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Trichothiodystrophy (TTD) is a rare hereditary neurodevelopmental disorder defined by sulfur-deficient brittle hair and nails and scaly skin, but with otherwise remarkably variable clinical features. The photosensitive TTD (PS-TTD) forms exhibits in addition to progressive neuropathy and other features of segmental accelerated aging and is associated with impaired genome maintenance and transcription. New factors involved in various steps of gene expression have been identified for the different non-photosensitive forms of TTD (NPS-TTD), which do not appear to show features of premature aging. Here, we identify alanyl-tRNA synthetase 1 and methionyl-tRNA synthetase 1 variants as new gene defects that cause NPS-TTD. These variants result in the instability of the respective gene products alanyl- and methionyl-tRNA synthetase. These findings extend our previous observations that TTD mutations affect the stability of the corresponding proteins and emphasize this phenomenon as a common feature of TTD. Functional studies in skin fibroblasts from affected individuals demonstrate that these new variants also impact on the rate of tRNA charging, which is the first step in protein translation. The extension of reduced abundance of TTD factors to translation as well as transcription redefines TTD as a syndrome in which proteins involved in gene expression are unstable.

The cancer-free photosensitive trichothiodystrophy (PS-TTD) and the cancer-prone xeroderma pigmentosum (XP) are rare monogenic disorders that can arise from mutations in the same genes, namely ERCC2/XPD or ERCC3/XPB Both XPD and XPB proteins belong to the 10-subunit complex transcription factor IIH (TFIIH) that plays a key role in transcription and nucleotide excision repair, the DNA repair pathway devoted to the removal of ultraviolet-induced DNA lesions. Compelling evidence suggests that mutations affecting the DNA repair activity of TFIIH are responsible for the pathological features of XP, whereas those also impairing transcription give rise to TTD. By adopting a relatives-based whole transcriptome sequencing approach followed by specific gene expression profiling in primary fibroblasts from a large cohort of TTD or XP cases with mutations in ERCC2/XPD gene, we identify the expression alterations specific for TTD primary dermal fibroblasts. While most of these transcription deregulations do not impact on the protein level, very low amounts of prostaglandin I2 synthase (PTGIS) are found in TTD cells. PTGIS catalyzes the last step of prostaglandin I2 synthesis, a potent vasodilator and inhibitor of platelet aggregation. Its reduction characterizes all TTD cases so far investigated, both the PS-TTD with mutations in TFIIH coding genes as well as the nonphotosensitive (NPS)-TTD. A severe impairment of TFIIH and RNA polymerase II recruitment on the PTGIS promoter is found in TTD but not in XP cells. Thus, PTGIS represents a biomarker that combines all PS- and NPS-TTD cases and distinguishes them from XP.

