Epigenetics and Non-Coding RNAs

Epigenetics

• Epigenetics entails the study of mechanisms leading to changes in gene expression without alterations to the DNA sequence itself.

Epigenetic Code

Chromatin Structure

• Encompasses DNA tagging, involving chemical modifications to DNA, and histone alterations, which impact how DNA is packaged and accessed.

Non-coding RNAs

• Involves RNA modifications which impacts the structure and function of RNA molecules.

Epigenetics and its Biological Impact

• Ongoing research focuses on the biological consequences and applications of epigenetic modifications.

Non-Coding RNAs (NCRs) in Gene Transcription Regulation

• The regulation of gene transcription via non-coding RNAs is recently discovered.
• Most human transcripts are not translated into peptides.
• NCRs are the most prevalent regulatory RNAs.
• NCRs play an important role in epigenetic control.
• NCRs include miRNAs, piRNAs, endogenous siRNAs, and long non-coding RNAs.

Processing of Non-Coding RNAs and the Role of RISC

• Regulatory functions of NCRs necessitate processing through the RNA-induced silencing complex (RISC).
• Argonaute (AGO) protein family members are needed for this process.
• An empty Argonaute (AGO) protein loads a small RNA duplex, forming pre-RISC.
• One strand, the passenger strand, is ejected from AGO leaving a complex consisting of AGO and a guide strand.
• AGO and a guide strand together is then called mature RISC, or simply RISC.
• RISC directs small RNAs to target complementary RNAs, repressing their expression via mRNA cleavage, degradation, or translational repression.

Silencing Mechanism

• The silencing mechanism is mediated by micro RNA (miRNA) and silencing RNA (siRNA).
• These are two types of non-coding RNAs that both participate in gene regulation.
• MiRNA originates from endogenous genes with 65–70 nucleotide pre-miRNA hairpin structures.
• These hairpin structures are processed into pre-miRNA in the nucleus.
• Pre-miRNA is then processed into mature miRNA by Dicer and loaded onto Argonaute (AGO2) protein.
• SiRNA is produced when long double-stranded RNA is cleaved into 21–25 nucleotide fragments by Dicer.
• These fragments are then loaded onto Argonaute (AGO2) protein.
• Loaded Argonaute proteins recognize and degrade mRNA complementary to the loaded guide strand.

Dr. Rayner's Discovery

• Dr. Rayner found microRNAs (specifically microRNA-33) regulates high-density lipoprotein and its atheroprotective effects.

Polyteny

• Polyteny occurs when chromosomes undergo multiple rounds of DNA replication without cell division, resulting in giant polytene chromosomes.
• Polytene chromosomes are easily visible under a microscope and exhibit distinct banding patterns reflecting regions of gene activity.
• Polytene chromosomes are studied in fruit fly (Drosophila spp.) salivary glands, as a model for understanding chromosome structure and gene regulation.
• Polytene chromosomes serve as a clear example of how chromatin structure epigenetically influences gene expression.

Genetic Research with Polyteny

• Polyteny can be a model for genetic research.
• There are variations of polyteny across species.
• Polytene chromosomes have particular structural and functional features.
• Regulatory control of polyteny occurs in Drosophila spp.
• Polyteny has functional significance.
• Labs use FlyBase in order to study polyteny.

Learning Outcomes

• Keywords: Polyteny, chromocentre, (a)synapsis, chromosomal band/interband/puffing, FISH, gene mapping, FlyBase, endocytosis/endomitosis/polyteny.
• List and describe five peculiarities of polytene chromosomes.
• List and briefly explain five applications of polytene in genetic research.
• Create a diagram of a polytene chromosome with zoomed-in sections highlighting five features.
• Explain why the number of visible polytene chromosomes corresponds to the haploid number.
• List the main molecular factors regulating polyteny in Drosophila spp. and briefly explain their functions.
• Discuss the pros and cons of polyteny in tumor prevention in mammals.
• Draw a puff on a polytene chromosome, highlighting key molecules present, and explain its effect on gene expression.
• Use a diagram to list and briefly explain the steps involved in the FISH protocol.

