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Plant Molecular and Cellular BiologyLecture 9: Nuclear Genome Organization: Chromosome Structure, Chromatin, DNA

Packaging, Mitosis

Gary Peter

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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

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DNA in the Nucleus is Organized into Chromosomes

Chromosomes –One very long linear dsDNA molecule/chromosome with

Single copy, Repetitive, and Highly repetitive sequencesCentromere sequencesTwo teleomere sequencesMultiple origins of replication

Proteins that fold and pack the long DNA strand into more compact chromatin

HistonesNonhistone chromosomal proteins

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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 diameterA maize cell contains 2 m of DNA stretched end to end that must fit in a nucleus that is <10 uM in diameterCompaction is ~1000 fold for interphase chromosomes and 10,000 fold between dsDNA and mitotic chromosomes

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Packing of DNA into the Nucleus: Multiple Levels of Compaction

Interphase Mitotic

3-fold

27 fold

700 fold

~1000 fold

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Evidence for Nucleosomes as the Basic Unit of Chromosome Structure

Histone mass = the mass of DNA in chromatinGentle lysis of nuclei and TEM analysis shows that chromatin is a 30nm wide threadDecondensation of chromatin reveals beads on string

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Nucleosome Isolation & Organization

Unfolded chromatin is digested with micrococcal nuclease

Limited digestion leaves histone H1 + nucleosomal core with an average of 200bp of DNAMore extensive digestion releases H1 and yields core particles with 146bp of DNA protected from nuclease digestionThe 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

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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 coilHistones are basic proteins rich in lysine and arginine that make salt bridges with the backbone phosphatesExtensive hydrogen bonds (146) between histones and DNA with ~1/2 forming between amino acids and phosphates on the DNAHydrophobic 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

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Structure of Nucleosome Core Histones

Histones are highly conserved across all eukaryotic organismsHistones are small basic proteins (102-135 aa) rich in lysine and arginineEach histone contains a region that folds in a characteristic structure called the histone fold and a tail regionTail region is post translationally modified in various ways to control many aspects of chromatin structure

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Histone Octamer Assembly

Dimers of H3-H4 form and then two dimers assemble into a very stable tetramerTwo H2A-H2B dimers associate with the H3-H4 tetramer

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Nucleosome Packing into 30nm FibersZigzag and solenoid models for packingHistone H1 plays a role by possibly altering the path of DNA that exits from the histone core helping to pull nucleosomes togetherHistone tails may help attach nucleosomes together

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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) = 1360nm1360/50 = 27.2

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Heterochromatin StructureHeterochromatin 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

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Heterochromatin: CentromeresCentromeres 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 arrangementsCentromere 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

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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.

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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

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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

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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

FEBS Letters 567 (2004) 15–19

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Nucleosome PositioningNucleosome spacing is irregular due to the local sequence of DNA and proteins bound in the vicinityA-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

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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

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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

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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 DNAMany sets of different chromatin remodeling enzymes existThese activities are involved in most all aspects of DNA repair, DNA transcription, DNA replication

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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 transferasesDeacetylated by histone deacetylasesPhosphorylated by histone kinasesDephosphorylated histone phosphatasesMethylated by methylasesDemethylated by demethylases

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Position of Postranslational Modifications on Histone Tails in a Histone Octamer

Cell, Vol. 116, 259–272, January 23, 2004,

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Histone Code Hypothesis

Distinct markings of histone tails confers particular “meanings” by attracting those proteins involved with appropriate functions

Gene expression should not take placeDNA has been recently replicated

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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 silencingIt 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 patternsEpigenetic inheritance of hypomethylated DNA occurs

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Telomeres & Telomere ReplicationReplication of the ends of linear DNA molecules are problematic for the replication machinery and loss of sequences from the ends occurs through multiple cyclesTelomeres are located at the ends of the chromosomes, and they have unique repeated sequences and a 3’ overhanging single stranded DNATelomerase is a DNA polymerase that completes replication of telomere sequences

Specialized reverse transcriptase

TRENDS in Genetics Vol.19 No.8 August 2003

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Telomerase

A ribonucleoprotein complex that adds repeated DNA nucleotides to the end of a 3’OHThe ribonucleotide provides the complementary bases for synthesis

Cell, Vol. 95, 963–974, 1998

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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 transcriptionChromatin is very dynamic Some of the mechanisms for regulating chromatin reorganization are now being dissected