Molecular Biology. BIO3001(Summary), Schemi e mappe concettuali di Biologia

Scarica Molecular Biology. BIO3001(Summary) e più Schemi e mappe concettuali in PDF di Biologia solo su Docsity! Running head: Molecular Biology — BIO3001 Molecular Biology BIO3001 Summary Name Institution Course Tutor Date Molecular Biology — BIO3001 BIO3001 - Molecular Biology Table of Contents Animal Models & Molecular Techniques 3 Signal Transduction 15 Transcriptional Control of Gene Expression 30 Epigenetics and Non-coding RNA Error! Bookmark not defined. Cancer (24.4 & 24.5) 56 Stem Cells, Asymmetry & Cell Death 68 Molecular Biology — BIO3001 5 them to a nonpermissive temperature for analysis of the mutant phenotype. e Inbreeding: used to study recessive lethal mutations in diploids. Mutations can be maintained in heterozygotes, of which the progeny contains recessive homozygotes. Consequences of the mutation can be analyzed in the homozygotes. e Complementation analysis: restoration of the wild-type phenotype by mating two different mutants. If two recessive mutations, A and B, are in the same gene, then a diploid organism carrying one A allele and one B allele will exhibit the mutant phenotype because neither allele provides a functional copy of the gene. In contrast, if mutations A and B are in separate genes (complementing), then heterozygotes carrying a single copy of each mutant allele will not exhibit the mutant phenotype because a wild-type allele of each gene is also present. e Double mutations: can be used to determine the order in which proteins function (biosynthetic pathways, signaling pathways) and their functionally significant interactions. o Suppressor mutations: when a mutation in protein A disrupts the ability to associate with protein B in the same cellular process. Similarly, mutations in protein B lead to small structural changes that inhibit its ability to interact with protein A. normal functioning of protein A and B thus depends on their interaction. o Synthetic lethality: the deleterious effect of one mutation is increased by a second mutation in a related gene. Mutation A for example inactivates a process, of which another process can take over. Mutation B inactivates the Molecular Biology — BIO3001 6 other process, of which the original one can take over. If both happen simultaneously, the mutations become lethal. e Genetic mapping studies: genetic analysis based on gene position, the less frequently recombination (crossing over) is observed to occur between two genes on the same chromosome, the closer together they must be (linked/same gene vs unlinked/different gene). Genetic markers allow for determination of the position of unmapped mutations, determined by assessing its segregation during meiosis. The more markers available, the more precisely a location can be mapped. Isolation: obtain discrete, small regions of an organism’s DNA that constitute specific genes. Needed to perform detailed studies of the structure and function of a gene at molecular level. Uses recombinant DNA technology to obtain large quantities of DNA in pure form. e DNA cloning: the genome is cut into restriction fragments by restriction enzymes/DNA ligases (endonucleases), DNA fragments of interest are then linked to a vector (plasmid) DNA molecule that can replicate within a host cell. The inserted DNA is replicated along with the vector, generating a lot of identical DNA molecules. The isolated DNA fragment can be used for subsequent analysis (sequencing/manipulation). o Cutting: performed by restriction enzymes which recognize 4-8bp restriction site sequences. Restriction enzymes generate fragments that have a single-stranded tail at both ends (sticky ends), which are complementary to those on other fragments and can therefore base-pair. Molecular Biology — BIO3001 The frequency of the cutting and the fragment length depends on the length of the restriction site. Insertion: DNA fragments are inserted into vector DNA with the aid of DNA ligase, which covalently joins the ends of a restriction fragment and vector DNA that have complementary ends (easier with sticky ends than with blunt ends). Plasmids are circular, bacterial dasbNA molecules that replicate separately from a cell’s chromosomal DNA, which are often used as vector. They contain three regions essential for DNA cloning: a replication origin (ORI), a marker that permits selection ' sent \ to bo cionod | Enzymaricaliy Insere DIA Into plasmid vector ci gar E. coli pletos containing ampiciln chremosome o Transformed cell Celle that do not tak up pla n ampici lin platos | rise scor BW | siii BD& 0 SB 9 o ZE Coleny of cells, each containing copiss Gf the same recombinont piasmici (drug resistant gene), and an insertion region for exogenous DNA fragments. The versatility if an E. coli plasmid vector can be increased by the addition of a polylinker (restriction site), which is a synthetically generated sequence containing one copy of each different restriction site that is not present elsewhere in the plasmid sequence. This restriction site is cleaved by two restriction enzymes in order to accept an exogenous DNA fragment with the right sticky ends (eliminates unwanted by products). Shuttle vectors are capable of propagation in different hosts. Bacterial cells will take up the plasmid DNA by the process of transformation, which happens when cells are mixed with recombinant DNA and Molecular Biology — BIO3001 10 hybridizing the fixed DNA to a labelled DNA probe complementary to the gene of interest, removal of un-hybridized probe by washing, and detection of the specifically hybridized probe by autoradiography. e DNA microarray analysis: monitors relative level of expression of thousands of genes at once. Organized array of thousands of closely packed, gene-specific DNA sequences attached to the surface of a glass microscope slide (DNA chips). ecDNAs are prepared from mRNAs expressed by cells and fluorescently labeled. cDNA is then applied to microarray, DNA spots representing genes that are expressed will hybridize to the complementary DNAs in the probe mix and can subsequently be detected in a scanning laser microscope. Expression levels of many genes can be measured simultaneously, allowing for comparison of sick and healthy cells e Cluster analysis: combines information from a set of microarray expression analyses to find genes that are similarly regulated under a variety of conditions over a period of time. Identifies co-regulated genes with similar changes in expression (participate in common cellular processes). A computer program can organize data and cluster genes that show similar expression Modulation: gene expression and protein function can be modulated to identify the role of such gene/protein in the cell. e Gene knockout: modified genes are incorporated into the germ line at their original genomic location by homologous recombination, thereby producing gene knockouts with a loss of function mutation, which provide a model for human Molecular Biology — BIO3001 ll genetic diseases. A DNA construct containing a disrupted allele of the target gene is introduces into embryonic stem (ES) cells. In a small fraction of transfected cells, the DNA undergoes homologous recombination with the target gene. The recombinant DNA construct includes two selectable marker genes (neoR, tkHSV) to enable selection for cells in which recombination occurred. neoR is incorporated within the target gene, and tkHSv is incorporated outside the target gene sequence. ES cells that undergo recombination will therefore contain neoR but will not incorporate thHSV, and can be selected by their ability to survive the presence of G-418 and ganicoclovir. ES ells heterozygous for a knockout mutation are injected into a wild-type mouse blastocyst, which is subsequently transferred into a pseudo pregnant female mouse. Progeny will contain tissues derived from both ES cells and host cells. Mating of heterozygous mice will produce progeny homozygous for the knockout mutation. o Conventional: for all cells o Conditional: knock out of genes in specific tissues/organs or at specific time points with use of the loxP-Cre recombination system to allow the study of essential genes. Antibiotics are used to activate cre protein, which catalyzes recombination. A loxP mouse (with loxP in intron of target gene) and a cre mouse (with gene X knockout and cre gene linked to cell-type specific promoter) are crossed. LoxP-Cre mice produce cre protein only in cells in which the promoter is active, which means that only in those cells homologous recombination occurs, leading to deletion of gene X. The other allele is a constitutive gene knockout, so deletion of the gene results in complete loss of function of gene X in all cells expressing Cre. Molecular Biology — BIO3001 12 e Transfection: genes are cloned in eukaryotic vectors and introduced in animals to allow expression of a genetically modified protein in animal cells and examine the function of a protein in an appropriate cellular context. Subjecting cells to electric shock (electroporation) or lipids makes them transiently permeable to allow uptake of DNA. Viral vectors can be used (retroviruses) to allow for stable gene expression without altering the cell viability. Toxins are used to select for cells that took up the gene. o Transient transfection: employs a plasmid vector engineered to carry replication origins derived from viruses that infect mammalian cells, which can be recognized by mammalian RNA polymerase. During cell division, however, such plasmids are not