Ch 3 - Differential Gene Expression

Genomic equivalence
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Terms in this set (111)
1. Every somatic cell nucleus of an organism contains the complete genome established in the fertilized egg (The DNA of all differentiated cells is identical)
2. The unused genes in differentiated cells are neither destroyed nor mutated (they retain the potential for being expressed)
3. Only a small percentage of the genome is expressed in each cell, and a portion of the RNA synthesized in each cell is specific for that cell type.
Central Dogma of Biology (simplified)DNA transcription RNA translation protein1. TranscriptionWhere: In the nucleus What: A chunk of genomic DNA accessed by RNA polymerase II, which transcribes a complementary copy of the gene into single-stranded pre-mRNA molecule. Result: The gene is said to be "expressed."2. ProcessingWhere: nucleus What: pre-mRNA transcript undergoes processing to make a finalized messenger RNA strand - excise the noncoding domains - add poly AAAA tail3. TransportationWhere: from the nucleus to cytoplasm What: mRNA transported from nucleus out into cytoplasm4. TranslationWhere: cytoplasm What: mRNA is fed through ribosome. The information is translated Result: polymer of amino acids.5. Protein folding and modificationWhere: cytoplasm What: polypeptide adopts 2° and 3° structures through folding and modifications Result: completed protein6. Carry out functionWhere: wherever protein was required What: expressed protein can now carry out it's determined functionPolytene chromosomesDrosophila DNA undergoes numerous rounds of replication without separation - different regions of the chromosomes were "puffed up" at different times and in different cell types, which suggested that these areas were actively making RNAThe cloned sheep taught us what about somatic cells?the nuclei of adult somatic cells in contain all the genes needed to generate an adult organism. - No genes necessary for development have been lost or mutated in the somatic cells; the DNA of their nuclei is equivalentWhy was Dolly (cloned sheep) sterile?Since she was cloned using a somatic cell, she cannot produce gametes since they lack the gamete information required to make eggsNucleosomeHistone core where DNA (Chromatin) is coiled aroundHistoneprotein molecule around which DNA is tightly coiled in chromatinChromatinClusters of DNA, RNA, and proteins in the nucleus of a cellHistone tailsextend from the core are the sites of acetylation and methylation, which may disrupt or stabilize, respectively, the formation of nucleosome assemblages.HeterochromatinChromatin that remains condensed throughout most of the cell cycle and replicates later than most of the other chromatin. Usually transcriptionally inactive.EuchromatinThe comparatively open state of chromatin that contains most of the organism's genes, most of which are capable of being transcribedHeterchromatin vs EuchromatinHeterchrom: cannot be transcribed Euchrom: can be transcribedIntronssequences between exons - Non-protein-coding regions of DNA within a geneExonsprotein coding sequences (interspersed amongst introns) - will be kept in the pre-mRNA (introns are removed)How was the Exon named?refers to a nucleotide sequence whose RNA "exits" the nucleus. It has taken on the functional definition of a protein- coding nucleotide sequence.Promoter; binds what enzyme?region of the gene where RNA polymerase II bindsTATA-binding protein (TBP)A general transcription factor that binds to the TATA box and assists in attracting other general transcription factors and RNA polymerase II to eukaryotic promoters.Cap sequencetranscription initiation site - is the DNA sequence that will code for the addition of a modified nucleotide "cap" at the 5′ end of the RNA - The cap sequence begins the first exon.5' UTRLeader sequence - determines the rate of translationTranslation initiation siteThe ATG codon (becomes AUG in mRNA), which signals the beginning of the first exon (protein-coding region) of a gene.Translation termination codonWhen a ribosome encounters this codon, the ribosome dissociates, and the protein is released.3' UTRis transcribed but is not translated into protein. This region includes the sequence AATAAA, needed for polyadenylationPolyadenylation; what is itthe insertion of a polyA tail (200-300 adenine residues on the RNA transcript)3 functions of polyA tail1. stability of the mRNA 2. allows the mRNA to exit the nucleus 3. permits mRNA to be translated into proteinTranscription termination sequence1000 nucleotides past the polyA tail, then transcription fully turns offpre-mRNAcontains: cap sequence, 5' UTR, introns, exons, 3' UTR, and PolyA tail - needs to be capped with methylated guanosine to protect from exonucleasesnoncoding regulatory elements (3)protmoter, silencers, and enhancersFunction of noncoding regulatory elementsnecessary for controlling where, when, and how actively a particular gene is transcribedcis regulatory elementspromoters on the same chromosome as the enhancer/ repressorbasal transcription factorsbind to DNA on the promoter (where RNA polymerase II will initiate transcription)Some transcription factors don't bind to the DNA, they instead bind to _____the transcription factors that have bound to the enhancer and promoter sequences. In this way, the chromatin loops to bring the enhancer to the promoter - they bind to TF in place and cause a folding + bind allowing transcription of sequences not regularly accessibleWhat makes up a CpG island?Cytosine-phosphate bond-guanineEnhancersignal where and when a promoter can be used and how much gene product to make - are needed to RNA pol-II doesn't bind EVERY promoter, only ones required. - enhancers