Lecture 7 (Cell Reprogramming)
Explain how complex traits, such as behaviour, can be reprogrammed epigenetically
Ant behavioural study - Carpenter ants have 2 main social classes: soldiers, and workers - Differences in morphology & behaviour were hypothesized to be somewhat due to differences in epigenetic regulation Experiment: - Workers were injected with inhibitory drugs for HAT or HDAC in the brain - Alternatively, siRNAs (small interfering RNAs) for the Rpd3 HDAC were injected in the brain - Minor and major ants were then examined in a foraging assay Results - Major ants injected with HDAC inhibitors or HDAC siRNAs displayed foraging behaviour - The effects of the HDAC inhibitors were abolished by co-injection of HAT inhibitors - The effects of the HDAC inhibitors were sustained for over fifty days following injection
Explain how Pioneering Factors are able to establish a state of "competence" in a cell
Case Study: - The FoxA and GATA transcription factors are detected on the promoter region of liver-specific genes in progenitor cells before expression of these genes (i.e. before differentiation) - These pioneer factor block CpG island methylation near the silent liver genes (passive process) - Thus, while not sufficient to trigger activation, the Pioneer Factor(s) establishes a state of "competence" for these genes that can then be activated in response to a stimulus (i.e. "primes" them) - The presence of a Pioneer Factor allows for a *rapid* recruitment of additional factors and activation of gene expression later on, in response to a stimulus
What is bookmarking?
Describes how Pioneer Factors are often able to remain bound to DNA, even during mitosis - Factors involved in Bookmarking are retained at their target loci, which can be re-expressed rapidly upon chromosome decondensation - Bookmarking is one of the mechanisms conveying information to progeny cells during mitosis
Explain how Pioneer Transcription Factors Can Reprogram Cells
Experiment: - Cardiomyocyte-inducing factors (Gata4, Mef2C, Tbx5) along with GFP were expressed in fibroblasts and the expression of muscle-specific markers was monitored by immunofluorescence microscopy Results: - Fibroblasts treated with the factors were shown to express cardiac genes and display sarcomeric organization
Give an example of how Transcription Factors can Convert Fibroblasts into Adipocytes
Experiment: - Overexpressed C/EBPb (a gene important for adipocyte differentiation) in fibroblasts - In response to C/EBPb, fibroblasts enter quiescence and begin to express genes promoting lipid accumulation (lipogenesis) and adipocyte differentiation - Resulted in accumulation of lipid vesicles - Thus, C/EBPb is a master regulator of adipogenesis (sufficient for this process) Note: C/EBPb does not act directly on genes promoting lipid accumulation (lipogenesis) but functions by inducing the expression of two direct activators of this program, namely C/EBPa and PPARg1
What is meant by the term "bivalency"?
Genes "poised" to be activated or repressed are often "marked" by both repressive and activating histone marks, including H3K4me3 and H3K27me3 - This is often observed in developmentally regulated genes - These genes are therefore regulated by *bivalent* marks - Removal of activating or repressive marks leads to rapid gene silencing or activation in a tissue / developmental stage-specific manner
What is Waddington's hypothesis?
If you take a specific pattern of differentiation & go over its "epigenetic barrier", you can turn it into a different type of cell - e.g. overexpressing Pdx1 in liver cells turns them into pancreatic cells, and overexpressing Cebpa in pancreatic cells turns them into liver cells
What are the passive and active roles of pioneer factors?
Passive: - speeding up inductive responses by occupying the promoter region to prevent CpG methylation (makes the process more rapid/ efficient) Active: - opening chromatin, enabling other factors to bind
What distinguishes Pioneer factors from other GTFs?
Pioneer factors: - The first factor that binds DNA (chrom. modification / remodeling complexes come afterwards) - Doesn't rely on other factors to bind to DNA (i.e. ATP-independent) - Can interact with condensed chromatin (binds to nucleosomes!)
What are the features of Pioneering Transcription Factors allowing them to bind in close chromatin?
The "Winged Helix" structure - DNA binding domain looks like winged helix structure, or HTH/helix turn helix similar to linker histones ("histone fold") - They also have a core histone binding domain
What is the Epigenome?
The epigenome is the set of all chemical compounds / modifications that have an influence on gene expression - Made up of chemical compounds and protein modifications that can attach to DNA, resulting in activation/repression of genes, controlling the production of proteins in particular cells
What is Epigenetics?
The study of changes in gene function that are mitotically and/ or meiotically heritable and that do not entail a change in the sequence of DNA.