Fiorenzo A. Peverali

Defective dorsal closure and loss of epidermal decapentaplegic expression in Drosophila fos mutants

Drosophila kayak mutant embryos exhibit defects in dorsal closure, a morphogenetic cell sheet movement during embryogenesis. Here we show that kayak encodes D‐Fos, the Drosophila homologue of the mammalian proto‐oncogene product, c‐Fos. D‐Fos is shown to act in a similar manner to Drosophila Jun: in the cells of the leading edge it is required for the expression of the TGFβ‐like Decapentaplegic (Dpp) protein, which is believed to control the cell shape changes that take place during dorsal closure. Defects observed in mutant embryos, and adults with reduced Fos expression, are reminiscent of phenotypes caused by ‘loss of function’ mutations in the Drosophila JNKK homologue, hemipterous. These results indicate that D‐Fos is required downstream of the Drosophila JNK signal transduction pathway, consistent with a role in heterodimerization with D‐Jun, to activate downstream targets such as dpp.

Modular Structure of the Human Lamin B2 Replicator

ABSTRACT The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.

Early mitotic degradation of the homeoprotein HOXC10 is potentially linked to cell cycle progression

Hox proteins are transcription factors involved in controlling axial patterning, leukaemias and hereditary malformations. Here, we show that HOXC10 oscillates in abundance during the cell cycle, being targeted for degradation early in mitosis by the ubiquitin‐dependent proteasome pathway. Among abdominal‐B subfamily members, the mitotic proteolysis of HOXC10 appears unique, since the levels of the paralogous HOXD10 and the related homeoprotein HOXC13 are constant throughout the cell cycle. When two destruction box motifs (D‐box) are mutated, HOXC10 is stabilized and cells accumulate in metaphase. HOXC10 appears to be a new prometaphase target of the anaphase‐promoting complex (APC), since its degradation coincides with cyclin A destruction and is suppressed by expression of a dominant‐negative form of UbcH10, an APC‐associated ubiquitin‐conjugating enzyme. Moreover, HOXC10 co‐immunoprecipitates the APC subunit CDC27, and its in vitro degradation is reduced in APC‐depleted extracts or by competition with the APC substrate cyclin A. These data imply that HOXC10 is a homeoprotein with the potential to influence mitotic progression, and might provide a link between developmental regulation and cell cycle control.

Phosphorylation of Drosophila Jun by the MAP kinase rolled regulates photoreceptor differentiation.

Drosophila Jun (D‐Jun) is a nuclear component of the receptor tyrosine kinase/Ras signal transduction pathway which triggers photoreceptor differentiation during eye development. Here we show that D‐Jun is a substrate for the ERK‐related Drosophila MAP kinase Rolled, which has previously been shown to be a part of this pathway. A D‐Jun mutant that carries alanines in place of the Rolled phosphorylation sites acts as a dominant suppressor of photoreceptor cell fate if expressed in the eye imaginal disc. In contrast, a mutant in which the phosphorylation sites are replaced by phosphate‐mimetic Asp residues, as well as a VP16‐D‐Jun fusion protein, can promote photoreceptor differentiation. These data implicate Jun phosphorylation in the choice between neuronal and non‐neuronal fate during Drosophila eye development.

Is DNA sequence sufficient to specify DNA replication origins in metazoan cells

DNA replication occupies a central position in the cell cycle and, therefore, in the development and life of multicellular organisms. During the last 10 years, our comprehension of this important process has considerably improved. Although the mechanisms that coordinate DNA replication with the other moments of the cell cycle are not yet fully understood, it is known that they mainly operate through DNA replication origins and the protein complexes bound to them. In eukaryotes, the packaging status of chromatin seems to be part of the mechanism that controls whether or not and when during the S-phase a particular origin will be activated. Intriguingly, the protein complexes bound to DNA replication origins appear to be directly involved in controlling chromatin packaging. In this manner they can also affect gene expression. In this review we focus on DNA replication origins in metazoan cells and on the relationship between these elements and the structural and functional organization of the genome.

