InteractiveFly: GeneBrief

hyrax: Biological Overview | References

BIOLOGICAL OVERVIEW

The Wnt pathway controls cell fates, tissue homeostasis, and cancer. Its activation entails the association of β-catenin with nuclear TCF/LEF proteins and results in transcriptional activation of target genes. The mechanism by which nuclear β-catenin controls transcription is largely unknown. A novel Wnt/Wg pathway component has been genetically identify that mediates the transcriptional outputs of β-catenin/Armadillo. Drosophila Hyrax and its human ortholog, Parafibromin, components of the Polymerase-Associated Factor 1 (PAF1) complex, are required for nuclear transduction of the Wnt/Wg signal and bind directly to the C-terminal region of β-catenin/Armadillo. Moreover, the transactivation potential of Parafibromin/Hyrax depends on the recruitment of Pygopus to β-catenin/Armadillo. These results assign to the tumor suppressor Parafibromin an unexpected role in Wnt signaling and provide a molecular mechanism for Wnt target gene control, in which the nuclear Wnt signaling complex directly engages the PAF1 complex, thereby controlling transcriptional initiation and elongation by RNA polymerase II (Mosimann, 2006).

The HRPT2 gene, encoding Parafibromin, the mammalian homolog of Hyrax, is ubiquitously expressed and encodes a predicted protein of 531 amino acids. The primary sequence of parafibromin neither closely resembles other known proteins nor reveals obvious structural motifs that might provide a direct clue as to its function. The exception is an ~200-amino-acid C-terminal segment of parafibromin, which displays modest homology (27%) to budding yeast Cdc73, a component of the Paf1 complex that functions at various stages during the yeast transcription cycle (Yart, 2005).

The Paf1 complex has been originally identified as an RNA polymerase II (RNAP II)-associated complex (Shi, 1997; Squazzo, 2002) and minimally contains Paf1 (see Drosophila Paf1), Cdc73, Rtf1 (see Drosophila Rtf1), Leo1, and Ctr9 (Mueller, 2002). It has been implicated in the regulation of genes whose products function in metabolism and cell cycle control (Betz, 2002; Porter, 2002). Genetic and biochemical evidence in yeast suggest key roles for Paf1 complex components at various stages of the gene expression pathway, including transcript site selection, transcriptional elongation (Pokholok, 2002; Rondon, 2004; Squazzo, 2002), histone H2B monoubiquitination and subsequent histone H3 methylation (Krogan, 2003; Ng, 2003a; Ng, 2003b; Wood, 2003), and more recently poly(A) length control and the coupling of transcriptional and posttranscriptional events (Mueller, 2004; Yart, 2005 and references therein).

The function of the Paf1 complex has also been intimately linked to site-specific phosphorylation events of RNAP II within its carboxy-terminal domain (CTD). Site-specific phosphorylation of RNAP II CTD is an important mechanism that contributes, at least in part, to the normal temporal coordination of the activities of the various protein assemblages involved in mRNA synthesis. For example, during the transition from transcription initiation to elongation, serine 5 of CTD is phosphorylated. As RNAP II elongates, serine 5 phosphorylation diminishes while serine 2 phosphorylation increases. The latter initiates the recruitment of factors involved in subsequent steps of RNA processing. For example, the protein URI is an unconventional member of the prefoldin (PFD) family of ATP-independent molecular chaperones. URI-associated proteins including the tumor suppressor parafibromin and human orthologs of the yeast Paf1 complex. Parafibromin is associated with the serine 5- and serine 2-phosphorylated forms of RNAP II CTD, and a naturally occurring tumor-derived mutant of parafibromin lacks PAF1 and RNAP II binding function. These data infer a potential role of the tumor suppressor parafibromin in transcriptional/posttranscriptional control pathways (Yart, 2005 and references therein).

Three lines of evidence argue for the notion that Hyx represents a component of the Drosophila Wg pathway. (1) The initial observation that increased expression of hyx can overcome the dominant-negative effect of overexpressed lgs^17E provides a first indication that Hyx positively influences Wg signaling outputs in vivo. lgs^17E encodes an altered form of Lgs which contains a mutation in its Arm-interacting domain that severely decreases binding of Lgs to Arm and consequently the recruitment of Pygo to Arm. When provided in excess, Lgs^17E protein likely impairs the function of nuclear Arm by outcompeting endogenous Lgs and thus disturbs the sensitive balance and/or sequence of factors normally recruited at Wg-responsive enhancers. Elevating the levels of a positively acting nuclear factor involved in Wg signaling, in this case Hyx, could readily explain the reversion of the Lgs^17E phenotype in genetic assays. (2) The subsequent observation that genetic reduction of hyx function in imaginal discs as well as the RNAi-mediated knock-down of hyx expression in S2 cells caused a severe decrease in Wg pathway activity is a strong argument for a requirement of Hyx in Wg signaling. (3) Ultimate confirmation of the above genetic claims was the discovery of Hyx as a direct binding partner of Arm. Together these observations provide a solid basis for a model in which Hyx plays a key role in mediating the transcriptional output of Arm in response to Wg pathway activation. In contrast to the Arm partners Lgs and Pygo, Hyx is most likely not a component dedicated solely to the Wg pathway. The phenotypes associated with hyx loss-of-function mutations indicate that Hyx is involved in other developmental processes, possibly in the transcriptional output of some other signal transduction pathway(s) (Mosimann, 2006).

