p8/TTD-A as a Repair-Specific TFIIH Subunit

Molecular Cell, Volume 21, Issue 2, 20 January 2006, Pages 215-226
Frédéric Coin, Luca Proietti De Santis, Tiziana Nardo, Olga Zlobinskaya, Miria Stefanini and Jean-Marc Egly

Summary

How subunits of the transcription/repair factor TFIIH cooperate to allow for the removal of DNA lesions or for the transcription of genes is crucial to understand the functioning of this complex. Here, we reveal that p8/TTD-A, the tenth subunit of TFIIH, has a critical role in DNA repair where it triggers DNA opening by stimulating XPB ATPase activity together with the damage recognition factor XPC-hHR23B. Fluorescent antibody labeling shows that such opening is needed for the recruitment of XPA to the site of the damage. By contrast, p8 is dispensable for RNA synthesis and doesn't interfere with the transcriptional function of CAK, although both interact with the XPD subunit. Interestingly, p8 overexpression in TTD-XPD cells counteracts the detrimental effect of XPD mutations by restoring the cellular TFIIH concentration. These findings resolve the primary functions of p8 and unveil how TFIIH components specifically direct the complex toward repair or transcription.

Nucleotide Excision Repair Driven by the Dissociation of CAK from TFIIH

Molecular Cell, Volume 31, Issue 1, 11 July 2008, Pages 9-20
Frédéric Coin, Valentyn Oksenych, Vincent Mocquet, Stefanie Groh, Christine Blattner and Jean Marc Egly

Summary

The transcription/DNA repair factor TFIIH is organized into a core that associates with the CDK-activating kinase (CAK) complex. Using chromatin immunoprecipitation, we have followed the composition of TFIIH over time after UV irradiation of repair-proficient or -deficient human cells. We show that TFIIH changes subunit composition in response to DNA damage. The CAK is released from the core during nucleotide excision repair (NER). Using reconstituted in vitro NER assay, we show that XPA catalyzes the detachment of the CAK from the core, together with the arrival of the other NER-specific factors. The release of the CAK from the core TFIIH promotes the incision/excision of the damaged oligonucleotide and thereby the repair of the DNA. Following repair, the CAK reappears with the core TFIIH on the chromatin, together with the resumption of transcription. Our findings demonstrate that the composition of TFIIH is dynamic to adapt its engagement in distinct cellular processes.

XPG Stabilizes TFIIH, Allowing Transactivation of Nuclear Receptors: Implications for Cockayne Syndrome in XP-G/CS Patients

Molecular Cell, Volume 26, Issue 2, 27 April 2007, Pages 231-243
Shinsuke Ito, Isao Kuraoka, Pierre Chymkowitch, Emmanuel Compe, Arato Takedachi, Chie Ishigami, Frédéric Coin, Jean-Marc Egly and Kiyoji Tanaka

Summary

Mutations in the human gene give rise toan inherited photosensitive disorder, xeroderma pigmentosum (XP) associated with Cockayne syndrome (XP-G/CS). The clinical features of CS in XP-G/CS patients are difficult to explain on the basis of a defect in nucleotide excision repair (NER). We found that XPG forms a stable complex with TFIIH, which is active in transcription and NER. Mutations in found in XP-G/CS patient cells that prevent the association with TFIIH also resulted in the dissociation of CAK and XPD from the core TFIIH. As a consequence, the phosphorylation and transactivation of nuclear receptors were disturbed in XP-G/CS as well as MEF cells and could be restored by expression of wild-type XPG. These results provide an insight into the role of XPG in the stabilization of TFIIH and the regulation of gene expression and provide an explanation of some of the clinical features of XP-G/CS.

p8/TTD-A as a Repair-Specific TFIIH Subunit

Molecular Cell, Volume 21, Issue 2, 20 January 2006, Pages 215-226
Frédéric Coin, Luca Proietti De Santis, Tiziana Nardo, Olga Zlobinskaya, Miria Stefanini and Jean-Marc Egly

Summary

How subunits of the transcription/repair factor TFIIH cooperate to allow for the removal of DNA lesions or for the transcription of genes is crucial to understand the functioning of this complex. Here, we reveal that p8/TTD-A, the tenth subunit of TFIIH, has a critical role in DNA repair where it triggers DNA opening by stimulating XPB ATPase activity together with the damage recognition factor XPC-hHR23B. Fluorescent antibody labeling shows that such opening is needed for the recruitment of XPA to the site of the damage. By contrast, p8 is dispensable for RNA synthesis and doesn't interfere with the transcriptional function of CAK, although both interact with the XPD subunit. Interestingly, p8 overexpression in TTD-XPD cells counteracts the detrimental effect of XPD mutations by restoring the cellular TFIIH concentration. These findings resolve the primary functions of p8 and unveil how TFIIH components specifically direct the complex toward repair or transcription.

