Chapter 7

Genome Structure, Chromatin, and the Nucleosome

Chapter OverviewI. Chromosome & Genome OverviewII. Overall Genome OrganizationIII. Introns and Intergenic DNAIV. RNAV. Chromosome ComponentsVI. The Cell Cycle: Chromosome Duplication

and SegregationVII. The NucleosomeVIII. Chromatin StructureIX. Regulation of Chromatin Structure

Chromosome & Genome Overview

• Chromosome – one molecule of DNA and associated proteins (half of the mass)

• Chromatin – complex of DNA and proteins that make up the chromosome– DNA– Nucleosome (histones)– Non-histone proteins – transcription,

replication, repair, recombination, topology

• Allows 2 meters of DNA to fit in the cell• Stability/protection from degradation• Means of transmission to daughter cells• Overall architecture contributes to

regulation of expression, recombination

I.

Prokaryotes• Millions of base pairs• 1 mm chromosome into 1 mm• Usually circular (although linear

has been found)• Have proteins similar to histones• Must be separated by

topoisomerase after replication• Usually 1 complete copy of

the chromosome in nucleoid• Plasmids

Ruptured E. coli

Eukaryotes• Billions of base pairs (compact,

restricted access)• 2 to 50 chromosomes • Usually diploid with 2 homologs

(thousands in protozoa Tetrahymena)• Haploid• Polyploid

– Allows for more RNA (and protein) generation

– Megakaryocytes (~32-64 copies of each chromosome) for platelet production

Humans• Autosomes – 22 pairs, 1 copy from each parent• Sex chromosomes (X and Y) – carry sex-determining genes.

Evolved ~200-300 million years ago after split from monotremes, evolved independently in reptiles, birds, plants

• Mitochondrial DNA – mostly from mother• Thousands of microorganisms – from mother, environment

http://www.nature.com/nrg/journal/v14/n2/abs/nrg3366.html

Genome size roughly correlated with an organism’s complexity, number of genes more closely linked to complexity

Genome Density• Viruses – some use both strands and have overlapping genes• E. coli genome – composed almost entirely of genes, with only a few

transcriptional regulatory regions, and operons to control many genes• More complex organisms have decreased gene density

– Intergenic sequences• Unique – mutated genes or pseudogenes• Repetitive • Junk DNA

– Regulatory sequences– Introns – Still thousands of overlapping genes

https://public.ornl.gov/site/gallery/gallery.cfm?topic=47

Veeramachaneni et. al., 2004

MutY Homolog

Target of Early Growth Response Factor 1Testis-specific kinase 2

Chromosome 1, P arm

Mitochondria

“stop” (TAA) only after polyA tail is added

Overall Genome OrganizationII.• Very similar• Chimpanzees, gorillas,

orangutans– 24 chromosomes – MRCA 14 mya

• Humans– 23 chromosomes– Chromosome 2 is a

fusion of two other chromosomes, homologues of which are found in other hominidae

– Vestigial centromere and telomeres

– In addition, ~2-6% Neanderthal DNA

Genome Organization – Humans vs. Mice

Cut human genome into ~150 pieces to piece together the mouse genome.Location usually irrelevant, but clustering is often required. Example: “On chromosome 11, there are five functional and two nonfunctional beta globin genes in a row. If the beta globin genes are removed from their surroundings, they are not properly regulated. Also, if you mix up the order of the genes, they are expressed at the wrong times.” --Dr. Barry Starr, Stanford University

http://genome.cshlp.org/content/13/6a/1169.full

New Genes on Chromosome 2 at Fusion Site• 15,000 new base pairs, seem to have come from Chromosome 9 prior to fusion event

Transcription control, growth/development

Non-functional

Unknown function

Bacterial protein that synthesizes B12 (brain development?)

The 44 Chromosome Man

• Chromosome 14 and 15 fused• Perfectly normal• His children (with 46 female)

would carry 1 copy of this fusion, and 2 copies with a 44 female

http://www.thetech.org/genetics/news.php?id=124

Y Chromosome Evolution

http://www.nature.com/nrg/journal/v14/n2/abs/nrg3366.html

• Pseudoautosomal regions (PAR) – homologous to X chromosome, recombine during meiosis

• X-transposed – 3-4 million years ago in humans, contains 2 genes

• X-degenerate – 16 housekeeping genes, homologs on X, similar across primates

• Ampliconic – 60 male-specific genes, repetitive, palindromes (for repair)

• Y chromosome has been replaced in mice:– SRY – male-specific transcription factor– Eif2s3y – spermatogonial proliferation factor

http://www.nature.com/nrg/journal/v14/n2/abs/nrg3366.html

Extensive Adaptive Evolution Specifically Targeting the X Chromosome of Chimpanzees

• Genetic mutations that boost an individual's adaptability have greater chances of getting through to X chromosomes, whereas only a few adaptations on the autosomes have occurred (most of which are related to immunity gene clusters)

• A new beneficial variant on one X chromosome in the female can 'hide itself' if it is not expressed as strongly as the old variant sitting on the other copy of the X chromosome (i.e. recessive). A new beneficial recessive variant does not immediately provide a benefit for the females. On the other hand, the males only have one X chromosome and it is expressed immediately, thus enabling natural selection to 'catch sight' of it. This does not apply to the autosomes

• 30% of amino acid substitutions on the X chromosome since humans and chimpanzees diverged (4-6 mya) were adaptive/beneficial for the chimpanzee.

