Central Dogma Lecture 14 Histones Exam 2

Question 1

What is chromatid made up of?

Answer 1

DNA + associated proteins (includes nucleosomes, etc.)

Question 2

What histones are used in chromatin? What is the function of each type? How many copies of each are in a nucleosome?

Answer 2

-H1= linker histone (interacts with DNA coming off histone to keep it compact and rigid) (1 copy)
-H2A = core histone (makes up histone octamer) (2 copies)
-H2B = core histone (makes up histone octamer) (2 copies)
-H3 = core histone (makes up histone octamer) (2 copies)
-H4 = core histone (makes up histone octamer) (2 copies)

Question 3

How much DNA is in the body vs a cell?

Answer 3

The body has ~67 billion miles of DNA
A human cell has ~6 ft of DNA

Question 4

What type of structure is important for constraining chromosomes?

Answer 4

Multiple layers and multiple modes of tertiary structure

Question 5

What amino acids are important in histones and why?

Answer 5

-Lys and Arg
-Both positive amino acids
-Important for binding DNA backbone, which is negative

Question 6

Do histones bind DNA in a specific sequence?

Answer 6

No, the entire genome needs to be wrapped around histones

Question 7

How can histones be extracted in the lab?

Answer 7

0.5M NaCl, which interferes with electrostatic interactions

Question 8

What are nucleosomes and how are they connected?

Answer 8

-The repeating unit of chromatin, the eight histones + linking histone + DNA
-Strands of DNA, looks like beads on a string

Question 9

How often do chromatin fiber regular structures repeat

Answer 9

Every 10 nm

Question 10

What is a partial digestion of chromatin?

Answer 10

-Doesn’t cut between all nucleosomes
-Uses micrococcal nuclease or Dnase I
-Cleaves dsDNA chews up the DNA between the particles (chews up linker DNA between cut nucleosomes)
-Results in fragments that are multiples of ~200 bp
-Can only be cut between nucleosomes

Question 11

Extended digestion of chromatin

Answer 11

-Reduces ~200 bp fragments to only 147 with the four histones
-Chews up all exposed DNA

Question 12

What is Nucleosome Core particle (NCP)?

Answer 12

Histone octamer + 147 bp of DNA

Question 13

How does the nucleosome octamer come together (only protein portion)?

Answer 13

-H3 and H4 form two dimers
-The H3-H4 Dimers come together to form a tetramer
-H2A and H2B form two dimers (never form tetramer)
-One H2A-H2B dimer goes to each “side” of H3-H4 tetramer
-Forms octamer

Question 14

Nucleosome composition

Answer 14

-Histone octamer
-H1
-167 bp of DNA

Question 15

How does the nucleosome come together?

Answer 15

-Histone octamer forms
-147 bp of DNA wraps around octamer 1 and 1/3 times to form NCP
-DNA enters and leaves the nucleosome on the same side
-One molecule of H1 seals off nucleosome
-Octamer + H1 binds 167 bp of DNA
-DNA makes nearly 2 full turns with full complex

Question 16

What are some important characteristics of nucleosome

Answer 16

-DNA enters and leaves nucleosome on the same side
-Left-handed supercoil around nucleosome
-Octamer + H1 binds 167 bp of DNA
-DNA makes almost 2 full turns around full complex of nucleosome

Question 17

What is chromatin usually organized into?

Answer 17

-Chromatin loops, held together by protein cohesion
-Topologically associating domains (TADs)
-Compartments (one compartment might be on, while another is off)
-Territories
-These all play key roles in gene regulation

Question 18

Chromatid in metaphase

Answer 18

-Chromosomes only assume familiar condensed form during mitosis
-Metaphase chromosome is compacted by ~10,000x

Question 19

H1 histone

Answer 19

-Linker histone
-Interacts with DNA to keep it compact and rigid, with the ends going in a specific direction
-Allows for chromatid to be guided into tight conformations, such as chromosomes

Question 20

How does chromatin structure regulate transcription in prokaryotes?

Answer 20

-Prokaryotic genes: 1000x range in transcription rates (fairly similar in range)
-Genes are never fully off; always a basal level of expression

Question 21

How does chromatin structure regulate transcription in eukaryotes?

