BIOC 410 Term Paper Assignment - 2017
Santiago Ayala Perez, 18318139
Important Observations
In our paper, we determined that phase separation drives heterochromatin domain formation by liquid-liquid demixing into phase-separated compartments. Heterochromatin plays an essential role in nuclear architecture, genome repair and stability, and gene expression. These domains are transcriptionally silent, highly conserved in eukaryotes, and defined epigenetically by H3K9 methylation, and recruitment of HP1 (heterochromatin protein 1, a fundamental unit of heterochromatin packaging). From in vivo and in vitro experimentation, we found that HP1a domains exhibit liquid-like dynamics characteristic of phase-separation. These include spherical
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Hence, although H3K9me2/3 methylation mark may be required, nucleation could occur from the inherent demixing ability of HP1a.
HP1a droplet formation initially occurs in a liquid-like manner with maintenance of droplet circularity. However, upon embryonic maturation, HP1a foci display decreased circularity, characterized by 30% GFP-HP1a stabilization (corresponding to the immobile droplet fraction). HP1a is believed to play a key role in recruitment of factors which induce phase separation. Similarly, decreased circularity may be induced by HP1a association with specific nuclear structures critical for silencing. Hence, understanding HP1a’s role in factor recruitment is paramount to the study of transcriptional regulation of liquid-like domains.
Hypothesis
Due to the role of HP1a in transcriptional regulation, we hypothesize that HP1a droplet morphology may influence lineage development of eukaryotic cells by selective permeability of phase boundary to regulatory factors. We will focus on circularity which was observed to decrease as the cells differentiate. Loss of circularity may be related to the inclusion of lineage development factors. In this case, the initial loss of circularity - as factors begin populating the droplet - could be an early indicator of lineage differentiation.
Experimental approach
Cheutin et al. (2003), observed that the human heterochromatin protein domains were positionally stable in Chinese
The EZH2 (enhance of zeste homolog2) is an enzyme that in humans is fixed by EZH2 and its supply the information’s about making of enzyme called a histone me-thyltransferase. The ploycomb group (PcG) is a protein with the catalytic subunit of the PRC2 (polycomb repressive complex) which the transcriptional involved in maintaining the repressive of genes cells and also the EZH2 mainly performs as a silence gene. The EZH2 performs the role by addition of methyl groups on the ly-sine 27 of histone H3, a modification leading chromatin
A related phrase described by Waddington to help elaborate the phenomenon of epigenetics, the ‘epigenetic landscape’ attempts to explain how identical genotypes could result in a wide variety of phenotypic variation through the process of development. This epigenetic landscape can be dynamic – capturing genetic, environmental, and cell lineage effects – and has been shown to be at least partly heritable. (Szyf, M. (2015) Nongenetic inheritance and transgenerational epigenetics. Trends Mol. Med. 21, 134–144). The epigenetic code is hypothesized to be a defining code in every eukaryotic cell consisting of the specific epigenetic modifications in each cell. While in one individual the genetic code in each cell is the same, the epigenetic code is tissue & cell
Epigenetics is a field where advances are being made daily. Epigenetics is defined as “heritable changes in gene expression that occur without a change in DNA sequence,” as stated by Dr. Alan Wolffe. A way in which we can understand this definition is by taking the analogy of a card game. The cards, the DNA sequence, have been dealt and will not change, however we need to understand how to play the cards, the rules, which is epigenetics. The guidelines can vary and completely change the way the card game is played and who comes out on top. The rules that are studied and understood through this research paper are those of DNA methylation and chromatin. These changes can produce
Contrasted when you look at the three olds epigenome there were still completely the same with much of the yellow coloring shown, this illustrated that over our lifetime epigenome change, enforcing that old saying “you are what you eat” and reminding us all that we have more control than what we thought we did in what goes on in our bodies on a cellular level.
The study greatly helped in understanding the mechanism of nutrition and epigenetic inheritance to the child while in the womb. It has been shown through animal studies that a methyl rich diet of the mother gives a progeny with a highly methylated DNA. Specifically, studies in mice have shown that diet affects the Agouti gene (present in all mammals) when the gene is not methylated the mice shows a yellow coat, a fatter complexion and is likely to develop diabetes and cancer. Unlike the healthy mice
Nucleosomes are made of DNA wrapped around eight histone proteins and they are called a histone octamer. Each histone octamer has two copies of the histone proteins H2A, H2B, H3, and H4. The chain of nucleosomes is then wrapped into a 30 nm spiral called a solenoid, where additional H1 histone proteins are associated with each nucleosome to maintain the chromosome structure.
At the 12-cell stage of embryogenesis, new P1 decendants(MS and E) will become signalling cells. GLP-1/Notch still remains on the surfaces of the ABa descendants. According to genetic studies done by Shelton and Bowerman (2005), signals from MS and E are identified as products of embryonically-transcribed genes. Since MS will be in contact with 2 out of 4 ABa descendants GLP-1 will be activated. Thus, ref-1 will be expressed. On the other hand, for ABp descendants, they are refractive to second Notch interaction even though they contact MS or E but still continue the GLP-1/Notch
Several factors contribute to the formation of a repressive environment in the nuclear periphery. First, chromatin is organized such that active regions are localized around the center of the nucleus, whereas inactive regions
Aging: Aging results in the hypomethylation of the genome, while CpG islands are hypermethylated often.
