Epigenetics and Non-coding RNAs

• The slides are about Epigenetics, the Epigenetic code, Non-coding RNAs and Polyteny

Epigenetics

• The regulation of gene transcription by non-coding RNAs (NCRs) is a relatively recent discovery.
• This is a highly active area of research
• Most human transcripts are not translated into peptides, but most of these NCRs nonetheless serve vital functions.
• Originally it was theught that genes would only regulate gene expression at the post-transcriptional level.
• Now it is suggested that non-coding RNAs (NCRs) are the most prevalent regulatory RNAs, playing a significant role in epigenetic control
• NCRs include miRNAs, piRNAs, endogenous siRNAs, and long non-coding RNAs

RISC Complex

• To achieve regulatory functions, NCRs are processed through the RNA-induced silencing complex (RISC) together with a member of the Argonaute (AGO) protein family
• An empty Argonaute (AGO) loads a small RNA duplex to form pre-RISC
• One strand (passenger strand) is ejected from AGO
• The complex consisting of AGO and a guide strand is called mature RISC
• RISC then targets complementary RNAs and represses their expression through mRNA cleavage, degradation, and/or translational repression
• The silencing mechanism is mediated by micro RNA (miRNA) and silencing RNA (siRNA), which are two types of non-coding RNAs involved in gene regulation
• MIRNA is 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 and loaded to protein Argonaute (AGO2)
• SIRNA is derived from the long double-stranded RNA molecule and is cut into fragments of 21-25 nt by the Dicer enzyme, then loaded to protein Argonaute (AGO2)
• The loaded Argonaute protein can further recognize and degrade mRNA that is complementary to the loaded guide strand
• Dr. Rayner discovered a role for microRNAs – specifically microRNA-33 in the regulation of high-density lipoprotein and its atheroprotective effects
• Macrophages in the atherosclerotic plaque accumulate cholesterol, initiating inflammation that drives lesion progression
• microRNAs (miRNAs) are small non-coding RNAs that play important roles in normal and pathophysiology
• A number of important miRNAs control lipid metabolism and inflammatory activation in macrophages in the plaque
• New RNA therapies show success in clinical trials and miRNA-based therapy may be exploited to treat atherosclerosis

Polyteny

• Polyteny happens when chromosomes undergo multiple rounds of DNA replication without cell division, resulting in the formation of giant polytene chromosomes
• These chromosomes are easily visible under a microscope and exhibit distinct banding patterns that reflect regions of gene activity
• Polytene chromosomes are most famously studied in the salivary glands of fruit flies (Drosophila spp.), serving as a valuable model for understanding chromosome structure and gene regulation
• Polyteny may be used as genetic research
• Variations of polyteny across species
• Structural and functional features of polytene chromosomes
• Regulatory control of polyteny in Drosophila spp
• Functional significance of polyteny

Relevant Model for Genetic Research

• Polyteny refers to a special type of chromosome structure where chromosomes undergo multiple rounds of DNA replication without cell division
• This results in large, multi-stranded chromosomes (up to thousands of DNA strand copies) known as polytene chromosomes
• Polytene chromosomes are so large that they can be observed in light microscopy
• Cells with polyteny often appear to have a haploid number of chromosomes because the homologous chromosomes are tightly paired (synapsed) and aligned
• Polytene chromosomes stay connected via their chromocenter
• Polytene chromosomes are commonly found in the salivary glands of some insects, such as fruit flies (Drosophila spp.), and are also present in other organisms like protozoans, plants, and some mammalian cells
• Data from polytene chromosomes provided final proof for the chromosome theory of heredity
• Gene activity was observed directly under the microscope as puff formation
• Polytene chromosomes were used to analyze hormonal action on gene activity
• Polytene chromosomes were used to discover the heat shock phenomenon 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
• The formation of polytene chromosomes is associated with eliminating the entire or partial mechanism of mitosis after each DNA doubling.
• At the end of each replication period, sister chromatids do not segregate and remain paired to each other to different degrees
• Thick polytene chromosomes cannot undergo mitosis due to the copies sister chromatids and homologous chromosomes being bundled together

Variations of Polyteny

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

Common Features of Polytene Chromosomes

• Bands and interbands are distinct regions that can be observed under a microscope due to their different staining properties
• Bands are darkly stained regions that contain a high concentration of DNA
• Bands are rich in heterochromatin and are typically less active in gene transcription
• Interbands are lightly stained regions that contain less DNA compared to bands
• Interbands are rich in euchromatin, and are more active in gene transcription, appearing as thin, light stripes between the dark bands
• The banding pattern of polytene chromosomes is used to map genes and study chromosome structure and function
• Polytene chromosomes display synapsis of homologous chromosomes and sister chromatids
• Somatic synapsis is based on the association of homologous polytene chromosomes and sister chromatids
• Both homologues and chromatids synapse band to band resulting in the impression that the chromosome is single
• The number of polytene chromosomes in the nucleus is reduced to the haploid chromosome number in Drosophila spp

