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The PGBD3 piggyBac transposon inserted into CSB intron 5 early in the primate lineage. As a result of alternative splicing, the human CSB gene now encodes three proteins: CSB, a CSB-PGBD3 fusion protein that joins the N-terminal CSB domain to the C-terminal PGBD3 transposase domain, and PGBD3 transposase. The fusion protein is as highly conserved as CSB, suggesting that it is advantageous in health; however, expression of the fusion protein in CSB-null cells induces a constitutive interferon (IFN) response. The fusion protein binds in vivo to PGBD3-related MER85 elements, but is also tethered to c-Jun, TEAD1, and CTCF motifs by interactions with the cognate transcription factors. The fusion protein regulates nearby genes from the c-Jun (and to a lesser extent TEAD1 and CTCF) motifs, but not from MER85 elements. We speculate that the fusion protein interferes with CSB-dependent chromatin remodeling, generating double-stranded RNA (dsRNA) that induces an IFN response through endosomal TLR or cytoplasmic RIG-I and/or MDA5 RNA sensors. We suggest that the fusion protein was fixed in primates because an elevated IFN response may help to fight viral infection. We also speculate that an inappropriate IFN response may contribute to the clinical presentation of CS., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The CSB-PGBD3 fusion protein arose more than 43 million years ago when a 2.5-kb piggyBac 3 (PGBD3) transposon inserted into intron 5 of the Cockayne syndrome Group B (CSB) gene in the common ancestor of all higher primates. As a result, full-length CSB is now coexpressed with an abundant CSB-PGBD3 fusion protein by alternative splicing of CSB exons 1-5 to the PGBD3 transposase. An internal deletion of the piggyBac transposase ORF also gave rise to 889 dispersed, 140-bp MER85 elements that were mobilized in trans by PGBD3 transposase. The CSB-PGBD3 fusion protein binds MER85s in vitro and induces a strong interferon-like innate antiviral immune response when expressed in CSB-null UVSS1KO cells. To explore the connection between DNA binding and gene expression changes induced by CSB-PGBD3, we investigated the genome-wide DNA binding profile of the fusion protein. CSB-PGBD3 binds to 363 MER85 elements in vivo, but these sites do not correlate with gene expression changes induced by the fusion protein. Instead, CSB-PGBD3 is enriched at AP-1, TEAD1, and CTCF motifs, presumably through protein-protein interactions with the cognate transcription factors; moreover, recruitment of CSB-PGBD3 to AP-1 and TEAD1 motifs correlates with nearby genes regulated by CSB-PGBD3 expression in UVSS1KO cells and downregulated by CSB rescue of mutant CS1AN cells. Consistent with these data, the N-terminal CSB domain of the CSB-PGBD3 fusion protein interacts with the AP-1 transcription factor c-Jun and with RNA polymerase II, and a chimeric CSB-LacI construct containing only the N-terminus of CSB upregulates many of the genes induced by CSB-PGBD3. We conclude that the CSB-PGBD3 fusion protein substantially reshapes the transcriptome in CS patient CS1AN and that continued expression of the CSB-PGBD3 fusion protein in the absence of functional CSB may affect the clinical presentation of CS patients by directly altering the transcriptional program., Competing Interests: The authors have declared that no competing interests exist.

Cockayne syndrome is a segmental progeria most often caused by mutations in the CSB gene encoding a SWI/SNF-like ATPase required for transcription-coupled DNA repair (TCR). Over 43Mya before marmosets diverged from humans, a piggyBac3 (PGBD3) transposable element integrated into intron 5 of the CSB gene. As a result, primate CSB genes now generate both CSB protein and a conserved CSB-PGBD3 fusion protein in which the first 5 exons of CSB are alternatively spliced to the PGBD3 transposase. Using a host cell reactivation assay, we show that the fusion protein inhibits TCR of oxidative damage but facilitates TCR of UV damage. We also show by microarray analysis that expression of the fusion protein alone in CSB-null UV-sensitive syndrome (UVSS) cells induces an interferon-like response that resembles both the innate antiviral response and the prolonged interferon response normally maintained by unphosphorylated STAT1 (U-STAT1); moreover, as might be expected based on conservation of the fusion protein, this potentially cytotoxic interferon-like response is largely reversed by coexpression of functional CSB protein. Interestingly, expression of CSB and the CSB-PGBD3 fusion protein together, but neither alone, upregulates the insulin growth factor binding protein IGFBP5 and downregulates IGFBP7, suggesting that the fusion protein may also confer a metabolic advantage, perhaps in the presence of DNA damage. Finally, we show that the fusion protein binds in vitro to members of a dispersed family of 900 internally deleted piggyBac elements known as MER85s, providing a potential mechanism by which the fusion protein could exert widespread effects on gene expression. Our data suggest that the CSB-PGBD3 fusion protein is important in both health and disease, and could play a role in Cockayne syndrome., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3' terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1-5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein., Competing Interests: The authors have declared that no competing interests exist.

