Chromosomes - 1

MEDICAL CELL BIOLOGY2010

CHROMOSOMES

Andrew F. RussoDepartment of Molecular Physiology & Biophysics

Recommended Reading: Alberts, 5th edition; Ch 4 p 202-219, 223-245, Ch 7 p 450-

452

Key Concepts

1. Requirements for chromosome propagation and maintenance: replication

origins, centromeres, telomeres

2. Epigenetic control of DNA structure and function by chromatin proteins

3. Condensation state of chromatin controls gene expression

Key Words

chromosome aneuploidy

chromatin replication origins

histone centromere

heterochromatin telomere

euchromatin DNase hypersensitivity

epigenetic condensation state

karyotype locus control region

myotonic dystrophy toxic RNA

nuclear sequestration chromatin remodeling

I. Chromosome numberA. Human genome

1. Diploid genome is 2x 22 autosome chromosomes and 2 sex

chromosomes (46 chromosomes, 6 billion base pairs)

Chromosomes - 2

B. Karyotype

1. Mitotic chromosomes can be visualized by Giesma and other stains,

including chromosomal painting with fluorescently tagged oligos

2. Aneuploidy is deviation from the normal number of chromosomes

3. Usually only large rearrangements (deletions, translocations) detected

II. Requirements to be a chromosome

A. Chromosomal maintenance and propagation requires three DNA elements

Chromosomes - 3

1. Replication origins

a. many per chromosome

b. initiation point for DNA synthesis

2. Centromere

a. one centromere per unreplicated chromosome

b. two centromeres per mitotic chromosome, one per chromatid

c. attachment site of kinetochore proteins, allows segregation of

chromosomes

Chromosomes - 4

4. Telomere

a. repetitive element that defines and protects the ends of chromosomes

from loss of DNA due to “end

replication” problem

b. added by telomerase, both

protein and RNA subunits

c. decreased length correlates

with cell senescence (when

cells stop dividing)

B. Telomerase

1. Levels of telomerase vary in different cells

a. Most human somatic cells have little or no telomerase so that telomeres

shorten as cells divide

b. Exceptions: Stem cells (adult and embryonic), germline cells, & tumor

cells have high telomerase

2. Inhibition of telomerase as anti-cancer strategy

a. antisense against RNA subunit, other inhibitors cause tumor cell

death

b. small molecule inhibitors now in clinical trials

3. Overexpression of telomerase – fountain of youth or cancer?

Chromosomes - 5

a. Transfect human fibroblasts with telomerase —recall, telomerase

increased iPS production. With telomerase alone, most cells

immortalized and most are normal, but some

losing contact inhibition (recall iPS cancer

concerns) and others still undergo senescence.

b. Overexpress telomerase in transgenic mice

—mice live longer, fewer age-related diseases,

increased wound healing, but higher cancer

incidence following chemical carcinogenesis

and in younger mice

c. Human skin cells grown in the lab for grafting have shortened

telomeres and appear aged. Rescue?

d. Potentially useful tool for replacement therapies—but need better

control of telomerase

4. Telomeres in cloning and tissue replacement

a. Can old telomeres be restored?

b. Possibility lifestyle changes might increase telomerase activity in

peripheral blood mononuclear cells, but very preliminary.

III. Epigenetics and chromatin

A. Definitions.

1. Epigenetics= “above the genome”.

2. Chromatin = DNA binding proteins

a. Roles of chromatin proteins: Package DNA in

the nucleus. Regulate DNA replication and transcription

B. Histone proteins

1. Histone octamers

Chromosomes - 6

a. form an 11 nm chromatin fiber

b. always present on DNA

c. “beads on a string” 11 nm fiber never seen in

vivo

2. Histone H1 increases packing

1. forms a 30 nm chromatin fiber with

core histones and nonhistone

proteins

C. Higher order folding

1. Multiple steps of looping and folding

a. loops on loops allow compaction of

length and width of chromosome

2. Loops of 30 nm fibers held together

by nuclear matrix

a. Forms domains for global control

of gene expression

3. Mitotic chromosome condensed from

5 cm to 5 micron long (10,000x)

4. Mitotic chromosome compacted by condensin

5. Sister chromatids held together by

cohesin

IV. Two fundamental types of chromatin

A. Heterochromatin

1. Highly condensed chromatin, about 10% of genome

2. Transcriptionally inactive

3. Plays specialized functions

condensinscohesins

Chromosomes - 7

a. centromeres- chromosomal movement

b. repetitive elements- recombination,

telomeres

c. mitotic chromosomes- packaging

d. boundaries- define loop domains

B. Euchromatin

1. Loosely folded chromatin, about 90% of

genome

2. Two transcriptional states

a. active-- only ~ 10% in any given cell, this is the least condensed

b. inactive-- this is more condensed

C. Packing of chromatin in the nucleus

1. Active genes in open, accessible areas

2. Inactive genes in closed, dense storage area

called a "fractal globule" = super-dense,

knot-free structure, not equilibrium-based

structure

a. DNA packed very tightly, yet avoids knots

and tangles

b. allows DNA to unfold/refold during gene expression & mitosis

c. based on proximity-based ligation, genomic sequencing (Science 2009)

