Regulation of Gene Expression in Eukaryotes

1. Regulation of Gene Expression inEukaryotes Dr. Jason R Mayberry Castle View High School

2. The Need for Regulation of Gene Expression Gene Regulation (Regulation of Gene Expression): factors that determine under what circumstances the information in genes are used to produce functional proteins. Why wouldn’t cells express all their genes all the time? Cell Cycle:Different genes are needed at different times in the cell’s cycle (e.g. for DNA Replication or for Mitosis) Energy Efficiency:Energy is wasted on producing proteins that aren’t always needed. Respond to the Environment:Cell Behavior often depends on which proteins are active at any given point in time; producing different proteins allows for different responses to environmental conditions. Cell Specialization in Multicellular Organisms:Different cell types in multicellular organisms have different functions, and therefore require a different set of proteins. 1 S G2 G1 M 2 3 4

3. Different Opportunities for Gene Expression Regulation Signal Eukaryotes Prokaryotes Chromatin Chromatin modification: DNA unpacking DNA Gene available for transcription Transcription Exon RNA Primary transcript Intron RNA processing Tail mRNA in nucleus Cap Transport to cytoplasm NUCLEUS CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypeptide Protein processing Active protein Degradation of protein Transport to cellular destination Cellular function (such as enzymatic activity or structural support)

4. Different Opportunities for Gene Expression Regulation Signal Eukaryotes • Every Step from DNA Structure to Functional Protein is a potential target for gene regulation • Generally: • Activators: Proteins that promote Transcription and Protein Production • Repressors: Proteins that inhibit Transcription and Protein Production • Major Targets for Gene Regulation • Epigenetics • Transcription Initiation • Alternate Splicing • Translation Initiation • mRNA Degradation • Protein Degradation • See Supplemental Video on Gene Regulation in Eukaryotes Chromatin Chromatin modification: DNA unpacking DNA Gene available for transcription Transcription Exon RNA Primary transcript Intron RNA processing Tail mRNA in nucleus Cap Transport to cytoplasm NUCLEUS CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypeptide Protein processing Active protein Degradation of protein Transport to cellular destination Cellular function (such as enzymatic activity or structural support)

5. Epigenetics Eukaryotic Gene Regulation

6. Epigenetics Epigenetics: Factors in addition to (on top of) nucleotide sequence that affect chromatin structure. 1) Histone Modification and Chromatin Packing 3) Chromosome Territories and Nuclear Regions 2) DNA Methylation CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 ...TTTGCCCTCGTACTGCTATT... ...AATAGCAGTACGAGGGCAAA... ...TTTGCCCTCGTACTGCTATT... ...AATAGCAGTACGAGGGCAAA...

7. Histone Modification and Chromatin Packing Nucleosome Structure Amino acids residues available for chemical modification Histone tails DNA Histone Histone Modification alters Chromatin Structure Acetylated histone tails Deacetylatedhistone tails = Acetylation(by Activator Proteins) De-acetylation(By Repressor Proteins) Compact: DNA not accessible for transcription Looser: DNA accessible for transcription

8. Methylation and Acetylation. Methylation of Histones (and Cytosine residues of DNA) causes nucleosomes to pack together tightly. Genes are inaccessible Acetylation of Histones causes nucleosomes to pack loosely Genes are accessible

9. DNA Methylation • DNA Methyl Transferase methylates cytosine residues • Causes nucleosomes to package tightly so genes are inaccessible • After DNA replication, enzymes recognize methylation on one strand and methylate the new strand • DNA methylation is only erased (usually) during sperm and oocyte formation.

10. Chromosome Territories • During Interphase, Chromosomes are arranged in Chromosome Territories • Chromosome Territories are dynamic and can be actively altered • Transcriptionally Inactive Regions are often found on the periphery of the nucleus • Transcriptionally Active Regions are often moved into centralized regions known as Transcription Factories Chromosomes in the interphase nucleus Chromosome territory 5 µm Chromatin loop Transcription factory

11. Transcription Eukaryotic Gene Regulation

12. Anatomy of a Gene (Basic) … … RNA DNA TF1 TF2 R-Pol Enhancer 1 Enhancer 2 Promoter Coding Portion For Cell Type “A” For Cell Type “B” • Regulatory Segments of Gene: Short DNA sequence that initiate transcription under the correct circumstances (not transcribed). • Transcribed Portion: entire transcribed region • Enhancers • Usually has multiple enhancers; one for each unique condition where it will to be expressed. • Can be in front of or behind the Coding Part of the Gene • Binds various Transcription Factors (TF) • Initiate Transcription under the right circumstances (time, place, conditions) • Remove Histones from Gene • Stabilize RNA polymerase at Promoter • Different cell types have different transcription factors and therefore make different proteins • Promoter • Bind RNA Polymerase which transcribes the Coding Part of the Gene • Initiation site for Transcription • Requires “activation” by proteins bound to Enhancers. • Coding Part of Gene: Sequence of nucleotides that is transcribed and which contains the information for making RNAs and Proteins. Different sequences code for different RNAs and Proteins Genes: Functional units of DNA that contains the information for making RNA and proteins.

