Monthly Archives: November 2016

Mammalian Genomic Imprinting

  • Small number of genes are marked on their parental allele
  • Only one parental allele is expressed
  • 1980: pronuclear transplantation experiments demonstrated that mammalian development requires contribution from both parents
    • Maternal uniparental embryos were exclusively embryonic tissues
    • Paternal uniparental embryos developed into extra-embryonic structures only
  • Engineering of offspring dome through combination of nuclei from nongrowing and fully grown oocytes with mutations in 2 different imprinted loci
    • Resulting bimaternal offspring had normal imprinted gene expression
  • Some methods used to identify imprinted genes:
    • Molecular characterization
    • Gene targeting experiments
    • Genome wide studies – more popular once sequencing of the whole genome possible
  • Igf2r: insulin-like growth factor type 2 receptor, first of 3 imprinted genes reported in 1991
  • H19 gene encodes a ncRNA, maternal-specific expression (identified via SNPs)
  • To date, ~100 imprinted genes have been found in mammals
  • ICRs: imprinting control regions, shows parent-of-origin specific epigenetic modifications that are set up in the germline
  • Many imprinted genes are dosage sensitive, with over/under expression resulting in consequences
    • e.g. prenatal growth control, brain function, postnatal energy homeostasis
  • Conditions that result from faulty imprinting:
  1. Prader-Willi syndrome: loss of expression of SNRPN implicated, but contribution unknown
  2. Angelman syndrome: caused by the absence of UBE3A transcript expressed from maternal chromosome
  3. Beckwith-Wiedemann syndrome:
  4. Silver-Russell syndrome:
  • Placenta and the brain are sites of widespread imprinted gene expression
  • Absence of Igf2r causes impaired nutrient transport to the fetus
  • Peg10, Rtl1 are imprinted genes required for placental development, originated from retrotransposons
  • In the brain, imprinted genes are implicated in metabolic axes, behaviour, learning, maternal care
  • G5a: gene expressed from the maternally inherited chromosome in the hypothalamus, controls melanocortin-mediated energy expenditure
  • Peg1, Peg3 paternally expressed imprinted genes, strongly transcribed in brain, KO leads to decreased maternal care

Properties of the Imprinting Mechanism

4 important properties of the genomic imprinting process:

  1. The mark must be able to influence transcription
  2. It must be heritable in somatic lineages
  3. The mark is likely to be placed on the chromosomes when the paternal and maternal chromosomes are located in different nuclei
  4. Must be a mechanism of erasure so that paternally inherited chromosomes in the female germline can establish a new mark and vice versa

 

Chromatin remodelling and bivalent histone modifications in ESCs. EMBO Reports.

  • Nucleosome formed by pair of each of the core histones – H2A, H2B, H3, H4
  • Linked histones (H1) between the nucleosomes forms higher-order chromatin structure
  • Histone modification can assist with transcriptional activation and repression
  • Combination of H3K4me3 (activating) and H3K27me3 (repressive) found in some promoters, called bivalent modifications
  • HCNEs (highly conserved noncoding elements) tend to be enriched with bivalent modifications
  • WT conditions there was little activation of these bivalent promoters
  • Depletion of PRC2 subunit leads to loss of H3K27me3, upregulation of genes
  • Concluded that the promoters are “poised” for gene expression
  • Bivalent domains tend to be developmentally regulated, although some have been found in terminally differentiated cells
  • In ESCs, bivalent domains tend to be associated with CpG islands
  • Challenge: have yet to develop single nucleosome resolution technique, hard to determine function of these domains conclusively
  • Trithorax group (TrxG) proteins deposit H3K4me3 – SETD1A, SETD1B, MLL complexes
  • SET1A/B – responsible for global deposition of H3K4me3
  • MLL2 is the main methyltransferase at bivalent domains
    • Contains CXXC and ZF-CXXC domains, which recognize unmethylated CpGs
    • MLL2 KO is embryonic lethal, shows developmentally important
  • PcG proteins (PRC2) mainly deposit H3K27me3 at bivalent promoters
    • Core PRC2 made up of EZH2 or EZH1, EED, SUZ12
    • EZH2: enhancer of Zeste, catalytic subunit of the complex, methyltransferase for H3K27me3
    • PRC2-specific bivalent domains are usually on promoters of not “bona fide” developmentally active genes
    • PcG proteins are vital for ESC differentiation, depletion of PRC2 in ESCs shows aberrant differentiation potential

 

Key takeaways:

Glossary:
HCNE: highly conserved noncoding elements, tend to be enriched with bivalent modifications
Bivalent modifications: combination of both repressive and activating histone modifications
Chromatin remodelling: any change or modification to chromatin
H2K27me3: trimethylated lysine 27 on histone 3, repressive mark
H3K4me3: trimethylated lysine 4 on histone 3, activating mark

Learning Journal 4

While I had previously been quite familiar with a variety of fluorescence-based molecular biology techniques (immunofluorescence, FACS, FISH), Fluorescence (Forster) Resonance Energy Transfer (FRET) was completely new to me. Learning about this technology during the Techniques Cafe made me realize the diversity of techniques that have been and will continue to be developed for a better understanding of molecular biology moving forward.

