Download Ostlund Farrants

Dept.
Wenner-Gren
of Molecular
Institute
Biosciences,
for
TheExperimental
Wenner-Gren
Biology
Institute
Chromatin, the packaging of
chromosomes, and the
epigenome is a regulator of
nuclear processes
Ann-Kristin Östlund Farrants
Stockholm University
[email protected]
Chromosomes
The chromosomes
take up most of
the space in the
eukaryotic
nucleus.
DNA is bound by
proteins =
chromatin
www.cellnucleus.org
Chromosomes
The DNA is packaged
with ptoteins to
condense it about
1000 fold
The chromosomes are
even more highly
packed at mitosis – 10
times more condensed
hromosomes.
The packaging of
chromosomes
DNA is wrapped around
histone cores to form
nucleosomes. 1.7 turns,
146 bp
The nucleosomes, in turn,
are further package by
association by H1 to form
the 30 mm fibre. Linker
reagion 30-60 bp
The 30 nm fibre is further
package with non-histone
protein.
Alberts et al.
Crystal structure
2.8 Å resolution
Luger et al.
The structure of the nucleosome
Histone octamer – tetramer of H3-H4
2 dimers of H2A-H2B
EM image of salt extracted chromosomes
Beads-on-a-string
Chromosomes have different regions with
different functions.
Heterochromatin – densly packed
Telomeres, centromeres, silent regions
Euchromatin – less densly packed
Regions with actively transcribed genes
What is specific for the different chromosome regions?
Different histone modifications
Different proteins associated with the different regions
RNA
At least 7 different chromatin structures
Histone variants
CEN-A – centromer, kinetochore H3
synthesised late in S-phase
H3.3 – H3-form synthesised outside of Sactive transcribed regions
phase, in
H2Az/v – H2A formed during G1 and G2, in transcription.
H2Ax – 2-25% of all H2A, phosphorylated
damage
upon DNA
Other proteins
Histone H1 – 30 nm fibre
Non-histone proteins –
HMG box proteins, structural
proteins.
Specific proteins for chromosome
region –
heterochromatin proteins, polycomb
proteins (silenced),
transcription factor (active)
The different states are dynamic and are
different in different cell type.
- different in different cell type
-
changes depending on cell state
Changes upon differentiation or on envronmental cues
Differentiation –
changes in gene
expression
Waddington’s epigenetic
landscape
Differentiation is following paths
Differentiation is tipping the balance
Saddled-node landscape:
Commitment is the
disappearing of valleys –
Ferell Jr
Processes in the nucleus
Replication
Transcription
RNA processing
(capping, splicing, polyadenylation of mRNA, rRNA
modification)
Recombination
DNA-repair
Export of RNPs, import of protein
Histone modifications
• 25% N-terminal part of the histones are not in
the core region.
• N-terminal tail – lysines, serines, argenines
• N-terminal tails are modified by;
acetylation, methylation, phosphorylation,
ubiquitinylation, ribosylaton
Histone code
• Modifications located in
heterochromatinmethylation of H3lysine9, H3K27,
H4K20me3, H3R2
Allis and Jenuwein
• Modifications in actively
transcribed regions –
Methylation of H3K4,
Acetylation of H3K9, H3K27
Acetylation of H4
DNA-methylation also affects chromatin structure
Methylation at the promoters inactivates transcription
slideshare
The dynamic chromatin structure
Inactive
Me
me
Enzymes:
DNA methylases/demethylases
Acetyl transfeases/deacetylatses
TF
Use energy to change the structure of the
nucleosome
A
c
Ac
mRNA
Active
Epignetic alterations
Enzyme puts on a modification, other protein
binds, and yet other removes the modifications-
modifier
Binding
protein
Eraser
Epignetic alterations
Enzyme puts on a modification, other protein
binds, and yet other removes the modifications-
HAT
Activator
HDAC
In addition -Move nucleosomes – nucleosome
remodellers
Histone modifications are differently
distributed along active genes (J. Mellor)
H3K4trimet
K3K4dimeth
H3K36
-metn
H3K-Ac
H4K-Ac
MODIFIERS
• Acetylation-Histon acetyltrasferases (HAT)
• Deacetylation-histone
deacetylases (HDAC)
• Methylation- Methyl transferases
• Demethylation- demethylases
• Ubiquitinylation – Ligases
• Deubiquitinylation deubiquitinylases
• Phosphorylation –kinases
• Dephosphorylation-phsosphtases
The histone marks are
decoded by proteins:
Bromodomains – Ac
Chromodomains – H3K9me3
PhD-domain – H3K4me3
Location of euchromatin and heterochromatin
in the nucleus - EM
S-phase
early
Replication is affected
by the chromatin
packaging, and the
location of the different
forms of chromatin
middle
late
very
late
Transcription is also affected by location
Between the chromosomes
Chromosomes
are organised in
territories
Chromosome loops –
functional domains:
Silent regions, active regions
Insulators
CTCF is involved
in mammalians cells
Insulators – separates functional domains
Prevent chromaitn domains from spreading
Locus control region
domains
Monoallelic expressed
locus -Imprinted genes
Silencing
Anchoring to
structures
Regulate Xchromosome activity
Matharu 2015
TADs provide a 3-D structure to the nucleus
Change in TAD usage – redistribution of TAD factors
Setting the
chromosome landscape
such that specific
genes are transcribed
The TADs at enhancers, insulators, promoters also
change histone modification
Active Enhancer – H3K4me1 and H3K27ac
Active Promoter – H3K4me3 and H3K27ac
Actively transcribed gene body – H3K36me3
Inactive enhancers and promoters – H3K27me3 and H3K9me3
Some also have dual marks – very accessible
Chromatin Immunoprecipitation
The active sites are more open, accessible –
They are regarded as nucleosome free
Enzyme such as DNase I or MNase can cut
The active sites exhibit a larger mutation rate than gene
bodies. Changes transcription factor binding.