control the efficiency and rate of transcription from a specific promoterSilencers / repressorscan prevent promoter use and inhibit gene transcription - can silence gene expression spatially (in particular cell types) or temporally (at particular times)mouse _____ gene (which is expressed in the lens, cornea, and retina of the eye; in the neural tube; and in the pancreas) has several enhancersPax6Enhancer Region Modularitymodel for gene expression where the same gene can have multiple enhancers throughout, each regulating the transcription of specific body partsDNA sequence that prevents a promoter's activation in any tissue except neurons. Found in many mouse genesneural restrictive silencer element (NRSE)Transcription factor which binds to NRSENeural restrictive silencer factor (NRSF) - expressed in EVERY cell that is not a mature neuronTranscription factors work in two nonexclusive waysa) recruit nucleosome-modifying proteins to change the chromatin format so it can be read by RNA pol-II b) recruit transcriptional co-regulators (enhancers or repressors) to form bridges to loop the chromatin near the promoterEpigenetic mechanisms most commonly involveadding or removing some of these factors, thereby altering the three-dimensional shape of DNA and its histones.Main Mechanisms of regulating gene transcription (2)a) epigenetic modification of chromatin b) control through transcription factorsEpigeneticsrefers to phenotypic changes caused by modifying how a gene is expressed, rather than modifying the DNA sequence itself.Histones are critical becausethey appear to be responsible for either facilitating or forbidding gene expressionWhich histone tails are typically modified in order to control the repression and activation of gene expression?H3 and H4histone acetylationAdding an acetyl group to the histone (makes the charge neutral) = loosening the histone wrap, making transcription easierhistone acetyltransferasesadd acetyl groups to histones (destabilizing the histone) breaking them apart easier - makes chromatin more euchromatichistone deacetylasesstabilize nucleosome packaging (chromatin becomes more heterochromatic) and prevent transcription.histone methylationis the addition of methyl groups to histones - more often results in heterochromatic states and transcriptional repressionhistone methyltransferaseadds methyl groups to histonesHistone methylation patterns can be heritable due tothe recruitment of certain proteins which possess a "memory" of the transcription and carry that info through mitosisDNA methylation: High CpG-content promoters (HCPs) are usually found indevelopmental control genes. Default state is ONDNA methylation: Low CpG-content promoters (LCPs) are usually found inproducts of mature cells (ex. hormones of pancreatic cells) default state is OFFEnzyme recruited to make DNA methylation pattern heritableDNA methyltransferase-3 (Dnmt3)Genomic imprinting and DNA methylationIt can matter if a gene was carried by the egg or sperm (for some genes) this may be altered if it was methylated differently than normalT / F : During development, transcription factors play essential roles in every aspect of embryogenesis. When in doubt, it is usually a transcription factor's faulttrueThe process by which miRNAs inhibit expression of specific genes by degrading their mRNAs is calledRNA Interference (RNAi)MicroRNA (miRNA)Small (about 22 nucleotide) RNAs complementary to a portion of an mRNA that regulates the translation of a specific message. MicroRNAs usually bind to the 3′ UTR of mRNAs and inhibit their translation.RNA-induced silencing complex (RISC)A complex containing several proteins and a microRNA, which can then bind to the 3′ UTR of messages and inhibit their translation.How does the embryo get rid of maternal RNAs once they have been used and the embryonic cells are making their own mRNAs?In zebrafish, this cleanup operation is assigned to microRNAs such as miR430How does miR430 work?there are about 90 copies of this gene in the zebrafish genome. So the level of miR430 goes up very rapidly. - miR430 hundreds of targets (about 40% of the maternal RNA types) - promotes maternal RNA degradation5 types of post-translational modifications1. Acetylation 2. Glycosylation 3. Methylation 4. Phosphorylation 5. UbiquitinationThree methods of characterizing gene expression1. In situ hybridization 2. Chromatin immunoprecipitation-sequencing 3. Deep sequencing (RNA-Seq)ChIP-Seq and CUT&RUN are techniques that allow the identificationof proteins that bind to specific DNA sequences"high-throughput" RNA analysis by RNA-Seq and whole genome sequencing has enabled researchers tocompare thousands of mRNAs, which when paired with computer programs can predict interactions between proteins and mRNAs.In situ hybridizationmain idea: take advantage of the single-stranded nature of mRNA and introduce a complementary sequence to the target mRNA that enables visualization. - antibody binds to specific complementary mRNA, can visualize where in the embryo/tissue its presentHow does one locate the places on the gene where a particular transcription factor binds or where nucleosomes with specific modifications are localized?the ability to identify protein-specific DNA-binding sites using chromatin immunoprecipitation-sequencing (ChIP-Seq), or a type of ChIP-Seq called CUT&RUN, showed that there are different types of promoters and that they use different scripts to transcribe their genesChIP-Seq is based on two highly specific interactions. What are they?a) binding of a transcription factor or a modified nucleosome to particular sequences of DNA b) binding of antibody molecules specifically to the transcription factor or modified histone being studiedSteps of ChIP-Seq (detailed)1. Chromatin is isolated from the cell nuclei. 