4/12/15&1&What&is&the&molecular&basis&for&cell&fate&determina8on?&1. Understand&the&structure&of&eukaryo8c&chroma'n&including&the&structure&and&role&of&nucleosomes&and&the&different&packing&states&of&chroma8n.&2. Understand&how&chroma8n&can&be&“remodeled”.&3. Know&how&modifica8ons&of&histones&influence&transcrip8on.&4. Be&able&to&explain&the&three&different&mechanisms&by&which&cells&can&“remember”&their&transcrip8onal&state&JJ&in&other&words&the&mechanisms&for&cell&fate&determina8on.&Learning0Goals:00Reading:&ECB3&184J192;279J281;287J288&ECB4&184J190;271J273;279J281&In&sec8on&this&week:&Gene8cs&and&development&of&Drosophila&4/12/15&2&Transcribed&region&Transcribed&region&Cell&A&Cell&B&Not&covered&in&class,&but&covered&in&the&book&or&synopsis&or&both:&Determina8on&(commitment&to&a&par8cular&cell&fate)&is&progressive.&Ectoderm0vs.0endoderm0vs.0mesoderm 0ventral000vs.000dorsal 0 0notochord00000vs.00000intermediate0mesoderm0000 0back0muscle000000000vs.000000000heart 00Determina8on&requires&that&cells&remember&their&history!&Epigene8c&landscape&**See additional slide for question about strategy for using mutants to trick bacteria into maximizing beta-galactosidase production. keep iclicker question: Mutation in the operator of the lac operon that prevents biding of the repressor Mutation in the CAP protein that makes CAP active in the absence of cAMP. CAP must bind DNA to turn operon on.4/12/15&3&Learning&Goal&1:&&Understand&the&structure&of&eukaryo8c&chroma'n&including&the&structure&and&role&of&nucleosomes&and&the&different&packing&states&of&chroma8n.&&&Eukaryo8c&DNA&is&packaged&along&with&proteins&into&chroma'n.&&There&are&two&classes&of&chromosomal&proteins:&&&histones&and&nonJhistone&chromosomal&proteins&&Figure 5-21 Essential Cell Biology (© Garland Science 2010) 30&nm&chroma8n&fiber&Beads&on&a&string&Beads&=&nucleosomes0String&=&DNA&&Linker&DNA&&from&a&few&nucleo8des&to&80&nucleo8des&4 histones 8 subunitsLength of DNA between nucleosomes = variable lengthbeads = nucleosomes string = DNA4/12/15&4&Figure 5-22 (part 2 of 2) 5-21 Essential Cell Biology (© Garland Science 2010) Histones0* get slide off of blackboardvideo on packagingimportant virtue of histones = amino N-terminal tails can interact with DNA, with other histones, and with other proteins in nucleus can faciliate the packing4/12/15&5&Histone&tails&The&histone&NJterminal&tails&can&interact&with&DNA,&with&other&histones&and&with&other&proteins&in&the&nucleus.&4/12/15&6&Figure 7-12 Essential Cell Biology (© Garland Science 2010) Ini8a8on&of&eukaryo8c&transcrip8on&requires&a&set&of&general0transcrip'on0factors&in&addi8on&to&RNA&polymerase.&(you&do&not&need&to&memorize&the&details&of&this&process.)&But&this&is&not&enough!&TATA0box&&J&sequence&of&DNA&rich&in&A&and&T&that&serves&to&ini8ate&binding&of&general&transcrip8on&factors&4/12/15&7&In&eukaryotes,&transcrip8onal&regulators&&can&bind&at&great&distances&from&their&targets&and&influence&transcrip8on&Modified&from&Figure&8J10&Essen8al&Cell&Biology&J&Garland&Science&©&Enhancer0=0the0DNA0sequence0that0binds0to0ac'vators0or0repressors&&Figure 8-10 Essential Cell Biology (© Garland Science 2010) enhancer0repressor0or0can be regulatory or repressors4/12/15&8&Modified&from&Figure&8J10&Essen8al&Cell&Biology&J&Garland&Science&©&prevented&Summary:&&1. RNAPol&II&can&only&bind&at&eukaryo8c&promoters&with&the&help&of&a&large&group&of&general&transcrip8on&factors.