// Raefa Abou Khouzam 1 , Chiara Molinari 2 , Samanta Salvi 2 , Monica Marabelli 1 , Valeria Molinaro 1 , Donata Orioli 3 , Luca Saragoni 4 , Paolo Morgagni 5 , Daniele Calistri 2 , Guglielmina Nadia Ranzani 1 1 Department of Biology and Biotechnology, University of Pavia, Pavia, Italy 2 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 3 Institute of Molecular Genetics, Consiglio Nazionale delle Ricerche, Pavia, Italy 4 Pathology Unit, Morgagni-Pierantoni Hospital, Forli, Italy 5 Department of General Surgery, Morgagni-Pierantoni Hospital, Forli, Italy Correspondence to: Guglielmina Nadia Ranzani, email: guglielmina.ranzani@unipv.it Keywords: CDH1 transcripts, digital-PCR quantification, intestinal-type gastric cancer, normal gastric mucosa Received: October 04, 2016 Accepted: November 09, 2016 Published: November 16, 2016 ABSTRACT E-cadherin is a cell-cell adhesion protein encoded by CDH1 tumor-suppressor gene. CDH1 inactivating mutations, leading to loss of protein expression, are common in gastric cancer of the diffuse histotype, while alternative mechanisms modulating E-cadherin expression characterize the more common intestinal histotype. These mechanisms are still poorly understood. CDH1 intron 2 has recently emerged as a cis -modulator of E-cadherin expression, encoding non-canonical transcripts. One in particular, CDH1a , proved to be expressed in gastric cancer cell lines, while being absent in the normal stomach. For the first time, we evaluated by digital PCR the expression of CDH1 and CDH1a transcripts in cancer and normal tissue samples from 32 patients with intestinal-type gastric cancer. We found a significant decrease in CDH1 expression in tumors compared to normal counterparts ( P = 0.001), which was especially evident in 76% of cases. CDH1a was detected at extremely low levels in 47% of tumors, but not in normal mucosa. A trend was observed of having less CDH1 in tumors expressing CDH1a transcript. The majority of tumors with both a decrease in CDH1 and presence of CDH1a also showed a decrease in miR-101 expression levels. On the whole, the decrease of CDH1 transcript, corresponding to the canonical protein, and the presence of CDH1a , corresponding to an alternative isoform, are likely to perturb E-cadherin-mediated signaling and cell-cell adhesion, thus contributing to intestinal-type gastric carcinogenesis.

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.

The extracellular matrix (ECM) is a highly dynamic and heterogeneous structure that plays multiple roles in living organisms. Its integrity and homeostasis are crucial for normal tissue development and organ physiology. Loss or alteration of ECM components turns towards a disease outcome. In this review, we provide a general overview of ECM components with a special focus on collagens, the most abundant and diverse ECM molecules. We discuss the different functions of the ECM including its impact on cell proliferation, migration and differentiation by highlighting the relevance of the bidirectional cross-talk between the matrix and surrounding cells. By systematically reviewing all the hereditary disorders associated to altered collagen structure or resulting in excessive collagen degradation, we point to the functional relevance of the collagen and therefore of the ECM elements for human health. Moreover, the large overlapping spectrum of clinical features of the collagen-related disorders makes in some cases the patient clinical diagnosis very difficult. A better understanding of ECM complexity and molecular mechanisms regulating the expression and functions of the various ECM elements will be fundamental to fully recognize the different clinical entities.

Defects in Cockayne syndrome type A (CSA), a gene involved in nucleotide excision repair, cause an autosomal recessive syndrome characterized by growth failure, progressive neurological dysfunction, premature aging, and skin photosensitivity and atrophy. Beyond its role in DNA repair, the CSA protein has additional functions in transcription and oxidative stress response, which are not yet fully elucidated. Here, we investigated the role of CSA protein in primary human keratinocyte senescence. Primary keratinocytes from three patients with CS-A displayed premature aging features, namely premature clonal conversion, high steady-state levels of reactive oxygen species and 8-OH-hydroxyguanine, and senescence-associated secretory phenotype. Stable transduction of CS-A keratinocytes with the wild-type CSA gene restored the normal cellular sensitivity to UV irradiation and normal 8-OH-hydroxyguanine levels. Gene correction was also characterized by proper restoration of keratinocyte clonogenic capacity and expression of clonal conversion key regulators (p16 and p63), decreased NF-κB activity and, in turn, the expression of its targets (NOX1 and MnSOD), and the secretion of senescence-associated secretory phenotype mediators. Overall, the CSA protein plays an important role in protecting cells from senescence by facilitating DNA damage processing, maintaining physiological redox status and keratinocyte clonogenic ability, and reducing the senescence-associated secretory phenotype-mediated inflammatory phenotype.

