transcription rings in repair

NATURE CELL BIOLOGY VOLUME 7 | NUMBER 6 | JUNE 2005 553

N E W S A N D V I E W S

Transcription RINGs in repairMary Ann Osley

A recent study has shown that the Ssl1 subunit of the general transcription/DNA repair factor TFIIH contains ubiquitin ligase activity that is dependent on a RING domain. The RING domain is required for transcription of a subset of yeast genes that are involved in DNA damage repair, thus uncovering a novel ubiquitination-associated function for TFIIH in the repair process.

The multisubunit TFIIH factor is the most structurally and functionally complex of the general factors required for transcription ini-tiation by RNA polymerase II (Pol II) (Fig. 1a). Besides its essential and global function in tran-scription, TFIIH also has important roles in DNA repair, namely nucleotide and base exci-sion repair1–3. Both roles are dependent on dis-tinct enzymatic activities that reside in TFIIH. The yeast cyclin-dependent kinase Ccl1-Kin28 (whose human homologues are cyclin-MO15) in TFIIH phosphorylates the largest subunit of Pol II and signals the transition from tran-scription initiation to elongation1,4. The same kinase also activates cyclin-dependent kinases that drive the cell cycle in human cells5. In addi-tion, two ATP-dependent helicases in TFIIH with opposite polarities — Ssl2 (XPB) and Rad3 (XPD) — unwind DNA at promoters during transcription initiation, as well as DNA at lesions that have been damaged by ultraviolet irradiation2,3. An exciting discovery made by Takagi et al., reported in Molecular Cell, reveals that TFIIH harbours enzymatic activity that links the transcription and DNA repair func-tions of TFIIH in an unexpected way6.

The authors note that TFIIH contains two subunits with a RING domain, Ssl1 (p44) and Tfb3 (Mat1), a domain that is commonly present in the ever expanding family of ubiq-uitin ligases or E3s7,8. They then present bio-chemical evidence that a five-subunit complex representing the core TFIIH in yeast (Fig. 1a) possesses ubiquitin ligase activity that is dependent, at least partly, on the RING domain of the Ssl1 subunit, marking the first time that such an activity has been identified in the general transcription machinery. Mutation of the RING domain confers sensitivity to DNA damaging agents in vivo but, surprisingly, does not impair nucleotide excision repair.

Moreover, although the Ssl1 RING domain is dispensable for the global transcription func-tion of TFIIH, it is required for optimal induc-tion of a number of genes with known roles in DNA damage repair. Thus, core TFIIH seems to contribute to DNA repair through differ-ent but complementary enzymatic activities: using a ubiquitin ligase to induce transcription of genes whose products are involved in DNA repair pathways, and using helicases to melt DNA at sites that have been damaged by agents such as ultraviolet light.

Ubiquitination occurs through a sequential series of enzymatic reactions that involve activa-tion of ubiquitin by an E1 ubiquitin-activating enzyme, transfer of activated ubiquitin to an E2 ubiquitin-conjugating enzyme, and then attach-ment of ubiquitin to a protein substrate8. The RING domains of E3 ubiquitin ligases perform essential roles in the last step of this pathway by binding E2s and targeting them to the correct substrates7,8. RING domains contain repeating cysteine and histidine residues that represent zinc-binding sites, and have been classified as RING-HC or RING-H2 on the basis of whether a cysteine or histidine residue occupies the fifth zinc coordination site7. The solution structure of this domain in p44, the human homologue of Ssl1, was recently solved and was shown to adopt a C4C4 RING structure, in which two zinc ions are coordinated by eight cysteine resi-dues9. Takagi et al. tested the importance of zinc binding in the ubiquitin ligase activity of Ssl1 by constructing alanine substitution mutations in the two conserved cysteine residues of the second zinc-binding domain. Consistent with disruption of the RING domain structure by these mutations, the ubiquitination activity of Ssl1 in vitro was abolished.

Many RING-E3s bind both an E2 and sub-strate, with the substrate-binding domain gen-erally residing in a region of the ubiquitin ligase that is separate from the RING domain7,8. Other E3s of the RING class show more extreme com-partmentalization and are present in multisub-unit complexes7,8. In this latter arrangement, a separate subunit interacts with substrate, and

adaptor proteins bring the RING subunit with its associated E2 into proximity of the substrate. The presence of Ssl1 as an integral component of core TFIIH suggests that it might fall in this second class of RING-E3s.

