All Wrapped UpChromatin-Level Gene Regulation

Genes can be regulated by chromatin organization

GE

NE

TIC

EP

IGE

NE

TIC

Pie

rce,

B. 2

005.

Gen

etic

s, a

con

cept

al a

ppro

ach.

2nd

Ed.

WH

Fre

eman

.

Chromatin Packaging

• Chromatin is the combination of DNA and proteins found in eukaryotic chromosomes.

• Proteins in chromatin are:– histones: small, positively charged– non-histone proteins: transcription factors,

enzymes

• Packaging of DNA with proteins leads to compacted structure, which facilitates fitting the DNA into the nucleus

Nucleosomes: the basic chromatin unit

• Nucleosomes are formed by winding about 200 base pairs of the DNA duplex around a core of histone proteins.

• The histone core is made of:– 2 molecules histone H2A– 2 molecules histone H2B– 2 molecules histone H3– 2 molecules histone H4

• Another histone, H1, binds outside the core.

Nucleosome organization

htt

p:/

/ww

w.c

bs.

dtu

.dk/

sta

ff/d

ave

/ro

an

oke

/ge

ne

tics9

80

21

8.h

tml

Higher orders of chromatin packing produce fibers with wider diameters

1. Nucleosome (11 nm)

2. Solenoid (30 nm)

3. Loops (300 nm)

4. Coiled loops (700 nm)

5. Metaphase chromosome (1400 nm)

Pie

rce,

B. 2

005.

Gen

etic

s, a

con

cept

ual a

ppro

ach.

2nd

Ed.

WH

Fre

eman

.

1

23

45

The DNA component of chromatin can be covalently modified

• DNA methylation of cytosines

CH3 CH3

CH3 CH3

CH3

• Only certain cytosines can be methylated.• Sequence context matters.

– In animals, CG– In plants, CG and CNG

Core histones can be covalently modified

• Example: Histone acetylation

N-ARTKQTARKSTGGKAPRKQLATKAARKSAP9 14 2318

H3 Acetylation

H-N-CH2-CH3

––N–C–C––

(CH2)4

OH

––N–C–C––

(CH2)4

NH3+

OH

Lysine Acetylated Lysine

Acetylation

Acetylation causes histones to lose some of their positive charge. This causes them to bind less tightly to the negatively charged DNA backbone.

Pierce, B. 2005. Genetics, a conceptual approach. 2nd Ed. WH Freeman.

Consequences of chromatin modificaton

• Histone modification can reduce the positive charge on the proteins, thus altering their attraction for negatively charged DNA and loosening chromatin packing.

• Modification of both histones and cytosines can provide recognition sites for binding of other regulatory proteins, which in turn can alter chromatin packing.

Acetylation

Pierce, B. 2005. Genetics, a conceptual approach. 2nd Ed. WH Freeman.

Relationship of chromatin organization and gene expression

Transcriptional Activity

Chromatin Configuration

Histone Modification

DNA Methylation

On

Active

Expressed

Open

Loosely packed

Accessible to transcription machinery

Acetylated Low

Off

Inactive

Silent

Closed

Tightly packed

Not accessible to transcription

machinery

Deacetylated High

Chromatin-level regulation of gene expression is called “epigenetic”

• Altered chromatin structure

• Altered expression

• Potentially reversible

• Independent of DNA sequence changes

• Frequently developmentally regulated

• Mitotically and / or meiotically heritable

Example:X-inactivation in mammals

• In mammals, there are many genes on the X chromosome.

• Females have two X chromosomes and males have only one.

• To compensate for possible differences in gene expression, due to the differences in X-chromosome number in females and males, one of the X’s in females is transcriptionally silenced by tightly packing the chromatin of one X.

Example:X-inactivation in mammals

XX

XX

XX

XX XX

XX

XX

XX

XX

X •

X •

X • X •

• X

• X

• X

• X

• In cats, the gene for fur color is on the X.

• There are two alleles, for orange and black.

X-inactivation fits the criteria for epigenetic regulation

• It is an example of altered gene expression (silencing) due to altered (more tightly packed) chromatin structure, not change in DNA sequence.

• It is developmentally imposed early in embryo formation.

• The new chromatin configuration is mitotically heritable, in that daughter cells maintain the chromatin state of the cell from which they came.

• It is reversible; during meiosis, the inactive X is re-activated, so that gametes inherit fully active X chromosomes.

Non-reversible epigenetic change in gene expression: paramutation

• In corn, synthesis of purple anthocyanin pigments requires a gene called booster (b), which codes for a transcription factor that activates the enzyme-coding genes in the pigment pathway.

• A strong allele, called B-I, is expressed at high levels and activates the pathway very well to produce deep purple coloration.

• Another allele, called B’, is expressed at lower levels and leads to weaker coloration.

B-I

B’

B’/B-I

X

B-I/B-IB’/B’

X

B-I/B-I

Paramutation exhibits non-Mendelian inheritance

all B’

Non-Mendelianpattern of inheritance

Images courtesy of Vicki Chandler, Univ of Arizona

Molecular explanation for paramutation

• Allelic interaction.• One allele (B’) transfers its low expression state

to another allele (B-I), converting B-I to B’.• The conversion is chromatin-based.

– B’ has a more closed chromatin configuration than B-I. – After association with B’, B-I assumes a closed

chromatin configuration and is expressed at low levels like B’.

• The B-I-to-B’ conversion is permanent. B’ never reverts to B-I.

Chemically-induced transgenerational alterations in sexual function

• Many chemicals are related to estrogens and can disrupt endocrine function.

• Developing fetuses are especially sensitive to these so-called endocrine disrupting chemicals (EDCs).

• Treatment of pregnant mothers with EDCs can lead to changes in sexual development in their male offspring.

• The males can then transmit the changes to their offspring.

Chemically-induced defect can be passed to offspring

pregnant mouse males; low sperm count

X

normal female

males; low sperm count

X

normal female

males; low sperm count

X

normal female males; low sperm count

transient exposure to EDC

F1

F2

F3

F4

Reference:Science (2005) 308:1466

Chemically-induced change is epigenetic

• Analysis of genes in sperm of the defective male mice revealed changes in DNA methylation of several genes.

• This altered pattern of DNA methylation was seen in mice from multiple generations (F1, F2, F3).

• Interpretation is that EDC induced a DNA methylation change in the germ line of male mice, and this change can be transmitted to offspring.

What we don’t know about chromatin-level regulation

• How many genes are targets for this level of regulation?

• How are those genes targeted?• How are the chromatin modifications

accomplished?• What determines whether chromatin changes

will be transient or permanent?

Summary

• DNA is packed into the nucleus by association with histone and non-histone proteins to form chromatin.

• Both the DNA and histone components of chromatin can be modified.

• Modifications change the configuration of chromatin and this in turn alters accessibility of genes for transcription.

• Some chromatin modifications can have transgenerational effects on gene expression.