Answer 21

-Gene transcription rates differ by as much as 10^9
-Genes can be completely turned off (like liver genes in brain)
-A cell type is defined by its pattern of gene expression (single-cell RNA-seq)

Question 22

How do cells maintain their identities/cell type over time?

Answer 22

-Identity is based on pattern of gene expression
-Cell type is defined by its pattern of gene expression
-Epigenetic marks: DNA histone modifications
-Marks are copied from one cell cycle to the next

Question 23

Euchromatin

Answer 23

-Transcriptionally active (less densely packed), considered on/open
-10x more susceptible to cleavage by DNase I than heterochromatin (in the lab)
-DNase I hypersensitive gene regions are free of nucleosomes

Question 24

Heterochromatin

Answer 24

Transcriptionally inert (more densely packed), off/silent

Question 25

Histone tail characteristics

Answer 25

-N-terminal tail of histones -Are intrinsically disordered -Are where most of the histone modifications -Post-translationally modified to control transcription (role in regulating gene expression) -Play a key role in forming contacts between nucleosomes in the chromatin

Question 26

The tail regions of histones are notoriously difficult to visualize. Why?

Answer 26

The tail regions of histones are intrinsically disordered, making it difficult to lock them into a conformation during the crystallization process

Question 27

How are histones post-translationally modified (PTMs)

Answer 27

-Histones can be reversibly modified, and each modification means something different to the cell -Acetylation -Methylation -Phosphorylation -Ubiquitination

Question 28

What do histone modifications do?

Answer 28

Alter interaction between histones and DNA (modulating charge-charge interactions)

Question 29

How do modifications of histones control transcription?

Answer 29

-The histone code -Different combinations of PTMs on tails

Question 30

Which/how amino acids are modified in histones?

Answer 30

-Lys and Arg methylation -Lys acetylation -Lys mono-ubiquitination -Ser, Thr, Tyr phosphorylation

Question 31

Meanings of modification by acetylation, monomethylation, trimethylation, methylation, phosphoylation, and ubiquitination?

Answer 31

-Acetylation: activation -Monomethylation: activation -Methylation: activation -Ubiquitination: activation -Trimethylation: repression -Phosphorylation: DNA repair

Question 32

Writers, readers and erasers of histone markers

Answer 32

-Effect covalent modifications to histone tail -Writer: adds modification -Eraser: removes modification -Reader: interprets modification

Question 33

What are general possible outcomes from post translation modifications (PTMs)?

Answer 33

-Change affinity of histones to DNA -Recruit transcriptional machinery -Initiate remodeling (moving the nucleosomes) of chromatin structure

Question 34

Histone acetylation result, writer, and eraser

Answer 34

-Writer: Histone acetyl transferases (HAT) -Eraser: Histone deacetylases (HDAC) -Reader: bromo domains

Question 35

Histone acetylation PTMs output/results

Answer 35

-Acetylation weakens histone-DNA interactions -Promote chromatin decondensation and transcription -Acetylation patterns are recognized by certain proteins -Histone becomes less positive -Reduces affinity for DNA -Usually an "active" mark

Question 36

Histone methylation writer, eraser, and reader

Answer 36

-Writer Histone methyl transferases (HMT) -Eraser: Histone demethylases -Reader: chromo domains -Output: methylatin increases hydrophobicity and can either repress or activate transcription (depending on where it is and surrounding PTMs)

Question 37

Histone ubiquitination writer, eraser, reader, and output

Answer 37

-Mono- -Mbiquitin has 76 amino acids -Writer: E3 ligase -Eraser: Deubiquitinases (DUB) -Reader: ubiquitin binding domain (UBD) -Output: Monoubiquitination of histones controls transcription by recruiting (binding site for other proteins) additional proteins

Question 38

How do proteins that bind DNA gain access to their target DNAs

Answer 38

-Chromatin remodeling complexes remodel nucleosomes by moving, ejecting, or restructuring histones along the DNA to create nucleosome-free regions -Use translocases that "walk up DNA strand, put torsional strain on the DNA, which lifts the segment of the DNA off the nucleosome surface -DNA distortion locally releases DNA from octamer -Equivalent of spinning/sliding the histone down the DNA chain