faithfully segregated into both daughter cells, and in time, a substantial fraction of the cells in a culture will not contain a plasmid. (few days) o Stable transfection: an introduced vector integrates into the genome of the host cell with help of endogenous enzymes of DNA repair/recombination, so the genome is permanently altered. Integration occurs at random sites in the genome, so clones will differ in rate is transcribing the inserted cCDNA. Cells are usually screened for level of expression as well. Creates a cell line expressing the gene of interest indefinitely. (complicated method) e Gene overexpression: inject foreign DNA into pronuclei (vectors), injected cells are then transferred in females, select progeny for genotype (10-30% of offspring will have DNA in all cells), mice expressing foreign DNA are used for breeding to propagate DNA, amount introduced cannot be predetermined. Molecular Biology — BIO3001 15 Signal Transduction Cells do not function in isolation. They perform cellular communication with use of signaling molecules released by one cell to induce a response in a different cell. Many types of chemicals act as signals: small molecules, gases, peptides, soluble proteins and tethered proteins. The signal produces a specific response only in target cells with receptor proteins that bind the signals. e (small) Hydrophobic signaling Vogt siga Sarpi sign cpr Rotte pa molecules: spontaneously diffuse Toso Lp a ° through the plasma membrane inaciv cl \ / n. i i recent MAI RAR ectrior and bind to receptors in the ua d cytosol. The receptor-hormone E gn n . Signa complex moves into the nucleus, pl ss sanduzion | I Ja binds to specific regulatory Ò ca o sequences in DNA, and activates re), function, movement or represses expression of target Nucleus n genes. Netmemin ol pars erro orecpraci e (large) Hydrophilic signaling molecules: cannot diffuse through the plasma membrane, bind to cell-surface receptors (integral membrane proteins) embedded in the plasma membrane. The signaling molecule acts as a ligand by binding to a structurally complementary site on the extracellular domain of plasma-membrane-spanning domain of the protein. This induces a conformational change in the receptor, which is transmitted to the intracellular domain and results in binding to and activation or inhibition of proteins in the cytosol or plasma membrane. Activated proteins catalyze synthesis of second messengers (small molecules/ ions), which carry the signal to effector proteins (enzymes/transcription factors). Molecular Biology — BIO3001 16 Signal transduction: the process of converting extracellular signals into intracellular responses (linking receptors to effectors). A signal transduction pathway is the chain of intermediates that transduce/convert information from into another. Two major types of cellular responses can be induced. e Short-term changes: take seconds to minutes, change the activity or function of specific enzymes and other proteins that preexist in the cell, often involves covalent modifications (phosphorylation, ubiquitinylation) or binding of molecules (cAMP, Ca2+). e Long-term changes: take hours to days, involves activation or repression of specific transcription factors by covalent modification (phosphorylation), changes the amounts of specific proteins contained in a cell by influencing gene expression leading to cell differentiation/division etc. The process of signal transduction: e Signaling molecules: ligands, first messengers, can act on short or long distance. Signaling by extracellular molecules can be classified into three types, however, some signaling molecules (epinephrine, EGF) can act on both short and long distance (hormone and neurotransmitter, membrane bound and soluble). The response of a cell to a signal depends on the cell’s receptors that recognize the signal, as well as the signal transduction pathways activated by those receptors. o Endocrine signaling: signaling molecules (hormones) are synthesized and secreted by signaling cells (endocrine glands) and transported through the circulatory system to act on target cells distant from their site of synthesis. Molecular Biology — BIO3001 17 o Paracrine signaling: signaling molecules (neurotransmitters, growth factors) released by cells affect only target cells in that are in close proximity. Many developmentally important signaling proteins diffuse away from the signaling cell, forming a concentration gradient and inducing different responses in adjacent cells depending on the concentration of the signaling protein. (same extracellular fluid) = Synaptic signaling: specific type of paracrine signaling happening between neurons. o Autocrine signaling: cells response to substance that they themselves release (growth factors, specifically tumor cells) o Contact dependent signaling: when a receptor of one cell directly binds to a ligand in the membrane of another cell, allowing them to communicate through direct contact. e Receptors: located on the surface of the target cells, bind only a single type of signaling molecule of a group of very closely related ones. Ligand binding depends on multiple weak, noncovalent forces and molecular complementarity (binding specificity) between receptor and ligand. Binding of the ligand to the receptor causes a conformational change in the receptor that initiates a sequence of reactions leading to a specific response inside the cell. However, a ligand can have different effects on different cells, since different cell types often have different receptors for the same ligand, which activation induces a different intracellular signal transduction pathway. This property is known as the effector specificity of the receptor- ligand complex. Molecular Biology — BIO3001 20 activate downstream signaling proteins. Conversion back to the inactive form is mediated by GTPase, which slowly hydrolyzes the bound GTP to GDP by releasing Pi, thereby altering the conformation of switch I and switch II so they are unable to bind the target effector protein. The slower the rate of GTP hydrolysis, the longer the proteins remain in the active state. The rate can be modulated by proteins, like GTPase-activating proteins (GAPs) and regulators of G protein signaling (RGSs), which accelerate the process. = Active (on) form: bound to GTP (guanosine triphosphate), modulated the activity of specific target proteins = Inactive (off) form: bound to GDP (guanosine diphosphate), cannot affect the activity of target proteins. o Second messengers: low-molecular weight intracellular signaling molecules, concentration increases/decreases as a result of the binding of ligands to cell surface receptors, bind to proteins and modify their activity. = Calcium ions (Ca2+): cytosolic Ca2+ concentration can be increased locally by signal-induces release from the ER lumen or extracellular environment by the opening of calcium channels in membranes. The rise in cytosolic Ca2+ is sensed by Ca2+ binding proteins (EF hand family, calmodulin), which contain a helix-loop-helix motif. Binding to Ca2+ causes a conformational change that permits the proteins to bind target proteins and switching them on/off. This triggers contraction in muscle cells, exocytosis of secretory vesicles in endocrine cells Molecular Biology — BIO3001 21 and exocytosis of neurotransmitter containing vesicles in neurons. = Cyclic adenosine monophosphate (CAMP): a rise in cAMP triggers the activation of protein kinase A, which in turn phosphorylates specific target proteins to induce changes in cell metabolism (activity of ion channels). The dissociation constant (kd), is a measure of the mp e cia: n ci _ [RI] affinity (tightness of binding) of the receptor for its Ka = [RL] ligand. The smaller the dissociation constant, the more stable the receptor-ligand complex. The total number of receptors is the total number of free receptors [R] plus the total number of ligand-bound receptors [RL]. The total number of ligand- bound receptors can never reach the total number of receptors. [RL] < [R]. Therefore, the maximal cellular response to a particular ligand is induces when much less than 100 percent of its receptors are ligand-bound. e Sensitivity of a cell for a specific ligand determines the concentration of ligand needed for a particular cellular effect. Since cellular response depends on the number of receptor- ligand complexes, the fewer receptors for a ligand present on the surface of a cell, the less sensitive the cell is to that ligand. If the level of a receptor is increased, the cell will become more sensitive to the ligand. e Desensitization of a cell happens when the number of cell surface receptors present is reduced (by endocytosis) and the cellular response if effectively eliminated. It is critical to the ability of cells to respond appropriately to external signals and can be caused by various mechanisms. Molecular Biology — BIO3001 22 Synthetic analogs of natural signaling molecules are widely used in research on cell-surface receptors and as drugs. e Agonists: mimic the function of a natural hormone by binding to its receptor and inducing the normal cellular response to the hormone. Synthetic agonists usually bind more tightly to the receptor than the natural signaling molecule. e Antagonists: occupies ligand-binding sites on a receptor to block binding of the natural signaling molecule (agonist) and reduce the natural physiological activity of the signaling molecule. They inhibit receptor signaling. In order to study receptor function, it is necessary to purify them and characterize