Nucleotide Excision Repair Driven by the Dissociation of CAK from TFIIH

Molecular Cell, Volume 31, Issue 1, 11 July 2008, Pages 9-20
Frédéric Coin, Valentyn Oksenych, Vincent Mocquet, Stefanie Groh, Christine Blattner and Jean Marc Egly

Summary

The transcription/DNA repair factor TFIIH is organized into a core that associates with the CDK-activating kinase (CAK) complex. Using chromatin immunoprecipitation, we have followed the composition of TFIIH over time after UV irradiation of repair-proficient or -deficient human cells. We show that TFIIH changes subunit composition in response to DNA damage. The CAK is released from the core during nucleotide excision repair (NER). Using reconstituted in vitro NER assay, we show that XPA catalyzes the detachment of the CAK from the core, together with the arrival of the other NER-specific factors. The release of the CAK from the core TFIIH promotes the incision/excision of the damaged oligonucleotide and thereby the repair of the DNA. Following repair, the CAK reappears with the core TFIIH on the chromatin, together with the resumption of transcription. Our findings demonstrate that the composition of TFIIH is dynamic to adapt its engagement in distinct cellular processes.

XPG Stabilizes TFIIH, Allowing Transactivation of Nuclear Receptors: Implications for Cockayne Syndrome in XP-G/CS Patients

Molecular Cell, Volume 26, Issue 2, 27 April 2007, Pages 231-243
Shinsuke Ito, Isao Kuraoka, Pierre Chymkowitch, Emmanuel Compe, Arato Takedachi, Chie Ishigami, Frédéric Coin, Jean-Marc Egly and Kiyoji Tanaka

Summary

Mutations in the human gene give rise toan inherited photosensitive disorder, xeroderma pigmentosum (XP) associated with Cockayne syndrome (XP-G/CS). The clinical features of CS in XP-G/CS patients are difficult to explain on the basis of a defect in nucleotide excision repair (NER). We found that XPG forms a stable complex with TFIIH, which is active in transcription and NER. Mutations in found in XP-G/CS patient cells that prevent the association with TFIIH also resulted in the dissociation of CAK and XPD from the core TFIIH. As a consequence, the phosphorylation and transactivation of nuclear receptors were disturbed in XP-G/CS as well as MEF cells and could be restored by expression of wild-type XPG. These results provide an insight into the role of XPG in the stabilization of TFIIH and the regulation of gene expression and provide an explanation of some of the clinical features of XP-G/CS.

Rapid tRNA Decay Can Result from Lack of Nonessential Modifications

Molecular Cell, Volume 21, Issue 1, 6 January 2006, Pages 87-96
Andrei Alexandrov, Irina Chernyakov, Weifeng Gu, Shawna L. Hiley, Timothy R. Hughes, Elizabeth J. Grayhack and Eric M. Phizicky

Summary

The biological role of many nonessential tRNA modifications outside of the anticodon remains elusive despite their evolutionary conservation. We show here that mG methyltransferase Trm8p/Trm82p acts as a hub of synthetic interactions with several tRNA modification enzymes, resulting in temperature-sensitive growth. Analysis of three double mutants indicates reduced levels of tRNA, consistent with a role of the corresponding modifications in maintenance of tRNA levels. Detailed examination of a double mutant demonstrates rapid degradation of preexisting tRNA accompanied by its de-aminoacylation. Multiple copies of tRNA suppress the growth defect, directly implicating this tRNA in the phenotype. These results define a rapid tRNA degradation (RTD) pathway that is independent of the /-dependent nuclear surveillance pathway. The degradation of an endogenous tRNA species at a rate typical of mRNA decay demonstrates a critical role of nonessential modifications for tRNA stability and cell survival.

Heterochromatin Formation in Drosophila Is Initiated through Active Removal of H3K4 Methylation by the LSD1 Homolog SU(VAR)3-3

Molecular Cell, Volume 26, Issue 1, 13 April 2007, Pages 103-115
Thomas Rudolph, Masato Yonezawa, Sandro Lein, Kathleen Heidrich, Stefan Kubicek, Christiane Schäfer, Sameer Phalke, Matthias Walther, Andreas Schmidt, Thomas Jenuwein and Gunter Reuter

Summary

Epigenetic indexing of chromatin domains byhistone lysine methylation requires the balanced coordination of methyltransferase and demethylase activities. Here, we show that SU(VAR)3-3, the homolog of the human LSD1 amine oxidase, demethylates H3K4me2 and H3K4me1 and facilitates subsequent H3K9 methylation by SU(VAR)3-9. mutations suppress heterochromatic gene silencing, display elevated levels of H3K4me2, and prevent extension of H3K9me2 at pericentric heterochromatin. SU(VAR)3-3 colocalizes with H3K4me2 in interband regions and is abundant during embryogenesis and in syncytial blastoderm, where it appears concentrated at prospective heterochromatin during cycle 14. In embryos of / females, H3K4me2 accumulates in primordial germ cells, and the deregulated expansion of H3K4me2 antagonizes heterochromatic H3K9me2 in blastoderm cells. Our data indicate an early developmental function for the SU(VAR)3-3 demethylase in controlling euchromatic and heterochromatic domains and reveal a hierarchy in which SU(VAR)3-3-mediated removal of activating histone marks is a prerequisite for subsequent heterochromatin formation by H3K9 methylation.