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Gene to function - Eukaryotes > Lecture 15 - Chromatin > Flashcards

Lecture 15 - Chromatin Flashcards

1
Q

What is expression of genes in higher organisms dependent upon?

A

DNA accessibility

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2
Q

In what three ways can histones be modified?

A

Acetylation
Phosphorylation
Methylation

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3
Q

What does DNA methylation influence?

A

chromatin structure and gene expression

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4
Q

How is DNA packaged within cells?

A

DNA is associate with proteins to form chromatin

-all chromatin is condensed and highly ordered but there are differences in the types of chromatin

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5
Q

What are the different types of chromain?

A

Interphase DNA not equally packaged -
Heterochromatin: (stains darkly) little transcriptional activity
-RNA pol necessary to bind for DNA transcription but cannot access DNA as it is tightly packaged
Euchromatin: (stains lightly) active gene expression
-loosley packaged

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6
Q

What does the differential packaging of chromatin have effects on?

A

-transcriptional activity
-DNA replication e.g.
Euchromatin is replicated earlier in S phase than heterochromatin

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7
Q

What are the differences between heterochromatin and euchromain?

A 
Hetero-
-condensed
-few genes
-telomeres
-centromeres
-transposable/repetive elements (areas that need more stability or are silenced)
Eu-
-less condensed
-genes that need to be expressed
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8
Q

How can an interphase nucleus be lysed to view the ‘beads on a string’ structure of chromatin?

A

Under low salt conditions

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9
Q

What is the composition of the nucleosome?>

A
  • made up of an octamer of histone proteins
  • 4 core proteins make dimers and come together to form an octamer (H2A, H2B, H3, H4)
  • and 146bp of DNA
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10
Q

What are the features of the four core particles of the nucleosome?

A
  • small (102-135 aa)
  • basic (rich in lysine (can be post translationally modified) and arginine (overall + charge) residues)
  • C terminal portion is well folded and involved in protein:protein interactions
  • N terminal is less structure and wheremodifications occur
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11
Q

What are linker histones and what is their purpose?

A
  • H1 histones
  • join nucleosomes together to form a compact chromatin fibre (30nm in diameter)
  • binds to the linker area between nucleosomes to allow it to fold and allows the fibre to be more tightly packaged
  • ~40 fold reduction
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12
Q

What is necessary to make the soleinoid fibre compact further?

A

scaffolding proteins to form chromosomes

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13
Q

Is the DNA that interacts with proteins less susceptible to DNase digestions?

A

Yes
Enzyme needs access to DNA
-harder if associatede with proteins

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14
Q

What evident is there that transcribed genee are in some way packaged?

A
  • DNA that is transcribed has the characteristic ‘beads on a string appearance’
  • active genes are resistant to mild nuclease tratment
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15
Q

Are there subtle differences in the resistance to mild nuclease treatment between inactive and active regions of DNA?

A

Yes.

There is a differential sensitivity to digestion with DNaseI

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16
Q

How can you experimentally show that there are subtle differences in the resistance to mild nuclease treatment between inactive and active regions of DNA?

A
  1. Digest chromatin with a range of increasing concentration of DNaseI
  2. Isolate the surviving DNA and perform a southern blot or PCR to detect the sequences we are interested in
  3. Visualise on a gel
    Show that:
    Inactive gene will form thick, constant bands at each concentration of DNase I treatment
    Active gene will show some level of resistance (bands at lower levels of DNaseI) that reduces as the concentration increasedd
17
Q

What has been determined about active genes through the technique of differential DNase I sensitivity?

A
  • actively transcribed gene are in a more open structure than non-transcribed gene
  • there may be regions even within the active genes that are more open than others (DNaseI hypersensitive regions - destroyed no matter what conc. of enzyme used)
18
Q

What influences chromatin structure?