• Overall the X chromosome is less variable than the autosomes between humans and chimps because natural selection works stronger on the X chromosome since variations have a harder time “hiding”

• Y chromosome is fastest evolving

http://www.sciencedaily.com/releases/2012/01/120130130841.htm

X Inactivation

A mouse’s retinas

http://www.nytimes.com/2014/01/21/science/seeing-x-chromosomes-in-a-new-light.html

Mouse brain

cornea, skin, cartilage and inner ear

How We Became Human• 98-99% identical (1.23% different, ~35 million), most

divergence is in Y chromosome • ~95% identical when insertions and deletions are

considered (~ 5 million)• 29% identical proteins, most differ by only two amino acids

on average (hemoglobin only differs by a single amino acid)• 1,576 apparent inversions between the chimp and human

genomes; more than half occurred sometime during human evolution

• ~580 of 25,000 common genes seem to have been positively selected for in humans

• ~1418 gene differences (689 human, 729 chimps) that were lost in each species, 30 million SNPs

• Chimps have more variation (even between siblings), rhesus macaque has three times as much genetic variation as humans

• Up to 40% difference in protein expression levels

http://www.time.com/time/magazine/article/0,9171,1541283,00.html

Humans vs. Primates• Tiny differences, sprinkled throughout the genome, have made all the difference.

Agriculture, language, art, music, technology and philosophy are somehow encoded within minute fractions of our genetic code. They give us the ability to speak and write and read, to compose symphonies, paint masterpieces and delve into the molecular biology that makes us what we are.

• FOXP2 – reading/writing (humans with defect have difficulty with both), evolved within past 200,000 years. Differs in only 2 of 715 amino acids from chimps.

• MYH16 – myosin variant in jaw muscles, found in all primates but humans. Allowed for the evolution of smaller jaw muscles 2 million years ago, and allowed the braincase and brain to grow larger.

• Protein (domain) DUF1220 – found in areas of the brain associated with higher cognitive function. 212 copies in humans, 37 or less in primates, 1 in mice/rats

• HACNS1 (enhancer) – 13 nucleotide difference between humans and chimps (many more changes than expected via random drift). The human, chimp, and macaque gene was inserted into mice. It was active in the hands, feet, and throat. Human version showed most activity

• Yet, only 19,000 genes, so likely the noncoding regions also play an important role in distinguishing humans from other primates, and affect expression/regulation

http://www.popsci.com/stuart-fox/article/2008-09/how-human-got-his-thumbshttp://www.time.com/time/magazine/article/0,9171,1541283,00.html

Video: Humans and chimps: http://www.youtube.com/watch?feature=player_detailpage&v=KeJoVeKSsyA#t=463s

48/3200 = 1.5%

III.

ENCODE project (2012) showed 80% of genome biochemically active, containing many gene switches and regulatory

elements

Introns and Intergenic DNA• Most of the genome is non-coding• Introns increase with complexity (S. cerevisiae only has introns in

3.5% of genes)• Average transcribed region is 27 kb, average gene is 1.3 kb (~5%)• Vinculin: 22 exons over 75,000 base pairs, 1066 amino acids (4.2%)• Introns

– Make coding sequences discontinuous– Removed by splicing– Allow for diversification– Ribozymes or miRNA?

Alternative SplicingExons and Domains

Unique Intergenic DNA• ~25% of genome

– Regulatory– Gene fragments– Nonfunctional mutant genes– Pseudogenes (not expressed, no regulatory region)

• Gene duplications (~5% of genome)• Unequal crossing over• Transposons, retrotransposons – 40-50%, most unable

to transpose• Reverse transcriptase (from a virus) incorporating

random mRNA into genome• Nearly complete viral genomes – 1.3%• Example: ~80 genes on Y chromosome, but 282

pseudogenes• Many pseudogenes are shut down via methylation• Some may encode proteins linked to cancer

– miRNA (Section IV)– Origins of replication, segregation, telomeres

(Section V)

Repeats, transposons

Unique, transposons

Genes

Gene-related

5%

20%

25%

50%

AAAA

AAAA

Unequal Crossing Over

Gene Duplications on Chromosome 16

Red – interchromosomalBlue - intrachromosomal

Repetitive Intergenic DNA• ~50% of the human genomic DNA is

repetitive – Microsatellite

• Repeating units 13 bp, tandem repeats, • Most common is dinucleotide repeat (3% of

genome) like CACACA…• Arise from mistakes in DNA synthesis

– Genome-wide repeats • >100 bp, some over 1 kb, dispersed or clustered• Rare, but over time have reached 45% of genome• Transposable elements (retroelements) – move to

new positions, often leaving original copy behind, such as Alu, LINE-1 (can silence nearby genes, leading to cancer, schizophrenia), SVA elements which are now 33% of genome