The most confusing aspect of this technique is understanding the energy transfer between the two chromophores. Many biologists do not have a strong background in quantum mechanics, which forms the basis of this technology. A strong understanding of the underlying mechanism is not necessary, however. One just needs to know that FRET involves the excitation of a donor fluorophore that is bound to a biomolecule. This donor then transfers its energy to an acceptor fluorophore in a non-radiative fashion – meaning, the transfer of energy does not actually occur through fluorescence. Instead, the energy is transferred through long-range dipole-dipole interactions and resonance energy transfer (RET). The excited fluorophore acts as an oscillating dipole, and transfers its energy to an acceptor with a similar resonance frequency. The measurement that is obtained is E, the FRET efficiency. This is the fraction of energy transfer that is occurring each time a donor chromophore is excited, and can give a significant amount of structural information regarding the donor and acceptor.

In order to assess someone’s understanding of a technique, I think they should be asked what sort of biological questions the technique could be used to investigate. This can show that they understand not only the underlying concept of the technology, but also have a practical understanding of how the technique can be used to investigate scientific hypotheses.

Some examples of questions that could be investigated using FRET:

  • Detect in vivo interactions between two proteins
  • Examine the distances between two different domains on a protein of interest
  • Identify changes in conformation of a single protein in response to a particular physiological event
  • Visualize the temporal and spatial localization of molecules within the cell, see how they change over time
  • Determine the rate of biochemical reactions (e.g. enzymes)

Learning Journal 3

The second midterm for this course was very different from the majority of midterms and final exams that I have taken during my time in university, in a refreshing way. Far too often in science, particularly in biology, there is an emphasis on rote memorization of facts and definitions. For example, in one of my fourth-year courses that I took last year, I ended up with a list of over 200 transcription factors, genes, and developmental pathways to memorize for the final exam. However, this obviously is not what doing biological research is like in the real world, especially with the internet enabling us to look up the name of any gene or protein whose name or function we might have forgotten.

Instead, the midterm for this course completely focused on conceptual understanding. Delving deep into the primary research paper did require some fundamental knowledge of epigenetics, X chromosome inactivation, and developmental biology, but beyond that it was really about understanding the scientific method. I realized that up until a year or so ago, I would take scientific papers at face value, assuming that everything the authors had written was completely accurate. I would often skim through the results and go straight to the discussion, often ignoring the figures altogether! This course, however, has taught me how to bring a healthy skepticism to scientific literature, and to use my own judgement and critical thinking skills to assess the scientific validity of a conclusion. This is a process that will be incredibly helpful for me as I continue to work on my thesis, reading through a large amount of primary literature and investigating my own scientific questions.

Learning Journal 2

During the course so far, I have learned a significant amount regarding enhancers and their function in gene regulation. I had previously only learned about enhancers in passing, but the papers and lectures in class have allowed me to delve deeper and develop a better understanding of how enhancers work to upregulate gene expression. I realized that I understood the concept better as I was reading a paper for another class and there was a passing mention of an enhancer. I immediately found myself picturing the “enhancer looping” that has been proposed as a model for enhancer interaction with promoters – something I had learned from the Plank and Dean paper assigned as a reading for class. I think that this is the best sign that you have understood a concept – when you find yourself applying the knowledge to another area or problem.

I would say that enhancer function is a concept, rather than a skill or technique, since it takes the application of several skills and techniques in order to actually study it. Many experiments are required in order to determine the mechanisms of enhancer action. The understanding of enhancers and their function is important for anyone who is studying gene expression and development. Enhancers are believed to be key in embryonic stem cell differentiation, as the enhancers of pluripotency genes are progressively inactivated as cells become committed to different fates. Furthermore, enhancers are a vast potential area for future research. The majority of enhancers in the mammalian genome and their specific targets are still unknown, although many of them are presumed to be involved in development, evolution, and disease (Stark et al., 2014),