Plant Molecular and Cellular Biology Lecture 9: Nuclear Genome Organization: Chromosome Structure, Chromatin, DNA Packaging, Mitosis Gary Peter

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1 Plant Molecular and Cellular Biology Lecture 9: Nuclear Genome Organization: Chromosome Structure, Chromatin, DNA Packaging, Mitosis Gary Peter 9/16/2008 1

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 2

DNA in the Nucleus is Organized into Chromosomes Chromosomes One very long linear dsdna molecule/chromosome with Single copy, Repetitive, and Highly repetitive sequences Centromere sequences Two

3 DNA in the Nucleus is Organized into Chromosomes Chromosomes One very long linear dsdna molecule/chromosome with Single copy, Repetitive, and Highly repetitive sequences Centromere sequences Two teleomere sequences Multiple origins of replication Proteins that fold and pack the long DNA strand into more compact chromatin Histones Nonhistone chromosomal proteins 9/16/2008 PMCB Lecture 11: G. Peter 3

Example of Human Genome A human cell contains 2 m of DNA

in diameter A maize cell contains 2 m of DNA stretched end to

Compaction is ~1000 fold for interphase chromosomes and 10,000

4 Example of Human Genome A human cell contains 2 m of DNA stretched end to end that must fit into a nucleus that is ~6 um in diameter A maize cell contains 2 m of DNA stretched end to end that must fit in a nucleus that is <10 um in diameter Compaction is ~1000 fold for interphase chromosomes and 10,000 fold between dsdna and mitotic chromosomes 9/16/2008 PMCB Lecture 11: G. Peter 4

5 Packing of DNA into the Nucleus: Multiple Levels of Compaction 3-fold 27 fold 700 fold ~1000 fold Interphase Mitotic 9/16/2008 PMCB Lecture 11: G. Peter 5

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 6

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 7

8 Nucleosome Core Structure: X-ray Crystal Structure Core is a histone octamer with 2 subunits of H2A, H2B, H3 and H4 with DNA wrapped around 1.65 turns in a left-handed coil Histones are basic proteins rich in lysine and arginine that make salt bridges with the backbone phosphates Extensive hydrogen bonds (146) between histones and DNA with ~1/2 forming between amino acids and phosphates on the DNA Hydrophobic bonds and salt bridges also hold the core together and the DNA The long amino terminal tails of each histone extend out from the central portion of the nucleosome 9/16/2008 PMCB Lecture 11: G. Peter 8

in a characteristic structure called the histone fold and a tail region Tail region is post translationally

9 Structure of Nucleosome Core Histones Histones are highly conserved across all eukaryotic organisms Histones are small basic proteins ( aa) rich in lysine and arginine Each histone contains a region that folds in a characteristic structure called the histone fold and a tail region Tail region is post translationally modified in various ways to control many aspects of chromatin structure 9/16/2008 PMCB Lecture 11: G. Peter 9

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 10

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 11

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 = /16/2008 PMCB Lecture 11: G. Peter 12

13 Heterochromatin Structure Heterochromatin is highly condensed and more compact than the loops of 30nm fibers Remains tightly condensed even in interphase Centromeres, pericentromeres and telomeres are organized in facultative heterochromatin Heterochromatin contains few genes Heterochromatin represses gene expression Facultative heterochromatin regions can spread and retract Histone 3 tails (H3K9Met) and Lys 27 are methylated and are underacetylated in heterochromatin 9/16/2008 PMCB Lecture 11: G. Peter 13

nucleosome arrangements Centromere nucleosomes have a unique histone H3 variant (CenH3) that together with centromere specific

14 Heterochromatin: Centromeres Centromeres contain sequence elements that are repeated (>1000X) Repeat sequences are variable in number and sequence composition between species Centromere sequences are highly compacted and contain particularly dense nucleosome arrangements Centromere nucleosomes have a unique histone H3 variant (CenH3) that together with centromere specific proteins combine to form the kinetochore that attaches the centromere to the spindle apparatus 9/16/2008 PMCB Lecture 11: G. Peter 14

the formation of telomeric heterochromatin, whereas acetylation of this lysine

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 15

Model for Higher Order Euchromatin Structure 30nm fibers are folded into loops of 20,000-100,000 bp that are attached to a scaffold

16 Model for Higher Order Euchromatin Structure 30nm fibers are folded into loops of 20, ,000 bp that are attached to a scaffold through matrix attachment regions (MARS) MARS are AT rich DNA sequence motifs bp in length 9/16/2008 PMCB Lecture 11: G. Peter 16

when visualized after gentle nuclear lysis by TEM

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 17

18 Chromatin is Highly Dynamic Interphase chromosomes are in constant flux controlled by small nuclear RNAs, DNA methylation and histone modification Chromatin remodeling unfolds 30nm fibers to expose the regions for other proteins to access and perform functions such as transcription and DNA replication Evidence comes from chromosome puff regions in Drosophila polytene chromosomes and the identification of protein complexes that remodel chromatin 9/16/2008 PMCB Lecture 11: G. Peter 18 FEBS Letters 567 (2004) 15 19

favorable to bend DNA tightly around the histone core Proteins bound to DNA at specific

19 Nucleosome Positioning Nucleosome spacing is irregular due to the local sequence of DNA and proteins bound in the vicinity A-T bases in minor groove make it more energetically favorable to bend DNA tightly around the histone core Proteins bound to DNA at specific sites can promote while others can inhibit nucleosome binding 9/16/2008 PMCB Lecture 11: G. Peter 19

Chromatin Remodeling Works at Multiple Levels Histone H1 controls 30nm chromatin fiber organization Multiple isoforms of H1 and their abundance are important for

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 20

Chromatin Remodeling: Dynamic Repositioning of Nucleosomes Chromatin remodeling complexes are multisubunit protein complexes that hydrolyze ATP to change the structure of the

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 21

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

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 22

Modification of Histone NH4 Terminal Tails Affect the Stability of 30nm Fiber and Higher Order Structures The NH4 tails of the histones in the nucleosomal core are reversibly Acetylated by histone

23 Modification of Histone NH4 Terminal Tails Affect the Stability of 30nm Fiber and Higher Order Structures The NH4 tails of the histones in the nucleosomal core are reversibly Acetylated by histone acetyl transferases Deacetylated by histone deacetylases Phosphorylated by histone kinases Dephosphorylated histone phosphatases Methylated by methylases Demethylated by demethylases 9/16/2008 PMCB Lecture 11: G. Peter 23

24 Position of Postranslational Modifications on Histone Tails in a Histone Octamer 9/16/2008 PMCB Lecture 11: G. Peter 24 Cell, Vol. 116, , January 23, 2004,

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 25

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 11: G. Peter 26

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 /16/2008 PMCB Lecture 8: G. Peter 27

28 Telomerase Cell, Vol. 95, , 1998 A ribonucleoprotein complex that adds repeated DNA nucleotides to the end of a 3 OH The ribonucleotide provides the complementary bases for synthesis 9/16/2008 PMCB Lecture 8: G. Peter 28

29 Summary DNA is folded in very precise ways to fit the long DNA molecules into a very small space, but still be able to access the DNA for replication and the genes for transcription Chromatin is very dynamic Some of the mechanisms for regulating chromatin reorganization are now being dissected 9/16/2008 PMCB Lecture 11: G. Peter 29

Chapter 5 DNA and Chromosomes

Chapter 5 DNA and Chromosomes DNA as the genetic material Heat-killed bacteria can transform living cells S Smooth R Rough Fred Griffith, 1920 DNA is the genetic material Oswald Avery Colin MacLeod Maclyn