• Major role in evolution and disease• Repetitive DNA exists in E. coli, but far less

common– Repair systems– Gene disruption due to lack of introns– Not as competitive with more DNA to copy

Repeats, transposons

Unique, transposons

Genes

Gene-related

5%

20%

25%

50%

Repetitive/Intergenic DNA Function• Junk DNA? Or DNA with an unknown function? • Regulation, genetic variation

• Onions have 12X the DNA of humans, retain more DNA than lose it• Comparison of fruit flies vs crickets (11X more DNA) showed crickets lose DNA much more slowly,

and have more variability• Although there is a plant that has rid itself of nearly all junk DNA – 97% of its 80 mbp encodes

proteins• Stable maintenance of these sequences over hundreds of thousands of

generations suggest that intergenic DNA confers a selective advantage• 80% of human/mice homology is not in proteins, but in noncoding DNA• Repetitive DNA:

– Different lengths of repeats sequences in dogs correlate with morphological differences in the dogs' skulls and limbs, 51 regions of the dog genome associated with phenotypic variation among breeds in 57 traits

– Link between vasopressin receptor gene (associated with pair-bonding) and repeat length

• Repeat expansion and contraction is a very useful mechanism for imposing rapid evolution. Tandem repeat units can be added or subtracted by slipped-strand mispairing during DNA replication. This type of mutation happens at least 100,000 times as often as simple point mutations. Repeat length changes can make subtle alterations to proteins, whereas point mutations are usually either neutral or fatal to the protein

Fondon, Garner, PNAS 2004http://www.ncbi.nlm.nih.gov/pubmed/20711490 http://www.pnas.org/content/105/37/14153.fullhttp://www.news.harvard.edu/gazette/2000/02.10/onion.htmlhttp://www.nature.com/nature/journal/vaop/ncurrent/full/

nature12132.html

Endogenous Retroviruses• ERVs, a repeat derived from ancient viral

infections• 8% of total genome (98,000 fragments)• Neanderthal and Denisovan ERVs found in

modern humans• Linked to diseases in humans such as MS,

possibly schizophrenia• Mouse study of ERVs:

– ERVs significantly disrupt gene expression, up to 12.5 kb away from transcription site

– 100 genes were found to be disrupted via premature polyadenylation, up to 50-fold change in expression

– A mouse gene containing an ERV inherited from the father produced only an incomplete, truncated form of messenger RNA (mRNA); if the ERV came from the mother, both the truncated transcript and nearly normal levels of the full-length mRNA were produced from the gene

– Can cause biological variation and diversityhttp://www.sciencedaily.com/releases/2012/02/120223182640.htm

Alu Element• Descended from signal recognition particle

which targets proteins to the ER• 300 bp, 1 million copies, ~11% of genome

41.5% of BRCA1 intronic DNA is Alu elements (138 copies)

“Alu sequences have often been regarded as genomic instability factors because they are responsible for recombinational "hot spots" in certain genes and are frequently involved in exon shuffling during meiosis as a result of non-homologous recombination. These sequences may also act as regulatory factors in transcription, with structural roles (as "physical separators" of protein-protein interactions during chromosome condensation in cellular division) and functional roles (in alternative "splicing" or as a connection between transcription factors) are being proposed.”

“BRCA1 exon 5-7 deletion described in German families results from a non-allelic homologous recombination between AluSx in intron 3 and AluSc in intron 7. Both Alu repeats share a homologous region of 15 bp at the crossover site. (Preisler-Adams et al., 2006)”

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572009000300003

Selfish Genes Disrupt Segregation

• Segregation disorder (Drosophila) – 99% inheritance

• Sd produces a truncated version of the RanGAP nuclear transport protein, and its presence interferes with the normal processing of Rsps-bearing sperm

http://www.nature.com/nrg/journal/v2/n8/fig_tab/nrg0801_597a_F3.htmlhttp://www.genetics.org/content/193/3/771.full.pdf

Why Can’t Humans Synthesize Vitamin C?

• 500 million year old process, scavenges reactive oxygen• Most plants and animals synthesize vitamin C from glucose• L-Gulono-gamma-lactone oxidase (GULO), which catalyzes the last step of

biosynthesis, missing in humans• Functional rat GULO has 12 exons, the human pseudogene on chromosome 8

only has 5 exons• The human gene has a deletion of a single amino acid, several mutations, two

stop codons, two single nucleotide deletions, and an insertion• In addition there are two Alu sequences inserted in the vicinity of a presumed

position of lost exon 11 during the same period as GULO lost its function

http://sciencelinks.jp/j-east/article/200324/000020032403A0784254.php

Primates: Suborder Strepsirrhines Suborder HaplorhiniSplit 63 mya

A Drug To Re-Awaken Ancient Human Genes And Fight HIV• Dr. Alex Cole’s lab, UCF• "Junk DNA" are inactive parts of your genome, switched off long ago

in evolutionary history. Now scientists say there's a junk gene that fights HIV. And they've discovered how to turn it back on.

