The Sox2 gene encodes a transcription factor active in stem/progenitor cells during the development of central nervous system in vertebrates. Heterozygous Sox2 mutations in humans cause a characteristic spectrum of CNS abnormalities, involving the hippocampus and the eye, and causing epilepsy, learning disabilities and defective motor control. In order to understand the role of Sox2 in neural development, our laboratory generated Sox2 conditional KO mutations in mouse. The consequences of Sox2 ablation at different developmental time points produced important brain defects, more serious when the ablation was early. Sox2 conditional KO allowed to observe an important function for Sox2 also in the maintenance of NSC self-renewal in long-term in vitro NSC cultures. Sox2-mut NSC, cultured as neurospheres from P0 mouse forebrain, self-renewed for several passages in culture, but then underwent a decrease in growth, with progressive culture exhaustion. Sphere formation could be rescued by lentiviral Sox2. This reveled an essential role for Sox2 in the development of multiple CNS regions and in the maintenance of NSC. To understand the mechanisms of Sox2 function, a central question is which genes Sox2 regulates as a transcription factor, by what mechanisms Sox2 acts in regulating them, and which Sox2-regulated genes are critical mediators of its function. A new way in which Sox2 regulates its targets has been recently observed in our laboratory: Sox2 maintains a high number of long-range interactions between genes and distal enhancers, that regulate gene expression. We determined, by genome-wide chromatin interaction analysis (RNApolII ChIA-PET) the global pattern of long-range chromatin interactions in normal and Sox2-mut mouse NSC. Sox2 deletion caused extensive loss of long-range interactions and reduced expression of a subset of genes associated with Sox2-dependent interactions. Expression of one of these genes, Socs3, rescued the self-renewal defect of Sox2-mut NSC. Our work identifies Sox2 as a major regulator of functional chromatin connectivity in NSC, and demonstrates the role of genes associated with Sox2-dependent interactions in NSC maintenance and, potentially, in neurodevelopmental disorders. We studied the differentiation of Sox2-mut cells into neurons and glia, as compared to controls: at advanced stage, very few β-tub-positive cells were observed in Sox2-mut cells differentiated, with poor differentiated morphology. This result showed the importance of Sox2 in the development into mature neurons. We also analyzed the changes in gene expression resulting from Sox2 deletion by RNA-seq analysis of three samples for both wt and Sox2-mut cells in undifferentiated cells, and two differentiation conditions (day 4 and day 11). Hundreds of genes were deregulated in mutant cells. The most down-regulated gene was Socs3, so we transduced Sox2-mut cells with a lentiviral Socs3–vector, coexpressing GFP. Socs-3 transduced mut cells initially grew as the untransduced cells (only a proportion of the cells had been tranduced), but continued to grow even after the untransduced mut cells were completely exhausted, and transduced cells were positively selected. These results suggested that Socs3 partially rescued the proliferation defect of mut cells. I also tested if the reintroduction of Socs3 could rescue the neuronal differentiation defect of mut cells and my initial experiments suggest that this might be the case: Socs3-transduced cells were all GFAP-negative, and they all appeared β-tub-positive, though they seemed to have a suffering morphology. I aimed to test the role of some of the other most deregulated genes as mediators of Sox2 function in self-renewal and differentiation, by rescuing experiments of mut cells. I aim to test if Sox2 reintroduction in mut cells could rescue the long-range interactions of a small number of identified target genes, lost in Sox2-mut cells, by 3C experiments.

