Wright Lecture 10: Eukaryotic Gene Regulation by Chromatin Conformation Flashcards
Chromatin
DNA and proteins
Not free floating in eukaryotes
Attached to internal protein sub-structure called the nuclear matrix (scaffold or cage)
Chromosomes visible by light microscopy during cell division
Bacteria nucleoid
Chromosomal DNA complexed with few basic proteins
Lysis of bacterial cells release uniform chromatin structure of twisted loop containing supercoiled DNA, ends of loops held in place by proteins
Histones
H1, H2A, H2B, H3, H4
Tightly bound to DNA: requires high salt concentration to extract the histones from DNA
High content of basic (positively charged) amino acids that interact with phosphate groups of DNA
Most abundant chromosomal proteins
Amino acid sequence is highly conserved
Structural proteins: NOT regulators
Non-Histone chromosomal (NHC) proteins
Major constituents: high mobility group (HMG)
Abundant chromosomal proteins but not specific regulators of gene expression
Loosely bound to chromatin, extracted by low concentrations of 0.35M salts
High in basic (positively charged) and acidic (negatively charged) amino acids
More diverse in amino acid sequence of individual HMG-proteins than from one species to another
HMG proteins exhibit different tissue-specific patterns of distribution
High mobility group
Major constituent of non-histonal chromosomal proteins
Exhibit different tissue-specific patterns of distribution
Other NHC proteins
Low abundance
Specific transcription factors
Ie. Steroid hormone receptor
Beads on string
One of two types of chromatin conformations in interphase nuclei
10 (11)nm
Solenoid
One of two types of chromatin conformations in interphase nuclei
30nm fibre
200bp organization of DNA
- Interphase nuclei purified and lysed to release chromatin fibres
- Nuclease digestion of chromatin to release particles
- Chromatin particles sentiment by centrifugation
- DNA and protein purified from sediment chromatin particles
- Assayed by gel-electrophoresis
Discovered DNA digested into integers of 200 bp by mild digestion of chromatin by nucleases, releases nucleosome from beads on the string: in ALL eukaryotic chromatin
Octameric histone surrounded by two loops, stabilized by H1
Longer digestion of 200bp
Destroys all linker DNA and releases attached H1 from core nucleosome
About 145-147 base pairs attached to core octameric histone
Solenoid vs. Zig zag
Nucleosomes in 30nm chromatin fibre appear to be organized in solenoid but it is debated
Solenoid: one start helix
Zigzag: two start helix
Structure of metaphase chromosome
Naked DNA: 10nm beads on a string: 30nm solenoid: loops: miniband: chromosome
Chicken RBC (relationship between chromatin and gene expression)
Most abundant mRNA in RBC are for alpha and beta globin peptides: actively transcribed
Very sensitive to nuclease: transcriptionally active chromatin is preferentially digested by nuclease over inactive chromatin
Seen by digesting RBC DNA and then isolated beta-globing gene and subjecting it to Southern blot hybridization with probe for beta-globin
Transcriptionally active DNA conformation
In beads on a string conformation, and therefore more rapidly digested by nucleases
Acetylation of histones
Acetylation and deacteylation controls transition of chromatin form condensed (30nm) to extended (10nm)
Acetylation of tails of histones (positive tails bond negative DNA) weakens their interactions with DNA and repel adjacent nucleosomes, allowing access of transcription factors