their structures (with and without ligand) and biochemical properties (elucidate conformational changes). In order to study signaling cascades, quantitative measurement of the activity of signal transduction proteins that affect the activity of target effector proteins, like kinases and GTP-binding proteins, is required. ® Purification: difficult, membrane must be solubilized with non- ionic detergent, structure of receptor must be maintained, receptors need to be separated from other cellular components. o Recombinant DNA techniques: often used to generate cells that express large amounts of receptor proteins. However, purification is then still needed. o Affinity chromatography: an antibody that recognizes either the receptor or a ligand for the receptor is chemically linked to beads used to form a column. Detergent-solubilized preparation of membrane proteins (of a specific organelle) is then passed through the column, Molecular Biology — BIO3001 25 e Heterotrimeric Gs protein: functions as a receptor-activated switch, switches between active and inactive form, contains three subunits: a, pf, and y. o Ga subunit: binds GTP or GDP, bound to plasma membrane. In resting state, Ga subunit is bound to GDP and forms a complex with the Gy subunit. Binding of a ligand causes the GPCR to bind to the Ga subunit, which results in release of the bound GDP and binding of GTP, which causes a conformational change in switch I and switch II of the Ga subunit. As a result, the binding to the Gy subunit and the GPCR receptor is weakened. Ga-GTP (short-lived) remains anchored in the plasma and interacts with and activates or inhibits effector proteins. The GBy subunit can also transduce a signal by interacting with effector proteins. Hydrolysis is the bound GTP to GDP is catalysed by the GTPase activity of the Ga subunit (built- in feedback mechanism), in order to block further activation of effector proteins (block cellular overreaction) and reassemble the heterotrimeric G protein. Effectors can speed up this process by acting as GTPase-activating proteins (GAPs). Regulators of G protein signalling (RGS protein, act on Ga on different levels) can also accelerate GTP hydrolysis. = When GTP is nonhydrolyzable, it remains permanently bound to Ga and permanently activates the effector. Some bacterial toxins contain a subunit that catalyzes a chemical modification of Ga proteins and prevents hydrolysis of bound GTP to GDP. o Gfpy subunit: always bound together, Gy bound to plasma membrane Molecular Biology — BIO3001 26 e Membrane bound effector protein: activated /inhibited by the GTP-bound Ga subunit. Are either membrane-bound ion channels or membrane-bound enzymes that catalyze formation of second messengers (CAMP). o Gat: activation induces formation of cGMP, PDE o Gas + Gai: activation induces formation of adenyl cyclase, activate K+ ion channels, have opposite effect (stimulatory and inhibitory) o Gag: activation induces formation of phospholipase C e Second messengers: not always involved, modulate ion channel activity, regulate cellular functions (metabolism, synthesis, muscle contraction, activation of protein kinases which phosphorylate proteins, etc.), examples include cAMP, IP3 and DAG. o CAMP: activates protein kinase A (PKA), which catalytic subunits translocate to the nucleus to activate CREB transcription factor by phosphorylation. Phosphorylated CREB (CREB-P) binds to CBP/P300 co- activator to initiate transcription. e Desensitization proteins: participate in desensitization of the signaling pathway. Most GPCRs are also down-regulated by feedback repression, in which an end product of a signaling pathway blocks an early step in the pathway. o G protein receptor kinases: phosphorylate GPCRs, leading to a stop in activation and subsequent desensitization. * Protein kinase A: phosphorylates serine and threonine residues in the cytosolic domain of the Molecular Biology — BIO3001 27 receptor when GPCRs are exposed to hormonal stimulation for several hours. Phosphorylated receptors can bind their ligands but cannot efficiently activate the Ga subunit. = f-adrenergic receptor kinase (BARE): phosphorylates cytosolic domains of the f-adrenergic receptor, but only when epinephrine or an agonist is bound to the receptor, meaning it is in its active conformation. o Arrestins: plays a role in silencing GPCRs by binding to phosphorylated GPCRs. = $-Arrestin: binds to specific phosphorylated GPCRSs to promote endocytosis of the receptor leading to desensitization. Kinase linked receptors: binding of a ligand to the receptor triggers activation of a receptor-associated kinase. They generally have a transmembrane domain and are activated through ligand-induced receptor dimerization. Kinases phosphorylate and activate a variety of signal-transducing proteins, including transcription factors in the cytosol, or activate GTP-binding proteins. e Receptor serine kinases (TGFp receptor subfamily): signaling molecules from the transforming growth factor fp (TGF”) are stored in an inactive form on the cell surface or in the Molecular Biology — BIO3001 30 Active JAK kinase phosphorylate multiple tyrosine residues in the receptor cytosolic domain. The resulting phospohtyrosine functions as docking site for signal- transduction proteins. Signaling from cytokine receptors is terminated by the phosphotyrosine phosphatase SHP1 and several SOCS proteins. Transcriptional Control of Gene Expression The regulation of gene expression is the fundamental process that controls the development of multicellular organisms from a single fertilize egg cell. It results in differential protein expression in different cell types or developmental stages or in response to external conditions. In eukaryotes, La the primary purpose of gene control is mmm} the execution of precise Pa developmental programs so that the proper genes are expressed in the proper cells at the proper times during embryologic development and cellular differentiation. Abnormal gene expression can therefore result in developmental defects and cancer. Control of transcription initiation and elongation is the most important mechanisms for determining whether genes are expressed and how much of the encoded mRNA/protein is produced. e Protein-binding regulatory DNA sequences, called transcription control regions, are associated with genes. Transcription factors (proteins) bind to these regions, either upstream or downstream of the gene, and determine where transcription will start and whether transcription is activated or repressed at the promoter. When transcription of a gene is Molecular Biology — BIO3001 31 repressed, the corresponding mRNA and encoded protein or proteins are synthesized at low rates. When transcription of a gene is activated, both the mRNA and encoded protein or proteins are produced at much higher rates. o Transcription of a single gene can be regulated by the binding of multiple different transcription factors to different control elements. This way, a gene can be expressed in different types of cells and at different times during development. Therefore, mutations in one control region can affect expression of the gene in only one cell type. For example, pax6 gene, which is responsible for eye, brain, spinal cord and pancreas development, is expressed from at least three different promoters that function in different cell types and at different times during embryogenesis o Transcriptional-control regions are often analyzed by recombinant DNA techniques (transgenic mice) that combine a fragment of DNA that is tested with the coding region for a reporter gene whose expression is easily assayed, like luciferase which generated light; Green Fluorescent Protein (GFP), which emits fluorescence; or p-galactosidase, which generates blue insoluble precipitate when incubated with the sugar X-gal. o A method for identifying distant control regions is to compare sequences of distantly related organisms. Transcriptional-control regions for a conserved gene are also often conserved and can be recognized in the background of nonfunctional sequences that change during evolution. Similarly, the transcription factors that bind to these transcription-control regions are presumably conserved during evolution as well. Molecular Biology — BIO3001 32 e Chromatin structure: the association of eukaryotic chromosomal DNA with histone octamers results in the formation of nucleosomes which associate into chromatin fibers and further associate into chromatin of varying degrees of condensation. Inactive genes are assembled into condensed heterochromatin, which inhibits binding of the RNA polymerases and transcription factors required for transcription initiation. Active genes are located on decondensed euchromatin, which allows binding of RNA polymerases and transcription factors required for transcription initiation. o Activator proteins: bind to transcription-control regions near the transcription start site of a gene kilobases away. Promote chromatin decondensation, binding of RNA polymerase to the promoter, and transcriptional elongation. o Repressor proteins: bind to other transcription-control regions. Cause condensation of chromatin and inhibition of polymerase binding and elongation. Eukaryotes contain 3 major RNA polymerases, all of which are multi-protein complexes containing two large subunits and 10-14 smaller subunits. Each RNA polymerase has several enzyme-specific subunits that are not present in the other two. AI the subunits are necessary for RNA polymerases to function normally. e RNA polymerase