Elisabetta Citterio

ATP-Dependent Chromatin Remodeling by the Cockayne Syndrome B DNA Repair-Transcription-Coupling Factor

ABSTRACT The Cockayne syndrome B protein (CSB) is required for coupling DNA excision repair to transcription in a process known as transcription-coupled repair (TCR). Cockayne syndrome patients show UV sensitivity and severe neurodevelopmental abnormalities. CSB is a DNA-dependent ATPase of the SWI2/SNF2 family. SWI2/SNF2-like proteins are implicated in chromatin remodeling during transcription. Since chromatin structure also affects DNA repair efficiency, chromatin remodeling activities within repair are expected. Here we used purified recombinant CSB protein to investigate whether it can remodel chromatin in vitro. We show that binding of CSB to DNA results in an alteration of the DNA double-helix conformation. In addition, we find that CSB is able to remodel chromatin structure at the expense of ATP hydrolysis. Specifically, CSB can alter DNase I accessibility to reconstituted mononucleosome cores and disarrange an array of nucleosomes regularly spaced on plasmid DNA. In addition, we show that CSB interacts not only with double-stranded DNA but also directly with core histones. Finally, intact histone tails play an important role in CSB remodeling. CSB is the first repair protein found to play a direct role in modulating nucleosome structure. The relevance of this finding to the interplay between transcription and repair is discussed.

The Cockayne syndrome B protein, involved in transcription-coupled DNA repair, resides in an RNA polymerase II-containing complex

Transcription‐coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair‐ and transcription‐competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10–15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell‐free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency.

Human USP3 is a chromatin modifier required for S phase progression and genome stability

Protein ubiquitination is critical for numerous cellular functions, including DNA damage response pathways. Histones are the most abundant monoubiquitin conjugates in mammalian cells; however, the regulation and the function of monoubiquitinated H2A (uH2A) and H2B (uH2B) remain poorly understood. In particular, little is known about mammalian deubiquitinating enzymes (DUBs) that catalyze the removal of ubiquitin from uH2A/uH2B. Here we identify the ubiquitin-specific protease 3 USP3 as a deubiquitinating enzyme for uH2A and uH2B. USP3 dynamically associates with chromatin and deubiquitinates H2A/H2B in vivo. The ZnF-UBP domain of USP3 mediates uH2A-USP3 interaction. Functional ablation of USP3 by RNAi leads to delay of S phase progression and to accumulation of DNA breaks, with ensuing activation of DNA damage checkpoint pathways. In addition, we show that in response to ionizing radiation, (1) uH2A redistributes and colocalizes in gamma-H2AX DNA repair foci and (2) USP3 is required for full deubiquitination of ubiquitin-conjugates/uH2A and gamma-H2AX dephosphorylation. Our studies identify USP3 as a novel regulator of H2A and H2B ubiquitination, highlight its role in preventing replication stress, and suggest its involvement in the response to DNA double-strand breaks. Together, our results implicate USP3 as a novel chromatin modifier in the maintenance of genome integrity.

Np95 Is a Histone-Binding Protein Endowed with Ubiquitin Ligase Activity

ABSTRACT Np95 is an important determinant in cell cycle progression. Its expression is tightly regulated and becomes detectable shortly before the entry of cells into S phase. Accordingly, Np95 is absolutely required for the G1/S transition. Its continued expression throughout the S/G2/M phases further suggests additional roles. Indeed, Np95 has been implicated in DNA damage response. Here, we show that Np95 is tightly bound to chromatin in vivo and that it binds to histones in vivo and in vitro. The binding to histones is direct and shows a remarkable preference for histone H3 and its N-terminal tail. A novel protein domain, the SRA-YDG domain, contained in Np95 is indispensable both for the interaction with histones and for chromatin binding in vivo. Np95 contains a RING finger. We show that this domain confers E3 ubiquitin ligase activity on Np95, which is specific for core histones, in vitro. Finally, Np95 shows specific E3 activity for histone H3 when the endogenous core octamer, coimmunoprecipitating with Np95, is used as a substrate. Histone ubiquitination is an important determinant in the regulation of chromatin structure and gene transcription. Thus, the demonstration that Np95 is a chromatin-associated ubiquitin ligase suggests possible molecular mechanisms for its action as a cell cycle regulator.

BMI1 is recruited to DNA breaks and contributes to DNA damage-induced H2A ubiquitination and repair.

ABSTRACT DNA damage activates signaling pathways that lead to modification of local chromatin and recruitment of DNA repair proteins. Multiple DNA repair proteins having ubiquitin ligase activity are recruited to sites of DNA damage, where they ubiquitinate histones and other substrates. This DNA damage-induced histone ubiquitination is thought to play a critical role in mediating the DNA damage response. We now report that the polycomb protein BMI1 is rapidly recruited to sites of DNA damage, where it persists for more than 8 h. The sustained localization of BMI1 to damage sites is dependent on intact ATM and ATR and requires H2AX phosphorylation and recruitment of RNF8. BMI1 is required for DNA damage-induced ubiquitination of histone H2A at lysine 119. Loss of BMI1 leads to impaired repair of DNA double-strand breaks by homologous recombination and the accumulation of cells in G2/M. These data support a crucial role for BMI1 in the cellular response to DNA damage.

