LECTURE 4: DNA packaging and some epigenetics

Question 1

List other nuclear structures to which chromatin are attached, and where the structures
are in the nucleus

Question 2

a. List the different ways a gene can be epigenetically regulated (you’ll be able to describe all of these later on in the course!)

Question 3

List the types of histone tail modifications that can occur (not the exact positions, just the general types of chemical modifications)
a. Describe what acetylation, methylation, and phosphorylation do to histones and DNA accessibility

Question 4

Describe what barrier DNA sequences do

Question 5

Describe what chromatin is

Answer 5

chromosomes consist of this

composed of DNA and associated proteins

Question 6

Euchromatin

Answer 6

is in a dispersed state during interphase. Where genes are mostly found and is transcriptionally active.

Question 7

Heterochromatin

Answer 7

is condensed during interphase (10% of chromatin). Not transcriptionally active:

Question 8

Constitutive heterochromatin

Answer 8

remains condensed all the time. Consists of repeat DNA, especially in the telomeres and centromere (α satellite DNA).

Question 9

Facultative heterochromatin

Answer 9

can be activated and inactivated dynamically as required.

Question 10

where is each type of chromatin

positioned in the nucleus?

Answer 10

Heterochromatin is located around the nucleus periphery.

Euchromatin, is located toward the interior

constitutive heterochromatin around centromere

facultative heterochromatin- around the edges, can be unwound to give access

Question 11

List the levels of DNA organization

Answer 11

DNA organization level 1: nucleosomes
DNA organization level 2: 30 nm fibers
DNA organization level 3: looped domains
DNA organization level 4: Mitotic chromosomes

Question 12

Describe level 1 of DNA organization

Answer 12

DNA wound around a ‘nucleosome core particle’ forms nucleosomes

The proteins that form the core
particle are a group of positively charged, highly conserved proteins called histones

DNA is held by the histones due to non-covalent bonds, especially ionic bonds between negatively charged phosphate backbone of DNA and positive charge of histones (from lysine and arginine amino acids)

Question 13

histones

Answer 13

proteins that form the core
particle are a group of positively charged, highly conserved proteins

*Histones are remarkably super conserved: only 2 amino acid differences (out of 102) between pea and cow H4
Because DNA is DNA, the function of histones is exactly the same in all
eukaryotes.

Question 14

What is the nucleosome core particle composed of?

Answer 14

Made up of four types of histone proteins
• two of each:
– Histones H2A, H2B, H3 and H4
• DNA wraps around nucleosome core particle 1.8 turns or 146 nucleotide bases per nucleosome

Question 15

What is the function of H1?

Answer 15

the linker histone
• links adjacent nucleosome core particles.
• A total of 168 nucleotide bases per unit
• 7:1 packing ratio, the chromatin fiber is 10 nm thick

*BRINGS CORE PARTICLES CLOSER TOGETHER TO COMPACT EVEN FURTHER

Question 16

What happens to chromatin with each level?

Answer 16

get shorter but fatter

Question 17

Describe DNA organization level 2

Answer 17

Spontaneous assembly of adjacent nucleosomes results in a 40:1 condensation in length, with 30 nm thick fibers
• H1 histone may bind adjacent nucleosomes
• N-terminal tails of H4 may extend far enough to reach other nucleosome histones

Question 18

Describe DNA organization level 3

Answer 18

This is the ‘normal’ state of DNA in an interphase cell
§ The 30 nm fibers gather into supercoiled loops that are tethered to protein nuclear scaffold
§ loops are 20,000 to 100,000 bases § Cohesin – ring-shaped protein
may maintain the loops
§ DNA strands also associate with
nuclear matrix
§ AT-rich domains of DNA form
MARs (matrix associated
regions)
§ a satellite and other non- coding DNA also matrix attached

*HIGHEST LEVEL OF DNA PACKAGING SEEN IN NORMAL INTERPHASE CELL

Question 19

Describe how MARs and chromatin remodelling factors facilitate translation

Answer 19

MARs – DNA sequence that has affinity for nuclear matrix proteins; note that DNA loops can be moved to facilitate transcription.
The transcriptional ‘machinery’ is also matrix associated. So if a gene is to be transcribed it’s actually moved to the proteins that form the transcription machinery!

Question 20

List the other things chromatin remodeling factors do

Answer 20

ATP-dependent chromatin remodeling complexes can:
1. move DNA along nucleosomes,
making DNA sequences more (or
less) accessible for transcription
2. Completely remove the core
particle
3. Swap in/out histone variants

Question 21

Nuclearmatrix

Answer 21

a protein fiber framework
• major organizing structure for
RNA polymerase (makes messenger RNA), RNA processing, DNA replication.

