Epigenetics and Chromatin Flashcards
Epigenetics def
modification of gene expression without changing the actual DNA sequence
3 mechanisms of epigenetics
- DNA methylation
- histone modification
- non coding RNAs
Mechanism of post replicative methylation
-catalyzed by DNA methyltransferase
-CG methylation occurring in cytosine nucleotides followed by a guanine nucleotide
-Addition of methyl group CH3 on carbon 5 of cytosine with SAM acting as the donor of the CH3 group
Reason for the lower than expected numbers of methylated CG dinucleotides find in genome
- spontaneous UNMETHYLATED cytosine deamination in DNA changes C into U –> since Uracil isnt present in DNA it is flagged and removed (changed back into C)
- spontaneous METHYLATED cytosine deamination in DNA changes C into T –> this is not flagged as an error and so the C base is NOT replaced, causing the CpG methylation to disappear
CpG islands definition and purpose
CpG islands: regions of genome that are enriched with CG dinucleotide sequences
!!! NOT METHYLATED: which is the reason why they are maintained in such high amounts because there are no spontaneous methylated cytosine deamination (hence CG is not erased from sequence)
DNA methylation levels regulation during development of zygote
- germ cells: sperm cells are hypermethylated and oocytes are hypomethylated
- loss of methylation during the zygotic phase: maternal DNA gradual reduction and paternal DNA sudden drop
- increase in methylation post zygote until embryonic phase
are there differences in in vitro and in vivo epigenetics?
yes, and this causes small percentage increase in imprinted gene disorders in vitro (IVF)
gene imprinting definition
only one copy of a gene is expressed (either maternal or paternal) and the other copy is supressed
Types of enzymes involved in methylation
3 DNA methyl transferases:
-1 maintenance DNMT1: found in high concentrations in dividing cells and cancerous growths
-2 de novo DNMT3a and DNMT3b
Function of maintenance DNMT1
methylate hemiethylated sites generated during DNA replication, where one strand is methylated and the other is unmethylated
Function of de novo DNMT3a/b
methylate during embryonic development
result of knock out organism creation to investigate DNMT1/3A/3B
DNMT1 knockout: did not survive past midgestation (around 20 weeks)
DNMT3a knockout: survived birth but died 4 weeks post birth
DNMT3b knockout: none were able to be born
RESULT: DNMT1 defect causes the most severe consequences
Demethylation process
ENZYMES: TET1/2/3
no enzyme alone is able to remove the CH3 methyl group, hence the DNA is slowly degraded until it is flagged as damage and is then degraded by damage control of cell
Roles of methylation (2)
- inhibits gene expression
- maintenance of genome integrity via inactivation of parasitic DNA
2 methylation models proposed and the accuracy of each
- DIRECT MODEL:
-methylated DNA blocks the groove of DNA that TFs bind to, hence causing inactivation of the previously active gene
EVALUATION: not accurate. Even in TF binding sites that are unmethylated (bcos there are low numbers of CG dinucleotides) the TF still cannot bind and the gene is still inactive - INDIRECT MODEL: methylation causes MBD (methyl binding domains) proteins to bind to methyl groups instead of CH3 groups, and it is these proteins that cause the steric hindrance blocking TF binding
EVALUATION: accurate model
MeCP2 gene structure and function
-MBP protein that generates compact chromatin structure
-contains a DNA binding domain and a TRD (transcription repression domain) - hence is classified as a TF
-lack of MeCP2 causes Rett syndrome
!! doesn’t bind to a specific consensus sequence, only is able to recognise methylated DNA
Pathologies stemming from defective methylation
- DNA damage caused by mutations of methylated bases
- Aberrant silencing: hypomethylation causing lower gene suppression which causes mainly neurological disorders like Rett
- Cancerous growths: hypomethylation of sequences that need to be suppressed for genetic stability, and hypermethylation of tumour suppressing genes
- diseases associated with imprinted genes
Size of the nucleus
10 micrometers
DNA diameter
2 nm
4 purposes of DNA compaction
- containing long DNA into the 10micrometer nucleus
- preventing DNA from damage