Mutations in the XPD subunit of the transcription/DNA repair factor (TFIIH) give rise to trichothiodystrophy (TTD), a rare hereditary multisystem disorder with skin abnormalities. Here, we show that TTD primary dermal fibroblasts contain low amounts of collagen type VI alpha1 subunit (COL6A1), a fundamental component of soft connective tissues. We demonstrate that COL6A1 expression is downregulated by the sterol regulatory element-binding protein-1 (SREBP-1) whose removal from the promoter is a key step in COL6A1 transcription upregulation in response to cell confluence. We provide evidence for TFIIH being involved in transcription derepression, thus highlighting a new function of TFIIH in gene expression regulation. The lack of COL6A1 upregulation in TTD is caused by the inability of the mutated TFIIH complexes to remove SREBP-1 from COL6A1 promoter and to sustain the subsequent high rate of COL6A1 transcription. This defect might account for the pathologic features that TTD shares with hereditary disorders because of mutations in COL6A genes.

UV-sensitive syndrome (UV S S) is a recently-identified autosomal recessive disorder characterized by mild cutaneous symptoms and defective transcription-coupled repair (TC-NER), the subpathway of nucleotide excision repair (NER) that rapidly removes damage that can block progression of the transcription machinery in actively-transcribed regions of DNA. Cockayne syndrome (CS) is another genetic disorder with sun sensitivity and defective TC-NER, caused by mutations in the CSA or CSB genes. The clinical hallmarks of CS include neurological/developmental abnormalities and premature aging. UV S S is genetically heterogeneous, in that it appears in individuals with mutations in CSB or in a still-unidentified gene. We report the identification of a UV S S patient (UV S S1VI) with a novel mutation in the CSA gene (p.trp361cys) that confers hypersensitivity to UV light, but not to inducers of oxidative damage that are notably cytotoxic in cells from CS patients. The defect in UV S S1VI cells is corrected by expression of the WT CSA gene. Expression of the p.trp361cys-mutated CSA cDNA increases the resistance of cells from a CS-A patient to oxidative stress, but does not correct their UV hypersensitivity. These findings imply that some mutations in the CSA gene may interfere with the TC-NER-dependent removal of UV-induced damage without affecting its role in the oxidative stress response. The differential sensitivity toward oxidative stress might explain the difference between the range and severity of symptoms in CS and the mild manifestations in UV s S patients that are limited to skin photosensitivity without precocious aging or neurodegeneration.

Rac3, a neuronal GTP-binding protein of the Rho family, induces neuritogenesis in primary neurons. Using yeast two-hybrid analysis, we show that Neurabin I, the neuronal F-actin binding protein, is a direct Rac3-interacting molecule. Biochemical and light microscopy studies indicate that Neurabin I copartitions and colocalizes with Rac3 at the growth cones of neurites, inducing Neurabin I association to the cytoskeleton. Moreover, Neurabin I antisense oligonucleotides abolish Rac3-induced neuritogenesis, which in turn is rescued by exogenous Neurabin I but not by Neurabin I mutant lacking the Rac3-binding domain. These results show that Neurabin I mediates Rac3-induced neuritogenesis, possibly by anchoring Rac3 to growth cone F-actin.

Abnormalities in keratinocyte growth and differentiation have a pathogenic significance in many skin disorders and result in gene expression alterations detectable by quantitative real-time RT-PCR (qRT-PCR). Relative quantification based on endogenous control (EC) genes is the commonly adopted approach, and the use of multiple reference genes from independent pathways is considered a best practice guideline, unless fully validated EC genes are available. The literature on optimal reference genes during in vitro calcium-induced differentiation of normal human epidermal keratinocytes (NHEK) is inconsistent. In many studies, the expression of target genes is compared to that of housekeeping genes whose expression, however, significantly varies during keratinocyte differentiation. Here, we report the results of our investigations on the expression stability of 15 candidate EC genes, including those commonly used as reference in expression analysis by qRT-PCR, during NHEK calcium-induced differentiation. We demonstrate that YWHAZ and UBC are extremely stable genes, and therefore, they represent optimal EC genes for expression studies in proliferating and calcium-induced differentiating NHEK. Furthermore, we demonstrate that YWHAZ/14-3-3-zeta is a suitable reference for quantitative comparison of both transcript and protein levels.

