Chromosomes and chromatinChromosomes organize and package genes inside cellsBands and specialized regions of human chromosomesHuman chromosomes, ideogramsHuman chromosomes, spectral karyotypeIdentifying translocationsDistinctive and common features of chromosomesChromatin StructurePrincipal proteins in chromatin are histonesHistone structure and functionHistone interactions via the histone foldNucleosomes are the subunits of the chromatin fiberGeneral model for the nucleosomal coreA string of nucleosomesDetailed structure of the nucleosomal coreHigher order chromatin structureAlterations to chromatin structure are key steps in regulationPhosphorylation of histonesAcetylation and Deacetylation of lysines in proteinsAcetylation and Deacetylation of histonesEffects of histone modificationsMatrix and scaffoldChromosome localization in interphaseGene activation and location in the nucleusChromosomes and chromatinChromosomes organize and package genes inside cells•Bind packaging proteins to DNA to make it more compact.–Histones +DNA = chromatin in eukaryotes–Virion proteins in viruses–HU (?) or other proteins in bacteria•Loop chromatin and attach it to a matrix in nucleiBands and specialized regions of human chromosomesp15Geimsa dark bandGeimsa light bandCentromere = CENTelomere = TELlong arm (q)short arm (p)nTEL(CCCTAA) or (AGGGTT)np11p12p14p1315.115.215.315.415.5ATA=ataxia telangiectasiaHRASHBBPTHMYODHuman chromosome 11:125 Mb, 180 cMHuman chromosomes, ideogramsMitotic chromosomes are spread and stained with Geimsa. Those that stain are shown in black.G-bands (more A+T rich).Human chromosomes, spectral karyotypeReagents specific to each chromosome.Chromosomepainting.Identifying translocationshttp://www.ncbi.nlm.nih.gov/disease/Distinctive and common features of chromosomes•Distinctive proteins and DNA sequences have been used to develop chromosome painting reagents.•Genomic DNA in vertebrates has long (megabase) stretches of G+C rich DNA, and other long stretches of A+T rich DNA–Called isochores•Virtually all this DNA is organized into chromatin, which has a common fundamental structure.Chromatin StructurePrincipal proteins in chromatin are histonesH3 and H4 : Arg rich, mostly conserved sequenceH2A and H2B : Slightly Lys rich, fairly conservedH1 : very Lys rich, most variable in sequence between speciesHistone structure and functionK5 K8 K12 K16α1 α2 α3L1 L2CNHighly charged N-terminal tail. Histone structure and functionGlobular, hydrophobic domain for histone-histone interactions and for histone-DNA interactions."Minimal" structure for a core histone, e.g. H4. Others have one additional alpha helix.Histone interactions via the histone foldα1L1α2L2α3CNThe histone fold flankedby N and C terminal tails.Dimer of histones joined by interactions at the histone fold.The alpha-helical regions of the core histones mediate dimerization.L1L2CNL1L2CNNucleosomes are the subunits of the chromatin fiber•Experimental evidence:–Beads on a string in EM–Micrococcal nuclease digestionGeneral model for the nucleosomal coreA string of nucleosomesDetailed structure of the nucleosomal coreHigher order chromatin structureHistone H1 associates with the linker DNA, and may play a role in forming higher order structures.Alterations to chromatin structure are key steps in regulationPhosphorylation of histonesCOCHNHCH2CONH......CH2GlySerOHATPADPphosphateprotein kinaseprotein phosphataseH O2COCHNHCH2CONH......CH2GlySerOHPOOO-Negative charge onphosphoserineSerines in histones can be modified by phosphorylationAcetylation and Deacetylation of lysines in proteinsAcCoACOCHNHCH2CONH......CH2CH2CH2CH2NH3+COCHNHCH2CONH......CH2CH2CH2CH2NHGlyLysCCH3OCoAAcPositive charge on amino groupNo charge on amide groupAcetylation and Deacetylation of histonesK5K8K12K16α1α2α3L1L2CNK5K8K12K16K5K8K12K16AcAcNuclear histone acetyl transferase (HAT A), e.g. yeast Gcn5p and its mammalian homolog PCAFE.g., Histone H4Cytoplasmic HAT B, e.g. yeast Hat1p + Hat2p and mammalian chromatin asssembly factor 1 (CAF1)AcAcHistone deacetylases, e.g. yeast Rpd3p and mammalian HD1Histone deacetylasesEffects of histone modifications•Highly acetylated histones are associated with actively transcribed chromatin–Acetylation of histone N-terminal tails may affect the ability of nucleosomes to associate in higher-order structures–The acetylated chromatin appears to be more “open”, and accessible to transcription factors and polymerases–HATs are implicated as co-activators of genes in chromatin, and HDACs are implicated as co-repressorsMatrix and scaffoldScaffoldloopsof duplexDNAMatrixNucleusMitotic chromosomes, withsome DNA releasedIn interphase chromosomes, at least some DNA is attached to a matrixChromosome localization in interphaseIn interphase, chromosomes appearto be localized to a sub-region of thenucleus.Gene activation and location in the nucleus•Condensed chromatin tends to localize close to the centromeres–Pericentromeric heterochromatin•Movement of genes during activation and silencing–High resolution in situ hybridization–Active genes found away from pericentromeric heterochromatin –Silenced genes found associated with pericentromeric