- guaranteeing correct transmission of genetic info from parent to daughter cell in cell division
- regulates gene expression
overview of the levels of organisation of chromatin
- 10nm fibre
- 30nm fibre
- further supercoiling
- final chromosome
1st organisation of chromatin description
10nm fibre:
-made of DNA wrapped around histone proteins (nucleosome)
-chromatin spheres are a regular distance apart and are linked by LINKER DNA that are almost identical in length
Components of a nucleosome
- octameric histone core containing 2 copies each of: H2A, H2B, H3, H4
- DNA wrapped around histone of length 146 base pair
How were the components of a nucleosome dissociated and analysed? + results
- Individual nucleosome was isolated from long linked structure
- increase in salt concentration to dissociate the core histone protein from the DNA wrapped around it
- Running of an SDS-PAGE (protein electrophoresis) to separate the components of the octameric protein
RESULTS: 4 bands of equal thickness meaning 4 core histone proteins of equal concentration all present in 2 copies to make up the total molecular weight of the octamer
describe the investigation process to see if the DNA around histones was externally or internally placed
exposure to hydrolytic enzymes
DNA showed degradation upon this, so the conclusion is that DNA is exposed, wrapped externally and does not receive protection from the histone proteins
2 domains of histone proteins
- Globular core domain
- N terminal tails (+ve)
structure and function of the 2 histone protein domains
- Globular core: made of 3 alpha helixes arranged into HISTONE FOLD and used for histone-histone interaction to form the octamer proteins
- N terminal tail: rich in lysine and arginine which creates an accumulation of positive charges. used for higher order packaging and regulatory functions in epigenetics
final way of arrangement of the nucleosome
- octamer proteins with associated components
- DNA wrapped 1.7 times around histone
- N terminal tails of each histone core protein protruding
!! tails are highly positively charged and negative DNA associates with them to allow compaction
Compaction factor achieved at the first 10nm fibre level
7 times more compact
2nd level of chromatin organisation
30nm fibre:
-components: nucleosome (core histones + wrapped DNA) and the H1 protein (one copy per nucleosome meaning it is at half of the concentration of the other H proteins)
-formation of a zig zag conformation
Compaction factor achieved at the first 30nm fibre level
30nm fibre compacts by another 7 times
hence: total compaction is a maximum of 50 times
structure and binding of H1 protein
-contains 1 globular domain and 2 N terminal tails
2 BINDING SITES:
1. binding to linker DNA
2. binding to central DNA that is associated with nucleosome
2 complexes that attempt to deal with nucleosomes as a transcription inhibitor
- ATP dependent remodelling complexes: use ATP to either remove nucleosomes or slide them along DNA so TF can find a sufficient region of naked DNA to bind to
- Post translational histone modification: methylation, acetylation and sometimes phosphorylation
!! ATP dependent remodeling complexes are not epigenetic modifications whereas histone modifications are
what amino acid residues do acetylation and methylation target?
acetylation: lysine
methylation: lysine and arginine
!! both of these residues are present in high abundance in the N terminal tails of histones
Histone code definition
a hypothesis that the transcription of genetic information encoded in DNA is in part regulated by chemical modifications to histone proteins, primarily on their unstructured ends
ICF pathology information
-usually caused by a heterozygous mutation to the catalytic domain of DNMT3b
-this leads to hypomethylation being the cause of the disease
-symptoms include: intellectual, cognitive and motor problems
MOLECULAR PLAYERS INVOLVED IN EPIGENETICS:
1. writers
2. readers
3. erasers
WRITERS: enzymes adding epigenetic markers
!!! DNMT1/3A/3B
READERS: enzymes recognising and responding certain epigenetic markers
!!!! methyl binding proteins
ERASERS: enzymes removing or modulating existing markers
!!! TET1/2 enzymes
role of miRNA in gene expression
-miRNA is a part of ncRNA
-downregulates gene expression
-REASON: responsible either for translation inhibition or degradation of mRNA