I (pol I): transcribes inE Bi sli genes encoding precursor rRNA (pre- n i n i rRNA), which is processed into 28S, 5.8 a BE BO BE © 80 © ® e © o n È n n è RMOO a 7 Molecular Biology — BIO3001 35 regions and coding regions of DNA. Transcription from these elements is initiated by equal number of polymerases in both directions, however only sense strand transcription yields mRNA and anti-sense strand transcription stops/pauses after 0.5-3 kb. = Chromatin immunoprecipitation (ChIP): a technique that uses an antibody specific to RNA polymerase II and can detect differences in sense and anti-sense transcription. For example, Pol II can be detected more than 1 kb from the start site only in the sense direction. ChIP-seq combines ChIP with DNA sequencing to identify the binding sites of DNA- associated proteins. e Proximal promoter elements/enhancers/upstream activating sequences (UAS): located close to (PPEs, <200 bps) or far from (enhancers/UAS, 200bp — 50kb) the genes they regulate, either upstream or downstream (within introns), and work even when inverted. They control in which type of cell the gene is transcribed and how frequently it is transcribed by RNA polymerase (0) 5. cerevisiae gene II. They are often e i cell-type specific, ° functioning only in specific differentiated cell types, driving specific gene programs. Genes are usually controlled by multiple enhancers that function in different types of cells. Activators bound to a distant enhancer can interact with transcription factors bound to a promoter because chromatin is flexible, and the intervening chromatin can form a large loop. Molecular Biology — BIO3001 36 o Linker scanning mutagenesis: can pinpoint the sequences within a PES fe regulatory region that function to Sela de Lu . = vu - control transcription. Constructs with 1 contiguous overlapping mutations (small deletions and insertions) are assayed for their effect on expression of a reporter gene or production of a specific mRNA. Has shown that promoters tolerate considerable flexibility in the spacing between promoter- proximal elements (20bp), but that separations of several tens of base pairs may decrease transcription(30-50bp). General transcription factors (GTFs) position RNA polymerase II at start sites and assist in transcription initiation by helping to separate the DNA strands so that the template strand can enter the active site of the enzyme. The complex of RNA polymerase II and its associated GTFs bound at a promoter is called a preinitiation complex (PIC). 1. TBP subunit of TFIID recognizes and binds the TATA box promotor and interacts with the DNA in order to bind and bend its helix. 2. Once TFIID is bound, TFIIA and TFIIB can bind which help RNA polymerase II to melt the DNA helix at the start site. 3. After TFIIB binding, a preformed complex of TFIIF and RNA polymerase II binds, thereby positioning the RNA polymerase over the transcription start site (TSS). Molecular Biology — BIO3001 37 4. Then, two additional general transcription factors, TFIIE and TFIIH, bind the complex, which completes assembly of the transcription preinitiation complex (PIC). 5. Helicase activity of the TFIIH subunit uses energy from ATP hydrolysis to help unwind the DNA at the start site, thereby allowing Pol II to form an open complex in which the DNA is melted, and the template strand is bound at the polymerase active site. 6. As Pol Il begins transcription, CTD is phosphorylated on serine 5 by the TFIIH kinase domain. Phosphorylated CTD Pol II transcribes the DNA and GTFs dissociate from the PIC, except for TBP, which remains bound to the TATA box, thereby allowing the next RNA polymerase II to be activated. DNA binding transcription factors (TFs): bind promoter-proximal elements and o è enhancer sequences. Transcription of a gene Sf, AR; is independently regulated by combinations of specific TFs (unique for every gene). This i combinatorial complexity results from Si alternative combinations of transcription factor monomers forming heterodimeric transcription factors, and from cooperative binding of different (structurally unrelated) transcription factors to control sites. This w de increased number of DNA sequences one family of transcription factor can bind. The binding of multiple activators in a multiprotein complex to the enhancer leads to formation of the enhanceosome, which binds to multiple binding sites of the enhancer simultaneously. Transcription factors are usually composed of separable functional domains: a N-terminal DNA -binding domain, and a C-terminal activation/repression domain, which interacts with other proteins to Molecular Biology — BIO3001 40 e Regulation of transcription factor activity: Whether or not a specific gene is expressed in a particular cell at a particular time is a consequence of the nuclear concentrations and activities of the transcription factors that interact with the transcription- control regions of that gene. Transcription factor activity is influenced by ligands, hormones or post-translational modifications (dephosphorylation/ phosphorylation), leading to a conformational change that modifies their ability to interact with other proteins/DNA. o Extracellular proteins/peptides: bind to cell-surface receptors, which activate intracellular signal transduction pathways. The intracellular signal then regulates the activities of enzymes that modify transcription factors by post-translational protein modifications (phosphorylation, acetylation, etc), which activate or inhibit transcription factors in the nucleus. o Lipid-soluble hormones. extracellular signals that ME =_= Chaperones ‘ Syiosci iii» diffuse through the plasma GV 6 al (4) Hormone and nuclear membranes "(A % \ and bind to receptors Response element located in the cytosol or \ ue G ia a À / nucleus. The ligand- è P A receptor complex functions as a transcription activator. Transcription factors regulated by these hormones are called nuclear receptors, which function as transcription activators only when bound to the ligand. They contain three functional regions: a N terminal region of variable length which functions as the activation domain, a DNA-binding domain (same in all receptors), and a hormone-binding domain containing a hormone-dependent activation/repression domain. The Molecular Biology — BIO3001 41 DNA sites to which nuclear receptors bind are called response elements and they contain repeats and are highly similar. Specificity is determined by spacing between the repeats. = Heterodimeric receptors: located exclusively in the nucleus, repress transcription when bound to DNA in absence of their ligand by directing histone deacetylation by associating with histone deacetylase complexes, undergo conformational change when binding their ligand which results in binding histone acetylase complexes. =" Homodimeric receptors: found in the cytoplasm in absence of their ligand, binding of a hormone leads to translocation to the nucleus, cannot be transported into the nucleus and interact with target genes to activate transcription in the absence of ligand. For example, the glucocorticoid receptor (GR), which is trapped in the cytoplasm by molecular chaperones. It can enter the nucleus when hormones bind, which promotes change from Hsp70 to Hsp90 resulting in refold of the ligand binding domain, thereby increasing the affinity for hormone and releasing Hsp70. In the nucleus, the hormone-receptor complex can bind to response elements associated with target genes and act as an activator by interacting with chromatin-remodeling and histone acetylase complexes and mediators. e Heat shock genes: encode molecular chaperones (like Hsp70) that help to refold (denatured) proteins that help the cell to deal with the effects of heat shock. They are always Molecular Biology — BIO3001 42 paused in a state of suspended transcription and regulated by control of transcription elongation. When heat shock occurs, the heat- shock transcription factor (HSTF) is activated, which binds to specific sites in the promoter proximal region of the genes and stimulates paused polymerase to continue chain elongation and promotes rapid reinitiation by additional Pol II molecules, leading to many transcription initiations per minute and permitting a rapid response. Therefore, when an emergency arises, no time is required to remodel and acetylate chromatin at the promoter and assemble a transcription preinitiation complex. e Modulation of chromatin structure: influences whether TFs can access regulatory regions of genes or not (epigenetic control). Post-translational modifications of histones are associated with activation or repression of genes. Histone tails, which are residues within the N-terminal region of all histone, and the C-terminal regions of histones H2A and H2B, extend from the surface of the nucleosome and can be reversibly modified. o Acetylation: The interactions of several proteins with one another and with the hypoacetylated N-terminal tails of histones H3 and H4 are responsible chromatin-mediated repression. (positive/negative charge) = Heterochromatin: densely packed nucleosomes, inaccessible for TFs, contains inactive genes, especially present in telomers. Caused by hypoacetylation/deacetylation, which is initiated by Molecular Biology — BIO3001 45 elongation. Trithorax complexes oppose repression by polycomb complexes by methylation of histone H3 lysine 4. Post-Transcriptional Gene Control Post-transcriptional gene control involves all mechanisms that regulate gene expression following transcription. This is regulated at every