• They have re-awakened the human genome's latent potential to make us all into HIV-resistant creatures.

• Old World monkeys had a built-in immunity to HIV: a protein called retrocyclin (18 amino acids, only circular proteins in body), which can prevent HIV from entering cells and starting an infection.

• Humans have the gene but it contains a nonsense mutation that had turned it off

• Dr. Cole’s lab used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts and therefore start making retrocyclin again

• In preliminary tests the human cells made retrocyclin, fended off HIV, and effectively became AIDS-resistant. And it was done entirely using the latent potential in the so-called junk DNA of the human genome.

http://io9.com/5227470/a-drug-to-re+awaken-ancient-human-genes-and-fight-hiv

Horizontal Gene Transfer and Genomic Duplications

• Transformation, transduction, conjugation• Bacterial resistance to antibiotics• Mitochondria/chloroplast DNA into plant/human

genomes• Millions of years ago, a cluster of 23 genes jumped

from one strain of mold commonly found on starchy foods like bread and potatoes, Aspergillus, to another strain of mold that lives in herbivore dung and specializes in breaking down plant fibers, Podospora, which encodes a toxic compound sterigmatocystin (for protection)

• Arabidopsis (which replaces pea plants for genetic studies) duplicated its entire genome 38 million years ago, about 1/3rd of duplications remain intact

• 11% of Neisseria gonorrhoeae have human LINE-1 retrotransposon DNA

• Cancer

http://www.eurekalert.org/pub_releases/2011-02/vu-doj020411.php http://www.springerlink.com/content/j53040702v9601x3/http://www.eurekalert.org/pub_releases/2011-02/nu-gaa021111.php

Animal Eats DNA to Obtain New Enzymes

• bdelloid rotifer – found in lakes, can survive many years without water, tolerate high levels of radiation

• Ingests DNA from environment• Up to 10 per cent of the active genes of

an organism that has survived 80 million years without sex

• Of ~29,000 matched transcripts, ~10% were inferred from blastx matches to be horizontally acquired, mainly from eubacteria but also from fungi, protists, and algae

http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1003035

Approximately 80% of horizontally acquired genes expressed in bdelloids code for enzymes, and these represent 39% of enzymes in identified pathways. Many enzymes encoded by foreign genes enhance biochemistry in bdelloids (toxin degradation or generation of antioxidants and key metabolites)

RNA• Coding: messenger RNA (mRNA)

– 5’ UTR• 7methyl-G cap bound by cap

binding proteins• Translation regulation

– 3’ UTR• Stability elements• Subcellular localization (zip

codes)• poly(A) tail

• Non-coding RNAs:– Ribosomal RNA (rRNA)– Transfer RNA (tRNA)– Micro RNA (miRNA)– Short interfering RNA (siRNA)

IV.

RNA Conductome (or Transcriptome)• Traditional view: only proteins are important, most of the genome is nonfunctional

– 1.2% of genome encodes proteins– Huge portion of genome intergenic DNA (transposons, repeats)

• Today it is known that RNA plays an important role– Nematode has 1000 cells, but encodes as many proteins (19,300) as humans– 3-8% of genome conserved between humans, mice, and dogs– 33% to 93%* of the genome is transcribed (*from an intensive 1% of genome study called ENCODE, which

took 44 sections of genome of low/high gene density and low/high conservation to mice). Much of the genome is comprised of “gene switches” to turn genes on and off

– 4520 of 158,807 mouse transcripts form antisense pairs with exons• MicroRNAs – small non-coding RNAs that regulate gene expression by interfering with

mRNA function• 3000 ncRNAs found, predicted to be at least 12,000 – increase gene transcription• 400 long ncRNAs found in the livers of mice, which were found to prevent maturing red

cell death (a step to leukemia)• RNA is involved in all aspects of regulation of cellular processes, including chromatin

remodeling and epigenetic memory, transcription factor nuclear trafficking, and transcriptional activation or repression

• dsRNA linked to heterochromatin silencing, keeping transposons in check• Millions of genes, as opposed to 20,000?

Melissa Lee Phillips, John S. Mattick, The Scientist, October 2007 http://www.sciencedaily.com/releases/2011/12/111207175623.htm

Long Noncoding RNA

http://www.nature.com/nmeth/journal/v8/n5/pdf/nmeth0511-379.pdf

Signal for viral infection

Sense and Antisense RNA• Bidirectional transcription is more common than previously thought

and has implications for understanding the complexity of gene regulation

• RNA polymerase is often present at antisense location and transcription is initiated although not often elongated

• Some genes may be self-regulated by antisense transcription• RNA polymerase may induce negative supercoiling upstream

to help regulate transcription

MicroRNA / RNA Interference• Discovered in 1993 (lin-4)• ssRNA that is not translated, but

forms a hairpin secondary structure• Originate from precursors,

eventually processed to ~21 nt• Regulates post-transcriptional gene

expression via down-regulation• Often not 100% complementary to

the target• Important in development• Plant RNAs found in mammals:

