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During the heat shock response (HSR), heat shock factor (HSF1 in mammals) binds to target gene promoters, resulting in increased expression of heat shock proteins that help maintain protein homeostasis and ensure cell survival. Besides HSF1, only a relatively few transcription factors with a specific role in ensuring correctly regulated gene expression during the HSR have been described. Here, we use proteomic and genomic (CRISPR) screening to identify a role for RPRD1B in the response to heat shock. Indeed, cells depleted for RPRD1B are heat shock sensitive and show decreased expression of key heat shock proteins (HSPs). These results add to our understanding of the connection between basic gene expression mechanisms and the HSR.

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA polymerase II, causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions. However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR–Cas9 screen, we identified the elongation factor ELOF1 as an important factor in the transcription stress response following DNA damage. We show that ELOF1 has an evolutionarily conserved role in transcription-coupled nucleotide excision repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair transcription-blocking lesions and resume transcription. Additionally, ELOF1 modulates transcription to protect cells against transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage via two distinct mechanisms.

The heat shock (HS) response involves rapid induction of HS genes, whereas transcriptional repression is established more slowly at most other genes. Previous data suggested that such repression results from inhibition of RNA polymerase II (RNAPII) pause release, but here, we show that HS strongly affects other phases of the transcription cycle. Intriguingly, while elongation rates increase upon HS, processivity markedly decreases, so that RNAPII frequently fails to reach the end of genes. Indeed, HS results in widespread premature transcript termination at cryptic, intronic polyadenylation (IPA) sites near gene 5′-ends, likely via inhibition of U1 telescripting. This results in dramatic reconfiguration of the human transcriptome with production of new, previously unannotated, short mRNAs that accumulate in the nucleus. Together, these results shed new light on the basic transcription mechanisms induced by growth at elevated temperature and show that a genome-wide shift toward usage of IPA sites can occur under physiological conditions.

Correct transcription is crucial for life. However, DNA damage severely impedes elongating RNA Polymerase II (Pol II), causing transcription inhibition and transcription-replication conflicts. Cells are equipped with intricate mechanisms to counteract the severe consequence of these transcription-blocking lesions (TBLs). However, the exact mechanism and factors involved remain largely unknown. Here, using a genome-wide CRISPR/cas9 screen, we identified elongation factor ELOF1 as an important new factor in the transcription stress response upon DNA damage. We show that ELOF1 has an evolutionary conserved role in Transcription-Coupled Nucleotide Excision Repair (TC-NER), where it promotes recruitment of the TC-NER factors UVSSA and TFIIH to efficiently repair TBLs and resume transcription. Additionally, ELOF1 modulates transcription to protect cells from transcription-mediated replication stress, thereby preserving genome stability. Thus, ELOF1 protects the transcription machinery from DNA damage by two distinct mechanisms.

Heterochromatin Protein 1 (HP1) and the Mre11-Rad50-Nbs1 (MRN) complex are conserved factors that play crucial role in genome stability and integrity. Despite their involvement in overlapping cellular functions, ranging from chromatin organization, telomere maintenance to DNA replication and repair, a tight functional relationship between HP1 and the MRN complex has never been elucidated. Here we show that the Drosophila HP1a protein binds to the MRN complex through its chromoshadow domain (CSD). In addition, loss of any of the MRN members reduces HP1a levels indicating that the MRN complex acts as regulator of HP1a stability. Moreover, overexpression of HP1a in nbs (but not in rad50 or mre11) mutant cells drastically reduces DNA damage associated with the loss of Nbs suggesting that HP1a and Nbs work in concert to maintain chromosome integrity in flies. We have also found that human HP1α and NBS1 interact with each other and that, similarly to Drosophila, siRNA-mediated inhibition of NBS1 reduces HP1α levels in human cultured cells. Surprisingly, fibroblasts from Nijmegen Breakage Syndrome (NBS) patients, carrying the 657del5 hypomorphic mutation in NBS1 and expressing the p26 and p70 NBS1 fragments, accumulate HP1α indicating that, differently from NBS1 knockout cells, the presence of truncated NBS1 extends HP1α turnover and/or promotes its stability. Remarkably, an siRNA-mediated reduction of HP1α in NBS fibroblasts decreases the hypersensitivity to irradiation, a characteristic of the NBS syndrome. Overall, our data provide an unanticipated evidence of a close interaction between HP1 and NBS1 that is essential for genome stability and point up HP1α as a potential target to counteract chromosome instability in NBS patient cells.

Helicases are ubiquitous enzymes that unwind or remodel single or double-stranded nucleic acids, and that participate in a vast array of metabolic pathways. The ATP-dependent DEXH-box RNA/DNA helicase MLE was first identified as a core member of the chromatin remodeling MSL complex, responsible for dosage compensation in Drosophila males. Although this complex does not assemble in females, MLE is present. Given the multiplicity of functions attributed to its mammalian ortholog RNA helicase A, we have carried out an analysis for the purpose of determining whether MLE displays the same diversity. We have identified a number of different proteins that associate with MLE, implicating its role in specific pathways. We have documented this association in selected examples that include the spliceosome complex, heterogeneous Nuclear Ribonucleoproteins involved in RNA Processing and in Heterochromatin Protein 1 deposition, and the NuRD complex.

