosomes are subjected to a variety of post-translational modifications which play an important role in the regulation of transcription through chromatin. Particularly, the N-terminal histone tails are targeted by multiple enzymes that covalently modify specific residues. These modifications include acetylation, methylation, ubiquitination, ADP-ribosylation, sumoylation and phosphorylation (reviewed by Shilatifard, 2006 ; Li et al., 2007). Genes that are transcriptionally active have been associated with specific modifications such as acetylation of histone H3 and H4 (H3ac, H4ac), di- and trimethylation of H3 at lysine 4 (H3K4me2/3), lysine 36 (H3K36me2/3) and lysine 79 (H3K79me) or phosphorylation of H3 at serine 10 (H3S10p). Most of these modifications are deposited sequentially at the histone tails during transcription, and are distributed in a distinct pattern throughout the transcription unit (Fig. 5B). These modifications are notably involved in the maintenance of an open chromatin structure and modulate specific elongation events by promoting the recruitment of multiple transcription factors (Fig. 6).

Histone H3 and H4 acetylation occurs at several lysine residues and is carried out by multiple histone acetyltransferase (HATase) complexes (reviewed by Brown et al., 2000).

Histone acetylation is an important prerequisite for productive elongation, and H4K16ac was notably shown to inhibit higher-order chromatin structure (Shogren-Knaak et al., 2006).

Histone acetylation occurs at active promoter prior to PIC assembly, and both HATase enzymes and chromatin remodelers such as SAGA (Spt-Ada-Gcn5-acetyltransferase complex) and SWI/SNF are concomitantly recruited to increase DNA accessibility for PIC formation in yeast (Hassan et al., 2001 ; Bhaumik and Green, 2001 ; Neely et al., 2002).

Recent genome-wide analysis of HATases binding and histone acetylation in yeast showed that H3ac and H4ac peaks sharply at the promoter region of active genes, and that both Gcn5 and Esa1 HATases are generally recruited to promoters (Robert et al., 2004 ; Pokholok et al., 2005). Some HATase complexes including Elongator in yeast and human cells (Winkler et al., 2002 ; Kim et al., 2002 ; Gilbert et al., 2004), as well as TAC1 (Trithorax acetylation complex 1) in Drosophila, which display both HATase and HMTase (histone methyltransferase) activities (Petruk et al., 2001, 2004), remain associated with the elongation complex during transcription. In Drosophila, histone acetylation can be detected across relatively large chromatin domains, which may be used for a long-term maintenance and modulation of patterns of gene expression (Turner, 2000). Particularly, high levels of H4K16ac along the body of dosage-compensated genes on the male X chromosome are

required to increase the rate of transcription relative to the female levels (Smith et al., 2001 ; Gilfillan et al., 2006 ; Bell et al., 2008).