step of the life of an mRNA (synthesis + degradation). In the nucleus transcription and posttranscriptional modulation leads to the production of a mature mRNA which is less prone to degradation and translocated to the cytosol. This makes it difficult to predict a mRNA sequence from the DNA. Pre-mRNA processing is co-transcriptional, meaning that it is performed whilst another part of the RNA is still being transcribed. e 5° cap addition: needed for 8° end of RNA eee Bo efficient OO NAZZZINI elongation/termination and vL x ® export to the cytosol, aBi Ba — . SO0O-O AZZ necessary for translation, st e. Guanylyl transferase By protects mRNA from nuclease O degradation, added to all ‘Oer RNAs made by RNA ia ii O, polymerase II, composed of 7- n'GOO@N ZZZ cn 2 »| svi [+CHy from sse | S-Ado-Met — methylguanosine and fi methylated ribose. CTD, n0@G®Nm phosphorylated by polymerase II, recruits and activates the capping enzyme. The capping enzyme then removes a 5° phosphate from the RNA and replaces it with GMP, thereby creating a guanosine 5° triphosphate structure. Enzymes then perform guanine methylation at the N7 position of the nucleotide. Molecular Biology — BIO3001 46 e Splicing: can happen before or after me rar polyadenylation, generates correct coding "4 ni region by deletion of introns and joining 3 exons, and allows production of alternatively "| ili spliced mRNAs, which leads to the production of proteins which are limited to specific functional domains, depending on the cell type. Exon-intron junctions (splice sites) in a pre-mRNA contain conserved short "n sequences, including a 5° GU donor site, a 3° AG acceptor site, a pyrimidine-rich region near the 3° end upstream of the 3° splice site and about 30-40 nucleotides at each end of an intron. Furthermore, SR » proteins can interact with exonic splicing n] enhancers to mediate binding of snRNAs to 10 a true splice site. Splicing of exons proceeds via two sequential transesterification reactions during each of which a phosphoester bond is exchanged for another (no energy consumed), leading to the linkage of the 5’ guanine of the intron to an adenosine (branch point) near the 3° end of the intron. As a consequence, two exons are ligated, and the intervening intron is released as a branched lariat structure associated with RNPs and rapidly degraded by RNases. Splicing requires the presence of 5 U-rich small nuclear RNAs (snRNAs), which interact with each other and base pair with the pre-mRNA at the splice site Molecular Biology — BIO3001 47 and form a spliceosome with other proteins, like splicing factor 1 (SF1). Exons can be constitutive exons (always present) or regulated exons (not always included in the final mRNA). The 5' cap is bound by a heterodimeric nuclear cap-binding complex (CBC), which protects it from 5' exonucleases and also functions in export of the mRNA to the cytoplasm. o Splicing repressors: bind intronic of exonic splicing silencers to block the binding of splicing factors at specific sites in pre-mRNAs or inhibit their function. o Splicing activators: bind intronic or exonic splicing enhancers to enhance splicing by interacting with splicing factors, thereby promoting their association with a regulated splice site. e 3° cleavage and polyadenylation: involves cleavage (leading to alternative splicing) and addition of a 3° poly(A) tail to the 3° end of the RNA, leads to the release of the RNA from the polymerase. A cleavage and polyadenylation specific factor (CPSF) composed of five polypeptides, forms an unstable complex with the AAUAAA polyadenylation signal upstream of the 3” end. 3 additional proteins bind to the CPSF- RNA complex, including cleavage stimulatory factor (CStF), cleavage factor I (CFI) and cleavage factor II (CFII). Symplekin forms a scaffold on which these factors assemble, and poly(A)polymerase (PAP) binds the complex so cleavage and polyadenylation are linked, and no RNA is lost. Following cleavage, polyadenylation proceeds in two phases. The first is the Molecular Biology — BIO3001 50 early or late in development. In females, Sxl bind to an intronic splicing silencer, resulting in splicing of exon 2 to 4 and skipping exon 3. As a result, a functional sex- lethal mRNA is produced, which reinforces its own expression. o Transformer (Tra) protein: encoded by the transformer gene. Sxl protein regulates alternative splicing of the pre- mRNA. In male embryos (no Sxl1) exon I is spliced into exon 2, which contains a stop codon that prevents synthesis of a functional protein. In females, Sxl binds to an intronic splicing silencer, and exon 1 is spliced to an alternative site, and exon 2 is skipped. Female transformer mRNA contains more exons and is translated into a functional protein. o Doublesex (Dsx) protein: Tra protein regulates the alternative processing of pre-mRNA transcribed from the doublesex gene. In females, Tra binds to an exonic splicing enhancer in exon 4, and forms a complex with two SR proteins, Rbp1 and Tra2, which directs splicing of exon 3 to exon 4 and promotes cleavage at the 3° end of exon 4, leading to a short, female-specific Dsx protein. In males, Tra is not present, and exon 4 is skipped, so that exon 3 is spliced to exon 5, and exon 5 is spliced to exon 6, which is polyadenylated at the 3’ end. This leads to a longer, male-specific Dsx protein. Both proteins are transcription factors with a common DNA -binding domain, but different effects. Females have a strong activation domain, while males have a strong repression domain; meaning that the female Dsx protein activates female genes, while male Dsx protein represses female genes. Molecular Biology — BIO3001 SI Self-splicing introns: are i spliced out by their own Adel, catalytic activity, even in the absence of any protein. They La; a are ribozymes (catalytic I I RNA), which perform K a° \ transesterification with no i Ù 0” input of energy, similarly to the spliceosome. The snRNAs in the spliceosome are thought to have an overall tertiary structure similar to that of group II self-splicing introns. e Group [Iintrons: present in nuclear rRNA genes, use guanosine as a cofactor and can fold by internal base pairing to juxtapose the two exons that must be joined. The guanosine cofactor (G) not part of the RNA chain associates with the active site and its 3’ hydroxyl group participates in transesterification with the phosphate at the 5’ end of the intron. Subsequently, another transesterification reaction links the 5° and 3’ exons. e Group II introns: present in protein-coding genes and some TRNA and tRNA genes in mitochondria and chloroplasts. They fold into a complex stem-loop containing secondary structure and catalyze two transesterification reactions similarly to the spliceosome. The 2° hydroxyl group of the branch-point adenine (A) participates in a transesterification reaction with the phosphate at the 5’ end of the intron. Subsequently, another transesterification reaction links the 5° and 3’ exons. Transportation: functional RNAs are exported to the cytoplasm as components of ribonucleoprotein (RNP) complexes, so mRNA never occurs in the cell as a free molecule. Most mRNPSs are exported Molecular Biology — BIO3001 52 from the nucleus by multiple heterodimeric mRNP exporters that consists of nuclear export factor 1 (NXF1) and nuclear export transporter 1 (NXT1). NXFI binds mRNPs through associations with RNA and proteins, like RNA export factors (REF) and SR proteins. mRNP exporters interact with unstructured protein domains that fill the central channel of the nuclear pore complex (NPC). The direction of transport towards to cytoplasm results from dissociation of the mRNP exporter-mRNP complex in the cytoplasm due to the phosphorylation of mRNP adapter proteins (SR) by cytoplasmic kinases and the action of an RNA helicase associated with cytoplasmic filaments (nuclear basket) of the nuclear core complex. As a result, the complexes diffuse down a concentration gradient across the NPC. Pre-mRNAs bound by a spliceosome normally are not exported from the nucleus, ensuring that only fully processed, functional mRNAs reach the cytoplasm for translation. e RNA surveillance mechanisms prevent export of incorrectly processed mRNASs to the cytoplasm and lead to protein degradation once they are exported. The amount of RNA that translocates reflects the amount of correctly processed mRNA. Human pv gni immunodeficiency virus (HIV): when the provirus infects a cell, 3 different mRNA (spliced, partially spliced, not spliced) are produced. During initial infection, the fully spliced mRNA translocates to the cytoplasm, which leads to the production of ref protein, which contains a leucine-rich nuclear export signal that interacts with the transporter CYTOPLASMIC mRNA® exportin 1. Ref protein binds to sequences of the other (partially/not Molecular Biology — BIO3001 55 poly(A) tail. Binding of a specific protein to regulatory elements in the 3° UTR&s represses translation of these mRNAs. Phosphorylation of the RNA binding protein, induced by an external signal, leads to lengthening of the 3” poly(A) tail and induces translation. o Oocyte translation stop: cocytes contain many mMRNAS encoding proteins, that are not translated until after the egg is fertilized by a sperm cell. In immature oocytes, mRNAs containing the U-rich cytoplasmic polyadenylation element (CPE) have short poly(A) tails. CPE binding protein (CPEB) mediates repression of translation through the interaction with other proteins (Maskin), which prevents assembly of an initiation complex at the 5’ end of