Trichothiodystrophy (TTD) is a rare, autosomal recessive neurodevelopmental disorder most commonly caused by mutations in ERCC2 (XPD), a gene that encodes a subunit of the transcription/repair factor IIH (TFIIH). Here, we describe two TTD cases in which detailed biochemical and molecular investigations offered a clue to explain their moderately affected phenotype. Patient TTD22PV showed new mutated XPD alleles: one contains a nonsense mutation (c.1984C>T) encoding a nonfunctional truncated product (p.Gln662X) whereas the second carries a genomic deletion (c.2191-18_c.2213del) that affects the splicing of intron 22 and generates multiple out-of-frame transcripts from codon 731. XPD mRNA from the second allele corresponds to 20% of the total. The predicted proteins, which are longer than normal, affect the cellular repair activity but only partially interfere with TFIIH stability, suggesting that the observed changes in the C-ter region of XPD cause minor structural changes that do not drastically compromise the transcriptional activity of TFIIH. Patient TTD24PV was compound heterozygous for a typical TTD allele (c.2164C>T, p.Arg722Trp) and for a new XPD allele with a mutation that partially affects intron 10 splicing, resulting in both mutated and normal XPD transcripts (that together represent 15% of the total XPD mRNA). Compared to the previously described TTD compound heterozygotes for the Arg722Trp change, Patient TTD24PV's cells show similar level of TFIIH but increased repair activity, suggesting that even low amounts of normal XPD subunits are able to partially rescue the functionality of TFIIH complexes.

Xeroderma pigmentosum group C (XPC) protein plays an essential role in DNA damage recognition in mammalian global genome nucleotide excision repair (NER). Here, we analyze the functional basis of NER inactivation caused by a single amino acid substitution (Trp to Ser at position 690) in XPC, previously identified in the XPC patient XP13PV. The Trp690Ser change dramatically affects the in vivo stability of the XPC protein, thereby causing a significant reduction of its steady-state level in XP13PV fibroblasts. Despite normal heterotrimeric complex formation and physical interactions with other NER factors, the mutant XPC protein lacks binding affinity for both undamaged and damaged DNA. Thus, this single amino acid substitution is sufficient to compromise XPC function through both quantitative and qualitative alterations of the protein. Although the mutant XPC fails to recognize damaged DNA, it is still capable of accumulating in a UV-damaged DNA-binding protein (UV-DDB)-dependent manner to UV-damaged subnuclear domains. However, the NER factors transcription factor IIH and XPA failed to colocalize stably with the mutant XPC. As well as highlighting the importance of UV-DDB in recruiting XPC to UV-damaged sites, these findings demonstrate the role of DNA binding by XPC in the assembly of subsequent NER intermediate complexes.

Two transchromosomic mouse embryonic stem (ES) sublines (ESMClox1.5 and ESMClox2.1) containing a human minichromosome (MC) were established from a sample of hybrid colonies isolated in fusion experiments between a normal diploid mouse ES line and a Chinese hamster ovary line carrying the MC. DNA cytometric and chromosome analyses of ESMClox1.5 and ESMClox2.1 indicated a mouse chromosome complement with a heteroploid constitution in a subtetraploid range; the karyotypes showed various degrees of polysomy for different chromosomes. A single copy of the MC was found in the majority of cells in all the isolated hybrid colonies and was stably maintained in the established sublines for more than 100 cell generations either with or without the selective agent. No significant differences from the ES parental cells were observed in growth characteristics of the transchromosomic ES sublines. ESMClox1.5 cells were unable to grow in soft agar; when cultured in hanging drops, they formed embryoid bodies, and when inoculated in nude mice, they produced teratomas. They were able to express the early development markers Oct4 and Nanog, as demonstrated by reverse transcription-polymerase chain reaction assay. All these features are in common with the ES parental line. Further research using the transchromosomic ES sublines described here may allow gene expression studies on transferred human minichromosomes and could shed light on the relationships among ploidy, pluripotency, cell transformation, and tumorigenesis. Disclosure of potential conflicts of interest is found at the end of this article.

