L1 - Epigenetics and disease

• What is epigenetics?

  The study of heritable changes in gene expression that do not involve alterations in the DNA sequence
• What is a key feature of stem cells?

  Pluripotent - can self renew and differentiate into any type of cell
• What is the development of a stem cell?

  Stem cell

  Progenitor cell

  Precursor cell

  Effector cell
• How do cells go from a stem cell to a progenitor cell?

  Turn off stem cell gene expression and turn on progenitor gene expression
• How do cells go from a progenitor cell to a precursor and then effector cell?

  Turn on precursor gene expression and keep progenitor gene expression on

  Then turn off progenitor gene expression leaving only the precursor gene expression on
• What is one mediator of epigenetic control?

  Chromatin - complex of DNA and proteins that forms chromosomes
• What is the structure of a nucleosome?

  Octomer - DNA is wrapped around 4 pairs of histones (H2A, H2B, H3 and H4)

  H1 is a linker histone which acts to bind linker DNA between nucleosomes
• How many base pairs of DNA can wrap around a single nucleosome?

  147
• What are some things that regulate chromatin?

  DNA modifications

  Histone modifications

  Nucleosome remodelling

  Chromatin looping

  Non-coding RNAs
• What is Euchromatin?

  Open chromatin - DNA is more loosely associated with the chromatin and nucleosomes are more spread apart

  This occurs for regions of the genome that the cell knows is needed and will have genes turned on and off
• What is heterochromatin?

  Closed chromatin - DNA is more tightly compacted around the chromatin and nucleosomes are close together

  This is for regions of the genome that is not usually needed (eg centromeres and telomeres)
• Can modifications be treated in isolation?

  No - one modification will effect modifications of other components
• Can you go between hetero- and euchromatin?

  Yes
• What is the major DNA modification in mammals?

  5 methyl cytosine (5mC)

  Essential for mammalian development

  Repressive to transcription

  Generally occurs in a CpG context
• What happens if 5mC is knocked out?

  No development occurs in mammals
• What are CpGs?

  Cytosine residue is next to a Guanine residue - p is the phosphate connector
• What are CpG islands?

  Used as regulators - less likely to be methylated (more resistant)
• What does methylation of CpG islands induce?

  Gene silencing - CpG islands that coincide with promoters of certain genes are methylated
• What can DNA modification induce/effect?

  X inactivation

  Genomic imprinting

  Genomic stability

  Repetitive element repression
• What enzymes are methyl groups added by?

  DNA methyl transferases - DNMT3A/B or DNMT1
• What is the difference between DNMT3A/B and DNMT1?

  DNMT3A/B - De novo transferases

  Can recognise a stretch of DNA or CpGs and will go and methylate it

  DNMT1 - maintenance methyltransferase

  Recognises if there is methylation on one allele but not the other (hemimethylated DNA) - will act to match up methylation to make pairs therefore methylation is heritable
• How does active demethylation of DNA occur?

  Via the use of TET enzymes (dioxygenase enzymes) for iterative oxidation and/or base excision repair
• What is iterative oxidation?

  TET enzymes oxidate 5mC to 5hmC (5 hydroxy methyl C)

  Then oxidates 5hmC to 5fC (5 formyl C) and then 5fC to 5caC (5 carboxy C) - goes back to just C via replication after becoming 5hmC
• What is base excision repair?

  Once formed, 5fC and 5caC can be recognised by DNA repair machinery - TDG enzymes

  These remove the methylated cytosine and replace it with an unmethylated one
• What is passive demethylation?

  Absence of DNMT1 and successive rounds of methylation

  Gradually lose the methylation as the cell goes through rounds of the cell cycle since it would be hemimethylated DNA
• What are the features of histone tails?

  Extend out from the core nucleosome

  Are subject to post-translational modification (acetylation, methylation, phosphorylation and ubiquitination)
• What post-translational modifications can lysine undergo?

  Acetylation or methylation - mutually exclusive
• How many methyl states can Lysine or arginine have?

  3 - each have different functions

  monomethyl, dimethyl or trimethyl
• How do we asses histone modifications?

  Via chromatin immunoprecipitation (ChIP)

  Cross link the cells - fixes the DNA and proteins together

  Chop genome into chromatin fragments via sonication or enzymes - DNA and proteins are still bound

  Use specific antibodies that are against histone modifications to bind to the chromatin fragments

  This will separate fragments that have that specific modification from those that don't - can then assess and analyse the levels of each
• What is the TSS and TES?

  TSS - Transcriptional start site

  TES - Transcriptional end site
• What will different marks from ChIP analysis show?

  Different marks will correlate with different gene activity states

  Still correlation not causation - doesn't show that the modifications have induced the active states
• How does histone acetylation affect chromatin structure?

  It neutralizes the positive charge on the lysine to open the chromatin structure

  Unmodified lysine is electrostatically attracted to the nucleosome and DNA's negative charge

  Acetylation of the lysine neutralizes the charge and reduces the electrostatic attraction - opens up the chromatin and makes it more accessible
• What do the acetylated regions on histones act as?

  Acetylated groups/lysine residues will act as a recruitment site for proteins containing the modifications - eg bromo domains will recognise and bind to the acetylated groups
• What do histones act as?

  Reader proteins

  They recognise specific modifications and mediate downstream processes

  Are often linked to human disease
• What are the 4 ways a nucleosome can be remodelled?

  Sliding - move the DNA strand along to expose the specific region required

  Ejection - eject the nucleosomes completely to gain access to the DNA strand

  H2A-H2B dimer ejection - removal of partial nucleosomes

  H2A-H2B dimer replacement - replace some nucleosomes with variants
• What is the remodel of nucleosomes?

  The movement of nucleosomes in relation to DNA and sequences within that DNA that are required - these regions bind to activators
• What activity is needed before activators can bind to regions of DNA around nucleosomes?

  Activators cannot bind to DNA wrapped closely around nucleosomes

  Need the activity of a chromatin remodeller - moves the nucleosome and exposes the DNA sequence
• What can bind to DNA whether it is wrapped closely around a nucleosome or not?

  Pioneer factors - very few

  Can bind anywhere
• What are the roles of ATP dependent chromatin remodelers?

  Changes in nucleosome positioning

  Facilitate exchange of nucleosomes

  Incorporation of histone variants
• What do remodellers generate at the 5' end of genes?

  A stereotypic nucleosome organization required for transcription

  ISW1a/Chd1 - regulate spacing between nucleosomes

  Nucleosome free region - responsible for nucleosome disassembly