The early elongation phase coincides with methylation of H3K4. In yeast, H3K4 is methylated by the Set1 HMTase subunit of the COMPASS (complex proteins associated with Set1) complex (Miller et al., 2001 ; Briggs et al., 2001 ; Roguev et al., 2001). Set1 is responsible for H3K4me3 around the transcription initiation site of active promoter and H3K4me2 across the coding sequence (Pokholok et al., 2005). Importantly, Set1 is recruited at the promoter region through its interaction with the RNAPII CTD that has previously been phosphorylated at Ser-5 by TFIIH (Krogan et al., 2003a ; Ng et al., 2003b). Functional homologues of Set1 have been identified in higher eukaryotes including the Trx (Trithorax, Petruk et al., 2004 ; Smith et al., 2004) and Ash1 (Absent, small, and homeotic complex 1) (Beisel et al., 2002 ; Byrd and Shearn, 2003) proteins in Drosophila, as well as the SET1 and the MLL (mixed lineage leukaemia) protein family in human (Milne et al., 2002 ; Nakamura et al., 2002). The recruitment of Set1 to the phosphorylated CTD is assisted by the PAF (RNA polymerase-associated factor 1) complex. PAF is an evolutionarily conserved elongation factor from yeast to human involved at multiple stages of the transcription cycle (reviewed by Rosonina and Manley, 2005). PAF function is notably required for the local H3K4me3 distribution at the promoter region and the wide-spread H3K4me2 pattern deposited by Set1 in yeast. Similarly, human MLL HMTases require their association with the WRD5 protein for efficient H3K4me3 activity (Wysocka et al., 2005). In Drosophila, the Ash2 protein was shown to specifically affect H3K4me3 (Beltran et al., 2007). Although Ash2 does not possess intrinsic HMTase activity, its association with Ash1 or a currently unknown HMTase is required for subsequent H3K4me3 at the promoter. The functional relevance of the H3K4 methylation pattern has not been yet fully elucidated. Disruption of PAF activity in yeast affects mRNA processing but not the rate of transcription elongation, suggesting that Set1-mediated H3K4 methylation is rather involved in signalling mechanisms than in transcription (Mueller et al., 2004 ; Mason and Struhl, 2005). This hypothesis is notably supported by the observation that several chromatin remodelers such as NURF (nucleosome remodeling factor) (Santos-Rosa et al., 2003) and Chd1 (Pray-Grant et al., 2005) selectively interact with H3K4 methylated nucleosomes.

Subsequently to RNAPII CTD phosphorylation at Ser-5, the P-TEFb kinase is recruited to the TEC to trigger productive elongation. As RNAPII travels towards the 3’ end of the coding sequence, nucleosomes became trimethylated at H3K36 (Pokholok et al., 2005).

In yeast, H3K36 methylation is mediated by the Set2 HMTase, which also associates with the

phosphorylated CTD (Li et al., 2002, 2003 ; Krogan et al., 2003b ; Schaft et al., 2003 ; Xiao et al., 2003). P-TEFb depletion in yeast results in the loss of Set2 recruitment and H3K36 methylation across the coding region of genes, demonstrating that Set2 preferentially interacts with the Ser-2-phosphorylated CTD (Krogan et al., 2003b ; Xiao et al., 2003). Similarly to Set1, the recruitment of Set2 to the CTD and its HMTase activity is dependent on the PAF complex (Krogan et al., 2003b). H3K36me3 is recognized by the yeast histone deacetylase Rpd3, which generates an hypoacetylated environment within the coding sequence (Carrozza et al., 2005 ; Joshi and Struhl, 2005 ; Keogh et al., 2005). This process is thought to prevent inappropriate binding of transcription factors within the coding sequence and the generation of cryptic transcripts initiated from internal promoter-like sequences (Carrozza et al., 2005 ; Joshi and Struhl, 2005).

As mention above, PAF complex is involved in the recruitment of both Set1 and Set2 HMTases onto the phosphorylated CTD. PAF function is also required for H2B ubiquitination, which is a prerequisite for H3K4me2/3 at active genes (Ng et al., 2003a ; Wood et al., 2003 ; Dehe et al., 2005 ; Shahbazian et al., 2005). At the promoter region, PAF is recruited to the polymerase through interaction with the Ser-5-phosphorylated CTD and DSIF (Qiu et al., 2006), and is thought to play a pivotal role in regulating the binding of various CTD-associated chromatin regulators. These factors include notably Chd1 (Simic et al., 2003), Spt6 and FACT (Adelman et al., 2006 ; Pavri et al., 2006).

