02 - Packaging & Organizing Genomes

In what ways are these genomes likely to differ or be similar?

Genomes are continually changing. What do we mean by change?

How/why does change happen? What could occur from parent to offspring/daughter cell?

Eukaryotes

Prokaryotic cells (Archaea and bacteria):

Human chromosome structure

are linear and we have 2 of each (23 pairs), meaning we are diploid

What can your cells do that a bacteria can do too?

does size correalate with number of genes

Genome size varies tremendously! But size does NOT necessarily correlate with number of genes (or chromosomes)

True or false: All eukaryotes have larger genomes than all prokaryotes

True or false: Compared to eukaryotes, the number of genes in prokaryotic genomes correlates more strongly with genome size

Rank the following in order of average increasing gene density: Prokaryotes, Mitochondria, Eukaryotes

What do you think this means about the composition of your genome?

• Non-coding DNA can make up a ____ of eukaryotic genomes

large %

Non-coding DNA is often ___

repetitive

From chromosomes to genes

Non -coding sequences can exist within genes

Once transcribed into RNA, introns are removed by the process of RNA splicing

The remaining protein - coding sequences are called exons

What do you notice about the spacing and sizes of genes and exons?

Packaging the eukaryotic genome

Genome must be packaged in order to fit in the nucleus:

Packaging must be compact but also accessible to enzymes and regulatory factors

DNA Supercoiling

Supercoiling is helpful to relieve the strain

• Increases stability

• Making DNA more compact

• Allows unwinding of sections

Why would we need to unwind (open) a section of DNA?

Positive supercoils →

helix is overwound

Negative supercoils →

helix is underwound

What might happen to a cell if too much supercoiling prevents helix opening?

Topoisomerases

are protein enzymes that regulate/relieve supercoiling

chromatin

Eukaryotic DNA associates with histones and other proteins to form chromatin

nucleosomes

Histones package DNA into repeating units called nucleosomes

• Repeating subunit of DNA + histones

• 8 histones proteins form a central core

• Octamer: two each of H2A, H2B, H3 and H4

• DNA is wound around the octamer core

Overall, packaging ratio of DNA in nucleosomes is

7:1

Histones

are highly conserved proteins rich in basic amino acids (Lys, Arg,) resulting in a high positive charge which interacts easily interact with the negative (-) charge of DNA

Where does the negative charge on DNA come from?

phosphate backbone

Histone dimer formation is mediated by

C-terminal domain histone fold

Each histone has an N-terminal histone tail that

protrudes outward and can be subject to covalent modifications

30 nm fiber

• Histone H1 (linker histone) binds linker DNA that connects one nucleosome to another

• Regulates how tightly these nucleosomes pack together

• Without histone H1, nucleosomes look like ‘beads on a string’

• Histone H1 can line up nucleosomes end-to-end into two stacks

• This “30 nm fiber” increases the DNA-packaging ratio 6-fold further (overall now ~40-fold)

– Looped domains

• 30 nm fiber may further be gathered into a series of large loops

• These loops are attached to protein scaffolds

Levels of chromatin organization

As cells prepare for mitosis, looped domains get further compacted

Euchromatin

DNA that is less compacted and functionally active (accessible for

protein binding and transcription). Genes here can be expressed

Heterochromatin

DNA that is highly compacted and has little to no functional activity

Types of Heterochromatin

Constitutive heterochromatin

Facultative heterochromatin

Constitutive heterochromatin

• Permanently silenced DNA

• Includes regions around telomeres and centromeres

• Contains DNA repeats and few genes

Facultative heterochromatin

• Inactivated during certain phases of organism’s life

Why is this necessary?

What do you think would happen if all your facultative

heterochromatin suddenly became euchromatin and vice versa?

Which of the following are true?

A) All DNA is facultative heterochromatin until it becomes constitutive heterochromatin

B) Centromeres are an example of constitutive heterochromatin

C) Genes in a region of euchromatin are more likely to be expressed than genes in a region of heterochromatin

D) After every round of cell division in females, one X chromosome is randomly selected for inactivation

E) X inactivation occurs in the female gamete (egg) before fertilization

Epigenetics

Covalent modifications (e.g. methylation) of DNA and histones

Influences heterochromatin vs euchromatin formation

Epigenetic regulation of gene expression can be inherited by daughter cells during cell division without changes to the sequence of nucleotides in DNA

Covalent modifications of histone tails can

disrupt or stabilize nucleosome assemblages

acetylation leads to what in histone code

more open structure and more transcription,

methylation causes what in histone code

compaction/repression

condensed nucleosomes

uncondensed nucleosomes

Histone acetyltransferases (HATs)

acetylate histone proteins by transferring acetyl group from acetyl-CoA to specific lysine residues.

Histone deacetylases (HDACs)

remove the acetyl group.

Histone methyltransferases (HMTs)

add methyl group(s) (1, 2, or 3) to lysine or arginine residues.

Histone demethylases

remove methyl groups

Most histone methylation contributes to

silencing (compacting) DNA

DNA methylation

DNA methylation works together with the histone code

Proteins that bind to methylated DNA can recruit enzymes involved in modifying histone tails

Methylation of cytosine can prevent proteins from binding to DNA sequences (often preventing initiating of transcription)

DNA methyltransferase

can add the methyl group to DNA at sites where a C is followed by a G (reading 5’ to 3’ = “CpG”) creating an epigenetic marker on the DNA

DNA demethylases

can remove these methyl groups.

DNA is methylated as it is

replicated so that methylation patterns can be passed on to daughter cells (The symmetry of CpG on both strands is critical for this!)

Maintaining epigenetic regulation

Silencing of genes by heterochromatin formation occurs in regions (“position effect”) and is maintained in replicated DNA

Does this mean that you inherit all your epigenetics from your parents?

Epigenetic regulation from gametes to offspring

Methylation patterns are copied to newly synthesized DNA and so can be inherited during cell division, but are mostly rewritten during initiation of offspring development

genomic imprinting

Some (very few) methylation patterns are passed on from parents to offspring (called genomic imprinting)

Maintaining heterochromatin after DNA replication

The epigenetic signals that regulate chromatin are propagated through space (along chromosome) and time (during cell division)!

Which of the enzymes mentioned so far are likely to contribute to creating a more condensed (heterochromatin) state? (select all that apply)

A) HATs (histone acetyltransferases)

B) Histone demethylases

C) HDACs (histone deacetylases)

D) Histone methyltransferases

E) DNA methyltransferases

Why doesn’t heterochromatin propagate across entire chromosomes?

Barrier DNA sequences can recruit protein complexes that block the spread of reader -writer complexes and separate chromatin into domains

Mechanisms of barrier action

Barrier proteins can bind to barrier sequences and create physical obstacles or actively recruit opposing chromatin modifying enzymes

Chromatin becomes separated into domains with different transcriptional activation/regulation.

If you wanted to understand how genomes evolve over time (many generations), would you look at the genetic code or the epigenetic modifications?

If you wanted to understand why your liver cell doesn’t behave the same way as a heart muscle cell, would you look at the genetic code or the epigenetic modifications?

Which of the following is TRUE?

A) You inherit all the epigenetic modifications in your genome from your parents

B) After each round of cell division, heterochromatin forms randomly in various regions of DNA

C) In your neurons and skin cells, the same set of genes are silenced by facultative heterochromatin formation

D) Your liver cell and heart cell contain the same genome of 46 chromosomes but are likely to differ in their patterns of epigenetic modifications

E) Epigenetic changes are permanent