The high degree of homology between Hyx and its single human ortholog suggested that Parafibromin serves the same function in Wnt signaling as Hyx in Wg signaling. Indeed, with the exception of genetic evidence for an in vivo requirement, equivalent lines of reasoning as those arrived at for Hyx argue for an important role of Parafibromin in human β-catenin signaling. What could this role be? It has recently been shown that Parafibromin/Hyx represents the Cdc73 subunit of a metazoan PAF1 complex (Rozenblatt-Rosen, 2005; Yart, 2005; Adelman, 2006). The yeast PAF1 complex has originally been found associated with initiating and elongating forms of RNAPII. Moreover, the PAF1 complex interacts genetically and physically with the histone H2B ubiquitination complex, the Set1 methylase-containing COMPASS complex, and Set2, thus conferring control over a number of distinct histone modifications on RNAPII. Together, these findings suggest important conserved functions of the PAF1 complex in coordinating histone modifications 'downstream' of chromatin preparation on target promoters to ensure proper initiation, elongation, and memory of transcription (Mosimann, 2006 and references therein).

To date, Cdc73p has not been reported to interact directly or indirectly with a sequence-specific DNA binding transcription factor, and it is not clear how the PAF1 complex is recruited to its target genes. However, the metazoan homologs Parafibromin and Hyx share an extended N-terminal region, not present in Cdc73p, which have been found to physically interact with the core Wnt/Wg component β-catenin/Arm. It is thus tempting to speculate that during metazoan evolution, Cdc73 homology proteins evolved in their N-terminal sequences interaction domains for certain signal transduction pathways, such as the Wnt/Wg pathway, while conserving C-terminal sequences for PAF1 complex and/or RNAPII association (Mosimann, 2006).

β-catenin/Arm has two 'branches' of transcriptional output, an N-terminal and a C-terminal branch, which can be separated experimentally. The N-terminal activity maps to Arm repeat 1 and can be attributed to the recruitment of Lgs and Pygo. The current results suggest that Parafibromin/Hyx mediates an important aspect of the C-terminal output of β-catenin/Arm. The significance of any transcriptional activity mapping to C-terminal sequences of β-catenin/Arm is seemingly undermined by the finding that C-terminally truncated forms of Arm (such as the product of the allele arm^XM19) are able to drive Wg target gene expression under certain experimental conditions. However, the arm^XM19 allele exhibits robust signaling activity only when its product is 'forced' into the nucleus by overexpression of a membrane-tethered form of Arm and most likely uses the N-terminal Lgs/Pygo-dependent branch for this activity. Under physiological conditions, Arm^XM19 is severely impaired for Wg signaling. Arm^H8.6, which lacks only a distal portion of the CTD, retains residual transactivation potential at 18°C. This apparent correlation between signaling activity and the extent of C-terminal integrity of Arm might reflect the capacity of Arm to recruit Hyx, a view consistent with protein–protein interaction results (Mosimann, 2006).

Recent advances in the understanding of how transcriptional activators modulate gene transcription suggest a sequential recruitment of histone acetylases (such as CBP/p300) and chromatin-remodeling complexes (like SWI/SNF) to target genes before RNAPII is contacted to initiate transcription on the prepared chromatin. The β-catenin region encompassing Arm repeat 11 to the C terminus has been implicated in being necessary for chromatin remodeling using in vitro assays. Parafibromin/Hyx interacts with a region of β-catenin/Arm (repeat 12-C) that overlaps with the CBP/p300 binding site (repeat 10/11-C) and the Brg-1/Brm binding region (repeat 7-12). This raises the intriguing possibility of a concerted or sequential recruitment of chromatin remodeling factors during the control of Wnt/Wg-responsive genes to the C-terminal portion of β-catenin/Arm, as is being reported for other transcription factors. In such a scenario, CBP/p300 and Brg-1/Brm would, in sequential or arbitrary order, mediate chromatin remodeling steps at β-catenin/Arm-dependent target genes before the Parafibromin/Hyx-mediated recruitment of a PAF1-like complex orchestrates later transactivation steps involving the preparation of RNAPII with histone methylase complexes. In a final step, the PAF1 complex, including Parafibromin/Hyx, may be transferred from β-catenin/Arm to RNAPII to travel with it through the actively transcribed gene (Mosimann, 2006).

What role does the Wnt/Wg pathway component Pygo play in such a model? In several readouts it was found that the Parafibromin/Hyx-enhanced transactivation activity of β-catenin is dependent on Pygo. The Pygo-Parafibromin/Hyx dependence is interpreted as an indication for a more general cross talk between Pygo and proteins interacting with the C-terminal region of β-catenin/Arm. Thus, Pygo could act as a flexible recruitment module to facilitate the exchange or stabilization of transactivating complexes that sequentially bind to the β-catenin C terminus. It is therefore proposed that Wnt/Wg target gene activation might be a concerted, Pygo-guided process, which dynamically coordinates the sequential action of transcriptional modulators at the central scaffold protein β-catenin/Arm (Mosimann, 2006).

