BICH/GENE 431 KNOWLEDGE OBJECTIVES

 

 

Chapter 7 – Genome Structure and Chromatin

Know relative genome sizes and gene densities between model organisms

Why is gene density less for “higher” organisms?

-       introns

-       more intergenic DNA: regulatory sequences, repetitive DNAs

Different types of repetitive DNA

-       microsatellite repeats: very short, tandemly repeated

-       genome-wide repeats: moderate size (100-1000bp) and interspersed

Essential features of eukaryotic chromosomes

-       origins of replication: many, typically 30-40 kbp apart

-       centromeres: only one per chromosome to direct proper segregation

-       telomeres: protein/DNA complexes; one at each end; contain short repeats that are characteristic of organism; 3’ overhanging ends fold back to make T-loops

Eukaryotic cell cycle

-       know four phases

-       ~24 hours long in mammals

-       significance of cell cycle checkpoints and cell cycle arrest

Interphase chromatin vs. chromosome condensation in M phase

Events at S phase – cohesin rings hold sister chromatids together

Events at M phase – formation of kinetochore complex; attachment to microtubules and

to microtubule organizing center to form mitotic spindle; cohesins are proteolyzed to allow migration of sister chromatids to opposite poles

SMC proteins (structural maintenance of chromosomes) – cohesins and condensins

-       form rings with other proteins to hold chromosomes together

-       condensins mediate chromosome condensation at M phase

You should review steps in mitosis and meiosis (Figs. 7-15 and 7-16) on your own.

Massive DNA compaction necessary to fit into nucleus – 2 meters of DNA into 10-15

micron size of nucleus

Nucleosome structure

            nucleosome core particle: 147 bp DNA + octamer of core histones; 1.65 negative

superhelical turns

            linker DNA: size varies between organisms – core + linker for humans ~200 bp

            basic properties of core histones

            H3/H4 forms tetramer; H2A/H2B form two dimers

            Histone fold motifs contain alpha helices and mediate histone-histone

interactions

            N-terminal tails stick out: are susceptible to protease treatment; sites of extensive

modification to control functions

            Dyad axis of core particle

            Only approx. 6-fold compaction of DNA

Subunit structure of chromatin discovered by Roger Kornberg (and others)

            Micrococcal nuclease assay to detect DNA repeat

Supercoiling caused by nucleosomes – understand supercoiling assay to detect

nucleosomes (Box 7-2)

Histone-DNA interactions are very strong

-       mostly via extensive hydrogen bonds to oxygens of phosphates on DNA

-       involve minor groove interactions

-       do not have DNA sequence specificity

-       positive charges on histones help to shield neg. charge on DNA to enable bending of DNA around histone core

-       requires high salt concentrations to break histone-DNA interactions

Higher-order chromatin structure

-       involves binding of histone H1 to linker DNA

-       formation of 30 nm fiber

-       two models for 30 nm fiber: solenoid vs. zigzag structures

-       need histone tails for 30 nm fiber

-       approx. 40-fold DNA compaction with formation of 30 nm fiber (still a long way to go)

Nuclear scaffold – large loops of DNA attached; contains topoisomerase II and SMC

proteins

Nucleosome remodeling – why needed?

-       sliding vs. transfer

-       nucleosome remodeling complexes – know some names, esp. SWI/SNF

-       requires energy of ATP hydrolysis

Nucleosome positioning – some nucleosomes are positioned at precise locations on

genomic DNA, especially near promoters

-       mechanisms that contribute to positioning: protein binding; DNA sequences that favor or disfavor DNA bending

Histone acetylation

-       on lysines, usually on histone tails, know structure, removes pos. charge

-       comes from acetyl-CoA

-       correlates with transcriptional activation, or needed for deposition of new histones, or for DNA repair

-       remember some locations: H3K9, H4K8, H4K16

Histone methylation

-       on lysines or arginines, usually on histone tails, know structure on lysines

-       can be mono-, di-, or tri-methylated

-       comes from SAM (S-adenosyl methionine)

-       correlates with either transcriptional activation or repression, depending on site

-       remember some locations: H3K4 (activating), H3K9 (repressive), H3K27 (repressive)

Histone phosphorylation

-       on serines or threonines, know structure, adds a negative charge

-       comes from ATP

-       correlates with mitosis, apoptosis, or activation/repression of transcription

-       remember some locations: H3S10 (activating), other H3 sites correlate with mitosis

histone code hypothesis

proteins containing specific domains bind to various histone modifications

-       bromodomains bind to acetylated histone tails

-       chromodomains bind to methylated histone tails

-       SANT domains interact preferentially with unmodified histones

Histone modifications are reversible

Enzymes that catalyze histone modifications

-       HATs (histone acetyl transferases); add acetyl groups; correlate with activation of transcription; nuclear HATs vs cytoplasmic HATs

-       HDACs (histone deacetylases); remove acetyl groups; correlate with repression of transcription

-       Histone methyltransferases; add methyl groups; generally more specific for a given site of modification; SET domain proteins

-       Histone demethylases; remove methyl groups

-       Kinases add phosphoryl groups

-       Phosphatases catalyze hydrolysis of phosphoryl groups

An example of how histone modification is coordinated with nucleosome remodeling to

control access to DNA in chromatin (Fig. 7-41)

Nucleosome assembly after DNA replication

-       old H3/H4 tetramers stay together

-       old H2A/H2B dimers stay together

-       nucleosomes reassemble randomly on two new daughter molecules

-       histone code is propagated because HATs and HMTs contain bromodomains or chromodomains to recognize old modification – then modify new histones in that region (see Fig. 7-43)

Histone chaperones are needed to assemble nucleosomes on DNA

-       CAF-1 binds to H3/H4 tetramers

-       NAP-1 binds to H2A/H2B dimers

-       There are other chaperones too.

-       CAF-1 interacts with PCNA in order to direct H3/H4 tetramers to newly replicated DNA (PCNA is proliferating cell nuclear antigen, a sliding clamp protein that binds to DNA polymerase at the replication fork)