In support of this view, Takagi et al. found that the in vitro ubiquitination activity of core TFIIH was more robust than that of Ssl1 alone, and that addition of the core subunit Tfb4 enhanced the E3 activity of Ssl1. A low-resolu-tion structure of core TFIIH has been solved10, and the general location of Ssl1 within the core complex has been modelled on the basis of immunolabelling experiments with p44 (ref. 11), the human counterpart of Ssl1 (Fig. 1a). The most prominent feature of core TFIIH is a toroidal, ring-like structure that contains the Ssl2 (XPB) and Rad3 (XPD) helicases. Ssl1 seems to be present close to this ring struc-ture, and although the position of the Ssl1 RING domain cannot be ascertained, Takagi et al. showed that it is dispensable for global transcription in vivo and thus must not affect TFIIH complex assembly. An implication of these results is that the RING domain could therefore be accessible for interaction with an E2 in the context of core TFIIH.

Ubiquitin is a potent signalling molecule that has an increasingly important role in almost every cellular process, and it is involved in both proteasomal and non-proteasomal pathways depending on whether a polyubiquitin chain (proteolytic if linked through Lys 48 of ubiq-uitin) or monoubiquitin (non-proteolytic) is appended to a target protein8. A key question raised by the Takagi et al. study is what the in vivo target of Ssl1/TFIIH is. Ubiquitination is associated with both transcription and nucle-otide excision repair (NER)2,12, and the DNA-damage-sensitive phenotype of an ssl1 RING domain mutant initially suggested that TFIIH attaches ubiquitin to a component of the NER system. However, no deficiency in NER was detected in the absence of a functional Ssl1 RING domain6. By process of elimination, this suggested that TFIIH targets a component of the transcription machinery. Although RNA

Mary Ann Osley is at the Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Cancer Research Facility 123, 915 Camino de Salud, Albuquerque, NM 87131, USA.e-mail: [email protected]

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554 NATURE CELL BIOLOGY VOLUME 7 | NUMBER 6 | JUNE 2005


polymerase II itself becomes polyubiquitinated and ultimately degraded during transcription-coupled NER, another ubiquitin ligase, Rsp5,

is known to fulfil this function13. Moreover, the absence of ubiquitin ligase activity from TFIIH does not affect global transcription in vivo, an

essential role of TFIIH, indicating that the E3 performs a nonessential function6.

To reconcile these results, Takagi et al.

Figure 1 The multifunctional general transcription factor, TFIIH. (a) TFIIH subunits in yeast and humans. TFIIH contains nine subunits with a total relative molecular mass of ~500,000, and a tenth subunit (designated Tfb5) was recently identified in yeast15,16. All nine subunits have been conserved from yeast to humans. Three different enzymatic activities are found in TFIIH: two DNA-dependent ATPase/helicases, Ssl2 (XPB) and Rad3 (XPD); a cyclin-dependent kinase or CDK, Ccl1/Kin28 (cyclin/MO15); and a ubiquitin ligase, Ssl1 (p44) (refs 1–6). Yeast TFIIH contains two subcomplexes, core TFIIH and TFIIK, with different functional properties. The helicase activities of core TFIIH subunit Rad3 and the associated Ssl2 protein melt DNA at promoters during transcription initiation and at DNA lesions during nucleotide excision repair. The CDK subcomplex phosphorylates the carboxy-terminal domain of RNA polymerase II during the transition from initiation to elongation, signalling the recruitment of elongation and chromatin modifying activities to the transcribing template. Figure adapted from ref. 15. (b) Potential roles of the TFIIH ubiquitin ligase in transcription of DNA-damage-inducible genes.

Transcription of a large number of genes with roles in DNA repair pathways is activated upon DNA damage6. A common activator (Ac) is postulated to bind to an upstream activating site (UAS) in front of each gene, and to lead to the recruitment of the general transcription machinery, including RNA polymerase II (Pol II) and TFIIH. The RING domain of the TFIIH Ssl1 subunit is then presumed to bind an unknown ubiquitin-conjugating enzyme (E2), which in turn either monoubiquitinates or polyubiquitinates the activator. Either form of ubiquitin attachment could lead to gene activation. Monoubiquitination could promote recruitment or activation of transcription initiation or elongation factors, with the ubiquitin moiety serving as a protein-interaction platform. Polyubiquitination is generally associated with protein destruction, and activation domains frequently overlap with degrons, sequences that signal protein turnover via the proteasome12. Activator destruction has been associated with transcriptional activation, although its role is not well understood12. It is also possible that a common corepressor could be polyubiquitinated and degraded, thus relieving inhibition of the activator.

