Transcript Histone Modifications

Andrea Baccarelli, MD, PhD, MPH
Exposure, Epidemiology & Risk Program
Department of Environmental Health
[email protected]
EH298 – Lecture 2
Histone Modifications
Epigenetic markings
DNA methylation
Methyl marks added to certain DNA
bases repress gene transcription
Histone modifications
A combination of different
molecules can attach to the
‘tails’ of proteins called histones.
These alter the activity of the
DNA wrapped around them
Lecture 2 Outline
Definition
&
Mechanisms
Methods
&
Analysis
Case Study
Applying Epigenetics to Human Studies
Histone Modifications:
Definition and Mechanisms
Delving into the histone code
A severe problem of packaging!
• Human cell has 2m of DNA
• Nucleus is 0.006 mm in diameter
• Two opposing requirements:
– 1. Compaction
– 2. Access – Transcription
Replication
Repair
Chromatin
• Euchromatin –
– Partially decondensed
– Transcribed genes
• Heterochromatin –
– Hypercondensed in
interphase
– Transcriptionally inert
– Formation of chromosomal
structures
• Centromeres, telomeres
Levels of chromatin structure
Euchromatin: transcribed
and less condensed
“Loops” of 30-nm fibers seen
at interphase
Heterochromatin: more
condensed, genes
silenced, replicated later
in S phase.
Chromosome Structure
• Nucleosome
– fundamental unit of chromatin
– 147bp DNA wound 1.75 turns around histone core
(octamer)
• 2(H2A/H2B) + (H3/H4)2
– 11 nm fiber (“beads on a string”)
Electron Micrographs of “chromatin preparations”
Beads on a string
30-nm fibers
• Histones
Histone Modifications
– Globular core domain
– Unstructured N- and C-terminal tails
• Post-translational modifications:
– Acetylation – Lys
– Methylation (mono-, di- and tri-) – Lys and
Arg
– Phosphorylation – Ser and Thr
– Ubiquitination (mono- and poly-) – Lys
– Sumoylation (Lys); ADP-ribosylation;
glycosylation; biotinylation; carbonylation
Histone modifications
Biochimie. 2006 Aug 4
Epigenetic Marks
Berger, Nature 2007
Something is writing this code…
Region with deacetylated
histones
Epigenetic markings
Existing histones partition
evenly between the newly
formed daughter strands
The newly deposited histones
have an acetylation pattern
on their tails associated
with deposition
The newly deposited
nucleosomes adopt the
modification pattern
of the preexisting
nucleosomes
This allows the
stable inheritance of
particular chromatin
organizations
Enzymes catalyzing histone modifications
• Acetylation: HATs - CBP,p300, GCN5, ATF2, Tip 60…
• Deacethylation: HDACs- class I and II
• Methylation:
– Lysine : SET-domain HMTase and non-SET domain HMTase
(Dot1)
– Arginine: PRMT family, CARM1
• Demethylation: LSD1
• Ubiquitination: ubiquitin conjugase Rad6/ligase Bre1for
H2B
• De-Ubiquitination: SAGA-associated Ubp10
Histone acetylation
– Histone acetyl transferases (HATs)
–
–
–
–
Add acetyl groups to histone tails
Reduces interaction of histones with DNA
Facilitates transcription
Reversible – associated with inducible expression
– Histone de-acetylases (HDACs)
– Remove acetyl groups from histone tails
– Increases interaction of DNA and histones
– Represses transcription (usually)
• May involve same Lys residues as for methylation
Histone acetylation
– Histone methyl transferases (HMTs):
Histone methylation
• Histone lysine methyl transferases (HKMTs)
– Methylate specific Lys (K) residues
» Specific histones, residues, methyl status
– Associated with activation or repression of transcription,
depending on specific residue and number of methyls
• Protein arginine methyl transferases (PRMTs)
– Methylate Arg (R) residues
– Mainly linked to transcription activation
– Varying number of methyl groups:
• Lys – mono- di- or tri-methylated (on e-amino group)
• Arg – mono- or di-methylated (symmetric or
asymmetric) (on guanidino-e-amino groups)
– Histone de-methylases
• Remove methyl groups
Histone phosphorylation