The yeast PAF1 complex shows cotranscriptional association with a wide range of genes and has therefore been considered a general transcription cofactor complex. However, deletion of individual components of this complex does not have a global effect on mRNA transcription but instead has a more selective impact on the transcription of only a subset of genes. Currently, aside from findings of an involvement in Wnt signaling, little is known about the target gene spectrum of metazoan PAF1-like complexes. Recently published data indicate that, as in yeast, the Drosophila PAF1-like complex is broadly associated with active genes but, functionally, Cdc73/Hyx seems only necessary for a subset of PAF1 complex targets. This would be consistent with a view that Parafibromin and Hyx provide an adaptor function only to certain transcription factors, such as, for example, β-catenin and Arm. Indeed, in vivo and in vitro assays indicate that in contrast to a cohort of other genes, whose expression is constitutive or controlled by other pathways, Wg targets are remarkably sensitive to reduction of Hyx levels. However, since the assays severely reduced but never abolished hyx expression, currently it is not possible to evaluate the extent to which Hyx activity is also required for the transcription of targets of other pathways, which potentially are more resilient to reductions in Hyx levels (Mosimann, 2006).

Adelman, K., Wei, W., Ardehali, M. B., Werner, J., Zhu, B., Reinberg, D. and Lis, J. T. (2006). Drosophila Paf1 modulates chromatin structure at actively transcribed genes. Mol. Cell. Biol. 26(1): 250-60. PubMed ID: 16354696

Betz, J. L., et al. (2002). Phenotypic analysis of PAF1/RNA polymerase II complex mutations reveals connections to cell cycle regulation, protein synthesis, and lipid and nucleic acid metabolism. Mol. Genet. Genomics 268: 272-285. PubMed ID: 12395202

Krogan, N. J., et al. (2003). The PAF1 complex is required for histone H3 methylation by COMPASS and DOT1p: linking transcriptional elongation to histone methylation. Mol. Cell 11: 721-729. PubMed ID: 12667454

Mosimann, C., Hausmann, G. and Basler, K. (2006). Parafibromin/Hyrax activates Wnt/Wg target gene transcription by direct association with β-catenin/Armadillo. Cell 125(2): 327-41. PubMed ID: 16630820

Mueller, C. L., and Jaehning, J. A. (2002). CTR9, RTF1, and LEO1 are components of the PAF1/RNA polymerase II complex. Mol. Cell. Biol. 22: 1971-1980. PubMed ID: 11884586

Mueller, C. L., et al. (2004). The PAF1 complex has functions independent of actively transcribing RNA polymerase II. Mol. Cell 14: 447-456. PubMed ID: 15149594

Ng, H. H., Dole, S. and Struhl, K. (2003a). The RTF1 component of the PAF1 transcriptional elongation complex is required for ubiquitination of histone H2B. J. Biol. Chem. 278: 33625-33628. PubMed ID: 12876293

Ng, H. H., Robert, F., Young, R. A. and Struhl, K. (2003b). Targeted recruitment of SET1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. Mol. Cell 11: 709-719. PubMed ID: 12667453

Pokholok, D. K., Hannett, N. M. and Young, R. A. (2002). Exchange of RNA polymerase II initiation and elongation factors during gene expression in vivo. Mol. Cell 9: 799-809. PubMed ID: 11983171

Porter, S. E., et al. (2002). The yeast PAF1-RNA polymerase II complex is required for full expression of a subset of cell cycle-regulated genes. Eukaryot. Cell 1: 830-842. PubMed ID: 12455700

Rondon, A. G., et al. (2004). Molecular evidence indicating that the yeast PAF complex is required for transcription elongation. EMBO Rep. 5: 47-53. PubMed ID: 14710186

Rozenblatt-Rosen, O., et al. (2005). The parafibromin tumor suppressor protein is part of a human Paf1 complex. Mol. Cell. Biol. 25: 612-620. PubMed ID: 15632063

Shi, X., et al. (1997). CDC73p and PAF1p are found in a novel RNA polymerase II-containing complex distinct from the Srbp-containing holoenzyme. Mol. Cell. Biol. 17: 1160-1169. PubMed ID: 9032243

Squazzo, S. L., et al. (2002). The PAF1 complex physically and functionally associates with transcription elongation factors in vivo. EMBO J. 21: 1764-177. PubMed ID: 11927560

Wood, A., et al. (2003). The PAF1 complex is essential for histone monoubiquitination by the RAD6-BRE1 complex, which signals for histone methylation by COMPASS and DOT1p. J. Biol. Chem. 278: 34739-34742. PubMed ID: 12876294

Yart, A., et al. (2005). The HRPT2 tumor suppressor gene product parafibromin associates with human PAF1 and RNA polymerase II. Mol. Cell. Biol. 25: 5052-5060. PubMed ID: 15923622

date revised: 2 January 2023

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