CMMB 411 - 2 Nucleosomes, Chromatin, Chromosome Structure

DNA organization in prokaryotes

one chromosome (predominantly circular) and other small independent circular DNA called plasmids in the cytoplasm

DNA associated with proteins and organized into chromosomes

DNA organization in eukaryotes

linear chromosomes enclosed in a nucleus, DNA associated with proteins

chromatin

a given region of DNA with its associated proteins on a chromosome

not uniform along a chromosome, different levels of packing

essential components of eukaryotic chromosomes

• origins of replication (multiple)

• centromere

• telomeres

origins of replication

DNA sequences along chromosome which initiate DNA replication, start of denaturation

centromere

DNA sequences required for correct segregation of chromosomes by directing formation of the kinetochore protein complex in which the mitotic spindle attaches (one per chromosome)

kinetochore attaches here

more than one or none can cause chromosome loss or breakage

telomeres

DNA sequences located at the ends of the chromosome that prevent degradation and allow proper replication of the chromosomal ends

chromosomal ploidy

most eukaryotes are diploid (paternal + maternal), humans have 23 pairs of homologous chromosomes

only sexually reproductive cells have a haploid genome

few eukaryotes are polyploid such as large protists (need more proteins for larger cells)

eukaryotic cell cycle

an ordered set of processes by which one cell grows and divides into two daughter cells

need to fully replicate DNA and organelles and properly segregate them to daughter cells

g1 and g2

gap phases, synthesis of proteins, RNA, metabolites, other than DNA

s phase

DNA replication (replicated chromosome = sister chromatids)

m phase

mitosis, nuclear division/chromosome segregation

cytokinesis

cell division, split of cytoplasm

g0 phase

resting phase or quiescence

mitosis

proliferation of cells where the genome content is identical to parental cell and two daughter cells

homologous chromosomes act independently throughout

meiosis

creation of gametes (sperm and egg)

number of homologous pair of chromosomes is reduced from two in the parental cell to one in the four daughter cells

genomic content (alleles) in all four daughter cells are unique due to recombination after homologous chromosomes align in prophase I

reasons for chromosomes

1. chromosomes compact DNA so they can fit into the cell/nucleus (10,000x)

2. chromosomal structure protects DNA from damage (naked DNA is unstable and prone to degradation by nucleases, fragmentation and oxidative damage)

3. chromosomes can be easily separated and transmitted to each daughter cell during cell division (compact structure prevents fragmentation from mechanic forces during segregation)

nucleosomes

the basic units of DNA compaction, form from histones

discovery of nucleosomes

half of the mass of chromatin/chromosomes is proteins, histones most abundant (form nucleosomes)

chromosomes treated with micrococcal nuclease (MNase)

light digestion produces a range DNA fragments, heavy digestion produces a single band (all linker DNA cut to yield only nucleosomes)

micrococcal nuclease (MNase)

a sequence-nonspecific nuclease, cleaves protein-free DNA sequences rapidly and protein-associated sequences poorly

nucleosome composition

two subunits each of the core histones H2A, H2B, H3 and H4 forming an octamer

146 bp of DNA wrapped in a left-handed solenoidal negative supercoil

appears as a beads on a string with linker DNA in between nucleosomes

histones

small highly basic proteins containing 20-32% lysine and arginine residues, highly conserved in all eukaryotes

linker DNA

variable, between nucleosomes

histone H1 (linker histone)

one per nucleosome or not present, not required for the formation of the 30 nm filament but facilitates and stabilizes its structure

two DNA binding sites

• linker DNA at one end of the nucleosome

• central region of DNA wrapped around the nucleosome

binding to linker DNA increases length of DNA wrapped tightly around the nucleosome and a more defined angle of DNA entry and exit from the nucleosome

promotes a more compact structure of nucleosomes in a zigzag pattern vs in absence

eventually condensed into a compact filament with width 30 nm

core histones

e.g. H2A, H2B, H3, H4 - have a histone-fold domain and N-terminal tails

histone-fold domain

three alpha helical regions separated by two short loops involved in formation of heterodimers of H3-H4 and H2A-H2B

involved in both histone-histone and histone-DNA interactions, a histone dimer contains three DNA binding sites

the domain contacts DNA by hydrogen bonds at the backbone and minor groove (non-sequence specific)

formation of octamer

requires DNA, if no DNA, the H3-H4 heterotetramer and the H2A-H2B heterodimers will form, but do not assemble into octamer

if DNA - H3-H4 tetramer binds to DNA and then recruits two H2A-H2B heterodimers with N-terminal tails extended away from nucleosome

histone chaperones

acidic proteins, function in nucleosome assembly by binding to H3-H4 heterotetramers and H2A-H2B heterodimers (does not require ATP)

