Sox2 encodes a transcription factor required for embryonic stem cell pluripotency. Heterozygous Sox2 mutations in humans cause defects in the development of eyes (anophthalmia, microphthalmia) and hippocampus, with neurological pathology including epilepsy, motor control problems and learning disabilities. Using a Sox2 conditional knock-out in mouse, we discovered that Sox2 is important for brain development and for neural stem cell maintenance. Recently, it was found that transcriptional regulatory elements are not always localized in proximity of the gene they control, but often they lie very far from it on the linear chromosome map. Mutations in these elements can cause pathology, due to the deregulation of the associated gene. In collaboration with Dr. C.-L. Wei’s lab (California), we compared long-range DNA interactions in chromatin of wild-type mouse neural stem/precursor cells (NPCs) and Sox2-deleted cells, using the ChIA-PET technique: out of a total of 7000 long-range interactions mapped in wild-type NPCs, 2700 were lost in Sox2-deleted cells. Many of the lost interactions involved genes important for neural development and sequences already identified as forebrain enhancers by p300 binding in mouse developing telencephalon. In parallel, we determined the genome-wide map of SOX2 binding sites in chromatin of wild-type NPCs, by ChIP-seq (in collaboration with Dr. F. Guillemot; London). At least half of the SOX2-dependent long-range interactions contain a SOX2 ChIP-seq peak, suggesting that SOX2 has a direct role in their maintenance. My project seeks to define if distal sequences, associated in a SOX2-dependent way to neural genes (candidates to be putative SOX2 targets), represent transcriptional regulatory elements active during embryonic brain development and if their activity is regulated by SOX2. We selected 13 putative distal regulatory elements (DREs), among the ChIA-PET interactions lost in Sox2-deleted cells, to functionally characterize them in transgenic experiments in zebrafish. I did the transgenesis experiments in Dr. P. Bovolenta’s lab in Madrid, supported by an EMBO short-term fellowship. We cloned the 13 DREs upstream of a minimal promoter and a GFP gene (in a “ZED” plasmid). The plasmid is injected in 1-cell stage embryos and the DNA is integrated into the fish genome. After injection, the embryos are observed during development to analyze if, and where, the tested sequences drive GFP expression. I found that 12 out of 13 DREs give rise to reproducible GFP expression in the developing forebrain and/or in more posterior neural regions, matching the expression pattern of the associated gene. This indicates that the selected DREs alone are able to guide reporter gene expression. I collected the transient GFP+ embryos (F0) of 8 DREs to obtain F1 stable transgenic lines. To test if the enhancer activity of DREs is regulated by SOX2, I used a loss of function experiment. I injected a morpholino antisense oligonucleotide, specifically directed against the Sox2 mRNA, in F2 zebrafish embryos at 1-cell stage. Two, out of 8, stable lines showed a reduced GFP expression specifically in forebrain in early developmental stages. We have also cloned some of the selected DREs in a luciferase vector to test them by transfection in cultured cells. One of the DREs showed a significant increase in luciferase activity if co-transfected with Sox2 and Mash1 expressing vectors, suggesting a regulatory mechanism operated by SOX2 on this element in presence of the cofactor MASH1. We can conclude that some of the tested DREs, involved in ChIA-PET interactions lost in Sox2-deleted cells, work as regulatory elements in in vivo experiments and are directly regulated by SOX2.