– 40 plant miRNAs found in blood– MIR168a (found at high levels in rice)

binds to LDLRAP1 mRNA, reducing the protein levels and ultimately impairing the removal of LDL from the blood

http://the-scientist.com/2011/09/20/plant-rnas-found-in-mammals/

MicroRNA• Tissue-specific. Involved in setting up the basic body plan, nervous system development, cholesterol

levels, bone formation, wound healing, pituitary hormone secretion, heart attacks, cancer

• Diabetes, cancer – miR-483-3p located in an intron within Insulin-like Growth Factor 2 (IGF2). Found elevated in liver, breast, colon cancer, and has oncogenic properties. Affected by maternal diet and suppresses Growth-Differentiation Factor 3 (GDF3), leading to weight gain

• Brain – miRNAs inhibit certain protein synthesis, when a synaspe is activated by a thought/sound/etc., miRNA is degraded and protein synthesis strengthens the synapse, allowing memory formation

• Ear – development, functioning. Conserved between humans and zebrafish• Eye – development. Flies missing Dicer have abnormally small eyes• Heart – Deletion of miR-1-2 causes heart defects, from deformation to electrical conduction to cell-

cycle control• Immune system – miR-155 knockout has faulty B and T lymphocytes and dendritic cells. Viruses

may also use miRNAs to regulate host genes (i.e. HSV-1 inhibiting apoptosis of infected neurons)• Blood cells – blood vessel formation by suppressing inhibitors. miR-15/16 found at chromosomal

region deleted in over half of B cell chronic lymphocytic leukemias (B-CLL)• Insulin secretion (miR-375)• Muscle – miR-1 mutation produces abnormally muscular sheep• Liver – Hepatitis C binds to miR-122 to stabilize its own RNA and promote replication• Angiogenesis – miR-296 elevated in primary tumor endothelial cells isolated from human brain

tumors. Directly targets the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, leading to decreased levels of HGS and thereby reducing HGS-mediated degradation of the growth factor receptors VEGFR2 and PDGFRbeta

• Atherosclerosis• p53 – regulated by 8 microRNAs in sperm production• Pancreatic cancer – 25 miRNAs expressed at different levels in 90% of samples• Polycystic Ovary Syndrome – over expression of miR-93 and decreased expression of GLUT4, a key

protein that regulates fat's use of glucose for energy

siRNA

• Small interfering RNA, similar to miRNA but originates from dsRNA

• Degraded by Dicer, RISC (RNA inducing silencing complex) forms

• Most commonly a response to foreign RNA (viruses, transposons) and is often 100% complementary to the target

RNA Interference• Roundworms infected with Flock House virus (only known infection)• Their progeny (who had RNAi machinery intentionally turned off) were exposed to

the virus and still able to defend themselves for more than 100 generations (nearly a year) after the initial infection.

• Extrachromosomal transmission• Lamarckism?

Rechavi et. al, Cell 2011http://www.sciencedaily.com/releases/2011/12/111205102713.htm

Chromosome Components

• Origin of replication – 30-40 kb apart (100k per chromosome) throughout entire chromosome in non-coding regions (Chapter 8)

• Centromeres – only 1 per chromosome, ~40 kb of repeats (only 200 bp and non-repetitive in yeast)– Kinetochore (protein) attaches to it, links to

microtubules• Telomeres – resistant to recombination and

degradation (associated proteins)– Single-stranded, repeat (TTAGGG in humans)– Telomerase

V.

22 pairs of autosomal chromosomes1 pair of sex chromosomes

46 total

p arm

q arm

Sister chromatids – different strands of DNA, not linked by phosphate backbone

Sister Chromatids and Homologous Chromosomes

The Centromere

(protein)

(DNA)

http://www.ted.com/talks/drew_berry_animations_of_unseeable_biology.html4:45-9:00

The Telomere

3’ end can be thousands of bases long

Telomeres (yellow) may anchor to cell edge during

division

The Cell Cycle: Chromosome Duplication and Segregation

• G0 – gap phase, resting (quiescent)• G1 – growth, preparation• S (synthesis) phase – synthesis, each

chromosome is duplicated – 2 sets of sister chromatids– 2 chromosomes x 2 copies = 4 copies of a gene– Histone replication – Induced at G1/S transition,

over a 25-fold mRNA increase in S phase

• G2 – growth, preparation• M (mitosis) phase

– Sister chromatids bound to mitotic spindle at kinetochore (centromere + protein complex)

– Spindles are microtubules attached to centrosomes/pole bodies

VI.

