1. Chapter 7
Genome
Structure, Chromatin, and the
Nucleosome

2. Chapter Overview
I. Chromosome & Genome Overview
II. Overall Genome Organization
III. Introns and Intergenic DNA
IV. RNA
V. Chromosome Components
VI. The Cell Cycle: Chromosome Duplication
and Segregation
VII. The Nucleosome
VIII. Chromatin Structure
IX. Regulation of Chromatin Structure

3. 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.

4. Prokaryotes
• Millions of base pairs
• 1 mm chromosome into 1 m
• 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

5. 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

6. 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

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

8. 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

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

10. Veeramachaneni et. al., 2004
MutY Homolog
Target of Early Growth Response Factor 1
Testis-
specific
kinase 2
Chromosome 1, P arm

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

12. 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

13. 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

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

15. 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?)

16. 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

17. Y Chromosome Evolution
http://www.nature.com/nrg/journal/v14/n2/abs/nrg3366.html

18. • 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

19. 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

20. X Inactivation
A mouse’s retinas
Mouse brain
cornea, skin, cartilage
and inner ear

21. 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

22. 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-thumbs
http://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

23. 48/3200 = 1.5%
III.
ENCODE project (2012) showed 80% of genome biochemically
active, containing many gene switches and regulatory elements

24. 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?

25. Alternative SplicingExons and Domains

26. 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%

27. AAAA
AAAA

28. Unequal Crossing Over

29. Gene Duplications on
Chromosome 16
Red – interchromosomal
Blue - intrachromosomal

30. 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%

31. 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 2004 http://www.pnas.org/content/105/37/14153.full
http://www.news.harvard.edu/gazette/2000/02.10/onion.htmlhttp://www.nature.com/nature/journal/vaop/ncurrent/full/nature12132.html

32. 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 diversity
http://www.sciencedaily.com/releases/2012/02/120223182640.htm

33. Alu Element
• Descended from signal recognition particle
which targets proteins to the ER
• 300 bp, 1 million copies, ~11% of genome

34. 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

35. 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.htmhttp://www.genetics.org/content/193/3/771.full.pdf

36. 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 Haplorhini
Split 63 mya

37. 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

38. 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

39. 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

40. 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)

41. 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.

42. 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

43. Long Noncoding RNA
http://www.nature.com/nmeth/journal/v8/n5/pdf/nmeth0511-379.pdf
Signal for viral infection

44. 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

45. 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/

46. 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

47. 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

48. 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

49. 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.

50. 22 pairs of autosomal chromosomes
1 pair of sex chromosomes
46 total 
p arm
q arm
Sister chromatids – different strands of DNA, not linked by phosphate backboneSister Chromatids and
Homologous Chromosomes

51. The Centromere

52. (protein)
(DNA)
http://www.ted.com/talks/drew_berry_animations
_of_unseeable_biology.html
4:45-9:00

53. The Telomere
3’ end can be
thousands of
bases long
Telomeres (yellow) may anchor
to cell edge during division

54. 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.

55. Animation
1 chromosome
(1 piece of dsDNA)

56. More compact
Beads (nucleosomes)
on a string

57. 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.

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

60. 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

62. Cohesin
mutation

63. Proteins Involved in Chromosome Alignment
Aurora B checks and regulates microtubule attachment, senses tension
http://www.nature.com/nrm/journal/v12/n8/pdf/nrm3149.pdf
http://www.nature.com/ncb/journal/vaop/ncurrent/pdf/ncb2440.pdf
Dynein controls alignment
http://www.nature.com/nature/journal/vaop/ncurrent/pdf/nature12057.pdf

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

65. S Phase
1 piece of dsDNA

66. 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

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

68. Condensin/cohesin may be
responsible for chromosome
condensation

69. Other models

71. 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

72. chiasma
2N
4N
2N
N
Cohesin cleavage

73. http://www.ncbi.nlm.nih.gov/pmc/articles/P
Shugoshin recruits PP2A to centromeres where it locally dephosphorylates Rec8,
rendering centromeric Rec8 resistant to separase

75. Cohesin cleavage

77. 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.

78. Extract DNA from proteins
Ladder in multiples of
nuclease length
Box 7-1

79. 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

80. Histones
• Histone-fold domain (three -helices)
– A conserved region in every core histone
– Mediates assembly of histones in absence of DNA
Modifications Structural

81. 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

83. Nucleosome Assembly Time
(cccDNA)

84. 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)

85. Box 7-2
Topoisomerase’s role in
nucleosome assembly

86. Histone-DNA Interactions
H3-H4 H2A-H2B
H3-H4 bind middle
and both ends of
DNA (essential)

88. 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)

89. Minor Groove Interactions

90. 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

91. Histone Tails

92. 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)

93. 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.

94. 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

95. 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

97. http://www.nature.com/nrm/jo
urnal/v13/n7/pdf/nrm3382.pd
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).

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

99. 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

100. Structure Based on Linker Length

101. 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

102. Active transcriptionCondensed
30 nm opens into 10 nm when passed by RNA polymerase

103. Protein
Scaffolding
Histones/nucleosomes
Topo II
ATPases (remodeling)
Linker proteins
Others

104. http://harvardmagazine.com/2010/01/dna-compacting-and-data-filing-abilities
Peano Curve (fractal)
Overall folding affects expression, splicing

105. 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.

107. 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”

108. 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

111. Bromo/Chromo/TUDOR/PHD/SANT domain recruitment

112. Can be cooperative or opposing

113. Bromodomain – acetylated lysines Chromodomain – methylated histones
TUDOR domain – methylated histones PHD domain – methylated lysines SANT domain – histone remodeling

116. Other interaction
sites:
http://www.nature.com/nrm/journal/v13/n7/pdf/nrm3382.pdf

117. Chromatin Remodeling

118. 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

120. 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

121. 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

123. Animation – Nucleosome Assembly
New histones
synthesized in
S phase

125. 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

127. 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.youtu
be.com/watch?v
=LcaRTDsLmiA

128. 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 affected
Dioxin (red)
Pesticide (light blue)
Plastic (pink)
Jet fuel (dark blue)

129. 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