Gene Regulation 4 - Regulation of eukaryotic gene expression Packing and unpacking of DNA

Question 1

Learning Outcomes

Answer 1

Students will be able to
➢ explain experimental evidence that shows that almost all cell have the same
genome and that this genome is sufficient to give rise to a fully functional
eukaryotic organism.
➢ relate development and differentiation of a multicellular eukaryote to the
regulation of gene expression.
➢ discuss why eukaryotic control of gene expression is more complex than
prokaryotic gene expression.
➢ list levels at which gene expression is regulated in eukaryotes
➢ describe eukaryotic DNA packaging into chromosome structures
➢ relate packaging of DNA to gene expression.

Question 2

If two cells share the same genome, how come they look
different and have different functionalities?

Answer 2

• Different cells differ in structure and
  function.
  ➢ A neuron cells from the retina
  receives electrical signals from
  many other neurons and carries
  them to many neighbouring
  neurons via neurotransmitters.
  ➢ A liver cell is involved in many
  metabolic processes. One of those
  is the detoxification of alcohol via
  the enzyme alcohol
  dehydrogenase
• The two cells have the same genes,
  same genome.
• They express different subsets of
  genes leading to different subsets of
  proteins which determine their
  different shapes and functions.

Question 2

Differentiation as a consequence of changes in gene expression

Answer 2

• In multicellular organisms, life begins as a single cell.
• In development, cells commit to specific fates & differentially express subsets of genes.
• Daughter cells may differ with respect to regulatory instructions & developmental fate, so new cells become different to
  their parent cell.

Question 2

Frog Embryo Development

Answer 2

Frog embryogenesis is characterised by cell division and cellular differentiation of pluripotent cells (in plants
meristem cells).
Pluripotent cell: immature stem cell that has the potential to differentiate into any of the three germ layers:
endoderm (gut, lungs and liver), mesoderm (muscle, skeleton, blood vascular, urogenital, dermis), or ectoderm
(nervous, sensory, epidermis), but not into extra-embryonic tissues like the placenta or yolk sac

Question 2

Are losses or gains of genes driving changes during development?

Answer 2

Question 3

Undifferentiated and Differentiated cells produce different specific mRNAs and proteins

Answer 3

Consider:
▪ A single fertilised egg cell develops into a multicellular organism with trillions of cells, ~ 200 different cell types.
▪ These cells are organised into tissues & organs performing different, specialised functions and morphologies.
▪ This requires that different cell types make different sets of proteins.
* The early embryo is characterised by rapid cell division followed by differentiation ➔ genes expressed will enable
the embryo to fulfil functions associated with division and differentiation appropriate to its developmental state.
* Differentiated cells will express genes that enables them to fulfil a specific function within an organism.

Answer 3

Answer 3

Question 4

Let’s look at levels of Gene Expression

Answer 4

• Control of gene expression in eukaryotes occurs at
  several levels:
  1) Packing or unpacking DNA
  2) Transcription
  3) mRNA processing
  4) mRNA export
  5) Translation, mRNA stability and degradation
  6) Post-translation protein modification
  7) Protein stability and degradation

Answer 4

Question 5

How is gene expression regulated in
Eukaryotes?

Answer 5

• Regulation of gene expression in eukaryotes is more complex than in
  prokaryotes.
• Control of gene expression in eukaryotes occurs at many levels:

Answer 6

Question 7

How long is my DNA ?

Answer 7

DNA in each of our cells is about 2 to 3 meters long based on 3.2 x 109 nucleotides and
a length of 0.6 nanometers (10-9 m) per nucleotide.
This DNA has to fit into the cellular nucleus which is about 6 µm in size (10-6 metres)
Image result for eukaryotic cell microscope
If we would magnify the nucleus 1000 x to 6 mm, the total length of all the DNA in the
cell’s nucleus would be 2 - 3 km long

DNA in our whole body is about 2.04 x 1010 km ➔ 66.5 trips from
the earth to the sun and back

Question 8

How to fit 2 meters of DNA into a nucleus of 6 µm diameter:
Package DNA around add histone H1

Answer 8

• The ‘string’ part of the beads on a string is formed by
  unbound linker DNA DNA ~ 20 bp between nucleosomes.
• The linker DNA is bound by the linker histone protein H1.
• The nucleosome plus the linker DNA and the linker histone
  form the chromatosome.
• The chromatosome contains about 166 bp of DNA.
• This condenses DNA from 2 nm to 11 nm.