RNA is involved in the setting of functional
domains
Many sites are transcribed – not just genes
Required for heterochromatin fomation
non-coding RNA is
transcribed and recruits
factors to specific sites –
Centromers in S. pombe
X-chromosome inactivation
LncRNA can act as
glue between sites in
cis or in trans
The expression of
nc-genes
determines the
spatial organisation
Enhancers and
promoters are
transcribed
The ncRNAs
binds proteins
that targets the
site
The ncRNA
hybridises to
other RNA
Transcription can also cause R-loops - RNA-DNA
hybrids
Result in replication forks and DNA breaks
Cause recombination in repetitive regions
Transcription of
ribosomal genes and
chromatin remodelling
Repetitive genes with
special chromatin
Hot spot for
recombination
Transcription
Actively
transcribed
chromosomes are
located in the
inside of the
nucleus, whereas
inactive
chromosomes
parts are located
at the periphery.
Does this always
hold true?
Inactive
active
Model:
LCR associates with
proteins – Ldb1
Leads to migration
Recruitment of the
transcription machinery
Moving
genes
depending
on their
state of
activity
Inter- and intrachromosomal chromatin loops
Schneider R, Grosschedl R Genes Dev. 2007;21:3027-3043
©2007 by Cold Spring Harbor Laboratory Press
Chromosomes translocate according to
functional requirement
RNA dependent mechanism?
Could explain the
movement of
chromosomes
during
differentiation
Different ncRNAs
are expresssed
ncRNA are seeding
points for proteins
Accumulate proteins in
subnuclear bodies.
1.ChIP-seq (Chromatin immunoprecipitation sequencing), aimed against different histone
modifications, can be used to identify chromatin states throughout the genome. Different
modifications have been linked to various states of chromatin.
2.DNase-seq (DNase I hypersensitive sites Sequencing) uses the sensitivity of accessible
regions in the genome to the DNase I enzyme to map open or accessible regions in the
genome.
3.FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing) uses
the chemical properties of protein-bound DNA in a two-phase separation method to extract
nucleosome depleted regions from the genome.[16]
4.ATAC-seq (Assay for Transposable Accessible Chromatin sequencing) uses the Tn5
transposase to integrate (synthetic) transposons into accessible regions of the genome
consequentially highlighting the localisation of nucleosomes and transcription factors
across the genome.
5.DNA footprinting is a method aimed at identifying protein-bound DNA. It uses labeling
and fragmentation coupled to gel electrophoresis to identify areas of the genome that have
been bound by proteins.[17]
6.MNase-seq (Micrococcal Nuclease sequencing) uses the micrococcal nuclease enzyme
to identify nucleosome positioning throughout the genome.[18][19]
7.Chromosome conformation capture determines the spatial organization of chromatin
in the nucleus, by inferring genomic locations that physically interact.
8.MACC profiling (Micrococcal nuclease ACCessibility profiling) uses titration series of
chromatin digests with micrococcal nuclease to identify chromatin accessibility as well as
to map nucleosomes and non-histone DNA-binding proteins in both open and closed
regions of the genome.[20]
Where does transcription take place
in the nucleus?
• Specific locations – Transcription
factories
In specific spots
In the nucleus
2000 in cell culture
200 in tissue
Moving
genes
depending
on their
state of
activity
Inter- and intrachromosomal chromatin loops
Schneider R, Grosschedl R Genes Dev. 2007;21:3027-3043
©2007 by Cold Spring Harbor Laboratory Press