Cockayne syndrome (CS) is a rare genetic disease characterized by severe growth, mental retardation and pronounced cachexia. CS is most frequently due to mutations in either of two genes, CSB and CSA. Evidence for a role of CSB protein in the repair of oxidative DNA damage has been provided recently. Here, we show that CSA is also involved in the response to oxidative stress. CS-A human primary fibroblasts and keratinocytes showed hypersensitivity to potassium bromate, a specific inducer of oxidative damage. This was associated with inefficient repair of oxidatively induced DNA lesions, namely 8-hydroxyguanine (8-OH-Gua) and (5'S)-8,5'-cyclo 2'-deoxyadenosine. Expression of the wild-type CSA in the CS-A cell line CS3BE significantly decreased the steady-state level of 8-OH-Gua and increased its repair rate following oxidant treatment. CS-A cell extracts showed normal 8-OH-Gua cleavage activity in an in vitro assay, whereas CS-B cell extracts were confirmed to be defective. Our data provide the first in vivo evidence that CSA protein contributes to prevent accumulation of various oxidized DNA bases and underline specific functions of CSB not shared with CSA. These findings support the hypothesis that defective repair of oxidative DNA damage is involved in the clinical features of CS patients.

Sek4 and Nuk are members of the Eph-related family of receptor protein-tyrosine kinases. These receptors interact with a set of cell surface ligands that have recently been implicated in axon guidance and fasciculation. We now demonstrate that the formation of the corpus callosum and anterior commissure, two major commissural axon tracts that connect the two cerebral hemispheres, is critically dependent on Sek4 and Nuk. While mice deficient in Nuk exhibit defects in pathfinding of anterior commissure axons, sek4 mutants have defects in corpus callosum formation. The phenotype in both axon tracts is markedly more severe in sek4/nuk1 double mutants, indicating that the two receptors act in a partially redundant fashion. sek4/nuk1 double mutants also exhibit specific guidance and fasciculation defects of diencephalic axon tracts. Moreover, while mice singly deficient in either Sek4 or Nuk are viable, most sek4/nuk1 double mutants die immediately after birth primarily due to a cleft palate. These results demonstrate essential and cooperative functions for Sek4 and Nuk in establishing axon pathways in the developing brain, and during the development of facial structures.

The Eph family of receptor tyrosine kinases and their cell surface bound ligands have been implicated in a number of developmental processes, including axon pathfinding and fasciculation, as well as patterning in the central nervous system. To better understand the complex signaling events taking place, we have undertaken a comparative analysis of ligand-receptor interactions between a subset of ligands, those that are tethered to the cell surface via a transmembrane domain, and a subset of Eph receptors, the so-called Elk subclass. Based on binding characteristics, receptor autophosphorylation, and cellular transformation assays, we find that the transmembrane-type ligands Lerk2 and Elf2 have common and specific receptors within the Elk subclass of receptors. The common receptors Cek10 and Elk bind and signal in response to Lerk2 and Elf2, whereas the Myk1 receptor is specific for Elf2. Elf2, however, fails to signal through Cek5 in a cellular transformation assay, suggesting that Lerk2 may be the preferred Cek5 ligand in vivo. A recently identified third transmembrane-type ligand, Elf3, specifically, but weakly, binds Cek10 and only induces focus formation when activated by C-terminal truncation. This suggests that the physiological Elf3 receptor may have yet to be identified. Knowledge regarding functional ligand-receptor interactions as presented in this study will be important for the design and interpretation of in vivo experiments, e.g., loss-of-function studies in transgenic mice.