Animation

1 chromosome (1 piece of dsDNA)

More compactBeads

(nucleosomes) on a string

http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1003019

Gap Phases

• Cell cycle checkpoints• Cells prepare for next cell cycle stage• Cells check that previous stage is

completed properly• Prior to entering S phase, a cell must

reach a certain size and level of protein synthesis to ensure there will be enough proteins and nutrients to complete next cycle

• Cells with DNA damage arrest in G1 before synthesis or G2 before mitosis to allow repair

http://www.nature.com/nrm/journal/v5/n5/fig_tab/nrm1365_F2.html

Movement through the cell cycle is driven by the activities of complexes of cyclins and cyclin-dependent kinases (CDKs), which phosphorylate retinoblastoma (RB)-family 'pocket proteins', thereby blocking their growth-inhibitory functions and permitting cell-cycle progression. Cyclins are shown as triangles.

Example: Protein signals prevent CDC20 from activating ubiquitin (which degrades proteins that are holding the cell at anaphase)

Mitosis• Maintains parental chromosome number• Prophase

– Cohesin/condensin• Metaphase

– Bivalent attachment causes alignment at metaphase plate, tension on chromosomes (essential to keep kinetochores attached)

• Anaphase– Cohesin cleavage by separase

• Telophase– Chromosomes decondense (loss of condensin)

• Cytokinesis

http://www.nature.com/nrm/journal/v12/n7/pdf/nrm3132.pdf

Ubiquitination and mitosis

Cohesin mutation

Proteins Involved in Chromosome Alignment

Aurora B checks and regulates microtubule attachment, senses tension

http://www.nature.com/nrm/journal/v12/n8/pdf/nrm3149.pdfhttp://www.nature.com/ncb/journal/vaop/ncurrent/pdf/ncb2440.pdf

Dynein controls alignment

http://www.nature.com/nature/journal/vaop/ncurrent/pdf/nature12057.pdf

http://www.nature.com/nrm/journal/v13/n12/pdf/nrm3474.pdf

S Phase

1 piece of dsDNA

Cell Cycle and Chromosome Duplication and Segregation

• Chromosome duplication, condensation• Cohesion and condensation are mediated by SMC (structural maintenance

of chromosome) proteins• Cohesin – two SMC + two non-SMC proteins, forming a ringed structure• Condensin – similar ring structure to cohesin• Mitotic spindles (microtubules) form, attach from centrosomes to

kinetochore• Cohesin keeps tension on chromatids, prevents migrating towards poles• Cohesin cleaved, chromatids pull apart

Spindle assembly checkpoint proteins prevent premature segregation by binding to kinetochores that are not attached to spindles or under tension

Condensin/cohesin may be responsible for chromosome condensation

Other models

Meiosis

• Two rounds of segregation, reduces parental number• Meiosis I

– Monovalent attachment – both kinetochores of each sister chromatid are attached to the same microtubule spindle

– Tension (essential) by crossing over (only 1 pair)– 30-40 crossovers. A sequencing study of 91 sperm showed ~23

recombinations per sperm– X and Y chromosomes cross over, and non-sex genes do swap (PAR),

so females could have Y chromosome DNA, and males could have DNA from their father’s X chromosome

• Meiosis II– Similar to mitosis without DNA replication– Most separase cleavage in anaphase II

chiasma

2N

4N

2N

N

Cohesin cleavage

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077332/pdf/cgr0133-0234.pdf

Shugoshin recruits PP2A to centromeres where it locally dephosphorylates Rec8, rendering centromeric Rec8 resistant to separase

Cohesin cleavage

The Nucleosome

• Nucleosomes are the building blocks of chromosomes, basic unit of compaction– Histone – scaffolding protein, building

block of nucleosome– Core DNA – wrapped 1.65 times around

core, ~147 bp (constant among eukaryotes)

– Linker DNA – 20-60 bp (varies among eukaryotes)

• DNA compaction at the nucleosome level is approximately 6-fold (far short of the 1000-10,000-fold compaction needed)

• Not all DNA packaged, some is expressing, replicating, or recombining

• Non-histone – transcription, replication, repair, recombination, topology

VII.

Extract DNA from proteins

Ladder in multiples of nuclease length

Box 7-1

Histones• H2A, H2B, H3, H4

(2 each = 8 total)– Core histones– Positively-charged amino acids

(K and R) make up 20% of protein– 11-15 kDa

• H1 – linker histone, one copy

Histones• Histone-fold domain (three -helices)

– A conserved region in every core histone– Mediates assembly of histones in absence of DNA

Modifications Structural

Nucleosome Assembly• Histone assembly in the presence of DNA

– H3-H4 tetramer followed by two H2A-H2B dimers– H3-H4 (big) bends DNA for H2A-H2B (small)

• Site of contact between histones and DNA– H-bonds with backbone and bases of minor groove– Not sequence-specific

Nucleosome Assembly Time

(cccDNA)

Negative Superhelicity

• Each nucleosome adds a -1.2 change in linking number (not -1.65 because histones change number of bases per turn from 10.5 to 10.2 bp/turn

• Topoisomerase relaxes remainder of DNA• Nucleosome essentially stores energy since negative

supercoiling favors unwinding• Nucleosome removal

– Initiation of replication– Transcription– Recombination

• Prokaryotes use gyrase to induce negative supercoils (requires ATP)

• Hyperthermophiles use reverse gyrase to induce positive supercoils (requires ATP)