the mRNA. Hormonal stimulation of oocytes activates a protein kinase that phosphorylated CPEB, causing it to release Maskin. The cleavage and polydadenylation specificity factor (CPSF) then binds to the poly(A)) site, interacting with bound CPEB, and the cytoplasmic form of poly(A) polymerase (PAP). After the poly (A) tail is lengthened, cytoplasmic poly(A) binding protein 1 (PABPCa) can bind to it and interact with initiation factors to initiate translation. Epigenetics: describes phenomena in which genetically identical cells or organisms express their genomes differently, causing phenotypic differences. Epigenetic tags are passed down to new generations of cells. However, when a zygote is formed, many epigenetic tags are removed, but some remain. e Chemical modifications at level of nucleotides Molecular Biology — BIO3001 56 o o DNA methylation: cytosine methylation, CpG islands, tissue specific, mechanism of gene silencing, alone not sufficient to silence genes, methylation pattern is not inherited but erased during early embryogenesis. RNA interference (RNAi): e Modifications at the level of histones: modification of histones and incorporation of histone variants to affects chromatin structure, very hypothetic. o o o Histone acetylation: acetylation open chromatin, performed by histone acetylases (HAT). Deacetylation closes chromatin, performed by histone deacetylases (HDAC). Histone methylation: maintains gene silencing, performed by HMTs Histone phosphorylation Histone ubiquitinylation Different types of histones e Nucleosome remodeling: eukaryotic DNA is packaged in nucleosomes, which is DNA wound around histone octamers, including five families of histones H1/H5, H2A, H2B, H3 and H4. ATP-dependent processes regulate associability of nucleosomal DNA o o Cancer Euchromatin: less densely packed Heterochromatin: densely packed, present around the centromere and at telomeres. Molecular Biology — BIO3001 57 Cancer occurs when the mechanisms that maintain normal proliferation rates in the cell malfunction to cause excess cell division, which results from genetic damage. Oncogenesis is an interplay between genetics and the environment; with most cancers arising after multiple gene alterations induced by carcinogens or faults in DNA replication/repair. Division of the altered cell will give rise to other altered cells, meaning they become more abundant, undergo further genetic changes and become progressively more dangerous. In carcinogenesis, six fundamental cellular properties can be altered. When all properties are present, the most destructive cancer phenotype is created. Less dangerous tumors rise when only some of these changes occur. e Resist cell death: cancer cells continue to live when they should die. e Sustain proliferative signaling: cancer cells acquire a way to proliferate without the need of an external inducing signal. e Evade growth suppressors: cancer cells fail to sense signals that restrict cell division. e Induce angiogenesis: cancer cells often induce the growth of blood vessels into the tumor, so they can grow bigger. e Enable replicative immortality: cancer cells fail to sense signals that restrict cell division. e Activate invasion and metastasis: cancer cells often change their attachment to surrounding cells or to the ECM, breaking loose to move away from their tissue of origin. Molecular Biology — BIO3001 60 Classification of tumors: e Benign (harmless): small and localized, cells closely resemble and may function as normal cells, cell-adhesion molecules hold tissue together, a fibrous capsule delineates the extent of the tumor, easy target for surgeons, only become a serious medical problem if they interfere with normal functions or secrete excess amount of substances (hormones). e Malignant (evil): not localized, potentially undergo metastasis o Carcinomas: derived from epithelia (endoderm/ectoderm). Most common type (90+%) o Sarcomas: derived from mesoderm (muscle, blood, connective tissue precursor) = Leukemias: grow as individual cells in the blood, do not need angiogenesis, metastasis happens only rarely. la) Primary tumor ss) Carcinoma cell Metastasis happens when cancer sa . la mentre cells invade surrounding tissues, PIE 5 Degradation of basement Ò membrane i by invadopodia TIFEGF secreted from macrophages and diffusod from blood vessels Carcinoma cell migrating on ECM fiber often breaking through the basement membranes that define the Degradation of basement membrane by invadopodia boundaries of tissues and spreading through the body to establish a Ce Ri secondary tumor. About 1 in “ Do O) 10.000 cells (very rare) escapes the a Mereto primary tumor and survives to colonize another tissue and form another metastatic tumor. e Epithelial-to-mesenchymal transition (EMT): the process by which metastatic tumor cells acquire migratory properties. The conversion of epithelial cells into mesenchymal cells requires Molecular Biology — BIO3001 61 changes in gene expression and results in fundamental changes in cell morphology, including loss of cell-cell adhesion; loss of cell polarity; and acquisition of migratory and invasive properties. EMT regulatory pathways are activated to produce a single migratory cell. Snail and Twist, 2 transcription factors, promote expression of genes involved in cell migration, trigger down-regulation of cell-adhesion factors (E-cadherin), and increase production of proteases that digest the basement membrane and allow penetration by tumor cells. Specific cancer cells leave the main tumor and can be attracted by signals (growth factors, EGF) secreted by macrophages so they reach blood vessels. Cancer cells can penetrate basement membranes using an invadopodium, which produces proteases, and enter the bloodstream to migrate to distant sites in the body. At the new location, they must adhere to the lining of the blood vessel, migrate through it and enter the underlying tissue by extravasation, and they must adapt to the foreign tissue environment. Multi-hit model of cancer: states that cancers arise by a process of evolutionary clonal selection. A mutation can give one cell a slight growth advantage. One of its progeny cells then undergoes a second mutation that allows its descendants to grow more uncontrollably and form a small benign tumor. A third mutation in a cell within this tumor allows it to outgrow the others and overcome constraints imposed by the tumor microenvironment. Its progeny forms a mass of cells, called a malignant tumor, of which each cell contains these three genetic changes. An additional mutation in one of these cells allows its progeny to escape into the bloodstream and establish daughter colonies at other sites, the hallmark of metastatic cancer. Molecular Biology — BIO3001 62 e Cancer cells have common gene alterations: Cells in a given tumor should have at least some genetic alterations in common. Systematic analysis from cells can prove that all cells in a tumor are derived from a single progenitor cell. e Cancerisage related: since it can take decades for the required multiple (5-6) mutations to occur, and the rate of mutations is roughly constant during a lifetime, the incidence of most cancers is dependent on age. The older you are the higher the chance of accumulating mutations. o Experiments with transgenic mice have shown that a variety of combinations of oncogenes can cooperate in causing cancer. Mice either carry the mutant rasV12 dominant oncogene or the MYC proto-oncogene under the control of a mammary-cell-specific promoter/enhancer from a retrovirus. Once the promoter is induced by endogenous hormone levels and tissue-specific regulator, overexpression of MYC or rasV12 is induced in breast tissue. By itself, MYC causes tumors only after 100 days and only in a few mice. RasV12 protein causes tumors earlier, but still slowly and in about 50% of the mice. When crossing transgenic mice, offspring produces both MYC and RasVI12 in increased amounts, tumors arise more rapidly, and all offspring acquires cancer. e Cancer cells can be studied in vitro and in vivo: Cultured cells and mice in which oncogenes and tumor suppressor genes can be expressed in a time- and tissue specific manner can teach us about how cancers arise and how these genes contribute to the development and progression of the disease. Molecular Biology — BIO3001 65 promoters/control elements can inactivate these genes as well. (APC, RB, p53) o Recessive loss-of-function mutations in tumor- suppressor genes are oncogenic (both alleles must be lost to promote tumor development). * Haplo-insufficient genes: loss of just one of the two gene copies can lead to cancer, since production of half the normal amount of gene product is not enough to control cell proliferation (dominant mutation). o Encode proteins that directly or indirectly control progression through the cell-cycle; like proteins involved in growth inhibiting signaling pathways, pro-apoptotic proteins, and checkpoint pathway proteins, which arrest cell cycle if a previous step has occurred incorrectly. o Imherited mutations in tumor-suppressor genes leads to increased predisposition for tumor development, since then only one mutation of the second allele facilitates tumor progression (accounts for 10% of cancers). The subsequent loss or inactivation of the remaining normal allele in a somatic cell, is referred to as loss of heterozygosity (LOH) and can happen by three mechanisms: = The normal allele becomes inactive by an inactivating mutation or deletion. Molecular Biology — BIO3001 66 = Chromosome mis-segregation