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BCHM 218 Part 2 > Module 3 Section 8 > Flashcards

Module 3 Section 8 Flashcards

1
Q

Cellular processes that rely on the modification of chromosomes

A
  • Regulation of gene expression
  • DNA replication
  • DNA editing and repair
  • Recombination events
  • The preservation of epigenetic tags
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2
Q

Epigenetic tags

A

-a specific modification on DNA or specific amino acids in the histone proteins that DNA is wrapped around

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3
Q

Chromatin remodelling complexes functions (3)

A
  1. repositions (slides) nucleosome to different position on DNA
  2. Eject nucleosome from the DNA
  3. Replace the nucleosome with one that contains a histone variant
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4
Q

Chromatin remodelling complexes mechanism

A
  • latch around histone octamer, pull out DNA associated with the nucleosome
  • “loop propagation,” slide DNA across, then tighten the histone around the new area of DNA
  • requires ATP (the enzymes contain ATPase domains)
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5
Q

Questions answered by ChIP for studying chromatin remodelling or modification

A
  • ChIP: chromatin Immunoprecipitation
    1. Across genome:
  • which segments of DNA are incorporated in nucleosomes?
  • What is nucleosome density in particular regions of the genome?
  • Can we use this info to identify actively transcribed regions of the genome?
    2. For gene of interest
  • is the promoter for my gene of interest associated with a nucleosome bearing a particular histone variant or histone modification?
  • What does this tell us about the accessibility of of this gene under different conditions?
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6
Q

ChIP steps (4)

A
  1. Cells treated with formaldehyde (this covalently crosslinks the nucleosomes to DNA.
  2. The genomic DNA is fragmented using non-specific endonuclease or mechanically (sheared)
  3. an antibody to a specific modified histone is used to immunoprecipitate the nucleosome-DNA complex. Any DNA not bound to a histone is washed away
  4. The protein-DNA crosslinks are reversed by heating. Released DNA is analyzed with PCR, qPCR, NGS or microarray
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7
Q

H3 histone variants

A

H3.3

CENPA (centromere protein A)

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8
Q

H2A histone variants

A

H2AX
H2AZ
macroH2AZ

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9
Q

H2AX function

A
  • associated with DNA repair/genetic recombination

- C-term has phosphorylation region, site for recruitment of proteins for DNA repair

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10
Q

H2AZ function

A
  • associated with nucleosomes located at actively transcribed genes
  • thought to stabilize the open state of chromatin to facilitate access of transcriptional machinery
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11
Q

macroH2AZ function

A
  • abnormally large, unique C-term domain
  • used for X-chromosome inactivation, keeps from double expression of X in females:
  • XIST RNA expressed by X-chromosome
  • macroH2AX substituted for normal H2A, serves as docking site for XIST RNA, capping off one of the X-chromosomes
  • occurs at blastysis stage of development
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12
Q

H3.3 function

A
  • found in transcriptionally active regions

- associated with transcriptionally active state

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13
Q

CENPA function

A

(centromere protein A)

-allows for kinetochore attachment (docking site for spindle fibres, separation of nucleosomes during cell division)

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14
Q

What do Histone Modification enzymes do?

A
  • covalently modify histones, primarily on the N-term tail
  • modifications are heritable
  • There are cis acting and trans acting modifying enzymes
  • NOT mutually exclusive, certain modifications can have both effects
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15
Q

Types of histone modifications (and the AA they happen on)

A
  1. phosphorylation of serine (S), threonine (T), tyrosine (Y)
  2. acetylation of lysines
  3. methylation lysines and arginines
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16
Q

Cis acting histone modifying enzymes

A
  • affect the the chromatin structure by direct modification of the histone
  • modifications may open or close (move histones closer or farther from each other) chromatin
  • ie. acetylation of lysine
17
Q

Trans acting histone modifying enzymes

A
  • involve other intermediary molecules

- attract other proteins such as transcription factors/chromatin remodelling factors, which produce chromatin change

18
Q

Writing conventions for histone modifications - specific examples (2)

