Lecture 5Chromosomes in the Cell CycleOutline:Basics of Chromosome OrganizationChromosome Organization and Function in InterphasePaper:Some Cell Biologists view of the Cell....the nucleus IS part of the cell and cell biologyChromosome Organization Changes Dramatically during the Cell CycleInterphase MitosisChromosome Organization Changes Dramatically during the Cell CycleHumans mitotic chromosomes are colorfulSKY-Spectral KarYotypingFISH with probes from FACs-sorted chromosomeseach labeled with unique mixture of fluorophoresOrganization of DNA in the NucleusOrganization of Eukaryotic Chromosomes/Genomesheterochromatin euchromatinmostly repeated DNAsome single copy genescondensed through cell cycle?stains brightly with DAPImostly single copy DNArepeat content depends on organismcondensed/decondensed through cyclestains weakly with DAPIHeterochromatic ‘Knobs’ in Cornmany levels of packagingmany levels of packagingmitotic chromosome - 10,000 fold shorter than length of DNA molecules‘beads on a string’ = nucleosomeslow salt treatment to remove ‘weakly bound’ proteins‘beads’ = core histones, ‘strings’ = DNAtop view side viewHistone N-terminal tails......NOT in the crystal structureHistones tails (and cores) are post-translationally modifiedHistone modifications are correlated with different properties (functions?)Proteins associated with Histone modificationsEnzymes:methyltransferases (HMTases)acetyltransferases (HATs)deacetylases (HDACs)demethylases Binding Proteins:Heterochromatin Protein 1 (HP1) H3K9me2 and 3Polycomb Proteins (Pc) H3K27me2 and 3bind modification via ‘chromodomain’different enzymes for each modificationSuvar3-9 - H3K9meEz - H3K27meTrx - H3K4me‘active’ or ‘open’ chromatin ‘silent’ chromatinmethylationacetylationphosphorylationSUMOylationubiquitinationD. AllisT. JenuweinThe ‘Histone Code’ HypothesisPosition Effect Variegation (PEV) = Silencingabnormal proximity to heterochromatin causes CLONAL, HERITABLE SILENCING of normally active genesheritable change in function without change in DNA sequence(EPIGENETIC)able to spread over Mbs of DNAMutations in Histone Modification Proteins Affect SilencingSuppressors and Enhancers of Variegation (Su(vars) and E(vars)Su(var)3-9 H3K9 HMTaseSu(var)2-5 HP1 K9me binding proteinproteins act cooperatively to propagate and spread modification stateEpigenetic inheritance of functional states depends on histone modifications and associated proteinsPOMBESWI6 = HP1 (binds H3K9me)Clr4 = H3K9 HMTaseThe RNAi Pathway and Post-transcriptional SilencingThe RNAi Pathway also Establishes Silent ChromatinGrewal and othersThe RNAi Pathway also Establishes Silent ChromatinGrewal and othersBeyond the beads....the 30nm fiberRabl configurationcentromerestelomeresChromosome Organization and Function in Interphasechromosomes are NOT randomly positioned in interphaseSpecific Regions are Associated with the PeripheryTelomeres HeterochromatinLaminIndividual Chromosomes are Organized as Domains or Territories1819Bickmore, Cremerearly replicatinglots of geneslate replicatinglower gene densityNucleolus(rRNA tx and processing)Cajal bodies(tx, processing)Replication‘factories’Speckles(splicing)PML bodies (tx)Order in the Interphase Nucleus:Nuclear ‘Organelles’ or ‘Protein Bodies’transcription and/or processing of mRNAs, snRNAs, histone mRNAs, and rRNAspatially associate with several U snRNA and histone gene loci (unusual 3’ processing)relationship to expression and nuclear metabolism of specific types of RNAs?The Cajal BodyThe Nucleolus:A Model for Formation of Nuclear OrganellesrDNA genes are the most highly transcribed genes in the genomedespite being embedded in heterochromatinMininucleoli form at ectopic sites of rDNA integrationModel for formation of a nuclear organelle:self assembly around nucleating elementeg transcribed rRNA recruits nucleolar componentsChromatin Structure and DNA ReplicationDNA replication is ‘simple’.......though the regulation of molecules (e.g. DNA polymerases, helicases) is not...Chromatin is also ‘Replicated’Epigenetic Patterns are Reestablished after Replicationpropagation of modifications in parental nucleosomesSu(var)3-9 : H3 K9 methyltransferaseHP1 / Su(var)2-5 : chromodomain, binds H3 K9 Me & 3-9JenuweinPropagation of a ‘Silent’ Epigenetic StateReplication is Initiated at ‘Origins’normally sequence dependent in cerevisiae, probably epigenetic in higher euksTemporal Control of Replication during S phasesynchronize cellsadd labeled nucleotides (e.g. BrdU) at different times in Slook at patterns in mitotic chromosomesTiming of Replication in Yeast is Chromatin-Dependentidentification of ARSs (Autonomously Replicating Sequences)Replication Timing and Activity is Chromatin-Dependent in Yeast(Ferguson and Fangman 1992; Weinreich et al. 2004)each rDNA copy (in tandem arrays) contain an ARS, but only 20% ‘fire’telomeric regions ‘silenced’ for gene expression-also replicate late in SGene expression is also dependent on chromosome structure and nuclear organizationLampbrush loops in Amphibian Oocytes1819Effects on expression in the context of nuclear architecturebw+Brown-Dominant: A model for effects of nuclear organization of chromosomes on gene expressionbw expressedbw expressed variablybw+bw+use FISH probes-mark positions of bw and heterochromatin (satellites)bwAAGAGbw normally basal, AAGAG apicalidentify position of bw-D by costainingBrown-Dominant associates with heterochromatinin tissues relevant to eye expressionlarval imaginal discs (eye precursor)association is specific to the SAME chromosome, not heterochromatin in generalbw+ normally in euchromatic ‘compartment’bw-D ‘loops’ in cis to associate with 2 heterochromatin, due to AAGAG insertionbw+ / bw-D - bwD associates in trans with bw+, ‘loops’ bw+ in trans to associate with 2 heterochromatin and silence gene expression of bw+ThursdayMitotic chromosome structure and