Sox2 codifica per un fattore trascrizionale necessario per la pluripotenza delle cellule staminali embrionali. Mutazioni eterozigoti in Sox2 nell’uomo causano difetti nello sviluppo dell’occhio (anoftalmia, microftalmia) e dell’ippocampo, con insorgenza di patologie come epilessia, problemi nel controllo motorio e difetti di apprendimento. Tramite “knock-out” condizionale di Sox2 in topo, abbiamo osservato l’importanza di Sox2 per lo sviluppo del cervello e per il “self-renewal” delle staminali neurali. Di recente è emerso che elementi regolatori possono trovarsi molto lontano dai geni che controllano lungo il cromosoma. Mutazioni in tali elementi talvolta causano patologie dovute a deregolazione del gene associato. In collaborazione con la Dr. C.-L. Wei (California), abbiamo comparato le interazioni “long-range” nella cromatina di cellule di precursori neurali (NPCs) di topi “wild-type” (wt) e Sox2-deleti, usando la tecnica ChIA-PET: su 7000 interazioni mappate in NPCs wt, 2700 erano perse in NPCs Sox2-delete. Tra queste 2700 interazioni, molte coinvolgevano geni legati allo sviluppo neurale e sequenze identificate come enhancer telencefalici per la presenza di siti di legame per p300. Abbiamo poi determinato la mappa genomica dei siti di legame per SOX2 nella cromatina di NPCs wt (in collaborazione con il Dr. F. Guillemot; Londra). Circa metà delle interazioni “long-range” SOX2-dipendenti presentava un picco di ChIP-seq per SOX2, suggerendo un ruolo diretto di SOX2 nel loro mantenimento. Il mio progetto di tesi intende definire se sequenze distali, associate in modo SOX2-dipendente a geni neurali (candidati “target” per SOX2), sono elementi di regolazione trascrizionale attivi durante lo sviluppo embrionale del cervello e se la loro attività è regolata da SOX2. Abbiamo selezionato 13 putativi elementi regolatori distali (DREs), tra le interazioni ChIA-PET perse nelle NPCs Sox2-delete, per caratterizzarli funzionalmente in esperimenti di transgenesi in zebrafish. Ho condotto questi esperimenti in vivo nel laboratorio della Dr. P. Bovolenta a Madrid, supportata da una “EMBO short-term fellowship”. Abbiamo clonato le 13 DREs in un plasmide (ZED), a monte di un promotore minimo e del gene GFP. Il plasmide è stato iniettato in embrioni allo stadio di 1 cellula e il DNA si è integrato nel genoma di pesce. Gli embrioni sono stati osservati durante lo sviluppo per analizzare se, e dove, la sequenza testata guidava l’espressione di GFP. La GFP era espressa riproducibilmente in 12 DREs su 13 nel cervello in via di sviluppo e/o in regioni neurali più posteriori, sovrapponendosi al pattern di espressione del gene associato. Ciò indica che i DREs da soli guidano l’espressione del gene reporter. Ho quindi selezionato embrioni GFP+ transienti (F0) di 8 DREs per ottenere linee transgeniche stabili F1. Per testare se l’attività enhancer dei DREs è regolata da SOX2, ho usato un approccio “perdita di funzione”. Ho iniettato un oligonucleotide antisenso (morfolino), specificamente diretto contro l’mRNA di Sox2, in embrioni F2 di zebrafish allo stadio di 1 cellula. In 2 linee stabili su 8, l’espressione telencefalo-specifica di GFP era ridotta a precoci stadi di sviluppo. Abbiamo anche clonato alcuni DREs in vettori luciferasi per esperimenti di transfezione in colture cellulari. Uno dei DREs mostrava un aumento di attività luciferasica in cotransfezione con vettori di espressione per Sox2 e Mash1, suggerendo un meccanismo di regolazione in cui SOX2 opera insieme al cofattore MASH1. Possiamo concludere che alcuni DREs testati, selezionati tra le interazioni “long-range” di ChIA-PET perse nelle NPCs Sox2-delete, agiscono come elementi regolatori in esperimenti in vivo e sono direttamente regolati da SOX2.

(2015). Functional characterization of regulatory sequences targeted by the transcription factor SOX2, identified by studies of long-range chromatin interactions in brain-derived neural stem/precursor cells. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).

