lecture 10: epigenetic regulation of differentiation Flashcards
1
Q
What regulates whether a cell remains a stem cell or differentiates?
A
- signals from the niche?
- intracellular determinants?
- ultimately, different patterns of gene expression cause cells to behave differently
2
Q
How do cells generally change gene expression patterns?
A
- without altering the nucleotide sequence of the DNA itself
- how do we know this? cloning!
- all of the instructions to regulate correct cell division and differentiation patterns of an oocyte into a frog are contained within the nucleus of a differentiated cell
3
Q
A cloned animal should be identical to the parent that donated the nucleus, right?
A
- what about the curious case of CC, the world’s first cloned cat
- “CC” (A) looks different to “Rainbow” (B), the donor
- the difference lies in the way the coat colour pattern is formed in calico cats (which are always female)
- the mixture of colours is due to random inactivation of one of the 2 x-chromosomes in each cell of a female mammal
- the cats have differenitiated in different ways due to changes in gene expression that are not associated with simple changes in levels of transcription factors
- X-chromosome inactivation is one example of what is called epigenetic modification of gene expression
4
Q
What is X-inactivation?
A
- female human cell with a condensed X-chromosome, or Barr body
- XXX female has two Barr bodies
- a mouse blastocyst derived from a father that had a lacZ transgene inserted on the X chromosome (all cells stain blue)
- at day 6 random inactivation of an X chromosome occurs in each cell of the embryo (so half the cells are pink) – in mice the trophoblast the paternally-derived gene is preferentially inactivated
5
Q
What is epigenetics?
A
- epigenetics can be defined as the study of heritable changes in gene expression that can be preserved through multiple cell divisions (or through generations of an organism) that are not the result of changes in DNA sequence (i.e. mutation)
- we wil discuss the role of DNA methylation and histone modification in this process and the consequences of genomic imprinting
- we will then relate this to stem cell biology and look at the relevance to iPS cell technology
6
Q
How do patterns of gene expression become fixed?
A
- e.g. how does a differentiated cell prevent accidental expression of inappropriate genes
- many genes are inactivated via DNA methylation
- methylation of cytosines “C’ only occurs when they are followed by “G”
- makes it very hard / the gene will not be expressed again
7
Q
How does cytosine methylation affect transcription?
A
- many enhancer sequences that are recognised by transcription factors are CG-rich and methylation can prevent factor binding
- DNA methylation can also recruit factors that modify histones and can result in nucleosomes forming tight complexes with DNA and not allowing access to transcription factors
- MeCP2 binds methylated C’s and recruits histone deacetylase and histone methyltransferase
8
Q
How can the pattern of DNA methylation be inherited through successive cell divisions?
A
- the importance of CG
- MeCP2 also recruits Dnmt3 (can methylate a block of nucleotides)
- this pattern is transmitted by Dnmt1 which recognises methyl-C and places methyl groups on the new synthesised strand opposite
- this is why G must follow a C – it allows stable inheritance of the pattern
9
Q
How much of the DNA codes for protein sequence?
A
- only a small percentage
- much of the “non-coding” DNA regulates gene expression
10
Q
How are these huge strands of DNA packaged into nuclei?
A
- chromatin is packaged as chromosomes
- chromosomes are most easily visualised during metaphase, when they are most highly condensed
- diploid organsms have two copies of each chromosome
- chromatin in interphase nuclei forms fibres 30nm thick
- when these fibres are experimentally decondesed nucleosomes become visible
- nucleosomes are packaged to form the 30 nm chromatin fibre
- DNA is packaged into nucleosomes by association with histones
- they are often represented as “building block” type structures (see H2A, H2B etc below) but they have “tail” regions that are crucial for regulating chromatin structure
11
Q
What causes changes in histone packing?
A
- methylation of histone tails condense nucleosomes and thus repress transcription
- acetylation uncondeses nucleosomes and allows access to RNA polymerase and transcription factors
- however… this is a little simplified
- methylation of lysine residues in the histone H3 tail at positions 4, 38, and 79 are associated with gene activation while methylation at positions 9 and 27 are associated with repression
- and histone tails can be phosphorylated and ubiquitylated as well
- general point: these methylation sites create binding sites for different sorts of proteins
- e.g. cell cycle regulation, transcriptional elongation, transcriptional memory, silent heterochromatin, transcriptional activation
12
Q
What is the take home message about modification of histone tails?
A
- affects the state of chromatin compaction
- provides binding sites for chromatin-modifying proteins
- take the example of the polycomb protein
- it binds to trimethylated histone 3 lysine 27 (H3Me3K27)
- polycomb is part of a group of proteins that act together as the Polycomb Repressor Complex (PRC1) to repress transcription
13
Q
What do we know about polycomb?
A
- Polycomb was first discovered because of its role in Drosophila development
- it is required to maintain repression of Hox gene expression in specific developing body segments
- Hox gene expression provides specific identity to each segment
- in an animal that is mutant for a member of the PRC Hox gene expression is set up normally but is not maintained through the cell divisions that occur as the body segments grow
14
Q
What are repressors that maintain the stem cell state?
A
- Rudolf Jaenisch (2006) Nature 441:349
- analysis of genes repressed by the polycomb complex in mouse ES cells
- found that 512 genes were specifically repressed by this complex and were mainly developmental regulators
- conclusion: genes that are expressed in stem cells are those required for cell proliferation and “housekeeping”
- differentiation and developmental genes must be kept silent
15
Q
What are the developmental consequences of DNA methylation?
A
- X chromosome inactivation occurs by chromosomal-wide methylation and this inactive chromosome is clonally inherited
- genomic imprinting breaks Mendelian rules
- in mendelian theory it should not matter if you inherit a gene from your mother or your father
- however, at a few specific chromosomal positions genes are imprinted
- this means that they function differently depending on the parent of origin
- imprinting is due to differential methylation
- primordial germ cells are actively demethylated around 12.5dpc and male PGCs are then remethylated during mitosis in the embryonic gonad while female germ cells are remethylated postnatally
16
Q
What is the result of imprinting?
A
- results in only one allele being expressed in offspring
- both parents express the same allele of gene A
- there is an imprinted allele and and an expressed allele in each of the parents
- removal of imprinting in cells followed by meiosis
- female and male imprints establish
- offspring differ in the allele of gene A that is expressed
- differential inheritance from either the mother or the father results in differential gene expression
17
Q
Why does imprinting occur?
A
- don’t really know
- one hypothesis:
- the “parental conflict hypothesis” suggests that imprint is due to the different interests of each parent:
- the father’s genes favour fitness of the offspring at the expense of the mother
- the mother’s drive is to conserve her fitness to provide resources to offspring
- maternal and paternal genomes are required for normal embryo development
18
Q
How many imprinted genes have been identified in mammals?
A
- insulin-like growth factor-2 is expressed from the male chromosome
- sperm mutant in Igf2 (plus a wildtype egg) produce small offspring – as only low levels of Igf2 are produced from the maternal chromosome
- wildtype sperm that fertilise a mutant Igf2 egg produce normal offspring