Several studies have reported a role for H3S10 phosphorylation in the regulation of early transcriptional events. H3S10 phosphorylation can be mediated by Snf1, the AMP-activated protein kinase (AMPK) homologue in yeast, which acts in concert with the Gcn5 HATase to activate expression of many stress-induced genes (Lo et al., 2001). Inhibition of H3S10 phosphorylation at such promoter eventually prevents the recruitment of TBP suggesting a role for this modification during PIC assembly (Lo et al., 2005). Snf1 physically interacts with P-TEFb (Ctdk1) (Van Driessche et al., 2005) and Mediator (Kuchin et al., 2000), and was recently shown to function in early elongation to alleviate transcriptional pausing (Tachibana et al., 2007). However the role of H3S10p in transcription remains elusive, and Snf1 can stimulate transcription independently of its H3S10-kinase activity. In mammalian cells, H3S10p also occurs on nucleosomes at the promoter and/or within the body of genes following induced activation, and is mediated by the mitogen- and stress-activated protein kinase (MSK)1 and MSK2 (reviewed by Davie, 2003 ; Soloaga et al., 2003).

A recent study also showed that human PIM1 kinase associates with the oncogene c-Myc and contributes to the activation of its target genes by phosphorylation of H3S10 at the

c-Myc-binding sites (Zippo et al., 2007). The function of H3S10p in Drosophila is more controversial. H3S10p is detectable at most euchromatic regions of polytene chromosomes, and the dynamic relocation of this modification at heat-shock genes along with the transcription machinery during the heat-shock response suggested a role for H3S10p in transcription activation (Nowak and Corces, 2000 ; Nowak et al., 2003 ; Ivaldi et al., 2007).

Ivaldi et al. (2007) have recently proposed that H3S10p mediated by JIL-1, the major Drosophila H3S10-kinase, is a hallmark for early transcription elongation and would be required for expression of the majority, if not all, genes in this species. The authors further proposed a model in which JIL-1 activity is required in the release of RNAPII from promoter-proximal pausing and that H3S10p is a prerequisite for P-TEFb recruitment. However, these data were not confirmed by others, and the group of Johansen claims that JIL-1-mediated H3S10 phosphorylation is actually required in the maintenance of euchromatin structure by counteracting the spreading of heterochromatin proteins (Zhang et al., 2006 ; Deng et al., 2008 ; Cai et al., 2008). It is conceivable that H3S10p is involved in both maintenance of chromatin architecture and transcription regulation, and further works in Drosophila are thus necessary to clarify the role of H3S10p in the transcriptional activation of certain stress-inducible genes such as the heat-shock genes.

2. P-TEFb : a key factor in the regulation of cellular processes and HIV-1 replication.

As discussed in the preceding section, P-TEFb behaves as a general transcription elongation factor whose kinase activity is required in early elongation to alleviate promoter-proximal pausing by phosphorylation of DSIF and NELF, and for pre-mRNA processing and chromatin remodeling through RNAPII phosphorylation at Ser-2. Many advances in the understanding of the cellular regulation of P-TEFb arise from studies in human cells, as abnormal Cdk9 function is associated with certain diseases. Particularly, P-TEFb was shown to be an essential factor for transcription and replication of the human immunodeficiency virus HIV-1. The mechanisms of P-TEFb regulation in mammals has been discussed in several excellent reviews (Garriga and Grana, 2004 ; Marshall and Grana, 2006 ; Zhou and Yik, 2006).

2.1. The Cdk9 protein kinases.

As mentioned previously, Cdk9 is a serine/threonine protein kinase member of the cyclin-dependent kinase (Cdk) family. Cdk9 was originally named PITALRE because of its PSTAIRE-like sequence, a cyclin-binding motif shared by Cdks and related kinases (Grana et al., 1994 ; Garriga et al., 1996a,b). Cdk9 is a highly conserved protein among eukaryotes (see annexe 1) and is expressed in all cell types tested to date. Although ubiquitous in both human and murine tissues, Cdk9 expression appeared upregulated in terminally differentiated cells (Bagella et al., 1998). Beside its PITALRE motif, several functional domains have been mapped within the Cdk9 protein sequence including a kinase-catalytic site, an ATP-binding site and a nuclear localization signal (Fig. 7A). Recently, a second Cdk9 isoform has been identified in human cell which contains a Proline-Glycine rich extension at its N-terminus, and was called Cdk9-55 referring to its apparent molecular weight (Shore et al., 2003) (Fig.