In Drosophila, dosage compensation is mediated by the MSL complex, which binds numerous sites on the X chromosome in males and enhances the transcriptional rate of a substantial number of X-linked genes. We have determined that topoisomerase II (Topo II) is enriched on dosage compensated genes, to which it is recruited by association with the MSL complex, in excess of the amount that is present on autosomal genes with similar transcription levels. Using a plasmid model, we show that Topo II is required for proper dosage compensation and that compensated chromatin is topologically different from non-compensated chromatin. This difference, which is not the result of the enhanced transcription level due of X-linked genes and which represents a structural modification intrinsic to the DNA of compensated chromatin, requires the function of Topo II. Our results suggest that Topo II is an integral part of the mechanistic basis of dosage compensation.

The receptor for advanced glycation endproducts (RAGE) mediates responses to cell danger and stress. When bound by its many ligands (which include advanced glycation endproducts, certain members of the S100/calgranulin family, extracellular highmobility group box 1, the integrin Mac-1, amyloid β-peptide and fibrils), RAGE activates programs responsible for acute and chronic inflammation. RAGE is therefore also involved in cancer progression, diabetes, atherosclerosis, and Alzheimer's disease. RAGE has several isoforms deriving from alternative splicing, including a soluble form called endogenous secretory RAGE (esRAGE). We show here that most soluble RAGE, either produced by cell lines or present in human blood, is not recognized by an anti-esRAGE antibody. Cells transfected with the cDNA for fulllength RAGE, and thus not expressing esRAGE, produce a form of soluble RAGE, cleaved RAGE (cRAGE) that derives from proteolytic cleavage of the membrane-bound molecules and acts as a decoy receptor. By screening chemical inhibitors and genetically modified mouse embryonic fibroblasts (MEFs), we identify the sheddase ADAM10 as a membrane protease responsible for RAGE cleavage. Binding of its ligand HMGB1 promotes RAGE shedding. Our data do not disprove the interpretation that high levels of soluble forms of RAGE protect against chronic inflammation, but rather suggest that they correlate with high levels of ongoing inflammation. © FASEB.

RNA hairpins are a common type of secondary structures that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi) and microRNA (miRNA) pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss) RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE was shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. Here we report that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, we show that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. We use two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway., Author Summary In virtually all RNA molecules, single stranded regions undergo complementary base-pairing with neighboring regions to form double-stranded structures called stem-loops or hairpins. During the fundamental processes of transcription and translation, these RNA structures are reshaped by helicases—enzymes that separate paired regions of nucleic acids. In addition, small non-coding RNA molecules involved in regulating gene expression rely on sequences that allow them to form hairpin structures. In this paper we present evidence that the Drosophila helicase MLE, well known for its role in dosage compensation, participates in the processing of hairpin RNAs in the pathway that leads to induced RNA interference.

Epigenetic regulation at the chromosomal level compensates for the difference in the dosage of X-linked genes between the sexes (dosage compensation). In Drosophila, this regulatory mechanism operates through the MSL complex that enhances the transcription of many genes on the X chromosome in males. The results of an investigation of the structural effects of various subunits of the complex confirmed that enriched, specific acetylation of histone H4 at lysine 16 by the histone acetyl transferase subunit MOF induced a more disorganized state of reconstituted single chromatin fibers. In addition, targeting of the MSL complex to plasmids by inclusion of the MSL assembly locus reduced the level of negative supercoiling. Similar targeting of incomplete complexes distinguished the roles that the various subunits of the complex play in this topological modification. Finally, the potential contribution of ISWI containing remodeling complexes to the architecture of compensated chromatin was analyzed and the results indicate a probable role for this remodeling factor in dosage compensation.

Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes and regulate terminal DNA replication. If telomeres are not properly capped, they are sensed as double-strand breaks, leading to the activation of cell cycle checkpoints and inappropriate DNA repair, which might result in end-to-end fusions (Palm and de Lange 2008). In most organisms, telomeres contain arrays of GC-rich repeats, which are added to chromosome ends by telomerase (Palm and de Lange 2008). Drosophila telomeres are elongated by transposition of three specialized retroelements, rather than telomerase activity; several studies indicate that Drosophila telomeres are epigenetic structures assembled independently of the sequence of terminal DNA (Cenci et al. 2005; Mason et al. 2008; Rong 2008). In organisms with telomerase, telomeres are protected by two well-characterized protein assemblies: the Cdc13–Stn1–Ten1 (CST) and shelterin complexes. These complexes are evolutionarily conserved, even if they vary in composition and architecture in different phyla. The three subunits of the CST complex all contain oligonucleotide/oligosaccharide-binding (OB) fold domains, and interact to each other to form an RPA-like complex that binds the telomere 3′ overhang. (Mitton-Fry et al. 2002; Gao et al. 2007; Gelinas et al. 2009; Sun et al. 2009). Shelterin has been thoroughly characterized in human cells; it is a six-protein complex that specifically associates with the telomeric TTAGGG repeats. Three of the shelterin subunits interact directly with the TTAGGG repeats: TRF1 and TRF2 bind the TTAGGG duplex, and POT1 binds the 3′ overhang. TRF1, TRF2, and POT1 are interconnected by TIN2 and TPP1, and TRF2 interacts with hRap1, a distant homolog of Saccharomyces cerevisiae Rap1. The shelterin subunits share three properties that distinguish them from the nonshelterin telomere-associated proteins. They are specifically enriched at telomeres, they are present at telomeres throughout the cell cycle, and their functions are limited to telomere maintenance (Palm and de Lange 2008). The Stn1 and Ten1 subunits of the S. cerevisiae CST complex are conserved in Schizosaccharomyces pombe, plants, and humans, while shelterin-like elements are found in S. pombe and plants but not in S. cerevisiae (Martin et al. 2007; Song et al. 2008; Linger and Price 2009; Lue 2009; Miyake et al. 2009; Surovtseva et al. 2009). S. pombe and plants have both a shelterin-like complex and a CST-like complex, both of which are required for telomere protection. The two complexes are present also in humans, and are thought to collaborate in telomere protection. However, the human CST complex does not share the shelterin properties, and appears to have a relatively minor role in telomere capping. (Miyake et al. 2009; Surovtseva et al. 2009). In addition to the shelterin and CST components, yeast, plant, and mammalian telomeres contain several conserved polypeptides required for proper telomere function. These polypeptides include many proteins involved in DNA repair, such as the ATM kinase, the Ku70/80 heterodimer, the MRE11/RAD50/NBS1 (MRN) complex, Rad51, the ERCC1/XPF endonuclease, the Apollo exonuclease, and the RecQ family members WRN and BLM. In addition, yeast and mammalian telomeres are enriched in proteins that are homologous to Drosophila HP1 (Heterochromatin Protein 1). All nonshelterin and non-CST proteins function not only at telomeres, but are also involved in several cellular processes that are not related with telomeres (Palm and de Lange 2008; Linger and Price 2009). The search for Drosophila telomere-capping proteins has relied mainly on the isolation of mutants that display frequent telomeric fusions in larval brain cells. Genetic and molecular analyses thus far have identified nine loci that are required to prevent end-to-end fusion in Drosophila. These are UbcD1, which encodes a highly conserved E2 enzyme that mediates protein ubiquitination; Su(var)205 and caravaggio (cav), which encode HP1 and HOAP (HP1/origin recognition complex [ORC]-associated protein), respectively; the Drosophila homologs of the ATM, RAD50, MRE11, and NBS1 genes; without children (woc), which specifies a transcription factor associated with the initiating form of RNA polymerase II (Pol II); and modigliani (moi, which encodes a novel protein that binds both HOAP and HP1 (for review, see Cenci et al. 2005; Ciapponi and Cenci 2008; Mason et al. 2008; Rong 2008; see also Raffa et al. 2009). HOAP and Moi have properties that distinguish them from the other Drosophila telomere-capping proteins: They localize only at telomeres, and appear to function only in telomere maintenance. These properties are similar to the properties of human shelterin, suggesting that the HOAP–Moi complex, which we called terminin, is a functional analog of shelterin (Raffa et al. 2009). Here we show that Verrocchio (Ver) is required to prevent telomere fusion. Ver interacts directly with both HOAP and Moi and localizes only at telomeres, suggesting that Ver is a novel terminin component. Bioinformatics analyses showed that Ver contains an OB fold domain that shares structural similarity with the OB fold of Stn1. Mutations in the predicted Ver OB fold cause telomere fusion, suggesting that the Ver OB fold is required for telomere protection.

Several proteins have been identified that protect Drosophila telomeres from fusion events. They include UbcD1, HP1, HOAP, the components of the Mre11-Rad50-Nbs (MRN) complex, the ATM kinase, and the putative transcription factor Woc. Of these proteins, only HOAP has been shown to localize specifically at telomeres. Here we show that the modigliani gene encodes a protein (Moi) that is enriched only at telomeres, colocalizes and physically interacts with HOAP, and is required to prevent telomeric fusions. Moi is encoded by the bicistronic CG31241 locus. This locus produces a single transcript that contains 2 ORFs that specify different essential functions. One of these ORFs encodes the 20-kDa Moi protein. The other encodes a 60-kDa protein homologous to RNA methyltransferases that is not required for telomere protection (Drosophila Tat-like). Moi and HOAP share several properties with the components of shelterin, the protein complex that protects human telomeres. HOAP and Moi are not evolutionarily conserved unlike the other proteins implicated in Drosophila telomere protection. Similarly, none of the shelterin subunits is conserved in Drosophila, while most human nonshelterin proteins have Drosophila homologues. This suggests that the HOAP-Moi complex, we name “terminin,” plays a specific role in the DNA sequence-independent assembly of Drosophila telomeres. We speculate that this complex is functionally analogous to shelterin, which binds chromosome ends in a sequence-dependent manner.