Chromosomes - 8

D. Epigenetic control of chromatin remodeling

1. Proteins bind DNA and change chromatin structure

a. Either relax (open) chromatin structure to activate or condense (close) to

inhibit gene expression

b. DNA methylation (condenses) and

histone acetylation (relaxes)

2. Dynamic chromatin condensation states

a. Changes in nuclear localization (genes in perinuclear heterochromatin

move and become euchromatic)

b. Changes during cell division (mitosis vs. interphase)

c. Changes during development (polytene chromosome during interphase

in Drosophila)

d. Changes in response to stimuli-- H3 histone kinase is activated by EGF.

The kinase is mutated in Coffin-Lowry Syndrome (mental retardation/

skeletal abnormalities)

e. Chromatin control is fully reversible

V. Chromatin control of gene expression

Chromosomes - 9

A. Occurs at global level often on an entire chromosomal loop (~100 kb)

B. Global control by a defined sequence called a locus control region (LCR)

1. LCR needed for correct expression patterns and levels

a. Best shown by studies on globin LCR

in transgenic mice

b. Globin LCR mutations seen in some

thalassemia patients

2. Local control still needed for gene

expression

C. Measuring chromatin condensation state

1. DNase hypersensitivity assay- measures accessibility of DNA to DNase in

an in vitro assay

a. Lightly treat nuclei with DNase (DNA

is still bound by chromatin proteins)

b. Tightly bound DNA is protected from

DNase

c. Treat DNA with restriction enzyme (RE), detect DNA fragments by Southern blot

(for high-throughput, PCR-based screening is done instead of Southerns)

d. DNase hypersensitivity is an indicator of relaxed chromatin

2. Hypersensitive site (HSS) often seen prior to detectable gene expression

Chromosomes - 10

unaffected affected

gene X

- + - + DNase

HSS siteR E site R E site

VI. Clinical Connection: Myotonic dystrophy (DM1)

A. Triplet repeat disorder

1. Most common form of adult-onset muscular dystrophy

a. Inherited autosomal dominant

b. Complex phenotype-- muscle wasting, mental retardation, cataracts, etc

2. In DM1, amplified triplet (CTG) repeat in 3’ UTR of DM protein kinase (DMPK) gene

a. unaffected 5-30 repeats; minimal to severe DM1 50-2000 repeats

b. effect of repeat in DMPK appears to involve multiple mechanisms

B. Original hypothesis—Phenotype due to chromatin condensation

1. Hypersensitive site adjacent to DM1 triplet is cleaved in DNA from fibroblasts of

unaffected individuals, but not cleaved from fibroblasts of DM1 patients

a. Small antisense RNAs near the triplet can induce histone deacetylation,

which causes chromatin condensation

b. Expanded triplets prevent insulator proteins from restraining spread of

heterochromatin

2. Reduced RNA from a nearby gene (SIX5), which encodes a transcription factor

involved in eye development

3. However, role in disease unresolved

DMPK RNA triplet PKPKRNA

Splicing protein

Chromosomes - 11

a. Paradox that DMPK and SIX5 knockout mice have only a minimal DM1

phenotype with no muscle wasting, which suggests another mechanism in

addition to or instead of the chromatin

model

C. New hypothesis— Toxic RNA model

1. Phenotype due to sequestration of

transcription and/or splicing factors by the

triplet repeat in nuclear DMPK RNA

a. Mutant DMPK RNA accumulates in the nucleus

b. Triplet RNA binds specific group of splicing proteins called muscleblind

(homologous proteins in Drosophila req’d for muscle and eye differentiation)

c. Muscleblind knockout mice—have DM muscle and eye phenotype

2. Muscleblind KO mice and DM patients have

aberrant Cl channel splicing.

a. Fewer channels cause muscle dystonia

(hyperexcitability) (Science 2003)

b. Excitable goats

3. Triplet RNA also binds transcription factors (e.g.

SP1) required for Cl channel expression.

a. Overexpression of SP1 restores Cl channel

in DM muscle cells (Science 2004).

D. Future therapies?

1. DM patient fibroblasts induced to

transdifferentiate into muscle cells in the lab

by MyoD expression (next lecture). Other therapies?

Chromosomes - 12

Review Questions

1. Consider the size of the genome. Why do you think we have so much non-

coding DNA in our genome?

2. What is your favorite gene? A patient has decreased expression of your favorite

gene. There are no mutations in the gene, so you suspect there is a distant

mutation that leads to condensed chromatin. Describe how you would screen for

DNase hypersensitive sites in the genome of the patient.

3. Give three possible reasons that might explain why the DMPK and SIX5

knockout mice do not show the full myotonic dystrophy phenotype.

4. Describe how you might use iPS cell therapy in an attempt to cure myotonic

dystrophy.