epigenetics- lecture 14 Flashcards
totipotent
each cell has the potential to develop into any cell type
gene regulation: epigenetics
epigenetic marks are heritable changes in gene expression caused by mechanisms other than changes in the underlying dna sequence
how is dna packed
coiled around 8 histone proteins to make an octamer protein complex, which form nucleosomes that are the basic repeating units of eukaryotic chromatin
dna accessibility
dna has to be accessible to transcriptional machinery
dna coiling can be modified to make genes accessible or inaccessible, controlling their expression
what do epigenetic marks do
they are chemical tags that affect local packaging of dna
Histone modification
Histone modification is defined as any covalent addition of a chemical group (acetylation, methylation, ubiquitination, phosphorylation
direct tagging of dna
methylation of cytosines
what motif tends to get methylated
cpg’s
rna molecules
interfere with transcription machinery access to the dna
writers/erasers
enzymes that modify tags
how are epigenetic marks passed on to new generations
how to we study the epigenome: methylation
methylation question
how to we study the epigenome: chromatin structure
x inactivation
what does xist rna do
xist wraps up x chromosome, inactivating it
genomic imprinting
the expression of a copy of a gene depends on its parent of origin
how can postnatal environment affect the epigenome
high nurturing in early development leads to high levels of glucocorticoid receptor gene, which allow for response to cortisol and quick recovery from stress
methylating agents
DNA methylation regulates gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factor(s) to DNA and adding methyl group
ex: methylate cpg islands associated with promoter
demethylation
remove methyl group, more transcription
acetyl transferase
acetyltransferase enzymes that act on particular lysine side chains of histones and other proteins are intimately involved in transcriptional activation. By modifying chromatin proteins and transcription-related factors, these acetylases are believed to regulate the transcription of many genes.
deacetylase
Histone deacetylases (HDACs) are enzymes that remove acetyl groups from lysine residues in the NH2 terminal tails of core histones, resulting in a more closed chromatin structure and repression of gene expression
thrifty phenotype response
Epigenetics
phenomenon by which changes above or beyond
differences in the DNA sequence are inherited, either from one generation to the next or from one cell generation to the next. Epigenetic changes affect the expression of DNA, but not the identity of the constituent DNA
sequences – the sequence of the DNA bases is unaffected
DNA packaging
the process of how accessible the DNA is to transcriptional machinery
DNA does not float freely inside the nucleus, but instead is
wrapped around histone protein complexes and packaged into repeating units known as nucleosomes. Collectively, these nucleosomes are referred to as chromatin. Each individual nucleosome consists of eight histone
proteins around which the DNA wraps 1.65 times. These nucleosomes then coil and fold around one another to produce a 30nm fiber, which subsequently undergoes progressive rounds of coiling and condensation
culminating in an extremely condensed and ‘super coiled’ chromosome. Coiling the DNA in this fashion results in the genome being condensed so tightly that it can fit into the tiny nuclear space
epigenome
the totality of epigenetic modifications on a
genome). Epigenetic modifications are made in response to particular stimuli; these modifications in turn specify a particular pattern of gene expression. This gene expression profile dictates the identity of a cell. A
stable form of gene regulation is critical to differentiation, as the identity of a cell must be maintained through all subsequent cell divisions
three well characterized epigenetic modifications
DNA methylation, histone acetylation and long non-coding RNAs (lncRNAs)
DNA methylation
refers to the addition of methyl (CH3) groups to the
nucleotide bases. The predominant form of DNA methylation is the methylation of the DNA base cytosine. DNA methylation often occurs to cytosines that are immediately adjacent to guanines. This combination is
commonly referred to as a CpG dinucleotide (‘di’ meaning two and ‘p’ representing the phosphate group that connects the C and G nucleotides). These CpG dinucleotides are distributed across the genome
non-randomly: they tend to cluster in “CpG islands” which are typically within the promoter regions of genes.
DNA methylation promotes coiling and condensation of chromatin so methylation of CpG dinucleotides in the
promoter region of a gene decreases transcription and reduces expression of the gene. Thus, cells can increase or decrease gene expression by demethylating and methylating DNA, respectively. Enzymes called DNA
methyltransferases are responsible for the addition of methyl groups, whereas DNA demethylases are responsible for the removal of methyl groups
bisulfite sequencing
genomic DNA is treated with the chemical sodium bisulfite, which chemically converts unmethylated cytosines to uracils, which are then detected as thymines during sequencing. Importantly, only unmethylated
cytosines are chemically altered during bisulfite sequencing. As such, methylated cytosines are unchanged (you can think of the methyl group as “protecting” the cytosine from conversion to uracil). By comparing
sequencing of DNA that has been treated with sodium bisulfite with untreated DNA, we can determine the locations of all methylated cytosines
in the genome
ATAC-Seq, Assay for Transposase-Accessible Chromatin via Sequencing
This uses an enzyme Tn5 transposase that evolved to facilitate the movement of segments of DNA around genomes. In essence, the enzyme attacks any piece of accessible DNA (it does not have sequence
specificity like we see with CRISPR), cuts, and inserts material at the cut site. Tn5 transposase can only successfully attack chromatin in an ‘open’
configuration; it cannot access tightly packed chromatin. The DNA fragments generated by Tn5 transposase therefore correspond to the parts
of the genome with open chromatin
enzymes that modify the epigenetic tags are classified as
either
‘writers’ or ‘erasers’.