– Kinases – adds phosphate to –OH of Ser/Thr
– E.g. aurora AIR2–Ipl1 kinase family
» Required for chromosome condensation
– E.g. ATM or DNA-PK
» H2AX variant phosphorylated in response to DNA
damage
– E.g. MSK1 and 2 or IKKa kinases
» Required for signal transduction leading to gene
activation
• Prevents nearby histone methylation due to (i)
steric hindrance or (ii) facilitation of competing
acetylation
• Alters recruitment of binding proteins
– Phosphatases – remove phosphate
• e.g. PP1 or PP2
Histone phosphorylation
Histone Ubiquination
– E3 Ubiquitin ligases
• Mono-ubiquitination or polyubiquitination and recruitment of
proteasome
– Alters chromatin structure
• Regulates H3 methylation
– E.g. Rad6-Bre1 – ubiquitinates H2B, opens nucleosome so accessible
to, or recruits, methyl transferases to H3-K4 and K79
• Regulates transcription
– E.g. PRC1/dRING – ubiquitinates H2A, directly or indirectly interferes
with transcription
– Ubiquitin hydrolases
– E.g. de-ubiquitination (by SAGA-associated Ubp8) regulates mono- vs
tri-methylation of H3-K4
Ubiquitin
Histone code hypothesis
• Developmentally active [B-globin] genes:
– H3-K4 di- and tri-methylated
– H3/H4 hyper-acetylated
– H3-S10 phosphorylation [Ifn-B]
• Condensed chromatin/inactive state:
– H3-K9 tri-methylated
– H3-K27 mono-methylated
– H4-K20 tri-methylated
• dependent on tri-methylation of H3-K9
Something is reading this code…
Reading the histone code
• Model 1 – structural role for modifications
– Based on charge density of histone tails
– Modulates protein-DNA interactions
• Model 2 – modifications as recognition sites
– Recruit effector molecules
– E.g. bromodomains – bind K-acetyl
– E.g. chromodomains, Tudor domains, WD40 repeats –
bind K-methyl
• Specific for particular residues
– E.g. HP1 binds di/tri-methylated H3-K9
– E.g. PC binds trimethylated H3-K27
Histone code hypothesis
Proteins that bind the modified histones
All together now!
Interaction between DNA methylation and Histone acetylation/
methylation
Rountree MR et al, Oncogene, 2001
Transcriptionally active DNA
unmethylated DNA with acetylated histones
Repressed (silent) DNA
methylated DNA with deacetylated histones
Methods for Histone
Modification Analysis
Deciphering the histone code
Histone Modification Analysis
• Step 1:
– Histone Purification & Isolation
• Step 2:
– Histone Analsyis (Several Methods)
• ELISA (Enzyme-Linked ImmunoSorbent Assay)
• ChIP (Chromatin ImmunoPrecipitation)
Histone Purification and Isolation
Hypertonic Solution
Any kind of analysis
Shechter et al. Nature Protocols 2, 1445 - 1457 (2007)
Types of measure
• ELISA
– Global genomic content of a certain modification:
• ChIP qPCR
– Gene specific measure of a certain modification
next to a specific gene
• ChIP-on-chip/ChIP sequencing
– Gene specific measure of a certain modification
next to many specific gene at the same time
Examples
• ELISA
• Histone: H3
• Modification: Di-Methyl-Histone
• Position: Lys4 (K4)
• ChIP qPCR
•
•
•
•
Histone: H3
Modification: Di-Methyl-Histone
Position: Lys4 (K4)
Gene: p15
• ChIP-on-chip/ ChIP sequencing
•
•
•
•
Histone: H3
Modification: Di-Methyl-Histone
Position: Lys4 (K4)
Genes: all the genes spotted on the chip/all DNA sequenced
ELISA
• Pros
– Can be performed in most labs with no need of
additional equipment
– Relatively low cost
• Cons
– Does not provide any information on specific
genes
– It needs quite large amounts of starting material
ChIP: ChIP-qPCR/ChIP-on-chip/ChIP Sequencing
ChipqPCR: All steps as above – quantitative PCR (qPCR) used instead of microarray
ChipSeq: All steps as above – Deep sequencing used instead of microarray
ChIP
• Pros
– Analysis of histone modifications linked with specific
genes
– Multiple methods can be used to measure enrichment
after CHiP
• Real-time PCR
• Microarrays
• Deep Sequencing
• Cons
– It needs large amounts of starting material for each
ChIP