Il gene Sox2 codifica per un fattore di trascrizione attivo nelle cellule staminali durante lo sviluppo del SNC nei vertebrati. Mutazioni eterozigoti di Sox2 nell'uomo causano uno spettro caratteristico di anomalie del SNC, che coinvolgono l'ippocampo e l'occhio, e che causano epilessia, disabilità di apprendimento e difettivo controllo motorio. Per comprendere il ruolo di Sox2 nello sviluppo neuronale, il nostro laboratorio ha generato KO condizionali di Sox2 nel topo. Le conseguenze della delezione di Sox2 in diversi momenti dello sviluppo producono importanti difetti cerebrali. Il KO condizionale permette di osservare una funzione importante di Sox2 anche nel mantenimento del self-renewal e delle colture a lungo termine di NSC in vitro. Sox2-mut NSC, coltivate come neurosfere, derivate dal prosencefalo di topi P0, si auto-rinnovano per diversi passaggi in coltura, ma poi vanno incontro a esaurimento della coltura. La formazione delle sfera viene recuperata da lentivirus Sox2. Questo rivela un ruolo essenziale per Sox2 nel mantenimento delle NSC. Per comprendere i meccanismi delle funzioni di Sox2, una questione centrale è quali geni Sox2 regola come un fattore di trascrizione, con quali meccanismi, e quali geni Sox2-regolati sono mediatori critici della sua funzione. Un nuovo modo in cui Sox2 regola i suoi targets è stato recentemente osservato nel nostro laboratorio: Sox2 mantiene un elevato numero di interazioni a lungo raggio tra geni ed enhancer distali, che regolano l'espressione genica. Abbiamo determinato mediante Chia-PET l’intero pattern di interazioni a lungo raggio in NSC wt e Sox2-mut. La delezione di Sox2 causa una vasta perdita di interazioni a lungo raggio e ridotta espressione di un sottogruppo di geni associati. L'espressione di uno di questi geni, SOCS3, recupera il difetto di self-renewal delle cellule mut. Il nostro lavoro identifica Sox2 come un importante regolatore della connettività funzionale cromatinica nelle NSC e dimostra il ruolo di geni associati con interazioni Sox2-dipendenti nel mantenimento delle NSC e, potenzialmente, in disturbi dello sviluppo neurologico. Abbiamo studiato il differenziamento delle cellule Sox2-mut in neuroni e glia, rispetto ai controlli: in stadio avanzato, poche cellule β-tub-positive sono state osservate nei mut differenziati, con scarsa morfologia differenziata. Questo risultato ha mostrato l'importanza di Sox2 nello sviluppo in neuroni maturi. Abbiamo anche analizzato i cambiamenti nell'espressione genica derivati dalla delezione di Sox2 mediante analisi RNA-seq di tre campioni per entrambe le cellule wt e Sox2-mut indifferenziate, e in due condizioni di differenziamento (giorno 4 e il giorno 11). Centinaia di geni sono deregolati in cellule mutanti. Il gene più down-regolato è SOCS3, quindi abbiamo trasdotto le cellule Sox2-mut con un lentivirus SOCS3. Le cellule mut trasdotte inizialmente crescono come le cellule non trasdotte (solo una parte delle cellule era stata trasdotta), ma continuano a crescere anche dopo che le cellule mut non trasdotte si sono completamente esaurite.Questi risultati suggeriscono che SOCS3 recupera parzialmente il difetto di proliferazione delle cellule mut. Ho anche provato se la reintroduzione di SOCS3 potrebbe recuperare il difetto nel differenziamento neuronale delle cellule mut e i miei esperimenti iniziali suggeriscono che potrebbe essere così: le cellule SOCS3-trasdotte erano tutte GFAP-negative e sembravano β-tub-positive, anche se sembravano avere una morfologia sofferente. Altro scopo è verificare il ruolo di alcuni degli altri geni più deregolati come mediatori della funzione di Sox2 nel self-renewal e nel differenziamento, con esperimenti di rescuing. Infine mi propongo di verificare se la reintroduzione di Sox2 nelle cellule mut potrebbe ripristinare le interazioni a lungo raggio, perse nei mutanti, di un piccolo numero di geni bersaglio identificati, con esperimenti di 3C.

(2017). Identification and functional characterization of Sox2-target genes involved in brain disease and abnormal brain development. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).