Ub

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SsI2Rad3Tfb1Tfb2SsI1Tfb4

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XPBXPDp62p55p44p34

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?

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NATURE CELL BIOLOGY VOLUME 7 | NUMBER 6 | JUNE 2005 555


performed transcriptional profiling with ssl1 RING domain mutants after cellular DNA was damaged. In a surprising but ultimately sat-isfying turn of events, they found that in the absence of Ssl1/TFIIH E3 activity there was a preferential reduction in the expression of many genes with roles in DNA damage path-ways. This neatly explains the sensitivity of ssl1 RING mutants to DNA damaging agents in the absence of an effect on NER, and it raises the very likely possibility that a transcriptional activator of DNA damage genes could be the biologically relevant target of the TFIIH ubiq-uitin ligase.

Ubiquitination has emerged as an impor-tant regulatory mechanism during Pol II transcription, having key roles in both the initiation and elongation phases of transcrip-tion12. Chromatin, transcription factors and the transcription machinery itself are ubiq-uitinated, leading to different effects on the transcription process. These effects can lead to factor degradation or inactivation, as well as more complex signalling roles. Ubiquitin ligase activity has been associated with elongating Pol II, and E3 complexes have been shown to target DNA-bound transcription factors and coacti-

vators for polyubiquitination, and histones for monoubiquitination12,14. However, unlike Ssl1, these ubiquitin ligases are not integral compo-nents of the transcription machinery but are targeted to genes through protein interactions. Assuming that the Ssl1 E3, which is constitu-tively present in core TFIIH, preferentially tar-gets activators that bind to promoters at DNA damage genes, this means that Ssl1 itself must be preferentially activated during the response to DNA damage. How could this occur? One way is by the selective recruitment of the E2 that interacts with the RING domain of Ssl1. Thus, it will be very interesting to identify the cellular E2 that interacts with the Ssl1 RING domain, and to determine whether this E2 is recruited to the same group of genes that are regulated by TFIIH upon DNA damage. One attractive candidate is the E2 Rad6, which is intimately involved in the repair of DNA dam-age lesions and is associated with at least three different ubiquitin ligases14. Moreover, Rad6 has been shown to have a role in gene activation in yeast, and is recruited to a number of highly expressed genes in an activator- and ubiquitin-ligase-dependent manner14. Whatever the E2, the key questions for the future are to identify

the cellular target(s) of the Ssl1/TFIIH ubiq-uitin ligase, the mode of ubiquitin attachment (mono- or polyubiquitination), and the func-tion of ubiquitination in activating DNA dam-age inducible genes. These questions promise to keep TFIIH in the forefront of gene expres-sion studies for some time to come.

1. Zurita, M. & Merino, C. Trends Genet. 19, 578–584 (2003).

2. Svejstrup, J. Nature Rev. Mol. Cell Biol. 3, 21–29 (2002).

3. De Laat W., Jaspers, N. & Hoeijmakers, J. Genes Dev. 13, 768–785 (1999).

4. Keogh, M.-C., Cho, E.-J., Podolny, V. & Buratowski, S. Mol. Cell. Biol. 22, 1288–1297 (2002).

5. Svejstrup, J., Vichi, P. & Egly, J. M. Trends Biochem. Sci. 21, 346–350 (1996).

6. Takagi, Y. et al. Mol. Cell 18, 237–243 (2005).7. Joazeiro, C. & Weissman, A. Cell 102, 549–552

(2000).8. Pickart, C. Cell 116, 181–190 (2004).9. Kellenberger, E. et al. J. Biol. Chem. (in the press).10. Chang, W. & Kornberg, R. Cell 102, 609–613

(2000).11. Schultz, P. et al. Cell 102, 599–607 (2000).12. Muratami, M. & Tansey, W. P. Nature Rev. Mol. Cell

Biol. 4, 1–10 (2003).13. Beaudenon, S. et al. Mol. Cell. Biol. 19, 6972–6979

(1999).14. Kao, C. et al. Genes Dev. 18, 184–195 (2004).15. Takagi, Y. et al. J. Biol. Chem. 278, 43897–43900

(2003).16. Giglia-Mari, G. et al. Nature Genet. 36, 714–719

(2004).



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