Chromatin

Question 1

Describe the organisation of DNA

Answer 1

Organised into nucleosomes about 11nm in diameter, allowing the DNA to be compacted.

146 bp of DNA is wrapped around an OCTOMER made up of 4 different histone proteins in a flat left handed SUPERHELIX.

HYDROGEN BONDS between DNA posphodiester backbone and LYS/ARG of the histone. These are needed as the DNA is not that flexible to wrap.

Question 2

List designs of the histone

Answer 2

1) Histone fold
2) Histone dimerisation
3) 4- Helix bundles

Increase in stability

Question 3

The histone fold

Answer 3

Found in the globular domain which consists of 3 helices connected by 2 loops;

the tails of the N and C terminals are verry flexible and usually have basic LYS, ARG

Question 4

Histone dimerasation

Answer 4

The histone fold motifs form the basis of a dimerization interface where the histones can interact with eachother via SPECIFIC PAIRINGS.

H3-H4 and H2a-H2b

Question 5

Histone helix bundles

Answer 5

TETRAMER: The H3-H4 dimers interact with each other via 4-helix bundle to form a (H3-H4)2 tetramer

OCTOMER: The H2A-H2B DIMER are bound to the H3-H4 TETRAMER via another 4-helix bundle interaction to form the histone octamer

Question 6

What is the relevance of the positioning of the nucleosome?

Answer 6

It affects access to the DNA; may interfere with sequence targeting.

Nucleases cleave in he MINOR GROOVE; DNA is rotated so outside is more accessible. This is done by phasing away from the octamer.

Question 7

What role do transcription factors have on DNA and histones.

Answer 7

DNA constantly unwrapping and rewrapping ; this means the DNA is only available at some time points (10% of the time available.

TF bind to the next base on the nucleosome so it cannot rewrap.

i) most factors bind to other factors
ii) Pioneer TF are independent usually PRECEDING other TF.

Question 8

What are histone tails

Answer 8

they extend out from the structure and make up 1/4 of the whole mass of the histone. They do not have homologous locations

They may be involved in dimer interaction and the assembly of the octomer

They also do gene regulation involving post-translational modifications

Question 9

Histone variants

Answer 9

Sometimes nucleosomes contain specialised variants of the core histones. A few examples are listed below:
H2az (gene activation in developmental cells)

Question 10

Linker histones

Answer 10

These are present in about half of nucleosomes., in particular less active regions (parts that are not as accessible.)

They have a WINGED HELIX

Question 11

Describe the organisation of DNA.

Answer 11

Chromatin fibers when viewed under the EM adopt an open 10nm diameter structure at low salt concentrations; native structure chromain is close but not organised.

At physiological salt concentrations 10nm fibers condense to form 30nm fibers.

Question 12

Where is chromatin in the nucleus?

Answer 12

distinct nuclear territories, where DNA is immobile. This changes alot in mitosis.

the membrane can attach to the DNA itself

Different chromosomes have different preferential locaions, creating regions of the nucleus that are conserved. These can be mapped and visualized.

The nucleolus has RNA machinery

Transcription sites around the nucleus;generally less transcription at the perifery as this is where the inactive chromatin is.

Question 13

List techniques that are used to examine chrimatin

Answer 13

1) An illuminated microarray (1 inch)used to identify gene sequences
2) High throughput sequencing maps changes in expression. It has a greater capaciy that microarrays

i) digest linker DNA
ii) remove protein
iii) amplify, make double stranded, amplify
iv) map chromatin genome
v) align start sites of every genome; nuclesomes organised with coding regions

3)Chromatin immunoprecipitation identifies DNA binding sites

This can recognize and PURIFY a target site (HoxA) and helps calculate the distribution across genome

4)Chromatin capture measures conformation and shows what regions are interacting on different or the same chromosome.

Topologically associating domains correlate with spacing

Question 14

Applications of genomics

Answer 14

Sequencing genome or exome (tumours can be sequenced)

RNA sequenced

Global accessibility e.g. to DNaseI

Nucleosome mapping

Chromatin Immunoprecipitation (ChIP)
.
HiSeq

Question 15

Give examples of chromatin modifiers

Answer 15

Modifications regulate ACCESS TO DNA; chromatin in different states in different regions of DNA.

All reversible

Different modifications, have different functional effects on histones

1) methylation (K4 becomes activated but K9 is repressed0
2) ATP dependant chromatin modellers
3) Histone modifying enzymes
4) Histone chaperones

Question 16

What happens hen chromatin is modified by acetylation?

Answer 16

A large part of the charge is neutralised

Question 17

What are HATs?

Answer 17

Histone acetyl trasferases (HAT’s) can function as COACTIVATORS in transcription initation, EG, P55 and homologous GCN3

Question 18

What is SAGA?

Answer 18

Histone modifying enzymes are normally present inside cells as components of multi-protein complexes (saga) which may modulate their specificity.

In saga there are deubquilating histones, transcription defects detecting proteins, transcription regulation; the taf proteins interact with the TATA box.

Many molecule components gives SAGA lots of functions. i) extending mRNA ii) modifying mRNA

Question 19

Histone Deacetylases

Answer 19

REMOVE ACETYL groups from histones.