can cause loss of the chromosome carrying the normal allele. = Mitotic recombination between a chromatid bearing the normal allele and a homologous chromatid bearing a mutant allele (most common). e Genome maintenance genes: maintain the genome?’s integrity, mutations that inactivate them cause the cell to acquire genetic changes at an increased rate (accumulation of mutations). o Recessive loss-of-function mutations in genome maintenance genes are oncogenic. Other drivers of tumorigenesis: e Hyper/hypomethylation: Mutations affecting epigenetic regulators, such as changes in the activity of histone- modifying enzymes or chromatin-remodeling complexes. Tumors typically harbor only a single mutated allele of a gene encoding a chromatin-modifying enzyme (haplo-insufficient). Losing both alleles would kill the cell. o TET DNA hydroxylases: catalyses conversion of 5- methylcytosine to 5-hydroxylmethylcytosine. Require a a- ketoglutarate as cofactor and are inhibited by oncometabolite 2-hydroxyglutarate. o SWISNF complexes: multiprotein complexes with a ATP-dependent helicase which control histone modification and chromatin remodelling. They are important in repressing the expression of E2F genes, which are involved in progression through the cell cycle. Loss of SWI/SNF function can lead to overgrowth and cancer. Molecular Biology — BIO3001 67 o SNS protein: e MicroRNAs can promote or inhibit tumorigenesis by affecting the expression of multiple oncoproteins with fundamental roles in cell proliferation, differentiation, and apoptosis. They can regulate multiple genes. Different genes are involved in tumorigenesis: (24.4) e Cell-growth and cell-cycle related genes: o PFGF-A: growth factor, acts on PDGF receptor, overexpression leads to proliferation o Cyeclins: lead to proliferation in separate stages of the cell cycle. Phosphorylation/dephosphorylation leads to progression in the cell cycle. Concentrations of different cyclins vary at each stage of the cell cycle. o Cdk4: activates cyclin cdk complexes, which leads to proliferation. o Cyclin D: interacts with Cdk4 to phosphorylate RB, which is then released from E2F, and can then bind target genes which leads to progression through the G1 phase. e Extracellular matrix genes: give the cell a certain structure, repression of them leads to loss of cell integrity and boosts invasion/migration. o Alpha 1 type V collagen o Fibronectin 1 o Desmin e Signal-transduction and transcription-related genes: Molecular Biology — BIO3001 70 cells receive different portions of the parent cell under the influence of distinct developmental or environmental signals and as a result the cells will have different fates. Cell polarity is the ability of cells to organize their internal structure, resulting in changes in cell shape and the generation of regions of the plasma membrane with different protein and lipid compositions. Cells have an intrinsic polarity program, meaning that they have the ability to polarize in the absence of external cues under influence of the GTPase Cdc42 (determined where the cell buds). This intrinsic program can be directed by external or internal cues and the polarity of cells is often maintained by intracellular mutually antagonistic complexes. e Cdc42 determines where the cell buds by generating a site highly and locally enriched for Cdc42-GTP on the cell surface, which is a process regulated by a positive feedback loop. Negative feedback loops ensure that the polarization at this site is not too strong, so that it can be redirected to another site on the cell surface upon receiving appropriate signals. Cell polarization happening before asymmetric cell division follows a general ° e° pattern. In order to know which direction to Ed . polarize, a cell senses specific cues that provide in with spatial information. The cell must have appropriate receptors on its surface to be receptive to these signals, which can be soluble signals from other cells or the ECM. Upon detection of the cues, the cell responds by activating the Molecular Biology — BIO3001 71 polarity program to define the orientation of polarity. Next, cytoskeletal reorganization (microfilaments, microtubules) happens locally, and once the cell has structural asymmetry, molecular motors direct trafficking of polarity factors (cytoplasmic proteins, membrane proteins) to their appropriate locations. Polarity is then maintained by moving the polarity determinants from sites of low concentration back to the polarization site. Asymmetric cell division then occurs in a plane perpendicular to the direction of polarization, to allow fate determinants (proteins, mRNA) to be segregated differentially between the cells. e Par proteins: asymmetric cell division in C. elegans happens when the zygote makes its first division, giving rise to 2 cells which create different lineages. Before cell division, P granules are concentrated at the cell end that will give rise to the posterior end of the embryo. The first asymmetric division of the PO cell gives rise to the P1 cell, containing the P granules, and the larger AB cell. When introducing mutations in partition defective (par) genes that result in incorrect portioning of P granules (not properly localized in the posterior), the mitotic spindles were not oriented correctly I preparation for the second division. The gene products of the par genes, called par proteins localize either at the cortex of the anterior half of the cell (par3, par6), or at the cortex of the posterior half (par2, parl). Antagonistic interactions between these par complexes (Cdc42-Par3-Par6- aPKC) exist, meaning that when Par6/Par6 is present, Par2/Parl cannot be present. The position of the sperm after fertilization determines the posterior end of the cell. The sperm centrosome releases a signal, which results in the local depletion active Rho, thereby lowering the activity of myosin II (contractile activity). This leads to actin-myosin network contraction toward the anterior of the cell, thereby dragging the anterior complex Molecular Biology — BIO3001 72 containing Par3, Par6 and aPKC to that end. Par 2 can then occupy the posterior cortex and cell asymmetry is established. Mammalian development: regulated by cell-cell interactions, the position of the cell is important in establishing cell fate. Cell compartments/tissues interact with/support/feed each other by signaling molecules in a process called embryonic induction. e Eight-cell stage: fertilization is quickly followed by cleavage, which is a series of cell division which take about one day each and happen before the embryo is implanted in the uterine wall. In this stage, each cell is totipotent, and has the potential to form every tissue (embryonic + extraembryonic) of a complete animal. e Sixteen-cell stage: three days after fertilization, the 8-cell embryo divides to form the 16-cell morula, after which the embryo undergoes compaction (increased cell-cell adhesion). Additional divisions produce a blastocyst composed of 64 cells that have separated into two cell types; each of which starts its own lineage to produce diverse populations of cells. o Trophectoderm (TE): will form extraembryonic tissues (placenta). Epithelial sheet forming a hollow ball around the ICM and blastocoel. o Imner cell mass (ICM): gives rise to the embryo. Loosely organized/attached pluripotent cell mass found on one side of the blastocoel. Source of embryonic stem cells (ESCs) Different cells are present in the (human) body during a lifetime: Molecular Biology — BIO3001 75 germ-line stem cell divides, one of the daughter cells remains adjacent to the cap cells and is therefore maintained as a stem cell; the other daughter cell is too far from the cap cells to receive signals, which causes it to differentiate. e Intestinal stem cells reside in the bases Absorptive of intestinal crypts, adjacent to Paneth "$ } ì dd cells, which form part of the niche, and ittit sez are marked by expression of the LgrS 4 ; î 7 receptor. Descendants of these LgrS- ami | ° } expressing cells give rise to all cells of 0 | the intestine epithelium, which is ò “ composed of four types of differentiated dio cells and continuously divides (fastest in n mammals, turn over every 5 days). Paneth cells produce antibacterial proteins (lysozyme) which degrade the bacterial cell wall and protect the intestine from infections; and Wnt and other hormones (EGF, Delta protein), which are essential for intestinal stem-cell maintenance. Wnt signals are essential for intestinal stem-cell maintenance (activates B- catenin), since they induce proliferation by activating certain genes, like Lgr5. Furthermore, +4 cells located in the crypts may be reverse stem cells, that can generate Lgr5-expressing stem cells following intestinal injury (irradiation). During periods of intestinal injury, some transient amplifying cells can dedifferentiate under the influence of Wnt signals. o Pulsechase experiments used radiolabeled thymidine to show that intestinal stem cells produce precursor cells that divide rapidly and then differentiate as they ascend the sides of crypts to form the surface layer of the gut (villi), across which absorption occurs. The time from cell birth to Molecular Biology — BIO3001 76 cell death is about 3-5 days and the production of new cells is precisely controlled (avoid cancer/breakdown of villi). o Lineage tracing studies: made use of genetically altered mice in which a version of the cre recombinant protein was placed under the control of the Lgr5 promoter. After addition of an estrogen analog, the