Developmental Biology Lecture Notes PDF

BIOL 413 – Developmental Biology Differential Gene Expression: The Gene’s Role in Development Genomic Equivalence “ Each somatic cell nucleus has the same chromosomes -and therefore the same set of genes- as all the other somatic nuclei” This is known as GENOMIC EQUIVALE...

BIOL 413 – Developmental Biology Differential Gene Expression: The Gene’s Role in Development Genomic Equivalence “ Each somatic cell nucleus has the same chromosomes -and therefore the same set of genes- as all the other somatic nuclei” This is known as GENOMIC EQUIVALENCE Genomic Equivalence If all cells have the same genetic material, how come only red blood cells make hemoglobin and insulin is provided by some of the pancreatic cells? Images from Wikipedia.org  THE ANSWER IS: Differential gene expression Differential gene expression Three important points: 1. The DNAs of all differentiated cells are identical. Differential gene expression Three important points: 2. The unused genes in differentiated cells are neither destroyed nor mutated. (But they keep their potential for being expressed) Differential gene expression Three important points: 3. Only a small percentage of the genome is expressed in each cell. A portion of the RNA synthesized in each cell is specific for that cell type. Gene expression Gene expression can be regulated at several levels: - Differential gene transcription regulation - Selective nuclear RNA processing - Selective messenger RNA translation - Differential protein modification Gene expression Gene expression can be regulated at several levels: - Differential gene transcription regulation - Selective nuclear RNA processing - Selective messenger RNA translation - Differential protein modification Studies in Drosophila Observations of polythene chromosomes, with no structural differences between cells, counted as proof of genomic equivalence. http://msg.ucsf.edu/sedat/Images/polytene2.gif One major question: Has the nucleus of a differentiated cell undergone an irreversible change? One major question: One major question: One major question: Cloning of an adult mammal Dolly (left) with Bonnie (via normal reproduction) Dolly with Professor Sir Ian Wilmut, who led the research which produced her. (Image credit: Photo courtesy of The Roslin Institute, The University of Edinburgh) Cloning of adult mammals Cc with her pups… (cloned in 2001) Gene expression Gene expression can be regulated at several levels: - Differential gene transcription regulation - Selective nuclear RNA processing - Selective messenger RNA translation - Differential protein translation Differential gene transcription regulation One of the main differences between most eukaryotic and prokaryotic genes: the presence of CHROMATIN to ‘store’ the eukaryotic genes. Differential gene transcription regulation CHROMATIN The nucleosome is made of octamer of histone proteins.  Methyl groups condense nucleosomes more tightly. They prevent access to promoter sites and prevent gene transcription.  Acetylation loosens nucleosome packing. This exposes the DNA to RNA polymerase II and transcription factors that will activate the genes. Anatomy of the gene: Active and repressed chromatin HISTONES act as on/off switch. Modifications on the ‘tails’ of histones H3 and H4 Anatomy of the gene: Active and repressed chromatin - Histone Acetylation Addition of negatively charged acetly groups (COCH3) - ACTIVATES TRANSCRIPTION Anatomy of the gene: Active and repressed chromatin - Histone Methylation Addition of methyl (CH3) groups to the histones. - CAN EITHER ACTIVATE OR FURTHER REPRESS TRANSCRIPTION (depending on the amino acid being methylated and the presence of other methyl or acetyl groups nearby). Histone Methylation on histone H3 Methylated lysines @ positions 4, 38, and 79  associated with gene activation. Methylated lysines @ positions 9 and 27associated with repression. The proteins binding these sites (not shown to scale) are represented above the methyl group.  Apart from histone methylation, there is also DNA metylation… DO NOT CONFUSE THE TWO!!! Anatomy of the gene: Exons and introns Exons- nucleotide sequence whose RNA ‘exits’ the nucleus. Introns- Intervening sequences between exons. Anatomy of the gene: Exons and introns Anatomy of the gene: Exons and introns Anatomy of the gene: Exons and introns Promoter region Transcription initiation site 5’ untranslated region (5’ UTR) Translation initiation site Anatomy of the gene: Exons and introns Translation termination codon 3’ Untranslated region (3’ UTR) Transcription termination sequence Anatomy of the gene: Exons and introns What is the original transcription product called? Anatomy of the gene: Exons and introns The original transcriptional product is called nuclear RNA Anatomy of the gene: Exons and introns The export of the mRNA from nucleus to the cytoplasm is a complex and a delicate process. (Tutucci and Stutz, 2011). Anatomy of the gene: Promoters and enhancer Regulatory sequences can be located on either end of the gene. PROMOTERS and ENHANCERS Necessary for controlling where and when a particular gene is transcribed. Anatomy of the gene: Promoters and enhancer PROMOTERS Many of them have the CpG islands. Anatomy of the gene: Promoters and enhancer ENHANCER Recruit and stabilized RNA Pol II on the promoters. Enhancers bind to specific TRANSCRIPTION FACTORS. The Mediator Complex: Linking enhancer and promoter In many genes, a bridge between enhancer and promoter is made by the MEDIATOR. Mediator is a large, multimeric complex. Enhancer Functioning How can we identify enhancer sequences for a gene of interest? Gene of interest Clone the flanking region Enhancer Functioning How can we identify enhancer sequences for a gene of interest? FUSE THIS REGION WITH A REPORTER GENE Enhancer Functioning How can we identify enhancer sequences for a gene of interest? GFP FUSE THIS REGION WITH A REPORTER GENE https://doi.org/10.1016/j.ydbio.2011.09.012 Emerson& Cepko (2011) What is meant by cloning? Cloning - the process of producing similar populations of genetically identical individuals