There are many different deacetylases often referred to as HDAC’s . Like HAT’s they can be found in large complexes.

histone deacetylases act as co-repressors (recruited to genes by repressor proteins) to acting to reduce transcription.

Question 20

enzyme recognition

Answer 20

Enzymes add and take away modifications to specific residues on the genome. Enzymes have multiple domains which are capable of recognising different histone modifications;

the bromodomain of Gcn5 enzyme (acetyltransferase) specifically recognises actylated peptide of hisstone

Question 21

What happens when certain residues are neutralised?

Answer 21

Basic Lysine residue, if neutralised ALTERS CHROMATIN STRUCTURE

acetylation causes neutralisation which in turn results in a LOOSE structure; unwinding the DNA of the core histone.

Histone tails were less effected.

Question 22

What is the histone code hypothesis?

Answer 22

Ability of modification PATTERNS/COMBINATIONs to exert downstream functions

modifications might determine whether genes are expressed and therefore these are a form of NON GENETIC INHERITANCE (or epigenetics.)

Question 23

How to chromatins proagate themselves? What is PEV?

Answer 23

Through Positive feedback of methylation and chromatin binding chromatin can SPREAD

Position effect variegation (PEV)

In fly eye colour PEV dictates that alleles in different positions show mutated phenotypes where the allele has not been expressed as a result.

Question 24

Locus Control Regions (LCR’s)

Answer 24

they have the ability to direct high level, tissue specific expression of linked genes at all sites of integration in transgeneic assays THROUGH LOOPING INTERACTION WITH GENES

ENHANCES from a distance upstream; sufficient to overcome all forms of repressive chromatin structure at the site of integration.

Question 25

Insulators

Answer 25

prevent heterochromatin spreading and enhancer action; these block the enhancer communication with the promoter like a barrier molecule, but remains neutral (no effect) on the promoter itself.

Question 26

What is surprising about the number of possible combinations of regulatory modifications

Answer 26

Far fewer combinations than predicted as the pattern of modification per histone is CONSERVED

Only 16 states exist, which can be grouped; there are 6 major chromatin groups/states

Question 27

Describe Methyl CpG binding proteins

which act to methylate DNA ITSELF

Answer 27

(MBD family: MeCP2) Some of these proteins (on the DNA) interact with the enzymes involved in histone modifications

Question 28

Describe a possible Pathway for DNA demethylation

Answer 28

1) Hydroxylation - add OH
2) deamination
3) glycosylation removes methylated base
2) repair/replace with an unmethylaed cytosine

Question 29

Describe link between transgenerational inheritance and mehylation

Answer 29

methylated patterns are passed down from the previous generation on imprinted genes unchanged by cell development

Question 30

Helicase motifs

Answer 30

are a type of DNA translocase where Helicase motors move along DNA

Question 31

What is the SW/SNF complex?

Answer 31

multiprotein complex that affects transcription (ATP dependent chromatin remodelling enzymes) in yeast. SNF2 has many sub families in most eukaryotes.

Subfamilies will have different properties.

Question 32

WHAT IS A CHAPERONE?

Answer 32

chaperones assist in the TRANSIT of histones to and from chromatin

Question 33

The b-Interferon enhanceosome

Answer 33

activating protein assemble at the enhancer in order to regulate the promoter.

Heterodimeric transcription factors, including the basic region-leucine zipper (bZIP) protein ATF-2-c-jun, activator protein such as IRF-3 are well-characterized components of this enhanceosome that cooperatively bind to the upstream enhancer region upon viral infection.

enhanceosome acts on the human interferon-beta gene, which is upregulated in cells that are infected by viruses.

The interaction is mediated by a fourth protein HMG-I, which assists in stabilizing the complex by promoting inter-protein interactions.

The assembled enhanceosome recruits transcriptional machinery such as RNA polymerase to the promoter region to initiate gene expression.

Question 34

Describe chromatin events prior to transcription

Answer 34

It is remodelled:

1) acticators recruit SWI/SNF and SAGA, co activators which remodel the chromatin

2 )PIC, initation complex binds

Question 35

What is epigenetics ?

Answer 35

The study of heritable changes in gene expression not controlled by changes in DNA sequence.

Gentically identical cells express different genes

Question 36

Give examples of epigenetic.NON genetical inherited effects

Answer 36

1) cancer
2) Different cell types in your body
3) Differences between identical twins
4) Stem cells
5) Position effect variegation
6) Cancer
7) Dosage compensation
8) Imprinting
9) drug addiction.

Question 37

Describe imprinted alleles

Answer 37

If the allele inherited from the father is imprinted and thereby silenced, then only (or primarily) the allele from the mother is expressed; this can be explained through chromatin and DNA methylation.

epigenetic marks are established (“imprinted”) in the sperm or egg cells of the parents and are maintained through mitotic cell divisions in the somatic cells of an organism.

Prader-Will syndrome

The X inactivation: where an X chromosome is SELECTED FOR INACTIVATION at random in embryo formation and through out

Question 38

Evidence for trans generational epigenetic inheritance

Answer 38

Environmental factors no food, malnurished small bodies, next gneration retained this phenotype despite nutrients.

Question 39

What are prions?

Answer 39

heritable protein filaments that also cause epigentics