Module 6 - Epigenetics/the Molecular Mechanisms of Cell Differentiation

included on exam 2

multipotent

having the ability to become many different types of cells

e.g. adult stem cells

i.e. hematopoietic

cell identity memory

cells must have memory of their differentiation (terminal fate) when they divide, so that daughter cells can take the same differentiation

the basis of epigenetics

epigenetics

the study of changes and variations in phenotypes that are potentially heritable, but are not caused by permanent changes in DNA base sequences

(think: roadblocks and traffic cones are placed, but the road itself is not repaved)

facultative heterochromatin

the regions of chromosomes that contain genes but can become heterochromatic depending on the cell type

e.g. regions that code for liver genes, in a neuron cell (liver genes are not needed in a neuron, so they condense but still have their genes)

amount in a cell increases as cells differentiate

found less in embryonic and adult stem cells than terminally differentiated cells

combinatorial controls regulating gene expression (in eukaryotes)

groups of transcription regulatory proteins work together to control the expression of single genes

transcription regulators bind to specific regulatory DNA sequences near genes and become of the transcription initiation complex

(regulators can be either activators or repressors)

effects of regulators add up to determine the final level of gene expression

because control is combinatorial, the removal of any one regulator from the complex can either significantly alter or completely inhibit transcription levels (because the complex is incomplete)

alternately: the addition of a single protein can complete a complex → switch a gene/several genes on

transcription initiation complex

a complex formed by transcription regulators binding to specific regulatory DNA sequences, general transcription factors, and RNA polymerase

the whole complex determines the final level of expression of a gene

if any regulator is removed, the complex is incomplete → transcription may be significantly reduced or inhibited

glucocorticoid receptor

an example of a transcription regulator that can activate the transcription of several genes when a hormone (cortisol) binds to it

genes transcribed are involved in the glucocorticoid response

combinatorial creation of different cell types

e.g. MyoD is a single transcription regulator that switches on all muscle-specific genes required for specification of skeletal muscle cells

MyoD commits cells to become myoblasts

myoblasts then proliferate and fuse → form multinucleated myotubes

myotubes become muscle fibers

muscle fibers express a number of muscle-specific products (thanks to muscle-specific genes switched on by MyoD), which gives muscle cells their specific properties

simplified: MyoD is the transcription regulator that turns on the genes that make cells become muscle cells

experimental induction of transdifferentiation

artificial expression of a certain transcription factor in a different kind of cell can lead to proliferation of the type of cell that the transcription factor activates

e.g. transdifferentiation of fibroblasts, or of liver cells

experimental induction of transdifferentiation of fibroblasts

MyoD is artificially expressed in fibroblasts

the fibroblasts start to behave as myoblasts, fuse to form muscle-like cells

meaning: fibroblasts already contain other transcriptional regulators required for combinatorial induction of terminal muscle fate; addition of MyoD completes a unique combination for muscle cells

summarized: fibroblasts and myoblasts have the same transcription initiation complex, except for MyoD, which is what differentiates myoblasts from fibroblasts

(other cell types not related to muscle cells do not respond the same way)

(in the picture, the yellow is muscle-specific proteins detected by a stain in fibroblasts)

fibroblasts

cells that belong to the same broad embryonic cell class as muscle cells, but are involved in wound healing instead of muscle formation

if MyoD is artificially expressed, though, will start behaving as myoblasts

the above happens because these cells already contain the other transcriptional regulators required for muscle fate, aside from MyoD naturally

experimental induction of transdifferentiation of liver cells

liver cells in culture can be converted to neurons (transdifferentiation):

3 neuron-specific transcription regulators are introduced to liver cells → form new combinatorial controls

induction of an entire organ by a single transcription regulator

a single protein can induce differentiation of all cell types needed to assemble a whole organ (since combinatorial effect can be triggered by a single transcription regulator)

e.g. regulator Ey in Drosophila

transcription regulator Ey in Drosophila

Ey controls expression of many genes that are expressed in all cell types of the eye of Drosophila