2. Chromatin is cross-linked to their DNA-binding sites, and the DNA is fragmented into small pieces. 3. Antibodies bind to specific chromatin proteins, and the antibodies are precipitated out of solution. 4. The DNA fragments are purified from the proteins and sequenced. 5. These sequences are compared with the genome maps to discover the precise locations of the genes these proteins may be regulating.Steps of ChIP-Seq (simple)1. Chromatin is glued to DNA w/ proteins 2. Complexes are cut up 3. DNA-protein complexes in solution 4. Antibody binding 5. Precipitation and purification 6. Sequence DNA 7. Compare with genome 8. Locate sequence on chromosomeRNA-seq (RNA sequencing)is a technique for gene expression analysis - sequence and quantify the RNA present in a biological sample.RNA-seq steps1. RNA is isolated from samples 2. converted to complementary DNA (cDNA) using reverse transcriptase. 3. The cDNA is broken up into smaller fragments 4. known adaptor sequences are added to the ends, which allow immobilization and PCR-based amplification of these transcripts 5. Sequences can be analyzed for nucleotide quantitiesCRISPR/Cas9-mediated gene editingused to cause targeted indel formation or insertional mutagenesis within a gene of interestSteps of CRISPR/Cas91. gene-specific guide RNA (gRNA) is introduced into cells together with the nuclease Cas9 (co-injection) 2. gRNA will bind to genome and Cas9 will cause a doublestranded break 3. gRNA can recruit plasmids carrying known insertions to be input at the break 4. Cell will attempt to fix the break through non-homologous end joiningWhat does CRISPR stand for?Clustered Regularly Interspaced Short Palindromic RepeatsEvidence from molecular biology, cell biology, and somatic cell nuclear cloning has shown that each cell of the body (with very few exceptions) carries the samenuclear genomeDifferential gene expression can occur at the levels of :gene transcription, pre-mRNA processing, mRNA translation, and protein modification.Differential gene expression from genetically identical nuclei createsdifferent cell types.The histone proteins form nucleosomes, and the _________ and __________ of specific histone residues can repress or activate gene transcription, respectively.methylation and acetylationHistone methylationis often used to silence gene expression. Histones can be methylated by histone methyltransferases and can be demethylated by histone demethylases.Acetylated histonesare often associated with active gene expression. Histone acetyltransferases add acetyl groups to histones, whereas histone deacetylases remove them.Eukaryotic genes contain promoter sequences to which RNA polymerase II can bind to initiate transcription. To do so, the RNA polymerases are bound by a series of proteins calledtranscription factorsEukaryotic genes expressed in specific cell types contain enhancer sequences that regulatetranscription in time and spaceEnhancers usually activate only genes on thesame chromosomeEnhancer sequences can be upstream or downstream or within introns; they can even be millions of base pairs away from thegene they regulateSilencers act to suppress thetranscription of a gene in appropriate cell types.Specific transcription factors can recognize specific sequences of DNA in the promoter and enhancer regions. These proteins activate or represstranscription from the genes to which they have boundEnhancers work in a ________ fashion, where the binding of several transcription factors can act to promote or inhibit transcription from a certain promoter.combinatorial. In some cases, transcription is activated only if both factor A and factor B are present; in other cases, transcription is activated if either factor A or factor B is present.Enhancers work in a _________ fashion, where a gene can contain several enhancers, each directing the gene's expression in a particular cell type.modularTranscription factors act in different ways to regulate RNA synthesis. List the two waysSome transcription factors stabilize RNA polymerase II binding to the DNA, and some disrupt nucleosomes, increasing the efficiency of transcription.A transcription factor usually has three domains:1. a sequence-specific DNA-binding domain 2. a trans- activating domain that enables the transcription factor to recruit histone-remodeling enzymes 3. protein-protein interaction domain that enables the transcription factor to interact with other proteins on the enhancer or promoter.Low CpG-content promoters are usuallymethylated, and their default state is "off," but they can be activated by transcription factors.High CpG-content promoters have a default state that is"on," and they have to be actively repressed by histone methylation.DNA methylation can block transcription bypreventing the binding of certain transcription factors or by recruiting histone methyltransferases or histone deacetylases to the chromatin.Differences in DNA methylation can account for genomic imprinting:wherein a gene transmitted through the sperm is expressed differently than the same gene transmitted through the egg. Some genes are active only if inherited from the sperm or the egg.MicroRNAs can act astranslational inhibitors - binding to the 3′ UTR of the RNA. The microRNA recruits an RNA-induced silencing complex that either prevents translation or leads to the degradation of the mRNA.A variety of molecular tools have enabled the study of differentially expressed genes; list the 4 and the main function of eacha) in situ hybridization for gene expression b) ChIP-Seq to identify regulatory regions of the DNA that proteins bind to c) gene knockdown (RNA interference) d) knockout (CRISPR/Cas9) to test gene function.