AT tracts

two or more consecutive A-T pairs, causes bending and narrowing of the minor groove, promote contact with histones

DNA wrapping around histone is not primarily base specific, but nucleosomes positioning favoured in regions with ~10 bp spacing of AT tracts

GC tracts

found at non-bending DNA regions not facing the nucleosome (no contact with nucleosome)

core histone tails

N-terminal extensions, flexible and disordered that can dynamically change conformation (lacks a stable 3D structure), not required for association of DNA with histone octamer

forms internucleosomal contacts to organize adjacent nucleosomes into higher ordered chromatin structures by a 6-7 fold (so not enough yet)

undergoes posttranslational modifications at KRSTY amino acids that influence internucleosomal contacts to control chromatin structure and gene expression (epigenetics)

tighter the nucleosomal contacts by histone tails, the less accessible DNA is to TFs and other proteins involved in replication and recombination

euchromatin

regions with lower DNA compaction and genes are actively expressed

heterochromatin

chromosomal regions of high DNA compaction, higher-order chromatin structure where gene expression is silenced

constituitive heterochromatin

DNA always highly compacted (centromeres and sub-telomeric regions), not genes

facultative heterochromatin

can switch to euchromatin depending on cell type and during development

30 nm filament

forms mainly due to histone tails that form internucleosomal contacts

compact filament with width 30 nm

two models - solenoidal and zigzag

both models likely form in the same chromatin filament (dynamic process), exist in different regions

solenoid model (one-start helix)

nucleosomes form a superhelix with 6 nucleosomes/turn, face of nucleosomes face each other, linker DNA circles but does not pass through the central axis (donut hole)

zigzag model (two-start helix)

nucleosomes form zigzag pattern with two stacks, linker DNA passes through central axis connecting two nucleosomes on opposite sides of the filament

higher-ordered chromosome structures

30 nm fiber forms loops that are tethered at their bases to a proteinaceous structure (chromosomal scaffold)

chromosomal scaffold

exposed by removal of histones and incubation in low salt bugger (hypotonic) causing chromosome swelling

major components include SMC proteins and topo II

eukaryotic topo II

hypothesized to prevent entanglements and torsional stress of 30 nm fiber loops and holds the loops together at the base during chromosome condensation

smc proteins (structural maintenance of chromosomes)

highly conserved key regulators of chromosome structure (also found in prokaryotes), head and hinge domains, also kleisin family proteins associated

head domains of smc

at both C- and N- terminal ends and contains a functional ATPase domain when fused

hinge domain of smc

connected to each head domain by an a-helical coil-coiled domain and required for dimerization

kleisin family proteins

binds to both head domains to fuse them and form a ring

smc heterodimers

cohesins and condensins

smc1-smc3

cohesin

smc2-smc4

condensin

cohesin

smc heterodimer, keeps sister chromatids together from DNA replication to onset of anaphase

removed by separase

condensin

smc heterodimer, compaction of chromosomes at mitotic entry by inducing loops and positive supercoils

position along chromosome and hinge domain binds to DNA

ATP binding fuses head domains and supercoiled DNA loop extrusions are created

arranged in a rosette-like appearance and condenses the DNA

separase

removes cohesins from sister chromatids ahead of anaphase

nucleoid

for bacterial chromosome compaction, a rosette of negative supercoiled DNA mega-loops that extend from a proteinaceous scaffold

scaffold proteins (bacterial nucleoid)

nucleosome associated proteins (NAPs), HU proteins, bacterial SMCs

loop structure and number are dynamic and influenced by amount of transcription

nucleosome associated proteins (NAPs)

in bacterial chromosome scaffold, are functionally analogous to histones

such as HU proteins (induce negative supercoiling in DNA)

bacterial SMCs

cause loop extrusions to form the DNA mega-loops in bacteria for chromosome compaction

chromatin remodeling of nucleosomes

nucleosomes not evenly distributed along chromosome and can either be ejected, repositioned by sliding or replace with a new nucleosome containing a variant histone subunit

chromatin remodeling complexes

have an ATPase domain that uses ATP energy to disrupt contacts between DNA and nucleosomes

3 main classes - SWI-SNF, CHD1/CHD and also ISWI and Mi2/NURD

SWI-SNF

chromatin remodeling complex for transcriptional activation

has a bromodomain, bind to acetylated histones

CHD1

chromatin remodeling complex for transcriptional activation

as a reader - binds to H3K4me3 at promoters and evicts nucleosomes

with a chromodomain

sliding mechanism

DNA is pulled, forms a loop that detaches from the nucleosome, the loop is propagated around the nucleosome until it reaches the linker DNA

histone variants

alter nucleosome function, histones that have different amino acid sequences at N or C terminal compared to WT versions

