The ATPase SRCAP is associated with the mitotic apparatus, uncovering novel molecular aspects of Floating-Harbor syndrome.

BACKGROUND: A variety of human genetic diseases is known to be caused by mutations in genes encoding chromatin factors and epigenetic regulators, such as DNA or histone modifying enzymes and members of ATP-dependent chromatin remodeling complexes. Floating-Harbor syndrome is a rare genetic disease affecting human development caused by dominant truncating mutations in the SRCAP gene, which encodes the ATPase SRCAP, the core catalytic subunit of the homonymous chromatin-remodeling complex. The main function of the SRCAP complex is to promote the exchange of histone H2A with the H2A.Z variant. According to the canonical role played by the SRCAP protein in epigenetic regulation, the Floating-Harbor syndrome is thought to be a consequence of chromatin perturbations. However, additional potential physiological functions of SRCAP have not been sufficiently explored. RESULTS: We combined cell biology, reverse genetics, and biochemical approaches to study the subcellular localization of the SRCAP protein and assess its involvement in cell cycle progression in HeLa cells. Surprisingly, we found that SRCAP associates with components of the mitotic apparatus (centrosomes, spindle, midbody), interacts with a plethora of cytokinesis regulators, and positively regulates their recruitment to the midbody. Remarkably, SRCAP depletion perturbs both mitosis and cytokinesis. Similarly, DOM-A, the functional SRCAP orthologue in Drosophila melanogaster, is found at centrosomes and the midbody in Drosophila cells, and its depletion similarly affects both mitosis and cytokinesis. CONCLUSIONS: Our findings provide first evidence suggesting that SRCAP plays previously undetected and evolutionarily conserved roles in cell division, independent of its functions in chromatin regulation. SRCAP may participate in two different steps of cell division: by ensuring proper chromosome segregation during mitosis and midbody function during cytokinesis. Moreover, our findings emphasize a surprising scenario whereby alterations in cell division produced by SRCAP mutations may contribute to the onset of Floating-Harbor syndrome.

RSPO1-mutated keratinocytes from palmoplantar keratoderma display impaired differentiation, alteration of cell-cell adhesion, EMT-like phenotype and invasiveness properties: implications for squamous cell carcinoma susceptibility in patients with 46XX disorder of sexual development.

BACKGROUND: Secreted R-spondin (RSPO) proteins play a key role in reproductive organ development, epithelial stem cell renewal and cancer induction by reinforcing canonical Wnt signaling. We have previously reported that palmoplantar keratoderma (PPK), predisposition to cutaneous squamous cell carcinoma (SCC) development and sex reversal segregate as autosomal recessive trait in patients carrying RSPO1-mutations. Although our previous findings suggested that RSPO1 secreted from fibroblasts regulates keratinocyte growth or differentiation, the role of this protein in the epidermis remains largely unexplored. Our study was aimed at expanding the phenotypic, molecular and functional characterization of RSPO1-mutated skin and keratinocytes. RESULTS: Cultured primary keratinocytes from PPK skin of a RSPO1-mutated XX-sex reversed patient displayed highly impaired differentiation and epithelial-mesenchymal transition (EMT)-like phenotype. Interestingly, RSPO1-mutated PPK skin expressed markers of increased proliferation, dedifferentiation and altered cell-cell adhesion. Furthermore, all these signs were more evident in SCC specimens of the patient. Cultured PPK patient's keratinocytes exhibited increased expression of cell‒matrix adhesion proteins and extracellular matrix remodeling enzymes. Moreover, they showed invasiveness properties in an organotypic skin model in presence of PPK fibroblasts, which behave like cancer-associated fibroblasts. However, the co-culture with normal fibroblasts or treatment with the recombinant RSPO1 protein did not revert or reduce the EMT-like phenotype and invasion capability of PPK keratinocytes. Notably, RSPO1-mutated PPK fibroblasts induced a hyperproliferative and dedifferentiated phenotype of age-matched normal control plantar keratinocytes. Wnt signaling has a key role in both PPK promotion and SCC development. Accordingly, Wnt mediators were differentially expressed in both PPK keratinocytes and skin specimens of RSPO1-mutated patient compared to control. CONCLUSIONS: Altogether our data indicate that the absence of RSPO1 in patients with 46XX disorder of sexual development affects the skin microenvironment and epidermal integrity, thus contributing to the risk of SCC tumorigenesis in palmoplantar regions exposed to major frictional stresses.