Question 8

How to fit 2 meters of DNA into a nucleus of 6 µm diameter:
Package DNA around core histones

Answer 8

Answer 9

Answer 9

Question 9

Chromosome structure is regulated

Answer 9

• Gene expression, DNA replication, DNA repair requires access of proteins to the DNA.
• Along an interphase chromosome we can find regions that are:
  – densely packed: heterochromatin ➔ DNA less accessible ➔ silent genes
  – less densely packed: euchromatin ➔ DNA is more accessible ➔ expressed genes.
• Eukaryotic cells have several ways to adjust the local structure of chromatin rapidly.
  – Chromatin remodelling complex
  – Reversible chemical modifications of histones

Question 10

How to fit 2 meters of DNA into a nucleus of 6 µm diameter:
Using scaffold proteins

Answer 10

• Multiple nucleosomes wrap into a 30 nm fibre forming
  heterochromatin or chromatin.
• During mitosis and meiosis DNA can be packaged even more
  into the metaphase chromosome (~1400 nm diameter). This
  requires other sets proteins:
  – Scaffold proteins determine and maintain the authentic
  chromosome shape
  – ensure that DNA winds up and unwinds without tangling.
  – Packaged DNA molecule in a mitotic chromosome is
  10,000 fold shorter than its fully extended length.

Answer 10

Answer 10

Answer 10

Question 11

Visualising transcribed areas of chromosomes

Answer 11

▪ Chromosomes transform into the
lampbrush form in the growing
oocytes of most animals, except
mammals, during the diplotene stage
of meiotic prophase I.
▪ Opened loops of varying length are
areas of active transcription of many
genes.
▪ The light line in the centre of the
chromosome is scaffold made up of
non-histone scaffold protein

Question 11

Chromatin Remodeling Complexes

Answer 11

• Chromatin Remodelling Complex: Regulatory proteins that use ATP (energy) to alter chromatin structure without
  changing histone chemistry.
• Bind directly to DNA and reposition nucleosomes ➔ DNA is accessible to proteins, e.g. to transcription factors.
• Example of ways in which remodeling occurs:
  – Nucleosome slides along DNA, DNA that would normally be wrapped around the histones becomes exposed.
  – Conformational change of nucleosomes, DNA or both, so the DNA is more exposed.
• May work in unison with histone acetylation (see next slides)

Question 12

Histone tail modifications determine active and repressive chromatin

Answer 12

• Histone tail modifications are Post-translational modifications (PTMs).
• PTMs are covalent modifications of a protein after protein biosynthesis (after translation).
• These modifications are done by enzymes (proteins).
• PTMs can regulate protein activities, localization, structure etc.
• PTMs of histone tails can lead to
• active, less densely packed DNA in euchromatin
• or to densely packed DNA in repressive heterochromatin

Question 13

How do histones interact with DNA?

Answer 13

• Chromatin: DNA packaged with protein
• Most abundant proteins are histones
• Histone proteins contain basic amino
  acids which are positively charged
  – Lysine
  – Arginine
• The positive charges of these amino
  acids can interact strongly with the
  negative charges of the phosphate
  groups in DNA = ionic interactions

Answer 13

Question 13

Visualising histone modifications and chromatin structure

Answer 13

Chromatin during meiosis prophase:
* Pink shows histone modifications, here methylation of histone
proteins at lysins (K) of histone protein 3
* LEFT (H3K4me3): active chromatin, radially, loop-like structures.
* Bottom (H3K27me3): inactive, repressive chromatin

Question 14

Acetylation of basic amino acids

Answer 14

Amino Acid Acetylation:
– Addition of an acetyl (CH3C=O-
) group to Lysine
▪ Acetylation of lysines is done by enzymes called
histone acetyltransferases = HATs
▪ Acetyl groups can be removed by other enzymes
called histone deacetylases = HDACs or HDs

➢Histone modification influences gene expression without changing the nucleotide sequence of the
DNA = type of epigenetic regulation
➢Epi means above or over

Answer 14

Answer 15

Decondensed chromatin
– Acetylated, active
– Acetylation neutralising positive charge of lysine
– Loose association of less positive histone with negative DNA ➔ prevents
ionic interaction of lysins with DNA
– DNA is accessible for proteins involved in gene transcription (RNA
polymerases, transcription factors…)

Question 15

Histone modifications are a form of Epigenetic Regulation of
eukaryotic gene expression

Answer 15

Epigenetic
Modifications:
Heritable alterations that
are not due to changes
in DNA sequence.
▪ DNA methylation
▪ Histone modifications

Answer 15

Question 16

Acetylation of histone protein tails
remodels chromatin

Answer 16

Condensed chromatin
– hypo-acetylated, inactive
– Tight ionic interaction of positively charged histone protein tails with
negative DNA
– Inaccessible DNA, not transcribed

Answer 16

Answer 16

Question 17

Can you …

Answer 17

➢ … relate regulation of gene expression to the creation of a fully functional eukaryotic organism with many
different cell types?
➢ … compare levels of the regulation of gene expression in pro – and eukaryotes?
➢ … list levels at which gene expression is regulated in eukaryotes?
➢ … explain the necessity of DNA packaging into chromosomes?
➢ … describe levels of DNA packaging?
➢ … relate packaging of DNA to gene expression?
➢ … describe different ways in which DNA packaging occurs for example by chromatin remodeling or histone
protein modifications?
➢ … explain the definition for “Epigenetic Regulation” and relate this to histone modifications?