Presentation is loading. Please wait.

Presentation is loading. Please wait.

RESCUE OF RE1/NRSE PATHOLOGY IN HUNTINGTON DISEASE and REGULATION OF THE STEM CELL EPIGENOME BY REST Chiara Soldati, Angela Bithell, Noel Buckley.

Similar presentations


Presentation on theme: "RESCUE OF RE1/NRSE PATHOLOGY IN HUNTINGTON DISEASE and REGULATION OF THE STEM CELL EPIGENOME BY REST Chiara Soldati, Angela Bithell, Noel Buckley."— Presentation transcript:

1 RESCUE OF RE1/NRSE PATHOLOGY IN HUNTINGTON DISEASE and REGULATION OF THE STEM CELL EPIGENOME BY REST Chiara Soldati, Angela Bithell, Noel Buckley

2 RE1/NRSE REST/NRSF First identified as a regulator of neuronal genes in non-neuronal cells Act as repressor of gene transcription exerts its function by binding to RE1-sites REST (RE1 Silencing Transcription factor ) NSRF (Neuron restrictive silencing factor) RE1 position-specific scoring matrices (PSSM)(Johnson, 2006) binds to over 2400 regulatory sites REST mice die around E10

3 REST and Stem cell Rest is not required for the maintenance of stem cell pluripotency (Yamada) REST is a component of the pluripotency network that includes Oct4,Sox2, a Nanog, which together control differentiation and pluripotency in ESC. (Johnson and Bukley) REST ablation causes delayed repression of pluripotent genes expression (Oct4,Sox2, a Nanog) during early differetiation (Yamada) In ESC REST bind and regulate several genes linked to neuronal function (neurotransmitter receptor subunits, neuronal adhesion-associated molecules, synaptic vesicle biology) (Johnson and Buckley)

4 Epigenetics Modifications A different layer of trascriptional regulation Influence gene expression Includes histone modifications and DNA methylation Heterochromatin and Euchromatin Nucleosome Basic unit = 147pb wrapped around Histone octamer 2X H2A H2B H3 H4 Histone C-terminal domain, N-terminal tail Chromatin

5 Histone modification regulates accessibility to gene promoter regions Transcriptionally active chromatin Transcriptionally repressive chromatin Acetylation and Methylation regulate accessibility to promoter regions of genes The developmental programme of embryogenesis is controlled by both genetic and epigenetic Mechanisms Regulation of chromatin structures is crucial for genome reprogramming during early embryogenesis and for tissue-specific gene expression and global gene silencing.

6 REST as epigenetic regulator recruits various histone-modifying and chromatin-remodelling complexes RE1 Silencing Transcription Factor Maintains a Repressive Chromatin Environment in Embryonic Hippocampal Neural Stem Cells Greenway and Buckley) Low level of H3K9ac and H4ac BRG1 SMCX LSD1 co-REST msin3a HDAC1/2 RE1/NRSE REST/NRSF

7 AIM Investigate the role of REST in genome and epigenome regulation of ESC Investigate the role of REST in neural precursor generation and in neuronal differentiation

8 Exon 2 Flped allele loxP Exon 2 Cre loxP Exon 2 Cre/loxP-Mediated REST Inactivation Knockout allele FloRES (D4, D2, C8) recombination of DNA between loxP site FloREScre(C18,C23,C11,C28) Oct3/4 DAPI Sox2 DAPI Oct3/4 DAPI Sox2 DAPI

9 REST protein by western blot with 2 different antibody: Santa Cruz (internal) Upstate (C-terminal) REST D4,C8= floRES C18, C23, C28 =floREScre UpstateSanta cruz

10 REST GENE by PCR on DNA

11 REST and transcription in Stem Cell REST and epigenome in Stem Cell REST and neural precursors Generetion

12 REST Regulates Distinct Transcriptional Networks in Embryonic and Neural Stem Cells Rory Johnson Real Time PCR gene regulated and not regulated by REST FloRES D4 FloREScre C18 Celsr13 Golga7b Unc13a Chga Snap25 Nps4a Vrk3 REST

13 TRANSCRIPTOME ANALYSIS FloRES D4 VS FloREScre C18 Microarray measure changes in expression levels RNA sequencing All transcript mRNA (non-coding RNA, small RNAs) Trascriptional structure (5and 3 end, splicing, and other post trascriptional modification) relative level of expression Novel trascribed reagions

14 REST and transcription in Stem Cell REST and epigenome in Stem Cell REST and neural precursors Generetion

15 Epigenome investigation by Chromatin Immunoprecipitatin assay (ChIP) H3K9 acetylated H4 acetylated Investigate the structure of chromatin around RE1

16 FloRES D4 FloREScre C18 ChIP with H3K9ac an H4ac antibody Snap25 Golga7b Celsr3 Unc13a Npas4 Vrk3 Chga Ctrl REST KO

17 FloRES D4 FloREScre C18 REST IgG Snap25 Golga7b Celsr3 Unc13a Npas4 Vrk3 Chga ChIP with REST antibody REST can affect histons modification independently from gene transcription i.e. Vrk3 and Npas4: gene expresion is not regulated by REST (in STEM CELL) But H3K9 and H4 acetylation change around RE1 Genome wide analysis by ChIP-seq

18 REST and transcription in Stem Cell REST and epigenome in Stem Cell REST and neural precursors Generation

19 ES aggregatesRosettes NSC Nestin Sox1 LIF N2B27 EGF bFGF Niche-Independent Symmetrical Self-Renewal of a Mammalian Tissue Stem Cell Conti and Smith 2005 RG Nestin Vimentin RC2 BLBP EGF bFGF EGF bFGF (P0)(nP)

20 Nestin Ki67 FloRES (P0) FloRES (P7) 99% Nestin % Ki67+ -Radial Glia marker 60-70% Radial Glia like cells 100% SOX2 + Blbp Dapi Vimentin Pax6 Dapi RC2 olig2 Dapi Sox2 Dapi GFAP bIIItub Dapi

21 Nestin Ki67 FloREScre (P0) Blbp Dapi Vimentin Pax6 Dapi RC2 olig2 Dapi GFAP bIIItub Dapi GFAP bIIItub Dapi GFAP bIIItub Dapi Sox2 Dapi FloREScre (P1) 99% Nestin % Ki67+ REST KO affect NSC maintaining By accelerate the switch between Neuroepithelial like to Radial Glia like cells By increase spontaneous differentiation

22 To summarize REST is a epigenetic regulator in Stem Cell It doesn't affect neural precursors generation It affect neural precursors maintaining

23 Work in progress Epigenome Analyze ChIP seq data HDAC1/2 ChIP and His tone deacetylase inhibitor Transcriptome Analyze Microarray and RNA seq data Differentiation Expression of pluripotency and differentional marker from ESC to RGC (Oct3/4, Nanog, Sox2, Gata4, Nestin, Sox1, Pax6, Vimentin, bIIItubulin) Cloning FloRES and FloREScre NSC to get a population of RGC to investigate late differentiation

24 Thanks to… Noel J Buckley Angela Bithell Cass Johnston Matthew Burney Alessandro Michelucci Genome Institute Singapore Kee-Yew Wong (microarray)


Download ppt "RESCUE OF RE1/NRSE PATHOLOGY IN HUNTINGTON DISEASE and REGULATION OF THE STEM CELL EPIGENOME BY REST Chiara Soldati, Angela Bithell, Noel Buckley."

Similar presentations


Ads by Google