Epigenetics and Gene Expression

• Epigenetics is the study of heritable changes in gene expression that do not involve alterations to the DNA sequence itself.
• The epigenetic code includes the following:

Chromatin Structure

• This involves the organization of DNA and proteins within the nucleus.

DNA Tagging

• This part of the epigenetic code involves the modification of DNA bases, such as the addition of a methyl group.

Histone Alterations

• Describes modifications to histone proteins can affect the accessibility of DNA for transcription.

Non-Coding RNAs

• This section describes the function of non-coding RNA molecules that play a role in regulating gene expression.

RNA Modifications

• Consists of chemical alterations in the RNA molecules can influence their stability, localization, and function.
• Epigenetics and its biological impact are areas of ongoing research.
• Regulation of gene transcription by non-coding RNAs (NCRs) has been recently discovered, and is a very active area of research.
• Most human transcripts are not translated into peptides, but the non-coding RNAs still serve vital functions.
• Non-coding RNAs (NCRs) were initially thought to only regulate gene expression at the post-transcriptional level, but recent studies suggest they are the most prevalent regulatory RNAs and play a significant role in epigenetic control.
• Non-coding RNAs (NCRs) include miRNAs, piRNAs, endogenous siRNAs, and long non-coding RNAs.
• Non-coding RNAs (NCRs) must be processed through the RNA-induced silencing complex (RISC) with a Argonaute (AGO) protein family, to achieve their regulatory functions.
• RISC assembly process:
  • An empty Argonaute (AGO) loads a small RNA duplex to form pre-RISC.
  • One strand, the passenger strand (blue), is ejected from AGO.
  • Final complex consisting of AGO and a guide strand (red) is the mature RISC.
• RISCs targets the complementary RNA and represses their expression via mRNA cleavage, degradation, and/or translational repression.
• Silencing is mediated by micro RNA (miRNA) and silencing RNA (siRNA), which are two types of non-coding RNAs involved in gene regulation.
  • MicroRNAs (miRNAs) are produced by endogenous genes containing hairpin structures of 65–70 nt pre-miRNA.
  • The hairpin structure is processed into pre-miRNA in the nucleus, where it is processed into miRNA by Dicer enzyme, and loaded to Argonaute (AGO2) protein.
  • Short Interfering RNAs (siRNAs) is derived from long double stranded RNA molecules, cut into fragments of 21–25 nt by the Dicer enzyme, and then loaded to Argonaute (AGO2) protein.
  • Loaded Argonaute protein recognizes and degrades mRNA complementary to the loaded guide strand.
• Dr. Rayner discovered that microRNAs – specifically microRNA-33 – regulate high-density lipoprotein and atheroprotective effects.