7A). The two Cdk9 protein isoforms are expressed from distinct promoters and are both targeted into the nucleus (Liu and Rice, 2000 ; Herrmann and Mancini, 2001 ; Shore et al., 2003 ; Liu and Herrmann, 2005). Although the two kinases coexist in different cell types, their relative abundance can change and the activity of the two independent promoters is likely to be regulated in a tissue specific manner (Liu and Herrmann, 2005). Indeed, whereas Cdk9-42 is predominantly expressed in spleen and testis, Cdk9-55 is upregulated in lung, liver and brain tissues (Shore et al., 2003 ; Shore et al., 2005). Interestingly, the larger form of Cdk9 appears to be specifically involved in the regulation of cell differentiation program of various tissues, including the hematopoietic system (Shore et al., 2003 ; Liu and Herrmann, 2005 ; Shore et al., 2005), myogenesis (Giacinti et al., 2006 ; Simone and Giordano, 2007 ; Giacinti et al., 2008), adipogenesis (Iankova et al., 2006) and possibly neurogenesis (De Falco et al., 2005).

2.2. The multiple Cyclin subunits that regulate Cdk9 activity.

In contrast to Drosophila in which only two distinct cyclin-regulatory subunits have been identified, CyclinT and CyclinK, several T-type cyclins can associates with Cdk9 in mammals and were named CyclinT1, T2a and T2b (Wei et al., 1998 ; Peng et al., 1998b).

These T-type cyclins are encoded by two separate genes, and CyclinT2a and T2b arise from alternative splicing of the same pre-mRNA (Peng et al., 1998b). CyclinT2a and T2b only differ in their C-terminus part, where CyclinT2b contains a C-terminal extension of 67 amino

acids (Peng et al., 1998b) (Fig. 7B). All three T-type cyclins share a highly conserved cyclin box at their N-terminus (81% identity), while the C-terminal part of CyclinT1 is more divergent to the one of CyclinT2 proteins (46% identity). As mentioned earlier, CyclinT1 is the major Cdk9-associated cyclin in HeLa cells and has been more characterized. CyclinT1 notably contains a putative coil-coiled motif, a PEST domain and a histidine-rich stretch (Fig.

7B). This latter motif was shown to mediate interaction with the CTD of RNAPII, and is partially conserved in the other CyclinT2 proteins (Taube et al., 2002). The CyclinT2 proteins however exhibits an additional CTD binding domain located upstream of the cyclin box (Kurosu et al., 2004). The function of the CyclinT1-PEST domain remains unknown, but could be involved in the recruitment of an ubiquitin ligase to mediate Cdk9 ubiquitination (Kiernan et al., 2001). Importantly, only CyclinT1 contains a Tat-recognition motif (TRM) required for interaction with the HIV-1 viral transactivator Tat (Wei et al., 1998 ; Garber et al., 1998 ; Bieniasz et al., 1998 ; Kwak et al., 1999) (see below). Consequently, only the human CyclinT1, but not the other CyclinT2 or murine CyclinT1 proteins, supports the

7B). This latter motif was shown to mediate interaction with the CTD of RNAPII, and is partially conserved in the other CyclinT2 proteins (Taube et al., 2002). The CyclinT2 proteins however exhibits an additional CTD binding domain located upstream of the cyclin box (Kurosu et al., 2004). The function of the CyclinT1-PEST domain remains unknown, but could be involved in the recruitment of an ubiquitin ligase to mediate Cdk9 ubiquitination (Kiernan et al., 2001). Importantly, only CyclinT1 contains a Tat-recognition motif (TRM) required for interaction with the HIV-1 viral transactivator Tat (Wei et al., 1998 ; Garber et al., 1998 ; Bieniasz et al., 1998 ; Kwak et al., 1999) (see below). Consequently, only the human CyclinT1, but not the other CyclinT2 or murine CyclinT1 proteins, supports the