Polytene Chromosomes

• Polyteny is a chromosome structure in certain cells, where chromosomes undergo DNA replication without cell division.
• Polyteny results in large, multi-stranded chromosomes (up to thousands of DNA strand copies) known as polytene chromosomes.
• Polytene chromosomes are easily observed in light microscopy.
• Cells with polyteny often appear to have a haploid number of chromosomes as homologous chromosomes are tightly paired (synapsed) and aligned.
• Polytene chromosomes are connected via their chromocenter.
• Polytene chromosomes are commonly found in the salivary glands of fruit flies, and are present in other organisms such as protozoans, plants, and some mammalian cells.

Uses for Polytene Chromosomes

• Proof that chromosome theory of heredity, was discovered through polytene chromosomes.
• Polytene chromosomes have been used to, analyze hormonal action on gene activity, which were the start of the heat shock phenomenon discovered in the early 1960s.
• Polytene chromosomes are essential for gene mapping and basic cloning techniques, such as microcloning chromosome regions using a micromanipulator to create a library of DNA clones.

Polytene Chromosomes and Banding Patterns

• Electron microscopy analysis of banding patterns has been performed in region 9F13-10B3 of polytene chromosomes in Sordaria melanogaster.
• Biochemical characterization of proteins and DNA elements in 9F13 – 10B3 region of nonpolytene chromosomes in Sordaria melanogaster also has been performed
• A & B describe the Physical map of positions of v and sev genes.
• F & G describe Histone localization in two cell types displaying differing transcriptional levels.
• H describes Chromatin state in two cell types and the colored areas, which correspond to regions of condensed chromatin.

Polytene Chromosome Formation

• Polytene chromosome formation is associated with the elimination of the entire or partial mechanism of mitosis after each DNA doubling.
• At the end of each replication period, sister chromatids do not segregate; rather, they remain paired to each other to different degrees.
• Thick polytene chromosomes cannot undergo mitosis because sister chromatids and homologous chromosomes are bundled together, preventing alignment and segregation.

Variations in Polyteny

• Polyteny/polyploidy occurs when cells proliferate through cell division without fully completing mitosis.
  • In A, G1, S, G2 and M follow as in a normal mitotic cell.
  • In B, there is no M-phase, so that the cell is in permanent 'interphase' and result in multistranded chromosomes.
• Intermediate instances of polyteny/polyploidy occur when various phases of the cell cycle are partially or completely bypassed.
  • Mouse hepatocytes (HPCs) skip only cytokinesis (acytokinetic mitosis).
  • Mouse megakaryocytes (MKCs) exit mitosis during anaphase, re-entering G1 from metaphase, a variant called endomitosis.
  • Drosophila melanogaster salivary gland cells (SGs) re-enter a G1-like phase before fully completing DNA replication in S phase.

Banding Patterns

• Polytene chromosomes have distinct regions that can be observed due to different staining properties.
  • Bands: These are darkly stained regions with high DNA concentration rich in heterochromatin and DNA strands enclosed in nucleosomes.
  • Interbands: Lightly stained regions with less DNA rich in euchromatin appear between dark bands where DNA strands are loose.
• The banding pattern of polytene chromosomes is used to map genes and study chromosome structure and function.

Chromatid Stabilization

• Polytene chromosomes display 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, making the chromosome appear single.
• In Drosophila spp., the number of polytene chromosomes in the nucleus is reduced to the haploid chromosome number.
• Factors that contribute to hold sister chromatids and homologous chromosomes together:
  • Topological entanglement caused by DNA coiling.
  • Cohesin and condensin complexes.
  • Packing of heterochromatin in dense bands.
  • Cohesins and condensins are like molecular pins holding DNA strands together.

Gene Amplification and Underreplication

• In gene amplification a single region on a polytene chromosome undergoes rereplication multiple times allowing a higher transcriptional output.
• Underreplication refers to when certain regions of the chromosome do not replicate as fully as others resulting in fewer copies of DNA than expected and is often observed in heterochromatic regions.