Polyteny Overview

• Polyteny occurs in chromosomes undergoing several rounds of DNA replication without cell division, leading to giant form.
• Polytene chromosomes:
  • Are easily seen under a microscope.
  • Exhibit distinct banding patterns showing regions of gene activity.
  • Are most famously studied in the salivary glands of fruit flies (Drosophila spp.).
  • Serve as a valuable model for studying chromosome structure and gene regulation.
  • Act as clear examples of the epigenetic influence of chromatin structure.
• Topics covered on polyteny:
  • As a study model for genetic research
  • Variations of polyteny across species
  • Structural and functional features
  • Regulatory control in Drosophila spp.
  • Functional significance
  • Lab 5 and FlyBase
• Key terms for polyteny:
  • Chromocentre
  • (a)synapsis
  • Chromosomal band/interband/puffing
  • FISH
  • Gene mapping
  • FlyBase
  • Endocytosis/endomitosis/polyteny
• Polyteny refers to a special type of chromosome structure in certain cells, where chromosomes undergo multiple rounds of DNA replication without cell division.
• Polytene chromosomes:
  • Results in large, multi-stranded chromosomes (up to thousands of DNA strand copies).
  • Are large enough to be seen in light microscopy.
  • Appears to have a haploid number of chromosomes because the homologous chromosomes are tightly paired (synapsed) and aligned.
  • Stay connected via their chromocenter.
  • Commonly occur in the salivary glands of some insects, such as fruit flies (Drosophila spp.).
  • Are also present in other organisms like protozoans, plants, and some mammalian cells.
• Data from chromosomes has provided final proof for the chromosome theory of heredity.
• The first time gene activity was seen directly under the microscope was known as "puff" formation.
• Hormone action on gene activity can be examined using polytene chromosomes.
• Thanks to polytene chromosomes, the heat shock phenomenon was discovered in the early 1960s.
• Polytene chromosomes are essential for gene mapping and basic cloning methods (like microcloning to create a library of DNA clones).
• Identical functional arrangement has been found in polytene & nonpolytene chromosomes of Drosophila melanogaster.
• Bands and interbands are observed under a microscope due to different staining characteristics:
  • Bands are darkly stained regions with a high DNA concentration that are rich in heterochromatin (tight DNA strands with nucleosomes), typically less active in gene transcription.
  • Interbands are lightly stained regions with less DNA, rich in euchromatin (DNA strands are loose), and more active in gene transcription.
• The pattern is essential for mapping genes and studying chromosome structure.
• Stabilizing sister chromatids:
  • Exhibits synapsis of homologous chromosomes and sister chromatids.
  • Somatic synapsis is based on the association of homologous polytene chromosomes and sister chromatids.
  • Homologues and chromatids synapse band to band, resulting in the impression that the chromosome is single.
  • In Drosophila spp., the number of polytene chromosomes in the nucleus is reduced to the haploid chromosome number.
• Sister chromatids and homologous chromosomes hold together because of:
  • Topological entanglement caused by DNA coiling
  • Cohesin and condensin complexes
  • Packing of heterochromatin in dense bands
  • Cohesins and condensins holding DNA strands together like pins
• Gene amplification occurs when a region on polytene chromosome is rereplicated several times, allowing a high transcription output when little transcripts are needed.
• Underreplication is when certain regions of the chromosomes don't fully replicate, which result in fewer DNA copies than what is expected.
  • This is often observed in heterochromatic regions, less active for gene transcription.
• Chromosome puffs occur when:
  • High transcription levels cause localized expansion of the polytene structure.
  • Polyteny (gene amplification) and chromosome puffing facilitates high transcription levels of intermolt genes during the third larval instar and prepupal stages of development.
• Puffing shows a direct relation to gene transcription levels, with larger puffs indicating greater transcriptional activity due to accessibility for RNA polymerase enzymes.
• S phase and M phase initiation are triggered by differing cyclin-dependent kinase (CDK) activity thresholds.
  • The S phase threshold is fulfilled by E- or A-type cyclins complexed with CDK2.
  • The M phase threshold is fulfilled by B- or A-type cyclins complexed with CDK1.
• In endocycling cells, only the S phase threshold is met periodically by cyclin B–CDK1 and A- or E-cyclins complexed with CDK2.
• Notch signalling leads to Hindsight(Hnt) activation.
  • Hnt inhibits String (Stg), a Cdc25 homologue needed to dephosphorylate and activate Cdk1.
  • Activates Hnt that prevents the Cut factor from inhibiting APC/C (anaphase promoting complex/cyclosome).
• Activated APC/C activates proteasomal breakdown of mitotic cyclins and Stg.
• Downregulation of Stg and mitotic cyclins degradation suppresses Cdk1 activity to form the Cdk1-B1 (or Cdk1-A/E complex) that triggers mitosis.
• Endoreplication is when the CDK activity doesn't reach the mitotic threshold.
• Polyteny arises in the presence of tissues, organs and developmental stages when there is a demand for rapidly developed organs at an unaltered high level of function.
• Organs that possess cells with polytene chromosomes usually have intense secretory capabilities.
• There is an unusually short amount of time against a background of rapid growth.
• Polytene puffs are where the chromatin has decondensed, and the DNA is made more accessible for transcription.
  • Puffs are sites for active gene express.
  • Relative gene expression within the puff is higher than non-puffed regions.
  • Increased activity is due to increased concentration of RNA polymerase and transcription factors, leading to robust transcription of genes found inside the puffs.
• Key facts about Polyteny:
  • Swollen segments are salient features on the third chromosome of D. melanogaster (seen during salivary gland dissection and observation in lab 5).
  • Third instar larvae must consume lots of food to build reserves for the metabolically demanding pupation phase meaning polytene chromosomes from salivary glands likely have essential digestive enzymes like amylase.
  • The saliva secreted by third instar D. melanogaster larvae has amylase as evidenced by the reduced starch levels.
  • In mammals, trophoblast giant cells show polyteny 4 hours post-fertilization.
  • Polyteny in giant trophoblasts:
    • Allows for amplification of genes.
    • Supports growth and metabolic activity essential for nutrient transport and hormone production during pregnancy.
    • Supports placental development and integrity.
  • 90% of rodent and ~50% of human hepatocytes have polyteny/polyploidy.
    • Making the liver one of the biggest polyploid organs in mammals.
  • Liver ploidy elevates substantially with the time of weaning and is sustained to grow with age.
    • Weaning leads to higher levels of polyteny/polyploidy, linked to reduced risk of hepatic tumors.
  • Liver exposure to conditions like oxidative stress and fatty liver disease:
    • Can further elevate polyteny/polyploidy.
    • Can potentially lower of the risk of developing tumors.
• Tumor suppressor mutations happen most often in liver tumors from diploid and polyploid mice.
  • Sequenced were 242 of the most commonly mutated genes in human and murine.
    • Red genes are oncogenes, Black genes are tumor suppressing genes.
• Polyploid hepatocytes, has protection over cancer using gene compensation.
• Process of gene compensation occurs when the tumor suppressor genes are buffer against their loss/mutation because of extra copies.

Lab 5 & FlyBase

• Final amount of ploid is achieved by developmentally programmed endocycling cells.
  • Salivary gland cells of D. melanogaster achieves 1,300C.
  • Fat body cells endocycle to 256C.
  • Midgut enocycles to 32C.
• Tissues and organs display asynchronous polyteny, resulting in different ploidy numbers within same cells.
• Key facts about the experiments on polyteny in Drosophila:
  • Lab 5 involves dissection and observing polytene chromosomes in salivary glands.
  • Sordaria fimicola exhibited synchronous polyteny with high C numbers.
  • Since 1992, National Center for Human Genome Research of the NIH has financed the FlyBase project in order to produce a database of genetic information.
  • FlyBase has genetic information (genes, alleles, and phenotypes), aberrations, clones, stock lists, and bibliographic references.
• Features on FlyBase:
  • Polytene chromosomes characterized, following chromosomes, Muller parts, and sections.
  • Division range: 1 to 102, Subdivisions range from A to F
• Using Flybase, researchers can:
  • Visualize to match certain chromosomal pattern to match
  • Locate genes and estimate transcription
  • Check genes to identify the type of allele.
  • Display the chromosomal position for a given gene