– One ChIP for each modification you may want to study
Case Study
Effects of Exposure to Metal-rich
Air Particles on Histone H3-K4
Dimethylation and H3-K9
Acetylation among foundry
workers
Histone modifications as biosensors of cumulative exposure
Background
• Ambient and occupational exposure to
particulate matter (PM) has been associated with
lung cancer risk
• Carcinogenic metals in PM may be responsible
for the excess risk:
– mechanisms are still poorly understood
– do not produce DNA adducts and are weak mutagens
• Epigenetic mechanisms may account for the
observed epidemiological associations with lung
cancer risk
Histone Modifications
• A combination of different
molecules can attach to the
‘tails’ of histones:
– control DNA condensation & gene
expression
– modify the activity of the DNA
wrapped around them
– modifications can be divided in
‘activating’ or ‘inactivating’
• In vitro studies* show that
chromium, arsenic, and
nickel induce ‘activating’
modifications such as H3K4
dimethylation
*Zhou X. Toxicol Applied Pharmacol 2009;
Costa M. Carcinogenesis 2008
Metal-rich air particles in foundry workers
•Foundry work has been
associated with lung
cancer risk
•Chemical exposures are
low in modern foundry
facilities
•Particulate Matter (PM)
Levels 10+ fold higher
than ambient levels
Objective of Study
• To determine whether exposure to PM or PM metal
components induces activating histone
modifications:
– Histone H3K4 dimethylation
– Histone H3K9 acetylation
Using ELISA (enzyme-linked immunosorbent assay)
on white blood cell samples
Study populations
Exposure Assessment
Day Off Day Off Work
Day 1
Work
Day 2
Work
Day 3
Work
Day 4
Blood drawing
•
•
•
60 workers in different tasks in the same steel plant
Differences in work tasks determined a wide and stable exposure
gradient (over time) among subjects
(11 work areas)
Mean age 44 years (from 27 to 55 years), worked at least one year in
the present job; 40% were current smokers
Cantone et al, Environ Health Perspectiv 2011
Exposure Assessment
• Exposure concentrations in 11 work areas:
Arsenic
Cadmium
Nickel
Lead
Manganese
Chromium
Total Quant
method
PM10
PM1
Dynamic Reaction Cell
method with ammonia
Micro-gravimetric
method on filter
• Exposure = area concentrations x time in the area
total time at work
• Exposure during the study reflects
usual
2
exposure (high correlation [r >0.90] in a
subset of the study group)
Cantone et al, Environ Health Perspectiv 2011
Laboratory Methods
• Histones were extracted from buffy coat separated
from peripheral blood samples
• We used a solid phase sandwich enzyme-linked
immunosorbent assay (ELISA) to detect endogenous
levels:
– dimethylated H3K4 (PathScan® Acetyl-Histone H3 (Lys9)
Sandwich ELISA)
– acetylated H3K9 (PathScan® Di-Methyl-Histone H3 (Lys4)
Sandwich ELISA)
– We used a spectrophotometer (Synergy HT-BioTek) to read
450nm absorbance, which was assumed proportional to the
amount of modified histone in the sample
Cantone et al, Environ Health Perspect 2011
Exposure Levels
Exposure (μg/m3)
Mean
SD
Min
Max
Arsenic
0.18
0.21
0.005
0.500
Cadmium
0.005
0.007
0.001
0.03
Chromium
0.09
0.03
0.02
0.20
Lead
6.98
6.63
0.52
18.0
Manganese
38.9
98.9
0.3
684.0
Nickel
0.43
0.26
0.10
0.90
PM10
233.4
215.0
74.0
1220.2
PM1
8.48
6.18
1.7
30.5
Coarse (PM10-1)
224.9
209.0
71.5
1189.7
Cantone et al, Environ Health Perspect 2011
Years of employment and histone modifications
H3K4 Dimethylation
H3K9 Acetylation
3.00
H3K9 Acetylation (Luminescence Units)
H3K4 Dimethylation (Luminescence Units)
3.40
p-trend=0.04
3.30
3.20
3.10
3.00
2.90
p-trend=0.006
2.80
2.60
2.40
2.20
2.00
2-8 yrs
(n=19)
9-21 yrs
(n=17)
>21 yrs
(n=20)
Job duration
2-8 yrs
(n=19)
9-21 yrs
(n=17)
>21 yrs
(n=20)
Job duration
Cantone et al, Environ Health Perspect 2011
Exposures and histone modifications
H3K4 Dimethylation
Exposure
β*†
(95% CI)*
H3K9 Acetylation
p*
β
95% CI
p*
Chromium
0.45
