Nuclear Genome Organization - Lecture Slides | PCB 5530, Study notes of Biology

Download Nuclear Genome Organization - Lecture Slides | PCB 5530 and more Study notes Biology in PDF only on Docsity! 9/16/2008 1 Plant Molecular and Cellular Biology Lecture 9: Nuclear Genome Organization: Chromosome Structure, Chromatin, DNA Packaging, Mitosis Gary Peter 9/16/2008 PMCB Lecture 11: G. Peter 2 Learning Objectives 1. List and explain how DNA is packaged in the nucleus 2. Explain how euchromatin and heterochromatin differ 3. Explain proposed mechanisms that maintain heterochromatin in its state 4. Explain the structure, functions of enzymes that mediate chromatin remodeling and their proposed mechanisms 9/16/2008 PMCB Lecture 11: G. Peter 5 Packing of DNA into the Nucleus: Multiple Levels of Compaction Interphase Mitotic 3-fold 27 fold 700 fold ~1000 fold 9/16/2008 PMCB Lecture 11: G. Peter 6 Evidence for Nucleosomes as the Basic Unit of Chromosome Structure Histone mass = the mass of DNA in chromatin Gentle lysis of nuclei and TEM analysis shows that chromatin is a 30nm wide thread Decondensation of chromatin reveals beads on string 9/16/2008 PMCB Lecture 11: G. Peter 7 Nucleosome Isolation & Organization Unfolded chromatin is digested with micrococcal nuclease Limited digestion leaves histone H1 + nucleosomal core with an average of 200bp of DNA More extensive digestion releases H1 and yields core particles with 146bp of DNA protected from nuclease digestion The 54bp on average is a linker DNA (Linker varies from 5-80bp) Nuclesome cores dissociated with high salt removes the 146 bp DNA from the octameric histone core 9/16/2008 PMCB Lecture 11: G. Peter 10 Histone Octamer Assembly Dimers of H3-H4 form and then two dimers assemble into a very stable tetramer Two H2A-H2B dimers associate with the H3- H4 tetramer 9/16/2008 PMCB Lecture 11: G. Peter 11 Nucleosome Packing into 30nm Fibers Zigzag and solenoid models for packing Histone H1 plays a role by possibly altering the path of DNA that exits from the histone core helping to pull nucleosomes together Histone tails may help attach nucleosomes together 9/16/2008 PMCB Lecture 11: G. Peter 12 What is the Fold Compaction in 30nm Fibers? Assuming that the 30nm chromatin fiber contains 20 nucleosomes (200bp/nucleosome) per 50nm of length what is the degree of compaction? It is compacted 27 fold in 30nm fibers relative to extended DNA dsDNA in 50nm is (20 nucleosomes x 200 bp/nucleosome x 0.34 nm/bp) = 1360nm 1360/50 = 27.2 9/16/2008 PMCB Lecture 11: G. Peter 15 Model for the Organization of a Chromosome End The telomere forms a t-loop which lacks nucelosomes In heterochromatin, unacetylated lysine 16 of histone H4 is required for the formation of telomeric heterochromatin, whereas acetylation of this lysine functions as a barrier to the spread of heterochromatin. 9/16/2008 PMCB Lecture 11: G. Peter 16 Model for Higher Order Euchromatin Structure 30nm fibers are folded into loops of 20,000-100,000 bp that are attached to a scaffold through matrix attachment regions (MARS) MARS are AT rich DNA sequence motifs 200-1000 bp in length 9/16/2008 PMCB Lecture 11: G. Peter 17 Evidence for Scaffold It appears that interphase and mitotic chromatin are attached to a scaffold when visualized after gentle nuclear lysis by TEM negative staining 9/16/2008 PMCB Lecture 11: G. Peter 20 Chromatin Remodeling Works at Multiple Levels Histone H1 controls 30nm chromatin fiber organization Multiple isoforms of H1 and their abundance are important for cell growth and proliferation ATP dependent chromatin remodeling works at the level of nucleosomes 3-fold 27 fold 700 fold 9/16/2008 PMCB Lecture 11: G. Peter 21 Chromatin Remodeling: Dynamic Repositioning of Nucleosomes Chromatin remodeling complexes are multisubunit protein complexes that hydrolyze ATP to change the structure of the nucleosome core so that the DNA becomes less tightly associated Movement of the H2A & H2B dimers in the nucleosome cores may be the mechanism 9/16/2008 PMCB Lecture 11: G. Peter 22 ATP Dependent Chromatin Remodeling ATP dependent protein remodeling is mediated by multiple large multisubunit complexes These complexes affect the interaction of DNA with the nucleosomes – opening the DNA for access by other factors The SWI/SNF complexes from yeast are required for viability and bind well with naked DNA Many sets of different chromatin remodeling enzymes exist These activities are involved in most all aspects of DNA repair, DNA transcription, DNA replication 9/16/2008 PMCB Lecture 11: G. Peter 25 Histone Code Hypothesis Distinct markings of histone tails confers particular “meanings” by attracting those proteins involved with appropriate functions Gene expression should not take place DNA has been recently replicated 9/16/2008 PMCB Lecture 11: G. Peter 26 DNA Methylation and Chromatin Organization: Epigenetic Control in Plants The DDM1 gene of Arabidopsis is required to maintain DNA methylation levels and is needed for transposon and transgene silencing It also is required for maintenance of histone H3 methylation patterns DDM1 is similar to the SWI/SNF family of ATP dependent chromatin remodeling genes DNA methylation patterns may depend on histone H3 methylation patterns Epigenetic inheritance of hypomethylated DNA occurs 9/16/2008 PMCB Lecture 8: G. Peter 27 Telomeres & Telomere Replication Replication of the ends of linear DNA molecules are problematic for the replication machinery and loss of sequences from the ends occurs through multiple cycles Telomeres are located at the ends of the chromosomes, and they have unique repeated sequences and a 3’ overhanging single stranded DNA Telomerase is a DNA polymerase that completes replication of telomere sequences Specialized reverse transcriptase TRENDS in Genetics Vol.19 No.8 August 2003