Identification and functional characterization of Sox2-target genes involved in brain disease and abnormal brain development

PAGIN, MIRIAM
2017

Abstract

The Sox2 gene encodes a transcription factor active in stem/progenitor cells during the development of central nervous system in vertebrates. Heterozygous Sox2 mutations in humans cause a characteristic spectrum of CNS abnormalities, involving the hippocampus and the eye, and causing epilepsy, learning disabilities and defective motor control. In order to understand the role of Sox2 in neural development, our laboratory generated Sox2 conditional KO mutations in mouse. The consequences of Sox2 ablation at different developmental time points produced important brain defects, more serious when the ablation was early. Sox2 conditional KO allowed to observe an important function for Sox2 also in the maintenance of NSC self-renewal in long-term in vitro NSC cultures. Sox2-mut NSC, cultured as neurospheres from P0 mouse forebrain, self-renewed for several passages in culture, but then underwent a decrease in growth, with progressive culture exhaustion. Sphere formation could be rescued by lentiviral Sox2. This reveled an essential role for Sox2 in the development of multiple CNS regions and in the maintenance of NSC. To understand the mechanisms of Sox2 function, a central question is which genes Sox2 regulates as a transcription factor, by what mechanisms Sox2 acts in regulating them, and which Sox2-regulated genes are critical mediators of its function. A new way in which Sox2 regulates its targets has been recently observed in our laboratory: Sox2 maintains a high number of long-range interactions between genes and distal enhancers, that regulate gene expression. We determined, by genome-wide chromatin interaction analysis (RNApolII ChIA-PET) the global pattern of long-range chromatin interactions in normal and Sox2-mut mouse NSC. Sox2 deletion caused extensive loss of long-range interactions and reduced expression of a subset of genes associated with Sox2-dependent interactions. Expression of one of these genes, Socs3, rescued the self-renewal defect of Sox2-mut NSC. Our work identifies Sox2 as a major regulator of functional chromatin connectivity in NSC, and demonstrates the role of genes associated with Sox2-dependent interactions in NSC maintenance and, potentially, in neurodevelopmental disorders. We studied the differentiation of Sox2-mut cells into neurons and glia, as compared to controls: at advanced stage, very few β-tub-positive cells were observed in Sox2-mut cells differentiated, with poor differentiated morphology. This result showed the importance of Sox2 in the development into mature neurons. We also analyzed the changes in gene expression resulting from Sox2 deletion by RNA-seq analysis of three samples for both wt and Sox2-mut cells in undifferentiated cells, and two differentiation conditions (day 4 and day 11). Hundreds of genes were deregulated in mutant cells. The most down-regulated gene was Socs3, so we transduced Sox2-mut cells with a lentiviral Socs3–vector, coexpressing GFP. Socs-3 transduced mut cells initially grew as the untransduced cells (only a proportion of the cells had been tranduced), but continued to grow even after the untransduced mut cells were completely exhausted, and transduced cells were positively selected. These results suggested that Socs3 partially rescued the proliferation defect of mut cells. I also tested if the reintroduction of Socs3 could rescue the neuronal differentiation defect of mut cells and my initial experiments suggest that this might be the case: Socs3-transduced cells were all GFAP-negative, and they all appeared β-tub-positive, though they seemed to have a suffering morphology. I aimed to test the role of some of the other most deregulated genes as mediators of Sox2 function in self-renewal and differentiation, by rescuing experiments of mut cells. I aim to test if Sox2 reintroduction in mut cells could rescue the long-range interactions of a small number of identified target genes, lost in Sox2-mut cells, by 3C experiments.
NICOLIS, SILVIA KIRSTEN
Sox2,; Chia-PET,; neurons,; glia,; socs3
Sox2,; Chia-PET,; neurons,; glia,; socs3
MED/04 - PATOLOGIA GENERALE
English
27-giu-2017
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET - 76R
29
2015/2016
open
(2017). Identification and functional characterization of Sox2-target genes involved in brain disease and abnormal brain development. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/170795
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