LECTURE 4: DNA packaging and some epigenetics Flashcards
List other nuclear structures to which chromatin are attached, and where the structures
are in the nucleus
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!)
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
Describe what barrier DNA sequences do
Describe what chromatin is
chromosomes consist of this
composed of DNA and associated proteins
Euchromatin
is in a dispersed state during interphase. Where genes are mostly found and is transcriptionally active.
Heterochromatin
is condensed during interphase (10% of chromatin). Not transcriptionally active:
Constitutive heterochromatin
remains condensed all the time. Consists of repeat DNA, especially in the telomeres and centromere (α satellite DNA).
Facultative heterochromatin
can be activated and inactivated dynamically as required.
where is each type of chromatin
positioned in the nucleus?
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
List the levels of DNA organization
DNA organization level 1: nucleosomes
DNA organization level 2: 30 nm fibers
DNA organization level 3: looped domains
DNA organization level 4: Mitotic chromosomes
Describe level 1 of DNA organization
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)
histones
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.
What is the nucleosome core particle composed of?
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
What is the function of H1?
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
What happens to chromatin with each level?
get shorter but fatter
Describe DNA organization level 2
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
Describe DNA organization level 3
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
Describe how MARs and chromatin remodelling factors facilitate translation
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!
List the other things chromatin remodeling factors do
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
Nuclearmatrix
a protein fiber framework • major organizing structure for RNA polymerase (makes messenger RNA), RNA processing, DNA replication.
TopoisomeraseII
an “un-tangling” protein
Insulator Proteins
keeps loops separate
Nuclear lamina(laminIFs)
Lamins on the inner part of
the nuclear envelope bind telomeres and a satellite DNA of the centromere
Describe the DNA organization level 4:
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
kinetochoreDescribe the difference between ‘normal’ genetics and ‘epigenetics’
‘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.
List the different histone variants that we actually discussed in class
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
list the different ways a gene can be epigenetically regulated
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
List mechanisms of histone modification
- Replacement with modified types of histone
– Chemical modification of the histones
List the types of histone tail modifications that can occur (not the exact positions, just the general types of chemical modifications)
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!
What can histone chemical modifications do?
i.) recruit other proteins to the chromatin, or ii.) change how the histones
interact, thus changing how compact (and accessible!) the DNA is.
Acetylated histones
looser DNA binding -> transcriptionally active
Phosphorylated histones
can increase or decrease DNA winding on histones
Methylated histones
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
Describe what barrier DNA sequences do
which
can block the spread of heterochromatin doing things
like recruiting histone-modifying
enzyme that acylate K9 on H3
Describe why X-chromosome inactivation must occur in females
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
List the molecular players and sequence of events that inactivate X chromosomes into Barr bodies
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.
