BIO 344 1st Edition Lecture 9 Outline of Last Lecture I. Stem Cell Researcha. Pluripotentb. Direct reprogrammingc. Metastable statesII. Eukaryotic Transcription—RNAP IIa. Promotersb. Enhancersc. Transcription factors III. Tethering a protein to DNAa. Persistence lengthIV. CombinatorialOutline of Current Lecture I. Chromatin and Gene Expressiona. NucleosomeII. Histonea. Structureb. Negative supercoilingIII. Chromatina. Transcription regulationCurrent LectureChromatin and Gene Expression- Experiment: micrococcal nuclease digestion of DNAo Grind, centrifuge, cell extracto As we treat with micrococcal nuclease, DNA gets digersted and stripped, but does not go all the way to a mononucleotideo This tells us that something in the extract is protecting our DNA- Chromatin-- DNA is wrapped in units called nucleosomeso Composed of DNA wrapped around a histone twice Histone is composed of a core octomer- 2 copies of H2A, H2B, H3, and H4These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o Forms “beads on a string” Only the “string” is exposed and gets digested by micrococcal nucleaseHistone Structure- Common pathway—assembly of nucleosomeo H3 and H4 form tetramer firsto Then H2A and H2B dimer forms- Histones are highly basico Aregenin and lysine amino acids are positively charged Neutralize DNA’s negative charge or else electrostatic potential would be very higho N- termini carry a positive charge Extends out from core and wraps DNA on the exterior- H1o Part of the histone, but not the core Serves as a linker Tightens the linkage between the core and the DNADNA is negatively supercoiled- Eukaryotes don’t have topoisomerase to drive supercoiling like bacteria do (with gyrase)- We get negative super coils through having histoneso When histones are removed, we get negative supercoils, and thus access to bps for replicationo When the DNA is closed covalently and relaxed, and one nucleosome is added, what happens? Negative supercoiling occurs at the nucleosome The torsinal strain causes supercoiling at the rest of the DNA and it will not accept another histone Topoisomerase then has the additional role of nicking the DNA to relieve the positive supercoild Net resultnegatively supercoiled DNAChromatin- 2 stateso Euchromatin= accessible, not condensed, active transcriptiono Heterochromatin= dense, highly condensed, transcriptionally silent- Chromatin and transcription regulationo Accessibility modulated by histone presenceo Even with transcript factor, it won’t have access to heterochomatin, and there will be no transcription Chromatin structure needs to be modulated (more details on this later)- Experiment: impact of histones on transcriptiono Primer extension o polyglutamate enhances histone binding ability in proper confermation, but has no impact on transctiption)o if transcription occurs, synthesized product will be seeno with zero histones, 100% activityo with 0.8 histones (=physiological level= 1 nucleosome/200 bp), 12-24% activity shows that they inhibit transcription- ground stateo bacteria: RNAP has access to promotero eukaryotes: restrictive—we need activators so how do promoters get access?- Those regions must be free of nucleosomes- How do we test this? - Experiment: DNAse hypersensitive map to site “nucleosome free” regions of actively transcribed geneso Treat with DNAse to “chop” promoters (if accessible)o With many histones, there are no cut regions—no available promoterso With spaces at the promoter or few histones, accessible areas will be readily cut by nucleaseo Cut with restriction enzymeso Will see 2 small fragments in the case of a lack of histones Run in gel and see that promoter region is missing- Genome- scale mappingo Chop all DNA with micrococcal nuclease o Random distribution of nucleosomes In different cells nucleosomes are in different placeso Positioned distribution of nucleosomes Over many different cells, nucleosomes are in the same place o We get a mix of each o On the map, high spikes= high probability of finding a nucleosome at a certain locationo Spikes close together= highly phased nucleosomes- In chromatin, how do transcriptional activators work? Experiment:o gal4-VP16= hybrid activatoro with none, we get a little transcription—basal levelo with activator, we get a lot—8 fold moreo with histone, RNAP gives no transcript and activator we get 200 fold more activating and reversing inhibition of nucleosomeo past the physiological level of histones, we get transcription but it begins to decrease even with the activator- Moving/ Modifying histones to go from repressed to active stateo What drives this dynamis process? Chromatin remodeling Histone modification- Histone code many residues are subject to methylation, acetylation, ubiquitination, and phosphorylation- Acetylation: acetylate lysineo HAT histone acetyl transferase Lysine is positively charged (giving its affinity for DNA), acetylation neutralizeso Acetylation loosens grip of histones on DNA Weakens histone affinity for DNA- Deacetylation—reversing acetylationo HDAC histone deacetylaseo Experiment: what is butyrate’s impact on chromatin remodeling Incubate Friend cells to differentiate via sodium butyrate HATs can be activated or HDAC can be inhibited to loosen DNA from the histones When treated with butyrate, we see an accumulation of H3 and H4 Graph B on slides is measuring HAT activity- No significant difference with or without butyrate Graph C measures HDAC activity- Without butyrate we see deacetylation- With we see ~70% reduction in HDAC activityo Shows that butyrate inhibits HDAC rather than activates HATReprogramming cells- See slide for experiment/ imageso By inhibiting HDAC and adding a transcription factor, the germ line is reprogrammed Shows that few modification can force expression and prevent deacetylation- Allowing access for transcription