Box 7-2

Topoisomerase’s role in nucleosome assembly

Histone-DNA Interactions

H3-H4 H2A-H2BH3-H4 bind middle and both ends of DNA (essential)

Minor Groove Interactions• Non-specific interactions

– 14 total contacts, one for each minor groove exposure– 40 hydrogen bonds between

proteins and phosphodiester backbone (2X a normal DNA-binding protein)

– 7 hydrogen bonds with bases in minor groove, none with any distinguishing base pairs

• Basic amino acids allow phosphodiester bending (generally unfavorable)

Minor Groove Interactions

Histone Tails

• N-termini• Exit nucleosome core at 11, 1, 4, 8

o’clock• Emerge between or on either side of

DNA• Form grooves of a screw, directing

DNA to wrap around the histone octamer in a left-handed manner

• Induces negative supercoils in DNA• Recent data suggests they inhibit

RNA pol II

Protease treatment shows

tails are accessible

Histone Tails

Histone Variants• Histones are some of the most conserved proteins• Variants change structure and function

– H2A.X – widely distributed, phosphorylated upon double-stranded breaks, recognized by repair enzymes

– H2A.Z – creates accessible region of the chromatin for transcription– MacroH2A – 3X larger than H2A with a leucine zipper region. Silencing?– CENP-A – replaces H3 in the centromere, binding sites for other kinetochore

proteins (aurora)

Chromatin Structure

• Euchromatin– Poor staining– Open, unorganized– High transcription– 30 nm fiber, 10 nm as

RNA pol passes• Heterochromatin

– Good staining– Condensed, very

organized (via nucleosomes)

– Low transcription– Still important– Telomere, centromere

VIII.

Higher-Order Chromatin Structure• Histone H1 binds to linker and middle of core DNA• H1 binding results in asymmetry of DNA and core structure, the final

outcome is that nucleosomes alternate on either side of linker DNA• Produces a zigzag

-H1 +H1

Higher-Order Chromatin Structure• H1 binding to nucleosome arrays forms the 30-

nm fiber– This structure is less accessible to DNA-binding

enzymes (RNA polymerase, transcription factors, etc.)– Models:

• Solenoid model– A superhelix with six

nucleosomes per turn– A helical pitch of 11 nm

• Zigzag model– Linker DNA passes through

center of the fiber

– Both have supporting evidence, but solenoid likely in most species

http://www.nature.com/nrm/journal/v13/n7/pdf/nrm3382.pdf

A heteromorphic fiber with predominant two-start (zigzag) type interspersed with one-start conformations was energetically more favorable than uniform zigzag or solenoid conformations under conditions that promoted the most compact folding (that is, the presence of linker histone and Mg2+ counter ions).

Robinson et. al., 2006. PNAS.Robinson et. al., 2006. PNAS.Robinson et. al., 2006. PNAS. Solenoid (one-start) Zigzag (two-start)

Robinson et. al., 2006. PNAS.

“We find that over the range of nucleosome repeat lengths analyzed, there are two discrete classes of fiber structure, one 33 nm in diameter and with ~11 nucleosomes per 11 nm, and the other 44 nm in diameter and with ~15 nucleosomes per 11 nm.” Data mostly supports one-start (solenoid) model, but linker DNA determines final structure.

Nucleosome More Compact Than Previously Thought

Solenoid Zigzag

Structure Based on Linker Length

Higher-Order Chromatin Structure

• Histone N-termini required for 30-nm fiber by interacting with adjacent nucleosomes (seen in crystal structure)

• The 30-nm fiber is a 40-fold compaction of DNA• Nuclear Scaffold

– Further folding of 30-nm fiber– Loops of 40-90 kbp held at the base– Base is made of non-histone proteins (mostly

unknown, and even debated as an artifact)• Topo II – about 50 kb apart, at base of loop for control and

topological isolation• SMC’s

Active transcriptionCondensed

30 nm opens into 10 nm when passed by RNA polymerase

Protein Scaffolding

Histones/nucleosomesTopo IIATPases (remodeling)Linker proteinsOthers

http://harvardmagazine.com/2010/01/dna-compacting-and-data-filing-abilities

Peano Curve (fractal)

Overall folding affects expression, splicing

Regulation of Chromatin Structure

• Histone association with DNA is dynamic to allow protein access

• DNA unwraps rather than just coming off and reattaching• Nucleosome-remodeling complexes

– Use ATP hydrolysis to slide DNA– Can also transfer nucleosome to another helix

IX.