A

1) Histone modifications (chemical groups added to the N terminal portion of histones e.g. to lysine)
- covalent modifications
- histone variants (different sort of packaging taking place in different places in the genome)
2) Chromatin remodelling complexed (potentially recurited by modifications of histones)
3) DNA methlyation status

19
Q

What is the structure of histones that allows them to be modified?

A
  • N terminal unstructured tail
  • C terminal histone fold
  • the histone fold is responsible for the dimerisation and is highly structured
  • the N terminal tails extend out from the DNA histone core and are subject to covelant modification
20
Q

What modifications can occur on the histone N terminal tails?

A

phosphorylation

  • acetylation (neutralise + charge)
  • methylation (normally repressive result in more closed structure)
  • ubiquitination (destruction)
21
Q

what occurs in histone acetylation?

A
  • the free amino group on specific lysine residues are modified by one od the hydrogen atoms in the amino group being subsituted to an acetyl group
  • modification is dynamic
  • neutralises + charge, makes the association with DNA looser and marked so that other proteins are recruited to the area
  • which could remove histones temporarily or shift them slightly away
22
Q

How does histone acetylation status affect gene expression?

A
  • Acetyl groups neutralise the positive charge of histones and therefore may weaken the electrostatic interactions between the negatively charged phosphodiester DNA backbone and the histones
  • may also disrupt histone structure by a mecahnism unrelated to charge
  • acetyl groups may also serve as signals to attract proteins that disrupt chromatin
23
Q

What are the features of histone variants?

A
  • histones are highly conserved in al eukaryotes
  • variants of H2A and H3 are incorperated into chromatin at a low freuqncy
  • variants have specific functions
  • suggest that the minor differences in amino acid sequence allows distinct modifications that could influence factor binding
24
Q

Give three examples of the inclusion of histone variants in the genome and what this results in

A

Example 1
-Histone H3.3 replaces H3 in a transcription coupled process
-thought to help maintain gene activity
Example 2
-H2AX is incorperated at low frequecy all over the genome
-when DNA is damaged it is phosphorylateede and participates in attraction of DNA repair enzymes
Example 3
-CENP-A replaces H3 at the centromeres
-binds spindle microtubules during mitosis

25
Q

Are histone acetylases activators or inhibitors of gene expression?

A

Activators

-promote the open complex of chromatin

26
Q

In what ways can DNA be methylated?

A
  • DNA bases can be modified by the addition of methyl groups
  • most common form is 5-methyl cytosine (2-7% of cytosine in mammalian DNA is modified)
  • implications in which regions are active
27
Q

What is the effect of DNA methylation?

A
  • unmethylated DNA usually adopts an open chromatin configuration (sensitive to DNase I digestion)
  • methylated DNA is associated with heterochromatin and inactive genes
28
Q

What processes are the basis of epigenetics?

A

-DNA methylation and/or histone modification

29
Q

What is the evidence that DNA methylation affects transcription?

A

Intoduction of methylated DNA into cells

  • templates that are methylated are transcribed at a lower frequency than unmethylated templates
  • unmethylated templates are more sensitive to DNAseI

Inhibition of DNA methyaltion

  • using a base analog e.g. 5-azacytidine that is incorperated into DNA but cannot be methylated acitvates previously silent genes
  • gene activation in DNA methyltransferase defective mutants
30
Q

What experiment can be done to visualise histones and other nuclear proteins?

A
  • remove soluble and loosely bound proteins
  • denature tightly bound proteins and DNA
  • separate by electrophoresis
  • stain protein bands
  • image and analyse results in relation to molecular weight markers
31
Q

What experiment can be done to reveal the periodicity of nucleosomes?

A

Isolate mammalian cell nuclei and digest chromatin with micrococcal nuclease (MNase)

  • remove soluble and loosely bound proteins
  • digest nuclei (MNase)
  • extract protein and recover DNA fragments
  • separate DNA by electrophoresis
  • image and analyse results in realtion to molecule weight markers

Gene to function - Eukaryotes (13 decks)

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