H3.3

an H3 variant, maintains a transcriptionally active open state in promoter regions by preventing H1 incorporation in chromatin - keeps chromatin open and less compact

keeps promoter exposed and active

CENPA

an H3 variant, incorporated in nucleosomes in the centromere (where kinetochore binds), contains an N-terminal tail that recruits kinetochore proteins to the centromere

for segregation of chromosomes

replaces H3 in nucleosomes in the centromere

H2AX

H2 variant, incorporates in nucleosomes at double strand breaks, is rapidly phosphorylated (y-H2AX) and recruits DNA-repair enzymes (DNA damage sensor) so it can be fixed

macroH2A histone variant in X chromosomes

in females, gene expression of one X chromosome is inactivated by heterochromatin formation (barr body)

mechanism depends on incorporation of the histone H2A variant macroH2A in nucleosomes and coating of noncoding Xist RNA on the entire inactive X chromosome

random and heritable

e.g. calico cats - heterozygous females may have black and orange patches depending on which X was inactivated, white colour is autosomal

posttranslational modifications of histone tails

acetylation, methylation, phosphorylation

acetylation of lysine

acetyl group added by histone acetyltransferases (HATs) neutralizes the positive charge of lysine and unfolding of the 30 nm fiber as well as nucleosome stability and eviction, transcriptional activation

e.g. H4-K16ac

histone deactylases (HDACs)

catalyzes the opposite reaction from HATs, makes DNA more compact around the nucleosome

methylation of lysine and arginine

histone methyltransferases can add mono, di, or trimethyl groups to activate or repress gene transcription depending on pattern

opposite reaction catalyzed by histone demethylases

H3K4me3

at promoters for transcriptional activation

H3K9me3

for heterochromatin formation

phosphorylation of serine, threonine, tyrosine

involved in chromosome condensation in mitosis (H3), DNA damage (y-H2AX) and transcriptional regulation (H3-S10 can promote H3-K14ac)

histone writer

adds PTM to histones, e.g. HATs, HMT

histone eraser

removes PTMs on histones, e.g. HDAC, HDM

histone reader

a protein that recognizes and binds to PTMs on histones and forms a complex with other proteins to regulate chromatin structure and gene transcription

also recruit writers/erasers to propagate/spread the PTM to neighboring histones for localized chromatin opening/closing

readers with bromodomains

bind to acetylated histones and recruit HATs, TFs and remodelers to promote/maintain an open chromatin state

e.g. SWI-SNF remodelers, HATs, TFs (TAF1/TFIID)

readers with chromodomains

bind to methylated histones and recruit heterochromatin-binding proteins, remodelers, HDACs to promote closed or opened chromatin states depending on the methyl group and on which amino acid residue

heterochromatin protein HP1 reader

binds to H3K9me3, has a bromodomain

promotes a closed state

CHD1 reader

a chromatin remodelling protein that binds to H3K4me3 at promoters and evicts nucleosomes

promotes an open state, has a bromodomain

epigenetics

genetics-independent phenotypic variation, no change in DNA sequence, but affects phenotype

histone PTMs, chromatin remodelling, DNA methylation are an example

can be transient or permanent over the lifespan of the organism

histone code

understanding the complex combination of PTMs on different histones in epigenetics

epigenetics diseases

aberrant gene expression in response to environment or altered physiology, genes could be linked to a disease but only shows due to environmental factors

anticancer therapeutics

targeting histone writers, readers, and erasers to activate/inactivate genes

assembly of nucleosomes after DNA replication

• one duplex and nucleosomes —> 2 duplexes and double the nucleosomes (where do they come from)

• old H3-H4 heterotetramers remain with parental nucleosomes and the CAF-1 histone chaperone assembles new H3-H4 heterotetramers on both daughter duplex DNA to restore normal nucleosome density

• NAP-1 histone chaperone adds old and new H2A-H2B heterodimers to the old and new H3-H4 heterotetramers

epigenetic inheritance of histones

PTM marks on the old histones are preserved and propagated/spread to unmodified histones in adjacent nucleosomes by histone-modifying protein complexes

active state of nucleosomes

HATs acetylate adjacent nucleosomes

repressed state of nucleosomes

HP1 recruits methyltransferases

trichostatin A

an antibiotic, an HDAC inhibitor

activates genes, inhibits removal of acetyl groups = activation → hyperactivation → apoptosis !!