Writers are responsible for the addition of the
modification (e.g., DNA methyltransferase)
erasers are responsible for the removal of the modification (e.g., DNA demethylases).
epigenetic tags are recognized by a third class of enzymes known as ‘readers’, which interpret the suite of epigenetic modifications to loosen or condense the DNA accordingly. When readers encounter DNA methylation they recruit
additional proteins that can, for example, act as transcriptional repressors through additional chromatin remodeling
histone modification
Histone modifications affect chromatin structure by chemically altering the positively charged tails of the histone proteins, which interact with the DNA.
The best understood histone modification is the addition of acetyl groups to the amino acids on the histone tails. The writers responsible for acetylation are called histone acetyltransferases and the erasers that remove this
modification are histone deacetylases. Whereas DNA methylation results in chromatin condensation and transcriptional repression, histone acetylation
relaxes the chromatin, which increases transcriptional machinery accessibility, resulting in increased gene expression
long non-coding RNAs
(lncRNAs)
a special type of RNA molecule that is transcribed but not
translated. These RNAs are typically greater than 100 nucleotides in length and lack an open reading frame. Instead of coding for a protein, lncRNAs perform their functions as RNA molecules. IncRNAs work by interacting with proteins that regulate transcription such as
transcription factors or chromatin binding proteins. Alternatively, they can directly bind to the nucleosomes, often in the process attracting additional
proteins involved in epigenetic modification
X-inactivation is an epigenetic phenomenon that is mediated by lncRNAs. Many epigenetic changes are dynamic and easily altered, but X-inactivation is remarkably stable. X-inactivation is necessary to ensure XX females express the appropriate amount of X chromosome gene product. The process occurs
in two steps: First, the cell ‘counts’ the number of X-chromosomes present to determine whether inactivation is necessary. Second, when two X chromosomes are present, one is randomly selected for inactivation, while
the other remains active. There are a number of lncRNAs involved in mediating X-inactivation; Xist (X-inactive specific transcript) is the best characterized. Xist expression is up regulated on the X-chromosome
selected for inactivation, and then it coats the chromosome, marking it for inactivation. The coated chromosome is then silenced via high levels of
DNA methylation and low levels of histone acetylation. This methylated chromosome condenses into an observable Barr body
approximately what percent of human genes on the X chromosome escape inactivation
in humans, approximately 10-15%
of human genes on the X chromosome escape inactivation, but enough genes are inactivated to prevent issues with gene dosage
cellular differentiation
cellular identity is specified by a specific set of
epigenetic modifications,
hemimethylated
After semiconservative replication, any cytosine that was methylated prior to replication will now only be methylated on one strand – the template
strand. The cytosine on the newly replicated strand will be unmethylated. We refer to this state as hemimethylated. Special methyltransferases recognize hemimethylated CpG dinucleotides and add methyl groups to
the unmethylated cytosine bases on the new strand (remember the CpG on one strand is paired with a complementary GC on the other strand),
thus restoring the methylation pattern to the same state as that of the parent cell.
reprogramming
Gametes are highly specialized and differentiated cells and, like other cell types, their gametic identity is specified by their unique epigenome. However, upon fertilization most or all of these epigenetic marks are removed to generate a totipotent zygote that can divide and
give rise to all tissues of the body. The removal of epigenetic marks is triggered by the cellular environment in the zygote; this process is referred to as reprogramming. As development and differentiation progress, cells acquire distinct epigenetic modifications that specify their cellular identity.
Some genes in gametes escape this reprograming. This is often associated with genomic imprinting. In the case of
genomic imprinting, the gene’s expression in the individual is mediated by the epigenetic markers set by the parent during the gamete’s formation.
Typically, the promoter of the silenced copy (whether maternal or paternal) is methylated
Epigenetic changes are often long-lived (in terms of the turnover of cells) but are typically reversible. One example relates to caste differentiation in social insects, in particular the honey bee
she is genetically indistinguishable from the workers: a queen is “created” by the provisioning of a bee larva with “royal jellyThis inhibits the action of a gene, Dnmt3, that causes genome-wide methylation of CpG sites, in the process reducing or preventing transcription of a large
number of genes. These methylation-repressed genes are demethylated in queens, and therefore transcribed. Larvae can be turned into queens without royal jelly by using genetic tools that specifically repress Dnmt3
activity
As we have seen, epigenetic marks are reset or reprogrammed upon fertilization. (The gametes combine to make a totipotent zygote, which then can differentiate into many different tissue types). Reprogramming of
DNA methylation also occurs at a second time point:
in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte.
Epigenetic marks are then laid down, based on the cellular context, to direct these PGC progenitors to develop into a sperm or egg. In each of these two reprogramming windows – in PGCs and at fertilization – a unique
set of mechanisms regulate DNA methylation erasure and re-establishment