13. Anatomy of a Gene (More Advanced) Poly-A signal sequence Distal control elements (Enhancers) Terminator Proximal control elements Transcription start site Exon Intron Exon Intron Exon DNA Upstream Downstream Promoter • RNA Polymerase binds to the promoter which marks the transcription start site. • Proximal Control Elements • General Transcription Factors bind to Proximal Control Elements, RNA Polymerase, and each other to stabilize RNA Polymerase and initiate low levels of – Form the Basal Transcription Complex • By themselves are only capable of initiating low levels of Transcription • Distal Control Elements (Enhancers) • At a significant distance upstream or downstream of the gene. • Bind to a small number of Specific Transcription Factors (Activators) that significantly enhance transcription under appropriate circumstances. • Each enhancer binds to a different set specific transcription activators and so enhances transcription under different circumstances.

14. DNA Bending Protein Promoter Activators DNA Gene Distal control element Enhancer TATA box DNA Bending Protein causes the DNA to bend so that Transcriptional Activators bound to Enhancers can interact with RNA Polymerase and General Transcription Factors. General transcription factors DNA-bending protein Group of mediator proteins RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis

15. Different Transcription Activators in Different Cell Types Enhancer for albumin gene DNA in both cells (activators not shown) Promoter Albumin gene Enhancer for crystallin gene Control elements Promoter Crystallin gene Liver cell DNA in liver cell Lens cell DNA in lens cell Liver cell nucleus Lens cell nucleus Available activators Available activators Albumin gene not expressed Albumin gene expressed Crystallin gene not expressed Crystallin gene expressed

16. Transcript Processing and Alternate Splicing Eukaryotic Gene Regulation

17. Alternate Splicing Exons 3 4 1 2 5 DNA Troponin T gene regulatory proteins control intron-exon choice by binding to regulatory sequences within the primary transcript Primary RNA transcript 1 2 3 4 5 RNA splicing 1 2 3 5 1 2 4 5 mRNA or

18. Beyond Transcript Processing Eukaryotic Gene Regulation

19. Regulation outside the nucleus CYTOPLASM mRNA in cytoplasm • Other Regulator Proteins also affect: • mRNA Degradation • Initiation of Translation • Protein Sorting • Degradation of Proteins • All as a means of regulating gene expression. • See Supplemental Video on Post Transcription Control Degradation of mRNA Translation Polypeptide Protein processing Active protein Degradation of protein Transport to cellular destination Cellular function (such as enzymatic activity or structural support)

20. non-coding RNA and Gene Regulation Eukaryotic Gene Regulation

21. Human Genome Composition • Only ~1.5 % of the human Genome codes for proteins. • The majority of the genome is made of transposable elements (i.e. selfish/parasitic DNA) making no contribution to the organism. • 20-35% of the genome is involved in Gene regulation. • A significant transcribed regions produces various types of regulatory non-coding RNA (ncRNA).

22. Ribonucleic Acids (RNA) A HN H O– O– O– O– H+ H+ H+ H+ C H3C C CH N O O O O P P P P O O O O C A NH2 CH C O H+ H+ H+ H+ O– O– O– O– C N N C N Thymine A HC O C C O Uracil N N H H+ C O– C H N H3C C C O H T N H3C C U P C CH O C N O H+ N CH2 O O– O U U OH H Cytosine Guanine G OH O C N OH C N H G HC C C N N OH N H H CH2 CH2 CH2 CH2 O O O O OH OH OH OH OH OH H H H H OH Purines Pyramidines • RNA differs from DNA: • RNA has the same nucleotides but replaces Thymine with Uracil. • RNA replaces and “H” with an “OH” on ones of its sugar’s carbons • RNA almost always remains single stranded • Can fold and behave like enzymes • Different types of RNA • mRNA (messenger RNA) sequence of nucleotides is read to determine amino acid sequence during translation. • tRNA (transfer RNA) “cipher” which converts mRNA codon sequence to an amino acid. • rRNA (ribosomal RNA) major component of Ribosomes, which carry out translation. • Some small RNAs help regulate gene expression • ATP, (also GTP etc.) provide chemical energy to fuel many reactions. OH OH Adenine OH OH mRNA tRNA

23. ncRNA • Examples of Types of Non Coding RNA • miRNA (micro RNA) • Degrade complementary mRNA • Block Translation • siRNA (small interfering RNA) • Degrade complementary mRNA • Participate in cromatin condensation at the centromere • piRNA (piwi-interacting RNAs) • Induce the formation of heterochromatin, blocking transposon expression • Alter methylation patterns in germ producing cells • lncRNA (long noncoding RNA) • Responsible for X-chromosome inactivation in women • May scaffold formation of DNA, proteins, and RNA complexes involved in chromatin remodeling • shRNA (Short hairpin RNA) • Artificial RNA molecule used in RNA interference (RNAi) • RNAi: when small ncRNAs are used experimentally to interfere with gene expression of specific genes

24. RNA interference (RNAi) via shRNA • Plasmid containing gene for shRNA complementary to target mRNA introduced via lipid-based or viral transfection Plasmid with gene for shRNA against target cell membrane • Transcription of shRNA plasmid results in a short RNA with a hairpin loop • Dicer removes loop yielding a double stranded, siRNA(silencing RNA) (siRNA can also be introduced directly with transient effect) Dicer • RISC(RNA Induced Silencing Complex) unwinds the RNA helix and Argonautedestroys one strand. RISC • siRNA/RISC complex associate with target mRNA via complementarity with guide RNA strand mRNA for Gene to be knocked down • siRNA/RISC complex degrades the mRNA – thus silencing gene expression.