(0.12; 0.77)
0.008
0.18
(-0.13; 0.05)
0.252
Lead
0.39
(0.07; 0.72)
0.019
-0.04
(-0.35; 0.28)
0.800
Arsenic
0.38
(0.03; 0.74)
0.034
0.17
(-0.16; 0.51)
0.308
Nickel
0.33
(0.00; 0.67)
0.049
0.10
(-0.21; 0.42)
0.516
Manganese
0.11
(-0.26; 0.47)
0.564
-0.10
(-0.43; 0.23)
0.533
PM10
0.14
(-0.16; 0.43)
0.351
-0.09
(-0.37; 0.19)
0.522
PM1
0.08
(-0.21; 0.37)
0.588
0.00
(-0.28; 0.28)
0.999
Coarse
0.14
(-0.15; 0.43)
0.346
-0.09
(-0.37; 0.19)
0.510
*Adjusted by age, BMI, smoking, cigarettes/day, % granulocytes, and education
† Standardized regression coefficients
Cantone et al, Environ Health Perspect 2011
Joint effect of employment length
and metal exposure on H3K4 dimethylation
Arsenic
H3K4 dimethylation
H3K4 dimethylation
Lead
years of employment
years of employment
Nickel
H3K4 dimethylation
H3K4 dimethylation
Chromium
years of employment
years of employment
Joint effect of employment length
and metal exposure on H3K4 dimethylation
Chromium
H3K9 acetylation
H3K9 acetylation
Manganese
years of employment
years of employment
Conclusions
• Exposure-related histone modifications:
– H3K4 methylation and H3K9 acetylation both increased with job
duration
– Arsenic, Nickel, Lead and Chromium exposure associated with
H3K4 methylation
– Evidence for joint effects of length of employment & metals
• Data interpretation
– Confirm in-vitro studies on chromium, arsenic and nickel toxicity
– Lack of short-term changes, job duration associations suggest
effects from chronic exposure
• Limitations:
– Long-term exposure is unmeasured
– White blood cells are a surrogate histone source
– Health-related outcomes to be explored
?
But wait…
There’s More!
Online resources for epigenetic research
Consortia & Initiatives
The Common Fund's Epigenomics Program
An NIH Initiative to generate new research tools, technologies, datasets, and
infrastructure to accelerate our understanding of how genome-wide chemical
modifications to DNA regulate gene activity without altering the DNA sequence itself
and what role these modifications play in health and disease.
https://commonfund.nih.gov/epigenomics/index#
The Human Epigenome Projects
A public/private collaboration to catalogue Methylation Variable Positions (MVPs) in
the human genome
http://www.epigenome.org/
NAME21
A German National Initiative to analyze DNA methylation Patterns of Genes on
Chromosome 21
http://biochem.jacobs-university.de/name21/
http://www.nature.com/nrg/focus/epigenetics/index.html
http://www.sciencemag.org/feature/plus/sfg/resources/res_epigenetics.dtl
http://es.landesbioscience.com/pub/
http://sbs.umkc.edu/waterborg/chromat/chromatn.html
https://www.encodeproject.org/
An atlas of human reference epigenomes and the results of their integrative and
comparative analyses
http://www.genboree.org/epigenomeatlas/index.rhtml
A searchable database for DNA methylation and environmental epigenetic effects
http://www.methdb.de/
An online repository of epigenetic data sets
http://www.ncbi.nlm.nih.gov/epigenomics/browse
A searchable database of information from experimental data to facilitate understanding of
histone modifications at a systematic level, with the current release incorporating 43 locationspecific histone modifications in humans
http://bioinfo.hrbmu.edu.cn/hhmd
http://www.mirbase.org/
http://www.comgen.pl/mirex2
Epigenetics
Editor-in-Chief
Manel Esteller
Cancer Epigenetics and
Biology Program
Barcelona, Spain
Impact Factor 4.78
http://www.landesbioscience.com/journals/epigenetics/
More focused on basic science
Impact Factor 5.33
First issue in October 2009
Impact Factor 4.64
http://www.futuremedicine.com/loi/epi
Environmental Epigenetics
Opened submissions in August 2015
No impact factor yet
http://eep.oxfordjournals.org/front.most-cited
http://www.geneimprint.com/
http://igc.otago.ac.nz/home.html
Next lecture
Lecture 3
Epigenetics: Environmental Instructions
for the Genome