The deubiquitination enzyme Fat facets negatively regulates RTK/Ras/MAPK signalling during Drosophila eye development

The Drosophila fat facets (faf) gene encodes a deubiquitination enzyme with a putative function in proteasomal protein degradation. Mutants lacking zygotic faf function develop to adulthood, but have rough eyes caused by the presence of one to two ectopic outer photoreceptors per ommatidium. Here we show that faf interacts genetically with the receptor tyrosine kinase (RTK)/Ras pathway, which induces photoreceptor differentiation in the developing eye. The results indicate that RTK/Ras signalling is increased in faf mutants, causing normally non-neuronal cells to adopt photoreceptor fate. Consistently, the protein level of at least one component of the Ras signal transduction pathway, the transcription factor D-Jun, is elevated in faf eye discs at the time when the ectopic photoreceptors are induced. We propose that defective ubiquitin-dependent proteolysis leads to increased and prolonged D-Jun expression, which together with other factors contributes to the induction of ectopic photoreceptors in faf mutants. These studies demonstrate the relevance of ubiquitin-dependent protein degradation in the regulation of RTK/Ras signal transduction in an intact organism.

Homeotic proteins participate in the function of human-DNA replication origins

Recent evidence points to homeotic proteins as actors in the crosstalk between development and DNA replication. The present work demonstrates that HOXC13, previously identified as a new member of human DNA replicative complexes, is a stable component of early replicating chromatin in living cells: it displays a slow nuclear dynamics due to its anchoring to the DNA minor groove via the arginine-5 residue of the homeodomain. HOXC13 binds in vivo to the lamin B2 origin in a cell-cycle-dependent manner consistent with origin function; the interaction maps with nucleotide precision within the replicative complex. HOXC13 displays in vitro affinity for other replicative complex proteins; it interacts also in vivo with the same proteins in a cell-cycle-dependent fashion. Chromatin-structure modifying treatments, disturbing origin function, reduce also HOXC13–origin interaction. The described interactions are not restricted to a single origin nor to a single homeotic protein (also HOXC10 binds the lamin B2 origin in vivo). Thus, HOX complexes probably contribute in a general, structure-dependent manner, to origin identification and assembly of replicative complexes thereon, in presence of specific chromatin configurations.

Hydrogen peroxide-mediated induction of SOD1 gene transcription is independent from Nrf2 in a cellular model of neurodegeneration.

BACKGROUND It is still unclear whether oxidative stress (OS) is a disease consequence or is directly involved in the etiology of neurodegenerative disorders (NDs) onset and/or progression; however, many of these conditions are associated with increased levels of oxidation markers and damaged cell components. Previously we demonstrated the accumulation of reactive oxygen species (ROS) and increased SOD1 gene expression in H2O2-treated SH-SY5Y cells, recapitulating pathological features of Amyotrophic Lateral Sclerosis (ALS). Since we observed a post-transcriptional regulation of SOD1 gene in this cellular model, we investigated the transcriptional regulation of SOD1 mRNA under oxidative stress (OS). RESULTS In response to H2O2 treatment, PolII increased its association to SOD1 promoter. Electrophoretic mobility shift assays and mass spectrometry analyses on SOD1 promoter highlighted the formation of a transcriptional complex bound to the ARE sequences. Western Blotting experiments showed that in our in vitro model, H2O2 exposure increases Nrf2 expression in the nuclear fraction while immunoprecipitation confirmed its phosphorylation and release from Keap1 inhibition. However, H2O2 treatment did not modify Nrf2 binding on SOD1 promoter, which seems to be regulated by different transcription factors (TFs). CONCLUSIONS Although our data suggest that SOD1 is transcriptionally regulated in response to OS, Nrf2 does not appear to associate with SOD1 promoter in this cellular model of neurodegeneration. Our results open new perspectives in the comprehension of two key antioxidant pathways involved in neurodegenerative disorders.

XPD mutations in trichothiodystrophy hamper collagen VI expression and reveal a role of TFIIH in transcription derepression

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.

GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy

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.