Feature Stabilization

• Factors contributing to holding sister chromatids and homologous chromosomes include 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 occurs when a single region on a polytene chromosome is rereplicated multiple times
• This allows extra transcriptional output when a small number of transcripts are needed in very large quantity
• Underreplication refers to certain regions of the chromosome do not replicate as fully as others
• This results in some parts of the chromosome having fewer copies of DNA than expected
• Underreplication is often observed in heterochromatic regions, which are typically less active in gene transcription
• Chromosome puffs occur when high levels of transcription at a particular location result in localized expansion of the polytene structure to accommodate the transcriptional machinery

Regulatory Control

• S phase and M phase initiation are triggered by quantitatively different thresholds of cyclin-dependent kinase (CDK) activity
• The S phase threshold would typically be fulfilled by E- or A-type cyclins complexed with CDK2
• The M phase threshold would be fulfilled by B- or A-type cyclins complexed with CDK1
• In endocycling cells, only the S phase threshold is achieved, periodically, and this could be executed by cyclin B-CDK1
• Upregulation of Notch signalling leads to activation of the transcription factor Hindsight(Hnt)
• The Hnt inhibits String (Stg), a Cdc25 homologue needed to dephosphorylate and activate Cdk1, and it activates Hnt that prevents the Cut factor from inhibiting APC/C (anaphase promoting complex/cyclosome)
• The activated APC/C promotes the proteasomal breakdown of mitotic cyclins and Stg
• Downregulation of Stg and degradation of mitotic cyclins both suppress Cdk1 activity needed to form the Cdk1-B1 (or Cdk1-A/E complex) that is needed to trigger mitosis
• Endoreplication occurs as the CDK activity cannot reach the mitotic threshold

Significance of Polyteny

• Polyteny arises in tissues, organs and at developmental stages when there is need for the rapid development of an organ at an unaltered high level of function
• Organs containing cells with polytene chromosomes are involved in intense secretory functions accomplished during a short time against a background of rapid growth
• Polytene puffs are regions of polytene chromosomes where the chromatin has decondensed, making the DNA more accessible for transcription
• These puffs are sites of active gene expression, and the relative gene expression level in polytene puffs is significantly higher compared to non-puffed regions
• This increased activity is due to the high concentration of RNA polymerase and other transcription factors in these areas, leading to robust transcription of the genes
• A series of swollen segments are the salient features of the third chromosome of D. melanogaster
• Third instar larvae need to consume as much food as possible to build up enough reserves for the metabolically demanding pupation phase
• Polytene chromosomes in the salivary glands are likely essential for secreting digestive enzymes like amylase
• The saliva secreted by third instar D. melanogaster larvae is rich in amylase, as evidenced by the reduced starch levels
• In mammals, trophoblast giant cells display polyteny within 4 hours post-fertilization
• Trophoblasts are specialized cells that form the outer layer of the blastocyst and display high metabolic activity
• Trophoblasts include the amplification of specific genes, support the growth and metabolic activity that are essential for nutrient transport and hormone production during pregnancy and support for placental development and integrity
• Up to 90% of rodent and about 50% of human hepatocytes display polyteny/polyploidy, making the liver one of the largest polyploid organs in mammals
• Liver ploidy significantly rises around the time of weaning and continues to grow with age
• Early Weaning leads to higher levels of polyteny/polyploidy, which is linked to a reduced risk of developing hepatic tumors
• Liver exposure to conditions such as oxidative stress and fatty liver disease promotes polyteny/polyploidy, and lower cancer risk
• Tumor suppressor mutations are prevalent in liver tumors from diploid and polyploid mice
• 242 of the most commonly mutated genes in human and murine hepatocellular carcinoma were sequenced, with red for oncogenes and black for tumor suppressing genes
• Polyploid hepatocytes have multiple sets of chromosomes and offer protection against cancer
• In the process of gene compensation, The presence of extra copies of genes, which can buffer against the loss or mutation of tumor suppressor genes, thereby reducing the risk of cancer development

Lab 5 and FlyBase

• The final maximal ploidy achieved by endocycling cells is developmentally programmed with salivary gland cells of D. melanogaster typically achieve ~1,300C, whereas fat body cells endocycle to ~256C and midgut to 32C
• Some tissues/organs display asynchronous polyteny with some cells experiencing different rounds of endocycling and resulting in different ploidy numbers among cells

FlyBase Database Navigation

• Beginning in 1992, the National Center for Human Genome Research of the NIH has funded the FlyBase project with the objective of designing, building and releasing a database of genetic and molecular information concerning this insect
• Genetic information on the FlyBase include genes, alleles (and phenotypes), aberrations, clones, stock lists and bibliographic references
• FlyBase is not a friendly-user database, but polytene chromosomes have been characterized following different systems, including chromosomes, Muller elements and sections
• Range division naming in FlyBase is as follows: 1) Division: 1 to 102 2) Subdivision: A to F 3)Band number: 1 to n, depending upon the particular subdivision
• 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