Lecture 6Chromosome Structure and Function in MitosisOutline:Chromosome Organization and Function in InterphaseChromatin effects on replicationEffects of nuclear organization on gene expressionChromosomes in MitosisSMC proteins:Condensins and CohesinsCentromeres and KinetochoresPaper: Loss of the Suv39h Histone Methyltransferases Impairs Mammalian Heterochromatin and Genome StabilityChromatin Structure and DNA ReplicationDNA replication is ‘simple’.......though the regulation of molecules (e.g. DNA polymerases, helicases) is not...Chromatin is also ‘Replicated’ Semi-conservativelyrequires energyremodeling factors (Swi/Snf helicases)assembly factorsCAFs-canonical histonesHIRA-histone variantsEpigenetic Patterns are Reestablished after Replicationparental nucleosomes and their modifications segregated to daughter strandsH3K9meHistones contain specific acetylated K residues prior to assemblyremoved after assemblyremoval required for other modifications of new nucleosomes, eg methylationpropagation of modifications on new nucleosomes based on parental nucleosomesvia binding protein / modification enzyme mechanism (e.g. HP1 and Su(var)3-9)Su(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 organizationnot able to be expressedLampbrush loops in Amphibian Oocytesunlooping observed in real time after tx activationchromosomes marked with lacO sitesbound by lacR-RFPAndy Belmont1819Effects 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) DernburgbwAAGAGbw normally basal, AAGAG apicalidentify position of bw-D by costainingBrown+ 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+Mitotic chromosome structure and functionHigh Salt extraction of chromosomes reveals ‘scaffold’Condensins and Cohesins~1990- cerevisiae screens for defective chromosome inheritancegenes required for Structural Maintenance of Chromosomes (SMC proteins)conserved from bacterial to human, ATPasescrucial roles in chromosome segregation (mitosis and meiosis)chromosome-wide gene regulation and recombinational repairnovel type of protein machine : function as dynamic linkers of the genomenucleotide binding sites at N and C terminilarge- 1000-1300 aamonomers fold back on themselves (antiparallel coiled-coil)exist as dimersrodsVsringsdifferent SMCs in complexes associated with different functionseach contains non-SMC proteins, associated with headsregulate catalytic activity and interactionsrodsVsringsrepairin?ATP binding drives head associationsassociations required for ATP hydrolysisATP hydrolysis required for disengaging headshinge associations strong, independent of ATPmany types of possible intra- and inter- molecular interactionscan form different higher order complexes and structuresintermolecular interactions require DNA?rodsVsringsStructures observed in vitro by EMdifferent for condensin and cohesinCondensintwo complexes:Condensin I and IIboth have SMC 2 and 4different non-SMC componentsbind DNA cooperatively, not ATP dependentcould also involve the stalkCondensin I + ATP drives positive superhelical tension on DNASpeculative model for chromosome condensation by Condensinacts on chromatin, not DNAroles of non-SMC subunits?Cohesinholds sister chromatids together through metaphaseINTERmolecular linking of two DNAs (compare to condensin)established at replication fork-preloaded in G1?degraded at onset of anaphase to allow sister separationcohesin in pericentromeric regions recruited by HP1/K9me, may be regulated differentlyRing model for CohesinCentromeres and Kinetochorescentromere vs. flanking pericentric heterochromatinprometaphase and anaphase movementspindle assembly checkpoint (SAC)there can only be one......Centromeric DNA sequences are not conservedS. cerevisiae and other budding yeast same specific, 125 bp sequence present at all 16 centromeresnecessary and sufficient for kinetochore formationeverywhere else, often repetitive, 10s-1000s of kb.....but.... sequences associated with centromeres are not conserved across species, or even among different chromosomes in the same organismbut centromere proteins are highly conservedCentromere PlasticityCentromere Identity and Propagation are Epigenetically RegulatedACTIVATION INACTIVATIONgain epigenetic marklose epigenetic markprimary sequence is not sufficient (dicentrics)‘marked’ DNA/chromatinEpigenetic Model for CEN Identitynon-centromeric sequence can acquire and propagate centromere function (neocentromeres)CEN Evolution: Hopping in terms of individual cell cycles, cerevisiae sequence specificity makes the most senseCentromere Plasticity Necessary for Chromosome Evolution ?Satellite expansion & contraction:CEN spreading & hoppingTranslocations:CEN inactivation & NeoCEN