that can occur in nature when organisms such as bacteria, insects or plants reproduce asexually. What is meant by cloning? OR Cloning (using modern biotechnology) processes used to create copies of: DNA fragments (molecular cloning), cells (cell cloning), or organisms (organismal cloning) in the lab or in a clinical setting. A reporter gene is a gene that researchers attach to a regulatory sequence of another gene of interest. Takechi et al. 2003  How does having these enhancers identified help us in developmental research? McWhorter et al. 2003 Enhancer Modularity The enhancer sequences on the DNA are the same in every cell type. Then why do we have different expressions? Different combination of transcription factor proteins. Differential Gene Transcription X chromosome inactivation Selective Nuclear RNA Processing Selective Nuclear RNA Processing regulating which processed RNAs are exported to the cytoplasm and become messenger RNAs (mRNAs). regulating how the transcribed RNAs are processed (spliced to remove introns and include specific exons) to produce a protein. RNA Selection Each nucleus has the same pool of RNA transcripts BUT different ones are selected and processed into RNA messages. This results in different proteins being expressed in the same cell. Gene expression Gene expression can be regulated at several levels: - Differential gene transcription regulation - Selective nuclear RNA processing - Selective messenger RNA translation - Differential protein translation Alternate nRNA splicing Alternate nRNA splicing is a means of producing a wide variety of proteins from the same gene. Most mammalian nRNAs contain numerous exons. Alternate nRNA splicing Based on the determination of which sequences will be spliced out as introns. Mediated through complexes known as spliceosomes that bind to the splice sites. Alternate nRNA splicing Spliceosomes are made up of small nuclear RNAs (snRNAs) and proteins called splicing factors. A sequence that is an exon in one cell type may be an intron in another. Alternate nRNA splicing examples Alternative splicing patterns are shown with V-shaped lines. Expressed in the nervous system. Disturbance sof this gene in humans may lead to the neurological defects of Down syndrome. (B) Dscam is required for self-avoidance between dendrites that fosters a dispersed pattern of dendrites (left). Loss of Dscam in Drosophila, however, causes crossing and fasciculated growth of dendrites from the same neuron (right; arrows). Dev Bio. 11th ed. Fig 3.25  About 92% of human genes produce multiple types of mRNA. 20,000 genes  100,000s different proteins. The PROTEOME is far more complex. https://www.nature.com/articles/d41586-021-02530-6 https://www.nature.com/articles/d41586-021-01994-w  Some splicing enhancers are specific for certain tissues. Mutations in splicing sites can lead to alternative developmental phenotypes. Muscle hypertrophy through mispliced RNA Control of Gene Expression at the Level of Translation It is not guaranteed that RNA reaching the cytoplasm gets translated. How though? Control of Gene Expression at the Level of Translation Ways gene expression can be controlled at the level of translation: Differential Stability/ mRNA longevity -dependent on the length of the polyA tail -proteins that affect specific mRNA stability Degradation of casein mRNA in the presence and absence of prolactin. Stored oocyte mRNAs: Selective inhibition of mRNA translation Prior to meiosis, the oocyte often makes and stores mRNAs that will be used only after fertilization occurs. These messages stay in a dormant state until they are activated by ion signals that spread through the egg during ovulation or fertilization. Stored oocyte mRNAs: Selective inhibition of mRNA translation Some of these stored mRNAs encode proteins that will be needed during cleavage, when the embryo makes enormous amounts of chromatin, cell membranes, and cytoskeletal components. Stored oocyte mRNAs: Selective inhibition of mRNA translation The stored mRNAs and proteins are referred to as maternal contributions (produced from the maternal genome) In many species (including sea urchins, Drosophila, and zebrafish), maintenance of the normal rate and pattern of early cell divisions does not require DNA or even a nucleus! Rather, it requires continued protein synthesis from the stored maternally contributed mRNAs. Maternal contributions to DNA replication in the zebrafish blastula. A) Wild-type blastulae (B) futile cycle mutants Although the correct number of cells is present in futile cycle mutants, they consistently show only two labeled nuclei, indicating that these mutants fail to undergo pronuclear fusion. Control of Gene Expression at the Level of Translation Ways gene expression can be controlled at the level of translation: Selective Inhibition of Translation -protein complexes needed to assemble properly for the positioning of the ribosome and for translation to begin Control of Gene Expression at the Level of Translation Translation Regulation in Oocytes Control of Gene Expression at the Level of Translation Ways gene expression can be controlled at the level of translation: Cytoplasmic localization -mRNA is selectively anchored by proteins to specific locations in the cell -mRNA is selectively protected against degradation -mRNA is actively transported along the cytoskeleton Control of Gene Expression at the Level of Translation Ways gene expression can be controlled at the level of translation: microRNAs Control of Gene Expression at the Level of Translation microRNAs inhibit translation 22 nucleotides made from longer precursors bind to 3’UTR of target mRNA block translation or promote mRNA cleavage Control of Gene Expression at the Level of Translation Differential protein modification regulates which proteins remain and are functional within a cell Examples: hemoglobin, insulin Proteins are cleaved, phosphorylated, or processed post-translationally in different ways and this modification can affect the function of the protein. Control of Gene Expression at the Level of Translation Differential protein modification