Central Dogma Lecture 14 Histones Exam 2 Flashcards
What is chromatid made up of?
DNA + associated proteins (includes nucleosomes, etc.)
What histones are used in chromatin? What is the function of each type? How many copies of each are in a nucleosome?
-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)
How much DNA is in the body vs a cell?
The body has ~67 billion miles of DNA
A human cell has ~6 ft of DNA
What type of structure is important for constraining chromosomes?
Multiple layers and multiple modes of tertiary structure
What amino acids are important in histones and why?
-Lys and Arg
-Both positive amino acids
-Important for binding DNA backbone, which is negative
Do histones bind DNA in a specific sequence?
No, the entire genome needs to be wrapped around histones
How can histones be extracted in the lab?
0.5M NaCl, which interferes with electrostatic interactions
What are nucleosomes and how are they connected?
-The repeating unit of chromatin, the eight histones + linking histone + DNA
-Strands of DNA, looks like beads on a string
How often do chromatin fiber regular structures repeat
Every 10 nm
What is a partial digestion of chromatin?
-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
Extended digestion of chromatin
-Reduces ~200 bp fragments to only 147 with the four histones
-Chews up all exposed DNA
What is Nucleosome Core particle (NCP)?
Histone octamer + 147 bp of DNA
How does the nucleosome octamer come together (only protein portion)?
-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
Nucleosome composition
-Histone octamer
-H1
-167 bp of DNA
How does the nucleosome come together?
-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
What are some important characteristics of nucleosome
-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
What is chromatin usually organized into?
-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
Chromatid in metaphase
-Chromosomes only assume familiar condensed form during mitosis
-Metaphase chromosome is compacted by ~10,000x
H1 histone
-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
How does chromatin structure regulate transcription in prokaryotes?
-Prokaryotic genes: 1000x range in transcription rates (fairly similar in range)
-Genes are never fully off; always a basal level of expression
How does chromatin structure regulate transcription in eukaryotes?
-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)
How do cells maintain their identities/cell type over time?
-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
Euchromatin
-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
Heterochromatin
Transcriptionally inert (more densely packed), off/silent
Histone tail characteristics
-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
The tail regions of histones are notoriously difficult to visualize. Why?
The tail regions of histones are intrinsically disordered, making it difficult to lock them into a conformation during the crystallization process
How are histones post-translationally modified (PTMs)
-Histones can be reversibly modified, and each modification means something different to the cell
-Acetylation
-Methylation
-Phosphorylation
-Ubiquitination
What do histone modifications do?
Alter interaction between histones and DNA (modulating charge-charge interactions)
How do modifications of histones control transcription?
-The histone code
-Different combinations of PTMs on tails
Which/how amino acids are modified in histones?
-Lys and Arg methylation
-Lys acetylation
-Lys mono-ubiquitination
-Ser, Thr, Tyr phosphorylation
Meanings of modification by acetylation, monomethylation, trimethylation, methylation, phosphoylation, and ubiquitination?
-Acetylation: activation
-Monomethylation: activation
-Methylation: activation
-Ubiquitination: activation
-Trimethylation: repression
-Phosphorylation: DNA repair
Writers, readers and erasers of histone markers
-Effect covalent modifications to histone tail
-Writer: adds modification
-Eraser: removes modification
-Reader: interprets modification
What are general possible outcomes from post translation modifications (PTMs)?
-Change affinity of histones to DNA
-Recruit transcriptional machinery
-Initiate remodeling (moving the nucleosomes) of chromatin structure
Histone acetylation result, writer, and eraser
-Writer: Histone acetyl transferases (HAT)
-Eraser: Histone deacetylases (HDAC)
-Reader: bromo domains
Histone acetylation PTMs output/results
-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
Histone methylation writer, eraser, and reader
-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)
Histone ubiquitination writer, eraser, reader, and output
-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
How do proteins that bind DNA gain access to their target DNAs
-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