Question 22

TopoisomeraseII

Answer 22

an “un-tangling” protein

Question 23

Insulator Proteins

Answer 23

keeps loops separate

Question 24

Nuclear lamina(laminIFs)

Answer 24

Lamins on the inner part of

the nuclear envelope bind telomeres and a satellite DNA of the centromere

Question 25

Describe the DNA organization level 4:

Answer 25

``` The ‘ultimate’ in packaging, only occurs during mitosis!! A further 20x decrease in length. Also have looped domain structure § No nucleus is present when we see chromosomes in this state § More compact than interphase chromatin § DNA loops on Condensin protein § Condensin is activated by phosphorylation via MPF (cell cycle lecture... waaay later on) Centromere is a site of extreme condensation § contains a-satellite DNA § binds to proteins, including the α-Satellite DNA: comprised of 171 nucleotide repeats microtubules kinetochore ```

Question 26

Describe the difference between ‘normal’ genetics and ‘epigenetics’

Answer 26

‘normal’ genetics is what we describe when different individuals have different DNA sequences for the same gene (i.e. different alleles), which may cause differences in phenotypes (in the encoded protein or at the organismal level). In other words, genotype effects the phenotype. phenotypic trait variations that are caused by external or environmental factors that switch genes on and off and affect how cells read genes instead of being caused by changes in the DNA sequence.

Question 27

List the different histone variants that we actually discussed in class

Answer 27

H2A.X – DNA being repaired H2AZ and H3.3 – DNA transcription by RNA polymerase II CNEP-A (a version of H3) – in the centromere, involved in kinetochore assembly • macroH2A – Included in heterochromatin (eg Barr bodies... more on this soon) – keeps chromatin condensed and inactive

Question 28

list the different ways a gene can be epigenetically regulated

Answer 28

Histones can be modified to alter chromatin states DNA is frequently being transcribed and (when in S-phase) duplicated Necessary machinery has to work around nucleosomes – The cell makes DNA more or less accessible to other proteins by modifying histones – Histone modification can be the difference between euchromatin an heterochromatin

Question 29

List mechanisms of histone modification

Answer 29

- Replacement with modified types of histone – Chemical modification of the histones

Question 30

List the types of histone tail modifications that can occur (not the exact positions, just the general types of chemical modifications)

Answer 30

N-termini can be chemically modified by enzymes - phosphorylation - acetylation - methylation (active lysine) - methylation (inactive lysine) - Ub These are all the possible modifications known. Different cells and different chromosome areas will have different combos of these histone modifications that causes different things to happen. Also many of these are dynamic and changed all the time!

Question 31

What can histone chemical modifications do?

Answer 31

i.) recruit other proteins to the chromatin, or ii.) change how the histones interact, thus changing how compact (and accessible!) the DNA is.

Question 32

Acetylated histones

Answer 32

looser DNA binding -> transcriptionally active

Question 33

Phosphorylated histones

Answer 33

can increase or decrease DNA winding on histones

Question 34

Methylated histones

Answer 34

Alter the accessibility of DNA to processing enzymes Alter binding to organizational structures such as nuclear matrix often tighter DNA binding e.g. methylation of histone H3 leads to HP1 binding, which helps form heterochromatin

Question 35

Describe what barrier DNA sequences do

Answer 35

which can block the spread of heterochromatin doing things like recruiting histone-modifying enzyme that acylate K9 on H3

Question 36

Describe why X-chromosome inactivation must occur in females

Answer 36

In humans, gender is determined by the sex chromosomes: • females have two X chromosomes, males have one X and one Y The X and Y chromosomes look very different: almost all the genes on the X have no counterpart on the Y To ensure that males and females have equal levels of gene expression derived from the X chromosome (gene dosage again!), one X in every female cell is inactivated during embryogenesis • the choice of which X is inactivated is apparently random *GENETIC MOSAIC

Question 37

List the molecular players and sequence of events that inactivate X chromosomes into Barr bodies

Answer 37

Inactivation of an X chromosome requires a gene on that chromosome called XIST. • XIST encodes a large non-coding RNA. • XIST RNA accumulates along the X chromosome containing the active XIST gene and proceeds to inactivate almost all of the other genes on that chromosome. • XIST RNA does not travel over to the other X chromosome in the nucleus. • Barr bodies are inactive X chromosomes "painted" with XIST RNA.