(all of these genes contain the regulatory DNA sequence recognized by Ey, even if they serve different functions in the eye (e.g. eye cone vs eye lens))

if Ey gene is ectopically expressed in larval cells on the area that will eventually become a leg, an eye-like structure will develop on the leg

summarized: Ey activates genes found in all eye cells. If Ey is moved to non-eye cells and forced to express, an “eye” will form (but non-functional)

epimorphic limb regeneration (epimorphosis)

an exception to the rule of terminal differentiation

observed in salamanders

when a limb is wounded, adult cells in the wound dedifferentiate → become neoblasts

neoblasts divide rapidly → become re-specified to form the missing adult limb

creation of different cell types by combinatorial controls

a few transcription regulators can combine in different combinations → form many different cell types

a few regulators in different combinations can control expression of a larger number of genes with shared regulatory sequences

different combination of regulators = different genes expressed

mechanisms of inheritance of gene expression patterns (3 of them)

1) feedback loop circuits involving transcription regulators

2) preservation of covalent histone modifications and chromatin condensation patterns

3) preservation of DNA methylation patterns

all 3 are examples of epigenetic inheritance, because they involve heritable changes in gene expression that are not caused by changes in the actual DNA sequence

positive feedback loops and cell memory

a signal (external or internal) triggers expression of a transcription regulator → the protein perpetuates expression of its own gene by activating its own transcription

cell expression the protein must preserve it through cell division so that it can be passed on to daughter cells

transcription regulator must be able to positively control its own transcription and the transcription of other cell type-specific genes (even if the signal that triggered the initial expression is no longer present)

organization

nucleosome: 147bp of DNA wrap around 8 histone proteins (a nucleosome core particle)

nucleosome is held in place by a 9th histone

adjacent nucleosomes interact, higher levels of organization occur → chromosome condenses

partial condensation of some regions of chromosomes is observed during interphase as heterochromatin

histone acetylation

lysine tail is acetylated

positive charge on lysine is eliminated

two Hs on the amino end (positive end) of lysine are replaced by an acetyl (Ac) group

forward reaction requires HAT (histone acetyltransferase) and adds Ac from the lysine

reverse reaction (deacetylation) requires HDAC (histone deacetylase) and removes Ac from the lysine

how can histone acetylation stimulate transcription initiation?

positively charged lysine tails in histones interacte with negative charges of phosphate groups in DNA (histone-DNA interactions are strong)

acetylation neutralizes lysine tails → weakens histone-DNA interactions, allows transcription factors/other proteins to bind to DNA and stimulate transcription

nucleosome replication (extra info, don’t worry about it unless you have brain space)

nucleosome partially disassembles into two half-nucleosomes

replication fork replicates past the half-nucleosomes, while building new DNA strands

nucleosomes reassemble from old half-nucleosomes and newly synthesized histones

inheritance of histone modification patterns

tissue-specific histone modifying enzyme is preserved throughout cell division and passed on to daughter cells

^so that enzyme can restore parental patterns of histone modifications and chromatin condensation (because only half of the daughter nucleosomes from a parent have modified histones, so they must be re-established by the enzyme)

see picture for better detail

DNA methylation

the addition of a methyl (CH3) onto a cytosine, if the cytosine is immediately followed by a guanine

only occurs at specific sites of CG sequences, depending on the gene expression needs of the cell (not every CG sequence will be methylated)

turns off gene expression (attracts proteins that block transcription)

must be maintained by an enzyme (does not maintain itself)

maintenance of DNA methylation

after DNA replication, only parental strands have methylated Cs

maintenance methyltransferase recognized already-methylated CG sequences, catalyzes methylation of corresponding CGs in the new complementary strand

maintenance methyltransferase is preserved throughout cell division and passed on to daughter cells to preserve this same methylation pattern (necessary for preserving DNA methylation patterns after every cellular DNA replication cycle)

de novo DNA methylation

new methylation patterns that are established during development, or in response to external cues (e.g. environment, behavior, diet)

established by de novo methyltransferases

e.g. in honeybees to make a new queen

DNA methylation in honeybees

queen bee: a female that is genetically identical to worker bees, but with significant differences in anatomy, physiology, behavior, and functions in the colony

(queen bee is the only reproductive female in the whole colony)

Dnmt3 gene results in a product: DNA methyltransferase 3 (a de novo methyltransferase)

Dnmt3 enzyme product methylates and silences queen-specific genes in worker bees

worker bees produce royal jelly, which gets fed to only a few larvae

royal jelly silences the expression of the Dnmt3 gene → DNA of the queen bee is less methylated

the largest royal jelly-fed larva eats the others and will become the new queen

summarized: in worker bees, Dmnt3 enzyme is made, silences the queen genes. Only in queen larvae (fed royal jelly), the silencer is removed → queen genes are expressed