Cockayne syndrome (CS) is an inherited neurodevelopmental disorder with progeroid features. Although the genes responsible for CS have been implicated in a variety of DNA repair- and transcription-related pathways, the nature of the molecular defect in CS remains mysterious. Using expression microarrays and a unique method for comparative expression analysis called L2L, we sought to define this defect in cells lacking a functional CS group B (CSB) protein, the SWI/SNF-like ATPase responsible for most cases of CS. Remarkably, many of the genes regulated by CSB are also affected by inhibitors of histone deacetylase and DNA methylation, as well as by defects in poly(ADP-ribose)-polymerase function and RNA polymerase II elongation. Moreover, consistent with these microarray expression data, CSB-null cells are sensitive to inhibitors of histone deacetylase or poly(ADP-ribose)-polymerase. Our data indicate a general role for CSB protein in maintenance and remodeling of chromatin structure and suggest that CS is a disease of transcriptional deregulation caused by misexpression of growth-suppressive, inflammatory, and proapoptotic pathways.

Infection by adenovirus 12, transfection with the Ad12 E1B 55 kDa gene, or activation of p53 cause metaphase fragility of four loci (RNU1, PSU1, RNU2, and RN5S) each containing tandemly repeated genes for an abundant small RNA (U1, U2, and 5S RNA). We now show that loss of the Cockayne syndrome group B protein (CSB) or overexpression of the p53 carboxy-terminal domain induces fragility of the same loci; moreover, p53 interacts with CSB in vivo and in vitro. We propose that CSB functions as an elongation factor for transcription of structured RNAs, including some mRNAs. Activation of p53 would inhibit CSB, stalling transcription complexes and locally blocking chromatin condensation. Impaired transcription elongation may also explain the diverse clinical features of Cockayne syndrome.

Adenovirus type 12 (Ad12) infection of human cells induces four chromosomal fragile sites corresponding to the U1 small nuclear RNA (snRNA) genes (the RNU1 locus), the U2 snRNA genes (RNU2), the U1 snRNA pseudogenes (PSU1), and the 5S rRNA genes (RN5S). Ad12-induced fragility of the RNU2 locus requires U2 snRNA transcriptional regulatory elements and viral early functions but not viral replication or integration, or chromosomal sequences flanking the RNU2 locus. We now show that Ad12 cannot induce the RNU1, RNU2, or PSU1 fragile sites in Saos-2 cells lacking the p53 and retinoblastoma (Rb) proteins but that viral induction of fragility is rescued in these cells when the expression of wild-type p53 or selected hot-spot mutants (i.e., V143A, R175H, R248W, and R273H) is restored by transient expression or stable retroviral transduction. We also observed weak constitutive fragility of the RNU1 and RNU2 loci in cells belonging to xeroderma pigmentosum complementation groups B and D (XPB and XPD) which are partially defective in the ERCC2 (XPD) and ERCC3 (XPB) helicase activities shared between the repairosome and the RNA polymerase H basal transcription factor TFIIH. We propose a model for Ad12-induced chromosome fragility in which interaction of p53 with the Ad12 E1B 55-kDa transforming protein (and possibly E4orf6) induces a p53 gain of function which ultimately perturbs the RNA polymerase II basal transcription apparatus. The p53 gain of function could interfere with chromatin condensation either by blocking mitotic shutdown of U1 and U2 snRNA transcription or by phenocopying global or local DNA damage. Specific fragilization of the RNU1, RNU2, and PSU1 loci could reflect the unusually high local concentration of strong transcription units or the specialized nature of the U1 and U2 snRNA transcription apparatus.