cre recombinase is transferred into the nucleus to excise a segment of DNA activating expression of a B-galactosidase reporter gene. At first, only the stem cells in the crypts express B- galactosidase, but after a few days, all of their descendant epithelial cells also express B-galactosidase. e Hematopoietic stem cells (HSCs): located in the embryonic liver and in bone marrow and form all cells of the blood-cell lineage. One HSC gives rise to two multipotent cell types; common myeloid progenitor and common lymphoid progenitor cells. Extracellular growth factors called cytokines regulate HSC self-renewal, proliferation and differentiation of the precursor cells. Each lineage has its own cytokines, thereby allowing exquisite control of the production of specific cell types. This system allows the body to specifically induce the replenishment of some or all of the necessary blood-cell types. Hematopoietic stem cells can be detected and quantified by bone marrow transplant experiments and they (and their niche) can be detected using a combination of marker surface Molecular Biology — BIO3001 7 proteins, since each type of precursor produces unique combinations of those. Transplantation of a single HSC is sufficient to restore the entire blood system, by taking residence in a niche in the bone marrow and dividing to make more HSCs as well as progenitors of blood-cell lineages. The niche of HSCs in the fetal liver is made by hepatocytes, and in the bone marrow its made of stromal cells, which express SCF and the receptor for cytokine leptin on their surface. Properties of stem cells: three different properties are needed for a cell to be classified as a stem cell. Since stem cells are very sensitive, these properties are hard to maintain in vitro. e Self-renewal: can reproduce themselves, can divide into 2 cells that maintain the same potential. They are not fully self- sustainable, since they need external output of ligands (secreted proteins) which bind to close cells (or the same cell) to activate a signaling cascade to influence gene expression. o Master transcription factors: produced shortly after fertilization, required Activate genes for self-renewal, l pluripotency Repress genes that induce specific differentiation pathways for the development of ICM cells in an embryo and specification of ESCs in culture. They have distinct roles in maintaining the developmental potential or pluripotent cells. Target genes encode a wide variety of proteins, including Oct4, Nanog, and Sox2, forming an autoregulatory loop in which each induced its own expression and that of others. They also induce transcription of genes involved in proliferation and self- renewal of ESCs. Molecular Biology — BIO3001 80 differentiate into all cells of the three primary germ layers, either in culture or after reinsertion into a host embryo. When cultured in suspension, ESCs form multicellular aggregates called embryonic bodies, that resemble early embryos. Upon treatment with specific growth factors and transfer to different conditions, they can produce a variety of differentiated cell types. e Induced pluripotent stem cells (iPSCs): somatic cells (fibroblasts, keratinocytes, etc.) that are reprogrammed into a pluripotent state, very similar to ESCs. They can be introduced into a blastocyst to form a complete organism, including germ cells. Furthermore, they be formed from cells of patients with many types of difficult-to-study diseases and then differentiated into the specific cell types affected by the disease. Study of such patient specific cells can illuminate crucial underlying causes of an individuals’ disease. They can also be used for in vitro drug screening. o Reprogramming: can be performed by transformation with retroviruses encoding four transcription factors (KLF4, Sox2, Oct4, Myc), which directly activate transcription of endogenous Oct4 gene, which activates demethylases which remove chromatin marks to activate pluripotency genes, thereby leading to induction of pluripotency. Over the course of several weeks, activation reprograms the somatic cells to an ES-like state. It involves major changes in epigenetic modifications. = Direct reprogramming: safer methods and quality controls. Molecular Biology — BIO3001 81 e Limitations: they have a bias of differentiation towards the cell type of origin due to remaining epigenetic patterns. This can be checked by bi-sulphite sequencing and RNA sequencing but is expensive. There is a risk of chromosomal aneuploidy. This can be checked by karyotyping but is expensive. And there is a risk of genetic mutations which are potentially oncogenic. This can be checked by next-generation sequencing but is also expensive. e Trophoblast stem cells: placenta cells, able to form the whole placenta, can be genetically manipulated, cannot contribute to germ cells. e Embryonic germ cells: e Cancer stem cells: found when dissecting a tumor, the tumor induces somatic cells which leads to re-acquiring stem-cell potential. Difficult to culture, can be genetically modified to study types of cancer. e Adult stem cells: multipotent stem cells, found in a whole range of organs, have a lot more restricted potential, can only form cell types from the tissue they are located at. Applications of stem cells: e Chimeric mice: when (genetically modified) ESCs are injected into the blastocoel of an early mouse embryo, the cell will participate in forming most tissues, so the mouse is made of 2 different types of cells. The injected cell can potentially give rise to functional sperm and eggs, which can generate normal live mice. When treating the host blastocyst with drugs that transiently block mitosis, making cells become tetraploid incapable of forming differentiated cells and tissues, all cells in Molecular Biology — BIO3001 82 the live mice are derived from the donor ESCs. They are essential for studying development and disease. e Organoids/blastoids: self-organizing structures made of a variety of differentiated cells derived from stem cells, that functionally act like an organ/blastoid. Used for in vitro disease modeling and drug screens. When using iPSCs a (patient specific) disease can be studied in vitro. o Limitations: they are not complex enough at the moment, meaning they have a limited complexity when compared to the real organ/blastocysts; and there are various ethical concerns for them (mini brains, blastoids). e Interspecies blastocyst complementation: obtain extremely complex organs, full of blood vessels and immune cells. Can be used for in vivo human disease modeling and drug screens. o Limitations: engraftment of the iPSCs, providing selective advantage for donor cells, developmental timing, evolutionary distance is a barrier to chimera formation, ethical concerns. e Regenerative medicine: restore damaged tissue Apoptosis is an essential process in metazoan development. All cells require trophic factors to survive, and in the absence of these, cells will undergo apoptosis. In the absence of trophic factors, direct interactions between pro-apoptotic and anti-apoptotic proteins lead to cell death. When trophic factors are present, they can trigger changes in these interactions, resulting in cell survival. Furthermore, hormone signals from other cells can induce apoptosis in developmental context. Cell corpses are ingested (engulfed) by neighboring cells, broken down into small molecules called apoptotic bodies, and Molecular Biology — BIO3001 85 allowing cytochrome c to enter the cytosol and bind to Apaf-1 to activate caspases. They regulate activity of Bcl-2 and Bax/Bak proteins. Besides absence of survival factors, apoptosis can also be stimulated by positively acting extracellular death signals, which are trimeric proteins present on the cell surface that bind to death receptors on an adjacent cell. By binding these receptors, they induce oligomerization of fas associated death domain (FADD) protein or TNF-receptor associated death domain (TRADD) protein, which triggers the caspase amplification cascade, leading to cell murder. Depending on the cell, death can result from apoptosis of necroptosis. e Tumor necrosis factor alpha (TNF-a): released by macrophages, triggers cell death and tissue destruction. e Fasligand: cell surface-protein produced by natural killer cells and cytotoxic T-lymphocytes, signal triggers death of virus- infected cells/tumor cells/foreign graft cells. A different form of cell death, called necrosis, occurs when cells are subjected to injury or excessive stresses (heat, absence of oxygen, infection my pathogens). In this process, cells swell and burst, so holes are created in the plasma membrane, causing leakage of intracellular contents and cell death, leading to inflammation and tissue damage. Necroptosis is triggered by extracellular hormones, like necrosis factor alpha (TNFa), but only in the absence of caspase- 8. Since several viruses/pathogens encode proteins that inactivate caspase-8, thus preventing apoptosis and allowing the virus to replicate and spread infection, necroptosis exist as an alternative method of apoptosis, which also prevents pathogen spread, but at a cost of the host mechanism (inflammation). Molecular Biology — BIO3001 86 TUNEL assay: method used to detect apoptotic (dead) cells. An enzyme called terminal deoxynucleotidyl transferase catalyses the addition of labelled (fluorescence/colour) AUTP nucleotides to the free 3’ end of fragmented DNA. One of the hallmarks of apoptosis if the nuclear fragmentation of DNA by nucleases, which are activated by caspases.