XAB2, a Novel Tetratricopeptide Repeat Protein Involved in Transcription-coupled DNA Repair and Transcription

Nucleotide excision repair is a highly versatile DNA repair system responsible for elimination of a wide variety of lesions from the genome. It is comprised of two subpathways: transcription-coupled repair that accomplishes efficient removal of damage blocking transcription and global genome repair. Recently, the basic mechanism of global genome repair has emerged from biochemical studies. However, little is known about transcription-coupled repair in eukaryotes. Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways. The XAB2 protein of 855 amino acids consists mainly of 15 tetratricopeptide repeats. In addition to interacting with XPA, immunoprecipitation experiments demonstrated that a fraction of XAB2 is able to interact with the transcription-coupled repair-specific proteins CSA and CSB as well as RNA polymerase II. Furthermore, antibodies against XAB2 inhibited both transcription-coupled repair and transcription in vivo but not global genome repair when microinjected into living fibroblasts. These results indicate that XAB2 is a novel component involved in transcription-coupled repair and transcription.

Translocation of Cockayne syndrome group A protein to the nuclear matrix: Possible relevance to transcription-coupled DNA repair

Transcription-coupled repair (TCR) efficiently removes a variety of lesions from the transcribed strand of active genes. By allowing rapid resumption of RNA synthesis, the process is of major importance for cellular resistance to transcription-blocking genotoxic damage. Mutations in the Cockayne syndrome group A or B (CSA or CSB) gene result in defective TCR. However, the exact mechanism of TCR in mammalian cells remains to be elucidated. We found that CSA protein is rapidly translocated to the nuclear matrix after UV irradiation. The translocation of CSA was independent of Xeroderma pigmentosum group C, which is specific to the global genome repair subpathway of nucleotide excision repair (NER) and of the core NER factor Xeroderma pigmentosum group A but required the CSB protein. In UV-irradiated cells, CSA protein colocalized with the hyperphosphorylated form of RNA polymerase II, engaged in transcription elongation. The translocation of CSA was also induced by treatment of the cells with cisplatin or hydrogen peroxide, both of which produce damage that is subjected to TCR but not induced by treatment with dimethyl sulfate, which produces damage that is not subjected to TCR. The hydrogen peroxide-induced translocation of CSA was also CSB dependent. These findings establish a link between TCR and the nuclear matrix mediated by CSA.

DNA damage stabilizes interaction of CSB with the transcription elongation machinery

The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active.

Cockayne syndrome: defective repair of transcription?

In the past years, it has become increasingly evident that basal metabolic processes within the cell are intimately linked and influenced by one another. One such link that recently has attracted much attention is the close interplay between nucleotide excision DNA repair and transcription. This is illustrated both by the preferential repair of the transcribed strand of active genes (a phenomenon known as transcription‐coupled repair, TCR) as well as by the distinct dual involvement of proteins in both processes. The mechanism of TCR in eukaryotes is still largely unknown. It was first discovered in mammals by the pioneering studies of Hanawalt and colleagues, and subsequently identified in yeast and Escherichia coli. In the latter case, one protein, the transcription repair‐coupling factor, was found to accomplish this function in vitro, and a plausible model for its activity was proposed. While the E.coli model still functions as a paradigm for TCR in eukaryotes, recent observations prompt us to believe that the situation in eukaryotes is much more complex, involving dual functionality of multiple proteins.

RNF168, a new RING finger, MIU-containing protein that modifies chromatin by ubiquitination of histones H2A and H2AX

BackgroundModulation of chromatin structure has emerged as a critical molecular device to control gene expression. Histones undergo different post-translational modifications that increase chromatin accessibility to a number of regulatory factors. Among them, histone ubiquitination appears relevant in nuclear processes that govern gene silencing, either by inhibiting or activating transcription, and maintain genome stability, acting as scaffold to properly organize the DNA damage response. Thus, it is of paramount importance the identification and the characterization of new ubiquitin ligases that address histones.ResultsWe identified and characterized RNF168, a new chromatin-associated RING finger protein. We demonstrated that RNF168 is endowed with ubiquitin ligase activity both in vitro and in vivo, which targets histones H2A and H2AX, but not H2B, forming K63 polyubiquitin chains. We previously described the presence within RNF168 sequence of two MIU domains, responsible for the binding to ubiquitinated proteins. Here we showed that inactivation of the MIUs impairs ubiquitin binding ability in vitro and reduces chromatin association of RNF168 in vivo. Moreover, upon formation of DNA double strand breaks induced by chemical and physical agents, RNF168 is recruited to the DNA damage foci, where it co-localizes with γH2AX and 53BP1. The localization of RNF168 at the site of damage highly increases the local concentration of ubiquitinated proteins and determines the prolonged ubiquitination signal.ConclusionThe RING finger protein RNF168 is a new ubiquitin ligase that functions as chromatin modifier, through histone ubiquitination. We hypothesize a dual function for RNF168. In normal condition RNF168 modifies chromatin structure by modulating ubiquitination of histone H2A. Upon DNA lesions, RNF168 is recruited to DNA damage response foci where it contributes to increase the amount of ubiquitinated proteins, thereby facilitating the downstream signalling cascade.