CSB329H1 Lecture Notes - Lecture 8: Fibroblast, Sox2, Neomycin
9 May 2018
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Gradient Represents Methylation
Lecture 8(b): Human ESCs & Induced Pluripotency
Questions–
1) What would have been the impact on the identification of pluripotency-inducing transcription
factors by Yamanaka’s laboratory IF they have accidently used an adult somatic cell line that
expresses the Fbx15 locus?
o Somatic cells will also be neomycin resistant – therefore, will be unable to distinguish
somatic cells from iPSCs
2) Why were the first somatic cell nuclear transplantation into enucleated oocytes performed
using the African clawed frog Xenopus Laevis?
o (1) accessible (2) large & high production of eggs when inducing ovulation
§ Can also be done in zebra fish due to advancement in technology
ð Different combinations can be used to induce pluripotency:
o (1) Oct4 (2) Sox2 (3) Nanog (4) Lin28
§ Lin28 is an RNA binding protein; function to increase translation
Epigenetic Modulators:
• Reprogramming somatic cells into iPSCs require DNA
methyl transferases:
a. Methylation of H3K4 markers increase =>
increase H3K4me2 & H3K4me3
§ Function to activate euchromatic
regions of pluripotent genes
b. Methylation of H3K27 markers decrease => decrease H3K27me3
§ Function to remove silencing of pluripotent gene regions
ð Modifiers are small molecules that are added into culture to augment the pluripotent state:
o AZA: promotes de-hypermethylation of pluripotency genes (removing H3K27me3)
Activating Transcription:
• Transcription factors are recruited to the enhancer and function to activate
transcription: (1) Oct4 (2) Sox2 (3) NANOG (4) Klf4 (5) cMyc
o … but enhancers for pluripotency genes are found in
heterochromatic regions in fibroblast cells (somatic cells)
• Pioneer Transcription Factors:
o (1) Oct4 (2) Sox2 (3) Klf4 (4) cMyc are capable to scan
heterochromatin and identify their target sequence – therefore, are capable of ‘reading’
the nucleosome structure of the DNA complex and bind to them EVEN if they are
found in a heterochromatic state
§ Will function to help promote factors require to opening the heterochromatic
region to allow endogenous expression of these factors
Document Summary
Can also be done in zebra fish due to advancement in technology. Different combinations can be used to induce pluripotency: (1) oct4 (2) sox2 (3) nanog (4) lin28. Lin28 is an rna binding protein; function to increase translation. Epigenetic modulators: reprogramming somatic cells into ipscs require dna methyl transferases, methylation of h3k4 markers increase => increase h3k4me2 & h3k4me3. Function to activate euchromatic regions of pluripotent genes. Gradient represents methylation: methylation of h3k27 markers decrease => decrease h3k27me3. Function to remove silencing of pluripotent gene regions. Modifiers are small molecules that are added into culture to augment the pluripotent state: aza: promotes de-hypermethylation of pluripotency genes (removing h3k27me3) Pioneer transcription factor properties: interact with their recognition sequences in heterochromatin prior to. Settler tfs can only bind to accessible copies of their dna target sites. Opening the heterochromatic region will allow the binding of other transcription factors.