Functional characterization of regulatory sequences targeted by the transcription factor SOX2, identified by studies of long-range chromatin interactions in brain-derived neural stem/precursor cells

BERTOLINI, JESSICA ARMIDA
2015

Abstract

Sox2 encodes a transcription factor required for embryonic stem cell pluripotency. Heterozygous Sox2 mutations in humans cause defects in the development of eyes (anophthalmia, microphthalmia) and hippocampus, with neurological pathology including epilepsy, motor control problems and learning disabilities. Using a Sox2 conditional knock-out in mouse, we discovered that Sox2 is important for brain development and for neural stem cell maintenance. Recently, it was found that transcriptional regulatory elements are not always localized in proximity of the gene they control, but often they lie very far from it on the linear chromosome map. Mutations in these elements can cause pathology, due to the deregulation of the associated gene. In collaboration with Dr. C.-L. Wei’s lab (California), we compared long-range DNA interactions in chromatin of wild-type mouse neural stem/precursor cells (NPCs) and Sox2-deleted cells, using the ChIA-PET technique: out of a total of 7000 long-range interactions mapped in wild-type NPCs, 2700 were lost in Sox2-deleted cells. Many of the lost interactions involved genes important for neural development and sequences already identified as forebrain enhancers by p300 binding in mouse developing telencephalon. In parallel, we determined the genome-wide map of SOX2 binding sites in chromatin of wild-type NPCs, by ChIP-seq (in collaboration with Dr. F. Guillemot; London). At least half of the SOX2-dependent long-range interactions contain a SOX2 ChIP-seq peak, suggesting that SOX2 has a direct role in their maintenance. My project seeks to define if distal sequences, associated in a SOX2-dependent way to neural genes (candidates to be putative SOX2 targets), represent transcriptional regulatory elements active during embryonic brain development and if their activity is regulated by SOX2. We selected 13 putative distal regulatory elements (DREs), among the ChIA-PET interactions lost in Sox2-deleted cells, to functionally characterize them in transgenic experiments in zebrafish. I did the transgenesis experiments in Dr. P. Bovolenta’s lab in Madrid, supported by an EMBO short-term fellowship. We cloned the 13 DREs upstream of a minimal promoter and a GFP gene (in a “ZED” plasmid). The plasmid is injected in 1-cell stage embryos and the DNA is integrated into the fish genome. After injection, the embryos are observed during development to analyze if, and where, the tested sequences drive GFP expression. I found that 12 out of 13 DREs give rise to reproducible GFP expression in the developing forebrain and/or in more posterior neural regions, matching the expression pattern of the associated gene. This indicates that the selected DREs alone are able to guide reporter gene expression. I collected the transient GFP+ embryos (F0) of 8 DREs to obtain F1 stable transgenic lines. To test if the enhancer activity of DREs is regulated by SOX2, I used a loss of function experiment. I injected a morpholino antisense oligonucleotide, specifically directed against the Sox2 mRNA, in F2 zebrafish embryos at 1-cell stage. Two, out of 8, stable lines showed a reduced GFP expression specifically in forebrain in early developmental stages. We have also cloned some of the selected DREs in a luciferase vector to test them by transfection in cultured cells. One of the DREs showed a significant increase in luciferase activity if co-transfected with Sox2 and Mash1 expressing vectors, suggesting a regulatory mechanism operated by SOX2 on this element in presence of the cofactor MASH1. We can conclude that some of the tested DREs, involved in ChIA-PET interactions lost in Sox2-deleted cells, work as regulatory elements in in vivo experiments and are directly regulated by SOX2.
NICOLIS, SILVIA KIRSTEN
FAVARO, REBECCA
Sox2; distal regulatory elements; zebrafish
Sox2; elementi regolatori distali; zebrafish
BIO/18 - GENETICA
English
25-giu-2015
Scuola di Dottorato in Medicina Traslazionale e Molecolare
SCUOLA DI DOTTORATO IN MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 72R
27
2013/2014
open
(2015). Functional characterization of regulatory sequences targeted by the transcription factor SOX2, identified by studies of long-range chromatin interactions in brain-derived neural stem/precursor cells. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/83922
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