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Chromatin remodeling protein HELLS is critical for retinoblastoma tumor initiation and progression.
Retinoblastoma is an aggressive childhood cancer of the developing retina that initiates by biallelic RB1 gene inactivation. Tumor progression in retinoblastoma is driven by epigenetics, as retinoblastoma genomes are stable, but the mechanism(s) that drive these epigenetic changes remain unknown. Lymphoid-specific helicase (HELLS) protein is an epigenetic modifier directly regulated by the RB/E2F pathway. In this study, we used novel genetically engineered mouse models to investigate the role of HELLS during retinal development and tumorigenesis. Our results indicate that Hells-null retinal progenitor cells divide, undergo cell-fate specification, and give rise to fully laminated retinae with minor bipolar cells defects, but normal retinal function. Despite the apparent nonessential role of HELLS in retinal development, failure to transcriptionally repress Hells during retinal terminal differentiation due to retinoblastoma (RB) family loss significantly contributes to retinal tumorigenesis. Loss of HELLS drastically reduced ectopic division of differentiating cells in Rb1/p107-null retinae, significantly decreased the incidence of retinoblastoma, delayed tumor progression, and increased overall survival. Despite its role in heterochromatin formation, we found no evidence that Hells loss directly affected chromatin accessibility in the retina but functioned as transcriptional co-activator of E2F3, decreasing expression of cell cycle genes. We propose that HELLS is a critical downstream mediator of E2F-dependent ectopic proliferation in RB-null retinae. Together with the nontoxic effect of HELLS loss in the developing retina, our results suggest that HELLS and its downstream pathways could serve as potential therapeutic targets for retinoblastoma
Regulation of BMAL1 Protein Stability and Circadian Function by GSK3β-Mediated Phosphorylation
Circadian rhythms govern a large array of physiological and metabolic functions. To achieve plasticity in circadian regulation, proteins constituting the molecular clock machinery undergo various post-translational modifications (PTMs), which influence their activity and intracellular localization. The core clock protein BMAL1 undergoes several PTMs. Here we report that the Akt-GSK3beta signaling pathway regulates BMAL1 protein stability and activity.GSK3beta phosphorylates BMAL1 specifically on Ser 17 and Thr 21 and primes it for ubiquitylation. In the absence of GSK3beta-mediated phosphorylation, BMAL1 becomes stabilized and BMAL1 dependent circadian gene expression is dampened. Dopamine D2 receptor mediated signaling, known to control the Akt-GSK3beta pathway, influences BMAL1 stability and in vivo circadian gene expression in striatal neurons.These findings uncover a previously unknown mechanism of circadian clock control. The GSK3beta kinase phosphorylates BMAL1, an event that controls the stability of the protein and the amplitude of circadian oscillation. BMAL1 phosphorylation appears to be an important regulatory step in maintaining the robustness of the circadian clock
Studio funzionale della transglutaminasi 3 e di p63 nel differenziamento epidermico mediante l'uso di modelli murini
RIASSUNTO BREVE
Il differenziamento epidermico prevede la migrazione dei cheratinociti dallo strato basale dellâepidermide a quelli sopra basali fino al raggiungimento dello strato piĂš superficiale dove formano lâinvolucro corneo. Durante questo processo i futuri corneociti subiscono una serie di cambiamenti nella loro struttura e tali modifiche sono mediate dagli enzimi transglutaminasi. Le transglutaminasi (TGasi) rappresentano una famiglia di enzimi funzionalmente e strutturalmente correlati che catalizzano la reazione di trasferimento acilico Ca2+-dipendente tra il gruppo Îł- carbossiamidico di residui di glutammina e il gruppo amminico di varie ammine primarie. In questo modo le transglutaminasi catalizzano la formazione di legami crociati (cross-linking) fra proteine che compongono lâinvolucro corneo. Quattro sono le TGasi espresse a livello epidermico (TGasi 1, TGasi 2, TGasi 3 e TGasi 5) e tutte, esclusa la TGasi 1, mostrano anche unâattivitĂ GTPasica. Nello strato basale dellâepidermide svolge invece una funzione molto importante il gene p63. Questâultimo è un membro della famiglia di p53, la cui trascrizione da due differenti promotori origina due diverse proteine:TAp63 che contiene un dominio di transattivazione allâN-terminale e âNp63che non lo possiede. Tale dominio è importante per lâattivazione dei geni bersaglio che presentano un motivo di legame per p63 di conseguenza TAp63 è considerato lâattivatore trascrizionale dei geni indotti durante il differenziamento, mentre lâespressione di âNp63 potrebbe essere correlata con il mantenimento dellâattivitĂ proliferante delle cellule staminali epidermiche localizzate nella membrana basale. Ognuna di queste due isoforme va inoltre incontro a splicing alternativo al C-terminale originando tre differenti varianti di splicing:Îą, β, Îł. Topi p63-/- presentano severe anomalie nella pelle e negli arti.