Chromosome Puffs

• Chromosome puffs occur when there are high levels of transcription at a location resulting in localized expansion to accommodate transcriptional machinery.
• Polyteny (gene amplification) and chromosome puffing facilitate high transcription levels of intermolt genes during the third larval instar and prepupal stages of development.
• The extent of puffing is directly related to gene transcription levels also depending on what polymerases are accessible to locations.

Cyclin Dependent Kinase (CDK) Model

• S phase and M phase initiation are triggered by different 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 achieved, periodically, potentially by cyclin B–CDK1 and A- or E-cyclins complexed with CDK2.

Notch Signaling

• Transitioning from mitotic to endocycling in Drosophila melanogaster needs Notch signalling.
• Upregulation of Notch signaling leads to activation of the transcription factor Hindsight(Hnt) that:
  • Inhibits String (Stg), a Cdc25 homologue needed to dephosphorylate and activate Cdk1
  • Activates Hnt that prevents Cut factor from inhibiting APC/C
• Activated APC/C promotes proteasomal breakdown of mitotic cyclins and Stg.
• Downregulation of Stg and degradation of mitotic cyclins both suppress Cdk1 activity to form the Cdk1-B1 (or Cdk1-A/E complex) needed to trigger mitosis.
• Endoreplication occurs as the CDK activity cannot reach the mitotic threshold

Polyteny

• Polyteny facilitates rapid organ development and high functional capacity.
• It predominantly occurs in cells which are involved in intense secretory functions accomplished during short time.
• Polytene puffs are regions of polytene chromosomes where the chromatin has decondensed.
• These puffs are sites of active gene transcription and the areas contain an abundance of of RNA polymerase and other transcription factors.

Salivary Gland Studies

• A series of swollen segments as describe the the third chromosome of D. melanogaster, that are seen when dissecting and observing the salivary glands in lab 5.
• Third instar larvae use as much food as possible so as to build proper reserves needed for pupation including the secretions of digestive enzyme amylase.
• The saliva of third instar D. melanogaster larvae is rich in amylase, as evidenced by the reduced starch levels.

Polygeny in Mammals

• In mammals, trophoblast giant cells that grow and develop following pregnancy display polyteny within 4 hours post-fertilization.
• Trophoblasts are specialized cells form the outer blastocyst specialized in;
  • amplifying specific genes.
  • Growth and metabolic activity that are essential for nutrient transport and hormone production during pregnancy.
  • Placental development.

Polyteny

• Rodent and human hepatocytes display polyteny/polyploidy, enabling liver composition and maintenance.
• Liver ploidy elevates around the time of weaning, which higher levels reducing the risk of cancer.
• Liver exposure to oxidative stress and fatty liver, reduces tumor growth.

Tumor Suppressor Mutations

• Tumor suppressor mutations are prevalent in liver tumors from diploid and polyploid mice including the genes identified
• Polyploid hepatocytes, offer protection against cancer due to the extra copies of genes with the potential to mutate through gene compenstation.

Ploid

• final maximal ploidy achieved by endocycling cells is developmentally programmed showing that D. melanogaster obtains 1300C where as other cells in the body dont get the same results.
• Some tissues/organs display asynchronous polyteny with different rounds of endocycling and different ploydy numbers.
• In Lab 5, polytene chromsomes were dissected in the salivary glands of Sordaria fimicola, that is synrconous wit hhigh C numbers.

Flybase

• The National Center for Human Genome Research of the NIH has funded Flybase so create public data base surrounding genetic and molecular genetic insect related studies.
• The database includes; genes, alleles and phenotypes, genes, aberrations, clones, lists, references etc.

Database Navigation

• FlyBase's is not user based navigation but is still useful show main options.
• Chromsomes, maller, and section elements are used within in it.
• The numbering structure also includes number ranges.

Lab Options

• Visualize and explore the map to match a pattern of bands/interbands/puffs.
• Identify genes found at a specific cytological position, and estimate their transcriptional levels based on the banding pattern.
• Visualize the cytological position of a given gene