&2. Ac8va8on&of&these&large&complexes&can&be&promoted&by&ac8vator&proteins&that&can&bind&to&enhancer&sequences&at&large&distances&from&the&promoter.&3. Ac8va8on&of&these&large&complexes&can&be&prevented&by&repressor&proteins&that&can&bind&to&enhancer&sequences&at&large&distances&from&the&promoter.&&4/12/15&9&How&does&the&transcrip8onal&apparatus&deal&with&this&packing?&(Cartoon&represen8ng&the&30&nm&chroma8n&fiber)&Figure 5-27 Essential Cell Biology (© Garland Science 2010) Chroma8n&can&be&“remodeled”&by&chroma'n0remodeling0complexes&to&expose&or&condense&appropriate&segments&of&DNA&for&gene&regula8on.&Newly&exposed&DNA&2 kinds of mechanism 1) chromatin remodeling complexes: use ATP. can remove nucleus cells and open up more of the DNA4/12/15&10&Figure 5-28 Essential Cell Biology (© Garland Science 2010) Histones&are&modified&by&histone0modifying0enzymes0to&regulate&chroma8n&packing&and&to&recruit&transcrip8onal&machinery.&M&&M0M&&A0M&&P0methyla8on&acetyla8on&phosphoryla8on&2) modification of the histone tails. mechanisms can be added to the tails. Adding the modifications changes the configuration of the chromatin. there is a code. Some modifications silence the genes (crunch the genes together) or opening the genes (opening the DNA)** slide below isn't in the book. List of enzymes that add or remove modifications4/12/15&11&Transcrip8onal&regulators&Chroma8n&remodelers&have&limited&access&Transcrip8onal&regulators&and&chroma8n&remodelers&have&access&Figure 8-10 Essential Cell Biology (© Garland Science 2010) enhancer04/12/15&12&Figure 8-11 Essential Cell Biology (© Garland Science 2010) Transcrip8onal&regulators&can&recruit&histoneJmodifying&enzymes&and&chroma8nJremodeling&complexes&to&specific&promoters.&Figure 8-12 Essential Cell Biology (© Garland Science 2010) The&transcrip8onal&status&of&any&gene&is&the&result&of&a&combina8on&of&transcrip8onal&&regulators&that&recruit&chroma8n&remodeling&proteins&and&histone&modifying&enzymes&to&ac8vate&or&repress&transcrip8on.&histone-modifying enzymes, chromatin remodeling complexes, and Mediatoriclicker question: Histone modification doesn't regulate transcription through changing the stability of the nucleosome4/12/15&13&Determina8on&(commitment&to&a&par8cular&cell&fate)&is&progressive.&Ectoderm0vs.0endoderm0vs.0mesoderm 0ventral000vs.000dorsal 0 0notochord00000vs.00000intermediate0mesoderm0000 0back0muscle000000000vs.000000000heart 00Determina8on&requires&that&cells&remember&their&history!&Epigene8c&landscape&1. Chroma8n&state&(via&heritable&histone&modifica8ons)&2. Posi8ve&feedback&loops&3. DNA&methyla8on&&Epigene'c0inheritance0Epigene8c&landscape&**Important slidesomething is happening in cell to allow cell to continue to express that expressionepigenetics: on top of genetics. positive feedback loops4/12/15&14&Figure 5-32 Essential Cell Biology (© Garland Science 2010) Histone&modifica8ons&and&hence&chroma8n&configura8on&can&be&inherited&from&mother&to&daughters.&Histone&modifying&enzyme&Figure 8-20