Nucleosome Positioning• Some are found in specific positions• Directed by DNA-binding proteins, which

preferentially assemble nucleosomes nearby• If two proteins bound within 150 bp,

nucleosomes cannot form (require 147 bp)• Nucleosomes are also attracted to bent DNA

(A:T)• At least 50% of nucleosomes “positioned”

Histone Tails• Histone N-terminal and C-terminal (H1) tails may

be modified (acetylases, methylases, ATPases)– Phosphorylation, acetylation, methylation,

sumoylation, or ubiquitination on

Ser, Thr, Lys or Arg– Affects chromatin structure and function (e.g.,

gene expression)– Acetylation – loosening– Methylation – silencing/repression

(occasional activation)– Ubiquitination

• “Histone Code” – proteins can read modifications, modify gene expression– Example: acetylation of lysines 8 and 16 of

H4 expression

Bromo/Chromo/TUDOR/PHD/SANT domain recruitment

Can be cooperative or opposing

Bromodomain – acetylated lysines Chromodomain – methylated histones

TUDOR domain – methylated histonesPHD domain – methylated lysinesSANT domain – histone remodeling

http://www.nature.com/nrm/journal/v13/n5/pdf/nrm3327.pdf

Other interaction sites:

http://www.nature.com/nrm/journal/v13/n7/pdf/nrm3382.pdf

Chromatin Remodeling

Nucleosome Assembly After Replication

• Nucleosome rapidly reassembled after replication

• H3-H4 tetramer, two H2A-H2B dimers, H1• Nucleosomes must double during each

chromosome duplication• Are old chromosomes all lost? If so, how

would modification memory carry over to the next cell? If not, how do they separate evenly and retain this memory?– Old and new on each daughter chromosome– H3-H4 tetramers and H2A-H2B dimers

either all old or all new– H3-H4 remains bound– H2A-H2B dimers released into the pool,

available for new assembly– Old nucleosomes recruit enzymes that add

similar modifications to adjacent nucleosomes, thus maintaining states of modification after DNA replication

– Critical role in inheritance

Nucleosome Assembly• Histone assembly requires chaperones

(not “folding” but “directing” chaperones), recognizing replicating DNA– Ex.: Rtt106 binds to H3 if acetylated at aa 56

and places H3-H4 on newly-replicated DNA

• CAF-I interacts with the sliding clamp (PCNA), which holds DNA polymerase in place during replication

• After polymerase finishes, the sliding clamp is released and interacts with CAF-I, which directs nucleosome assembly

New Evidence of a Pre-Nucleosome• ATP-dependent motor protein assembles/activates nucleosomes• Intermediate between naked DNA and mature chromatin?

http://www.cell.com/molecular-cell/retrieve/pii/S1097276511005417

Animation – Nucleosome Assembly

New histones synthesized in S phase

TrxG and PcG Proteins but Not Methylated Histones Remain Associated with DNA through Replication

• Study shows histones are completely removed in Drosophila• Trithorax and Enhancer-of-Zeste, which are H3K4 and H3K27 methylases, and Polycomb

continuously associate with their response elements on the newly replicated DNA. We suggest that histone modification enzymes may re-establish the histone code on newly assembled unmethylated histones and thus may act as epigenetic marks.

http://www.sciencedirect.com/science/article/pii/S009286741200935X#fx1

Epigenetics• Changes in gene expression (not sequence) based on environment via histone modification

(imprinting, silencing, inactivation). Most removed in zygote, but some can last generations for fast phenotypic adaptation

• ~100 genes known, relevant in development, cancer, disease, stem cells, exercise, drug abuse• Histone modifications play a role in autism, schizophrenia, depression, and other psychiatric

diseases, and the H3K4-specific histone demethylase, JARID1C/SMCX, has been linked to mental retardation and autism

• Disease – 15q11 maternal/paternal imprinting, variations cause Prader-Willi or Angelman Syndrome

• Starving Dutch mothers who gave birth during WWII famine had children who were more susceptible to obesity and other metabolic disorders – and so were their grandchildren

• Swedish grandsons (but not granddaughters) had lower cardiovascular disease if their grandfather had gone through famine

• Rats – chronic high-fat diets in fathers result in obesity in their female offspring; obese diabetic mice altered pancreatic/fat gene expression in offspring

• Mice – paternal stress at any point in their lives marks sperm via microRNA, causing hypothalamic–pituitary–adrenal (HPA) axis dysregulation in offspring

• Lamarckism?

http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1000506

Bees and methylation

Epigenetics: http://www.youtube.com/watch?v=LcaRTDsLmiA

Toxins and Epigenetic Changes via DNA Methylation• Exposed pregnant mouse to toxins, studied F1-F3• Early-onset puberty, egg/sperm reduction in F3 (promoter methylation)• Your great-grandparents’ exposure could be causing diseases epigenetically

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0031901

Hundreds of genes affectedDioxin (red)Pesticide (light blue)Plastic (pink) Jet fuel (dark blue)

Influenza Controls Gene Expression via Histone Mimicking• Immunosuppressive NS1 protein of the influenza A virus mimics a

core component of gene regulating machinery in order to block antiviral gene function

• NS1 protein of the H3N2 strain of influenza -- the "seasonal" flu -- contains the same sequence of amino acids as the "tail" domain of a DNA packaging protein in humans called histone H3. The histones are present in the cell nucleus and play an important role in gene activation. Chemical modifications of the histone "tails" allow recruitment of effector proteins that, in turn, determine which genes are switched on or off

• Impairs host antiviral response

http://www.sciencedaily.com/releases/2012/05/120506101543.htm