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Chromatin Structure & Genome Organization Overview of Chromosome Structure • Nucleosomes – ~200 bp DNA in 120Å diameter coil – 3.4Å/bp x 200 = 680Å – 680/120 = 5X compaction • 30nm fiber – Coil of nucleosomes w/ 6/turn – 1200bp/120Å vs 4080Å = 34X compaction • Chromatin loops – 30nm fiber is looped into 15100Kbp loops – 7-8 loops form rosette w/ bases of loops attached to central core of scaffold proteins – 300Å/rosette, 800Kbp/rosette = 800kbp/300Å vs 2720000Å 9000X compaction Nucleosomes • Core of 8 proteins, 2 of each – – – – H2A H2B H3 H4 • 166 bp of DNA • ~40bp between successive nucleosomes Nucleosomes • DNA associated with individual nucleosomes determined by DNAse I digestion Nucleosome • Each nucleosome contains 1 linker histone – H1 • Linker histones can vary with cell types and differentiation states Histone Post-translational Regulatory Modifications • Acetylation • Phosphorylation The Histone Song (To the tune of "Flintstones") With Accompaniment • Histones! Meet the Histones! They're a basic protein family. • There are Just four subtypes. They're conserved evolutionarily. • Two each Of each histone Do their thing. Forming Just like beads upon a string. • H1 Binds the complex. And it inhibits new transcription And the creation Of new mRNA. Transcriptional Regulation by Histone Modification • Histone acetyltransferase is component of transcriptional activation complexes H1 Promotes Nucleosome Compaction -H1 +H1 • H1 function thought to be regulated by phosphorylation on N and C terminal tails Heterochromatin vs Euchromatin • Constitutive heterochromatin – Never transcribed DNA • Facultative heterochromatin – DNA regions that can be switched between heterochromatin and euchromatin states – X inactivation • Chromo domain proteins – Bind to DNA keeping in heterochromatin state • Polycomb protein family in Drosophila Heterochromatin vs Euchromatin • Modifications to histones and DNA direct formation of heterochromatin •Deacetylation of H4 •Methylation of H3 •Methylation of CpG islan Loops & Chromosome Scaffolds • Chromosome scaffold – Proteins at base of loops – Potentially regulate formation of loops and/or regulate global gene expression • Components – DNA topoisomerase II – SMC proteins – HMGA • DNA elements – S/MARS • Sequences of DNA to which scaffold proteins bind – LCR • DNA elements known to regulate expression of large regions of DNA containing multiple genes – Boundary elements • scs elements flank puffs in Drosophila polytene chromosomes Chromosome Loops Chromosome Structure: The Metaphase Chromosome Centromeres • DNA sequence – Repetitive DNA elements – Constitutive heterochromatin • Protein components – CENP-A, B & C Centromeres • Repetitive DNA of mammalian centromeres – satellite DNA – 171 bp unit – Several specific unit sequences – Multiple repeats of these units form arrays Centromeres • Centromere of S. cerevisiae – CEN DNA is 125bp element – 3 regions - CDE I, II, & III – CDE's bound by special histone and other proteins and direct attachment of microtubule Telomeres Genome Organization • Sequence composition – Unique sequences • Genes – Low copy # elements • rRNA, 5S, tRNA genes – High copy # repetitive DNA • Retroelements – LINEs – SINEs • Simple Sequence DNA – Minisatellites (VNTR) – Microsatelites Retrotransposons, Retroelements & Transposons 23 – 9bp Repeats Mouse Satellite DNA Repeating unit given by restriction enzyme digestion 4 Imperfect Repeating Units VNTRs in DNA Fingerprinting