The food we eat gives us nutrients, and these nutrients enter pathways where they are turned into molecules the body can use. There is one pathway that is responsible for making methyl groups, which are epigenetic tags that make genes unreadable. To show how not only our diet, but our parent’s diet can affect the epigenome and how methyl affects our epigenome, researchers looked at the diet of pregnant mice and observed how diet and methyl consumption affected their offspring. They looked at the agouti gene in particular, since it is a gene all mammals have. When a mouse's agouti gene has no methyl in it, the mouse has yellow fur, is obese, and is at high risk for cancer and diabetes. When the mouse’s agouti gene has methyl in it, the fur is brown and the mouse has a lower risk for diseases. The fat yellow mice and skinny brown mice are genetically identical. However, the fat yellow mice are different because they have an epigenetic "mutation." When researchers fed fat yellow pregnant mice with a diet full of foods containing lots of methyl in them, most of the babies had brown fur and were healthy. This shows that our health is not only determined by what we eat, but also what our parents ate.
The proper division of cells is fundamental for growth and reproduction of organisms. Chromosomes must divide evenly and accurately between the two new cells so that harmful mutations are prevented from occurring. Cohesin is an important protein mediator in these precise divisive events. While preparing for mitosis, cohesin forms a ring around DNA to hold sister chromatids together in an organized fashion. Cohesin is comprised of 2 intramolecular coiled coil proteins, Smc1 and Smc3, which dimerize to form a circular protein. The dimerization domain of these proteins, also known as the hinge, keeps these proteins bound together. At the other end of cohesin, the ATPase head domains of Smc1 and Smc3 are held together by Scc1, a kleisin protein essential to the unloading of cohesin during anaphase. Scc1 is cleaved by separase once all chromosomes are attached to microtubules correctly and tension is sensed by the cell. Cleavage of Scc1 allows release of DNA from cohesin and separation of sister chromatids to opposite ends of the cell. The removal of cohesin from DNA is well studied and understood, but there is still controversy regarding the mechanisms of cohesin loading onto DNA.
Epigenetic modifications on histones and DNA exercise precise regulation of gene expression, and thus have a profound influence on cellular differentiation and function. The most widely studied modification on DNA is 5 methylcytosine (5mC) that frequently occurs in a CpG dinucleotide context. Initially, the de novo DNA methyltransferase (Dnmt) enzymes Dnmt3a and 3b mediate the transfer of the methyl group to cytosine, while the maintenance Dnmt1 ensures subsequent propagation of this mark through cell replication cycles. Because of prominent roles in gene repression, retro element silencing, X chromosome inactivation and gene imprinting, 5mC was initially thought to be irreversible. Thanks to burgeoning evidence, it is now clear that the methylcytosine can be removed through a number of mechanisms. Passive demethylation is proposed to remove 5mC over several cell replication cycles, through exclusion of the maintenance machinery from the replication fork. Active demethylation on the other hand constitutes removal of 5mC through enzymatic hydrolysis. The discovery of Ten Eleven Transclocation (TET) dioxygenase proteins was revolutionary. The TET proteins catalyze hydroxylation of 5mC to 5hmC, and conversion of 5hmC to 5-carboxylcytosine and finally to 5 formylcytosine. These 5mC derivatives enter the base excision repair pathway (BER), resulting in replacement of the methylcytosine with an unmodified cytosine. Second, although the role of deaminases is debated, these enzymes
The activity of genes is reliant on mostly on whether they are available to transcription factors; this is vastly controlled by the dynamics of chromatin restructuring. Epigenetic modifications on the chromatin play a vital role in regulating the construction of chromatin and thus the availability of DNA for transcription. Some of the sites at the DNA that are transcripted can be turned on or off by epigenetic changes. Moreover, it has previously been verified that environmental factors, for example diet, cigarette and alcohol use, stress, or exposure to chemical carcinogens and infectious agents, Sexuality and age effect the epigenome. Today, because of importance in epigenetic processes, most scientists prefer to work in this field of research.
Chromatin immunoprecipitation (ChIP) assay — The ChIP assay was performed using the SimpleChIP Enzymatic Chromatin Immunoprecipitation kit with magnetic beads (Cell Signaling Technology) as described previously (1). An equal amount of chromatin was incubated overnight at 4°C with antibodies against PPARδ, or against IgG as control (Santa Cruz Biotechnology). Then protein G magnetic beads were added and the chromatin was incubated for 2 h, at 4°C with rotation. An aliquot of chromatin that was not incubated with any antibody was used as the input control sample. Antibody-bound protein-DNA complexes were eluted and subjected to real-time PCR (1) with specific primers that amplify the PPRE and β-Actin in the promoter.
The FACT complex is a highly conserved general histone chaperone protein that is essential for transcription and DNA replication (Abe et al., 2011; Belotserkovskaya and Reinberg, 2004; Orphanides et al., 1998). The complex has also been shown to play important roles in DNA damage responses, centromere deposition, recombination and DNA methylation (Ikeda et al., 2011; Kumari et al., 2009; Okada et al., 2009; Oliveira et al., 2014). The FACT complex is thought to destabilize