The oncogenic kinase NEK2 regulates an RBFOX2-dependent pro-mesenchymal splicing program in triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most heterogeneous and malignant subtype of breast cancer (BC). TNBC is defined by the absence of expression of estrogen, progesterone and HER2 receptors and lacks efficacious targeted therapies. NEK2 is an oncogenic kinase that is significantly upregulated in TNBC, thereby representing a promising therapeutic target. NEK2 localizes in the nucleus and promotes oncogenic splice variants in different cancer cells. Notably, alternative splicing (AS) dysregulation has recently emerged as a featuring trait of TNBC that contributes to its aggressive phenotype. METHODS: To investigate whether NEK2 modulates TNBC transcriptome we performed RNA-sequencing analyses in a representative TNBC cell line (MDA-MB-231) and results were validated in multiple TNBC cell lines. Bioinformatics and functional analyses were carried out to elucidate the mechanism of splicing regulation by NEK2. Data from The Cancer Genome Atlas were mined to evaluate the potential of NEK2-sensitive exons as markers to identify the TNBC subtype and to assess their prognostic value. RESULTS: Transcriptome analysis revealed a widespread impact of NEK2 on the transcriptome of TNBC cells, with 1830 AS events that are susceptible to its expression. NEK2 regulates the inclusion of cassette exons in splice variants that discriminate TNBC from other BC and that correlate with poor prognosis, suggesting that this kinase contributes to the TNBC-specific splicing program. NEK2 elicits its effects by modulating the expression of the splicing factor RBFOX2, a well-known regulator of epithelial to mesenchymal transition (EMT). Accordingly, NEK2 splicing-regulated genes are enriched in functional terms related to cell adhesion and contractile cytoskeleton and NEK2 depletion in mesenchymal TNBC cells induces phenotypic and molecular traits typical of epithelial cells. Remarkably, depletion of select NEK2-sensitive splice-variants that are prognostic in TNBC patients is sufficient to interfere with TNBC cell morphology and motility, suggesting that NEK2 orchestrates a pro-mesenchymal splicing program that modulates migratory and invasive properties of TNBC cells. CONCLUSIONS: Our study uncovers an extensive splicing program modulated by NEK2 involving splice variants that confer an invasive phenotype to TNBCs and that might represent, together with NEK2 itself, valuable therapeutic targets for this disease.

The True Story of Yeti, the "Abominable" Heterochromatic Gene of Drosophila melanogaster.

The Drosophila Yeti gene (CG40218) was originally identified by recessive lethal mutation and subsequently mapped to the deep pericentromeric heterochromatin of chromosome 2. Functional studies have shown that Yeti encodes a 241 amino acid protein called YETI belonging to the evolutionarily conserved family of Bucentaur (BCNT) proteins and exhibiting a widespread distribution in animals and plants. Later studies have demonstrated that YETI protein: (i) is able to bind both subunits of the microtubule-based motor kinesin-I; (ii) is required for proper chromosome organization in both mitosis and meiosis divisions; and more recently (iii) is a new subunit of dTip60 chromatin remodeling complex. To date, other functions of YETI counterparts in chicken (CENtromere Protein 29, CENP-29), mouse (Cranio Protein 27, CP27), zebrafish and human (CranioFacial Development Protein 1, CFDP1) have been reported in literature, but the fully understanding of the multifaceted molecular function of this protein family remains still unclear. In this review we comprehensively highlight recent work and provide a more extensive hypothesis suggesting a broader range of YETI protein functions in different cellular processes.

eEF1Bγ binds the Che-1 and TP53 gene promoters and their transcripts.

BACKGROUND: We have previously shown that the eukaryotic elongation factor subunit 1B gamma (eEF1Bγ) interacts with the RNA polymerase II (pol II) alpha-like subunit "C" (POLR2C), alone or complexed, in the pol II enzyme. Moreover, we demonstrated that eEF1Bγ binds the promoter region and the 3' UTR mRNA of the vimentin gene. These events contribute to localize the vimentin transcript and consequentially its translation, promoting a proper mitochondrial network. METHODS: With the intent of identifying additional transcripts that complex with the eEF1Bγ protein, we performed a series of ribonucleoprotein immunoprecipitation (RIP) assays using a mitochondria-enriched heavy membrane (HM) fraction. RESULTS: Among the eEF1Bγ complexed transcripts, we found the mRNA encoding the Che-1/AATF multifunctional protein. As reported by other research groups, we found the tumor suppressor p53 transcript complexed with the eEF1Bγ protein. Here, we show for the first time that eEF1Bγ binds not only Che-1 and p53 transcripts but also their promoters. Remarkably, we demonstrate that both the Che-1 transcript and its translated product localize also to the mitochondria and that eEF1Bγ depletion strongly perturbs the mitochondrial network and the correct localization of Che-1. In a doxorubicin (Dox)-induced DNA damage assay we show that eEF1Bγ depletion significantly decreases p53 protein accumulation and slightly impacts on Che-1 accumulation. Importantly, Che-1 and p53 proteins are components of the DNA damage response machinery that maintains genome integrity and prevents tumorigenesis. CONCLUSIONS: Our data support the notion that eEF1Bγ, besides its canonical role in translation, is an RNA-binding protein and a key player in cellular stress responses. We suggest for eEF1Bγ a role as primordial transcription/translation factor that links fundamental steps from transcription control to local translation.