Histones and Nucleosomes Flashcards
How many nucleotides per nucleosome?
200 bp
How many nucleotides would be left after limit digestion (maximum, extreme digestion)?
146 bp
Which histone is not present in the 10 nm fiber and is required to form the 30 nm fiber?
H1
Which histones comprise the eukaryotic nucleosome?
H3, H4, H2A, H2B
Which histones are the most ancient and can be found in Archaea?
H3 and H4
How does the Archaeal nucleosome differ from eukaryotic nucleosomes?
Archaeal nucleosome is a tetramer and the eukaryotic is an octomer of two copies each of H4/H3 and H2A/H2B
Do Archaeal histones have tails? Why or why not?
does not have to have a histone code to control condensation of chromatin
explain how histones are related to 10 nm fibers
- composed of dsDNA and nucleosome
- beads on a string structure
- fundamental unit of structure for the chromosome
what is the general structure of the nucleosome?
- 200 bp of DNA
- two copies of each core histone, H2A and H2B form a dimer at the ends while H3 and H4 form a tetramer at the core
- histone fold (helix - turn - helix - turn - helix) located at the core
- histone tails at N terminal of both faces of the nucleosome
what is the structure of histone tails?
- N terminal
- flexible and disordered
- exit from both faces of the nucleosome between turns of DNA
- no set structure, no loops, or alpha helices
- site of the histone code
what is the difference between HAT and HDAC?
- HAT = histone acetyltransferase, associated with activation and adds acetyl groups
- HDAC = histone deactylase, associated with repression and removes acetyl groups
chromatin structure
- has compact organization in which most DNA sequences are structurally inaccessible and functionally inactive
- 60% protein (half = histones), 30 % DNA, and 10% RNA
how much DNA is recovered in nucleosomes or multimers when micrococcal nuclease cleaves DNA of chromatin?
more than 95%
- the length of DNA per nucleosome varies for individual tissues in a ranfe from 154 - 260 bp with an average of 200 bp
how is nucleosomal DNA divided?
- core DNA and linker DNA
- depends on the DNA’s susceptibility to micrococcal nuclease
low DNase (nicking)
- 200 bp
- mono-nucleosomes
high DNase (limit digestion)
- 146 bp
- core particles
what would the average size of the fragments of chromatin be if you added just enough nuclease to hit once per nucleosome?
200 bp
how many times does DNA wrap around the histone octamer?
1.67 times
- 165 bp
what are the most conserved proteins known?
H3 and H4
which proteins have species specific variations in sequence?
- H2A and H2B
- H1 but also tissue species specific variability
what is the role of histone H1 in chromatin?
- can form pre-nucleosome with mammalian H3/H4
- not in archaea
which proteins “cap” the two flat ends of the core nucleosome?
H2A and H2B
what are the types of modifications that occur with histone tails?
- acetylation
- methylation (methylases)
- phosphorylation (kinases)
- some modifications reduce the positive charge of the histone
what does acetylation do to the positive charge in histones?
neutralize it
does mono and di-methylation change the positive charge in histones?
do not not neutralize the positive charge, so no
what does phosphorylation do to histones?
introduces negative charges on serine
what occurs to lysine and arginine in methylation and acetylation in histones?
- the positive charge on lysine is neutralized upon acetylation
- methylated lysine and arginine retain their positive charges
what is the histone code?
pattern of modifications present on the tails of histones H3 and H4 that determines the activation state of genes
what do H3 histones do? H4?
- directly affect transcription state
- directly affect nucleosome assembly
H3 lysine 4
methylation - activation
H3 lysine 9
- methylation - repression
- chromatin condensation
- acetylation - activation
- transcription activation
H3 serine 10
phosphorylation - transcription activation
H3 lysine 14
acetylation - prevents methylation at lys-9
H4 arginine 3
methylation - affects nucleosome assembly
H4 lysine 5
acetylation - affects nucleosome assembly
H4 lysine 12
acetylation - affects nucleosome assembly
what happens during acetylation?
- acetylation during replication occurs on specific sites on histones before they are incorporated into nucleosomes
- acetylation is associated with gene activation and occurs directly modifying specific sites on histones that are already incorporated into nucleosomes
which enzyme removes acetyl groups from histons?
HDAC
what are the two pathways for nucleosome assembly?
- replication dependent (replication fork) and replication independent (transcription, repair)
which two proteins act as accessory proteins?
- nucleoplasmin and N1
- acidic and function as a general chaperons and may assist in wrapping DNA around nucleosome
what is the order in which histones are assembled?
- CAF1 binds to new H3/4 tetramer then H2A/B is added
- nucleosome is added as soon as new DNA appears
what is the role of CAF1 and ASF1?
- used in replication dependent assembly
- ASF1 and CAF1 act as a chaperone
- CAF1 recruited to replication fork by PCNA (associated with alpha and epsilon)
- replication fork displaces histone octamers from DNA
- ASF1 feeds new H3/4 dimers to CAF1 while CAF loads it as a tetramer onto new DNA
- FACT disassembles the nucleosome, ASF1 binds to new H3/4 for lagging strand
what is the role of FACT?
- ancillary factor
- acts to displace octamers during transcription by releasing H2A and B dimers, other proteins needed to re-assemble after transcription
- FACT participates in re-assembly after RNA pol passes
what is the role of HIRA?
- inserts H3 variant H3.1 in nucleosomes near he TSS and in transcribed regions
- occurs during transcription and in cells not undergoing replication
When are the histone 3 (H3) variants used?
- histone 3.3 slowly replaces H3 in non-differentiating cells and after transcription
- also used in repair
- centromeric DNA replicates early and uses Cse4p in yeast or CENP-A in mammals instead of H3 - does not use replication dependent pathway
What is the role of an “Insulator”?
- define transcriptionally independent domains
- an enhancer activates a promoter in its vicinity but can be blocked from doing so by an insulator located between them
- can block activation, repression, or both
- may block heterochromatin
BEAF
- heat shock loci 87A and C of drosophila are flanked by insulators
- a protein that binds to the insulator scs’ and is localized interbands in drosophila polytene chromosomes
specialized chromatin structures (scs and scs’)
- do not directly regulate gene activity but function to limit the crosstalk between promoters so that regions of very high activity can be located next to low activity regions of the genome
do RNA pol 2 and insulator proteins occupy the same regions?
they do not
what part of the histone is not conserved during replication?
histone ocramers
- H2A and H2B dimers and H3-4 tetramers are conserved
what is the structure of CAF1?
- 5 subunits
- mass of 238 kD
when are nucleosomes found at the same frequency when you digest with micrococcal nucleoase?
- transcribed genes
- nontranscribed genes
- some heavily transcribed genes appear to be exceptional cases that are devoid of nucleosomes
are histone octamers displaced by transcription?
- RNA polymerase displaces histone octamers during transcription in a model system
- octamers reassociate with DNA as soon as the polymerase has passed
- nucleosomes are reorganized when transcription passes through a gene
what are the relative sizes of RNA polymerase and nucleosomes?
- nucleosome = 300 kb, 6x11 nm
- RNA pol 2 = 500 kb, 14x13