Lo scopo di questo progetto di ricerca è sia quello di fornire nuove conoscenze sul ruolo svolto dalla TGasi 3 durante il differenziamento epidermico che quello di individuare nuovi geni la cui attivitĂ risulta essere regolata da p63. Per quanto riguarda la TGasi 3 abbiamo deciso di realizzare topi knock-out condizionali attraverso lâutilizzo della ricombinasi Cre e dei siti loxP. Tale sistema consente infatti lâeliminazione tessuto-specifica dellâespressione del gene bersaglio. Durante gli esperimenti preliminari per la creazione di tali topi abbiamo inoltre scoperto due nuove varianti di splicing per il gene TGM3. La prima, denominata â6â7, isolata nel topo manca della cisteina catalitica e sia lâattivitĂ di cross-linking che quella GTPasica risultano essere compromesse. La variante â9â10, isolata nellâuomo, pur conservando intatta la triade catalitica presenta una bassa attivitĂ transamidasica, mentre lâattivitĂ GTPasica risulta essere persa. Nella seconda parte di questo lavoro di tesi abbiamo invece identificato un nuovo gene bersaglio denominato Scotin, la cui espressione risulta essere regolata positivamente in vitro dallâisoforma TA, ma non da âN. Lâespressione di Scotin è localizzata nel reticolo endoplasmatico e nellâinvolucro nucleare. Questo gene è inoltre in grado di indurre apoptosi attraverso lâinduzione di stress del reticolo endoplasmatico. Esperimenti condotti su colture epiteliali primarie (murine ed umane) hanno evidenziato come lâespressione di Scotin durante il differenziamento sia accompagnata, come atteso, dallâincremento dellâisoforma TA e dalla dimuzione di âN. In conclusione i nostri dati hanno portato allâisolamento di Scotin come nuovo gene bersaglio indotto da p63 durante il differenziamento epidermico. Tale meccanismo attiva la risposta allo âstressâ da parte del reticolo endoplasmatico,di cui Scotin è un induttore, correlando cosĂŹ per la prima volta queste due differenti vie.SHORT ABSTRACT
Epidermal differentiation begins with the migration of keratinocytes from the basal layer and ends with the formation of the cornified layer. Cell proliferation, differentiation and death occur sequentially and each process is characterized by the expression of specific proteins. Transglutaminases (TGases) play an important role during the differentiation program. These enzymes catalyse post-translational modifications of proteins by the formation of isopeptide bonds (a cross-linking reaction). The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation. In mammals, nine distinct TGase isoenzymes have been identified at the genomic level. Four TGases are expressed in the epidermis (TGase 1, TGase 2, TGase 3 and TGase 5) and the last three TGases show also a GTPase activity. TGM1-/- knock-out mice show severe epidermal abnormalities and they dye soon after birth, while TGM2 -/- knock-out mice dontâshow any epidermal abnormality. During the complex mechanism that leads to epidermis formation an important role is played by the action of p63. This transcription factor, belonging to the p53 gene family, is transcribed from two different promoters, generating two classes of proteins: one containing an N-terminal transactivation domain (TA) and another that lacks this domain (âN). The first isoform is considered the transcriptional inducer whereas âN could exert a role of dominant negative. In addition alternative splicing at the 3â end of both transcripts (TA and âN) generates three different C-terminal splicing variants: Îą, β and Îł. Mice p63-/- show severe limb, cranofacial and skin defects and die soon after birth.
The aim of this work is to acquire new knowledges in the complex mechanism that leads to epidermal differentiation thanks to the investigation of TGase 3 and p63 function during the skin process formation. Regarding TGase 3 we plan to create conditional knock-out mice for this ezyme using the Cre/loxP system. During our work we isolated also two novel splicing variants for the TGM3 gene. The first one, isolated in mouse, lacks exons six and seven (â6â7), while the second one, isolated in NHEK cells, lacks of exons nine and ten (â9â10). The experiment demonstrated that the â9â10 is at least 4 times less active than the wild type one, while â6â7 is inactive because it lacks of the catalytic cysteine Regarding to the GTPase, it is lost in both splicing variants because several residues, that are important for the creation of the GTP pocket, are missed. In the second part of this work, we isolated a new p63-target gene: Scotin. This gene was previously isolated as a p53-inducible proapoptotic gene and the protein is located in the endoplasmic reticulum and in the nuclear membrane. Scotin expression is induced in response to endoplasmic reticulum (ER) stress in a p53 dependent or independent manner and this event lead to apoptosis. RT-qPCR showed that Scotin transcript is positively regulated by TAp63Îą in vitro, while âN isoform inhibits its expression. Western blot experiments, performed on differentiating mouse and human primary keratinocytes, revealed Scotin upregulation following the differentiation program. Immunofluorescence performed on mouse new born skin sections demontrated that Scotin expression is located in the suprabasal layer of epidermis where TAp63, but not âNp63 is expressed. In conclusion our data lead to isolation of a new p63 target gene induced during the epithelial differentiation program, a complex process that also involves endoplasmic reticulum stress induction