A

Acetylated histone 3, lysine 9:
H3K9ac
trimethylated histone 3, lysine 9:
H3K9me3

19
Q

Acetylation of lysine residues

A
  • neutralizes the pos charge on the amino group

- generally enhances transcription, alters DNA compaction through internucleosome interactions

20
Q

Methylation of lysine/arginine

A
  • thought to stabilize the open or closed state, often through recuitment of histone-binding protein complexes
  • can enhance or repress transription in context-dependent manner
21
Q

Specific Marks of activation on H3

A
  • Acetylation at lysine 9 (H3K9ac)

- Trimethylation at lysine 4 (H3K4me3)

22
Q

Mark of repression on H3

A
  • Trimethylation of lysine 9 (H3K9me3)
  • when it is methylated, it can’t be acetylated (H3K9ac is mark of activation), which stabilizes the open state, so this in turn stabilizes the closed state
23
Q

Enzymes that mediate reversible modification of N-term histone tails

A 
HATs (Histone acetyltransferases)
-add acetyl groups to histones
HDACs (histone deacetylases)
-remove acetyl groups
HMTs (Histone methyltransferases)
-add methyl groups to histones
Jumonji Family (KDMs)
-histone demethylases
-remove methyl groups
24
Q

Bromodomains

A
  • protein motifs that bind acetylated lysines
  • diverse functions depending on the complex
  • if found within a multiprotein complex that also has HAT (histone acetyltransferase) subunit, can be used to propagate open state by acetylating neighbouring histones
  • this typically increases gene expression
25
Q

Chromodomains

activated/repressed modification example

A
  • protein motifs that bind methylated lysines
  • can promote either activation or repression of genes
  • ie. H3K9me3 associated with repression, H3K4me3 associated with activation
26
Q

Phosphorylation Histone modification

A
  • adds a neg charge to the histone tail
  • critical step in chromosome remodelling
  • in cooperation with other modifications, it initiates recruitment/release of histone modifying enzymes or chromatin remodelling complexes
  • triggers a pathway of downstream modifications
27
Q

Multiple modifications on a single histone

A
  • can recruit histone modifying enzymes or chromatin remodelling complexes one step at a time
  • each modification contributes to a pathway
  • ultimately results in the activation/repression of gene expression
  • this is basis of the “histone code:” the stepwise accumulation of modifications
28
Q

Epigenetics

A 
-The study of heritable changes in gene function that do not involve changes in the DNA sequence
ie:
-chromatin remodelling
-histone modification
-nurturing influences
-mitochondrial effects
-DNA methylation
29
Q

Epigenetic inheritance

A
  • passage of histone marks from parent to daughter cells in mitosis and meiosis
  • intergenerational transfer of histone modifications
  • most epigenetic tags of adulthood are lost during gametogenesis
30
Q

Imprinting

A

The epigenetic silencing of maternal or paternal genes during gametogenesis

31
Q

Marked histones during replication

A
  1. The paternal (marked) H3-H4 heterotetramers are distributed randomly on the two new daughter duplexes, coat half the DNA after replication
  2. New H3-H4 heterotetramers that lack the particular modification pattern of those they are replacing are assembled onto the replicated DNA by CAF-1 chaperone protein
  3. Paternal H2A-H2B dimers remain in vicinity after being displaced by replication fork. They reassemble with H3-H4 heterotetramers and new H2A-H2B dimers on replicated DNA, chaperoned by NAP-1
  4. new octomers can be completely unmodified, completely newly modified, or mixture
32
Q

Steps for maintaining repressed state

A
  1. replication results in some octamers missing the original methylation mark
  2. HP1 (heterochromatin protein 1, has chromodomain motif) recruited, binds to methylated regions
  3. HP1 recruits HMP (histone methyltransferase), which propagates the methylation
  4. results in the maintenance of a closed state
33
Q

Steps for maintaining active chromatin state

A
  1. replication results in some octamers missing original acetylation mark
  2. Bromodomain HAT (histone acetyltransferase)
  3. acetylation is propagated to octamers missing the mark
  4. results in maintenance of open state

BCHM 218 Part 2 (13 decks)

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