25. Signal Transduction (Brief Introduction) Signal • If activators and repressors regulate gene expression… • What regulates the activators and repressors? Chromatin Chromatin modification: DNA unpacking DNA Gene available for transcription Transcription Exon RNA Primary transcript Intron RNA processing Tail mRNA in nucleus Cap Transport to cytoplasm NUCLEUS CYTOPLASM

26. Signal Transduction (a brief introduction) Eukaryotic Gene Regulation

27. General Aspects of Cell Signaling • Signalling Molecule: Molecule found in a cell’s environment (e.g. Hormones, pheromones, sugars, etc) • Receptor: Protein that binds reversibly to a specific signaling molecule and initiates a cellular response • Transmembrane (Integral) receptors detect signals that do not cross the membrane (usually hydrophilic molecules) • Cytosolic receptors detect molecules that cross the plasma membrane (usually lipophilic) • To respond to a signal, a cell must have a Receptor for the specific molecule • Types of Receptors • Chemoreceptors: respond to molecules from outside the cell known as the Ligand (depicted here) • Mechanoreceptors: respond to force, touch, or vibration • Thermoreceptors: respond to temperature changes • Photoreceptors: respond to light Gene Enhancer Prom.

28. General Aspects of Cell Signaling c) b) a) LatentEnzyme R-PII • Signal Transduction: sequence of events molecular interactions within a cell beginning with a signaling molecule binding to a receptor and leading to a cellular response to a signal • Binding and Conformational change: ligand binds reversibly to receptor causing a shape change affecting the intracellular portion • Signal Transduction Cascade/Pathway: • Intracellular molecules detect change in receptor shape • the “message” is often passed from one molecule to another until the final target is reached. • Cellular Response: cells can respond to a signal in three general ways: • Activation of Transcription Activators or Repressors • Activation of Enzymes in the cytosol • Alteration of the cytoskeleton to initiate shape changes or cell migration. Gene Enhancer Prom.

29. Types of Cell-Cell Signals in Multicellular Organisms Local Signaling Direct Cytosolic: Signals such as ions and small signaling molecules pass directly between cells through gap junctions Paracrine: Signals between neighboring cells where the signalling molecule is released into the extracellular space. Autocrine: When a cell signals to itself (makes both the signaling molecule and the receptor) Juxtacrine(Contact-Dependent): Signals between neighboring cells where the signaling molecules is bound to one of the cell’s surface OH O HO OH O e.g. Cardiac muscle cells e.g. Axon Growth, certain immune responses e.g. Neuron-neuron via neurotransmitters e.g. Density dependent growth. Long Distance Signaling Note: Cell Signalling is also essential for single cellular organisms, though we will generally focus on multicellular organisms Endocrine: Signaling molecules are released into the blood stream and affect cells over great distances throughout the body

30. Interpreting the Results of Cell Signaling Activation Repression

31. Differential Gene Expression • Gene Expression: the process of transcription and translation resulting in the production of proteins. • Differential Gene Expression: The expression of different genes in different cell types of multicellular organisms • Every cell comprising an individual has the same DNA • Cells differing in type, differ in which genes they express ∴ proteins • Results from differential gene regulation in different cell types.

32. Differential Gene Expression and Cell Specialization • All Cells in a multicellular organism have the same DNA • Differential Gene Expression: Cells specialize for a given task by expressing only those genes that are required for their function • Proteome: • The protein content of a cell • Includes both the types of proteins and the relative abundance of each • Represents only a subset of the possible proteins that could be expressed using the genome. 1n 1n 1n+1n Zygote Mitosis Embryo 2n Many Rounds of Mitosis Cell Type Genome Proteome Thousands to Trillions of Cells with the same genome Skin Keratin, Actin, etc. Keratin Hemoglobyn Actin Crystallin Eye Lens Crystallin, Actin, etc. Keratin Hemoglobyn Actin Crystallin Red Blood Cell Hemoglobyn, Actin, etc. Keratin Hemoglobyn Actin Crystallin

33. Differential Gene Expression and Cell Differentiation Embryonic Stem Cells Ectoderm Mesoderm Endoderm Internal Organs Mem-branes Muscle, Blood, Spleen, Kidney, Gonads Bone GI Tract Nerve, Neural Endo-crine Organs Skin Gametes Placenta Fertilization • Stem Cell: • Non-differentiated cell. • Capable of producing cells that can become a variety of different cell types. • Differentiation: • Process by which cells become specialized through differential gene expression. • Cell signaling during development initiates differential gene expression, differentiation, and specialization. Morula Blastocyst Trophoblast Inner Cell Mass Gastrulation