1. EH298 – Lecture 2 Histone Modifications
Andrea Baccarelli, MD, PhD, MPH
Exposure, Epidemiology & Risk Program
Department of Environmental Health
EH298 – Lecture 2 Histone Modifications

2. Epigenetic markings DNA methylation Histone modifications
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 2

3. Definition & Mechanisms
Lecture 2 Outline 1
Definition & Mechanisms 2
Methods & Analysis 3
Case Study
Applying Epigenetics to Human Studies

4. Histone Modifications: Definition and Mechanisms1
Histone Modifications: Definition and Mechanisms
Delving into the histone code

5. Published in PNAS
EH298 Environmental Epigenetics Day 1

6. A severe problem of packaging!
Human cell has 2m of DNA
Nucleus is mm in diameter
Two opposing requirements:
1. Compaction
2. Access – Transcription
Replication
Repair
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7. Chromatin Euchromatin – Heterochromatin – Partially decondensed
Transcribed genes
Heterochromatin –
Hypercondensed in interphase
Transcriptionally inert
Formation of chromosomal structures
Centromeres, telomeres 7

8. 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. 8

9. Nucleosome Chromosome Structure 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”) 9

10. Electron Micrographs of “chromatin preparations”
Beads on a string
30-nm fibers 10

11. Post-translational modifications:
Histones
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

12. Histone modifications
Biochimie Aug 4
Histone modifications

13. Epigenetic Marks
Berger, Nature 2007

14. Something is writing this code…

15. Epigenetic markings Region with deacetylated histones
Existing histones partition
evenly between the newly
formed daughter strands
The newly deposited histones
have an acetylation pattern
on their tails associated
with deposition 15

16. The newly deposited nucleosomes adopt the modification pattern
of the preexisting
nucleosomes
This allows the
stable inheritance of
particular chromatin
organizations
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17. 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

18. 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

19. Histone acetylation

20. Histone methylation Histone methyl transferases (HMTs):
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 methylation

22. 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

23. Histone phosphorylation

24. Histone Ubiquination E3 Ubiquitin ligases Ubiquitin hydrolases
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

25. Ubiquitin

26. 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

27. Something is reading this code…

28. 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

29. Histone code hypothesis
Proteins that bind the modified histones

30. All together now!
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31. 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
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32. Methods for Histone Modification Analysis2
Methods for Histone Modification Analysis
Deciphering the histone code

33. Histone Modification Analysis
Step 1:
Histone Purification & Isolation
Step 2:
Histone Analsyis (Several Methods)
ELISA (Enzyme-Linked ImmunoSorbent Assay)
ChIP (Chromatin ImmunoPrecipitation)
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34. Histone Purification and Isolation
Hypertonic Solution
Any kind of analysis
Shechter et al. Nature Protocols 2, (2007)
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35. ChIP-on-chip/ChIP sequencing
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
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36. Examples ELISA ChIP qPCR ChIP-on-chip/ ChIP sequencing Histone: H3
Modification: Di-Methyl-Histone
Position: Lys4 (K4)
ChIP qPCR
Gene: p15
ChIP-on-chip/ ChIP sequencing
Genes: all the genes spotted on the chip/all DNA sequenced
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37. 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
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38. 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
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39. 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
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40. 3
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

41. Carcinogenic metals in PM may be responsible for the excess risk:
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

42. 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

43. 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

44. 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

45. Study populations Exposure Assessment Blood drawing
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
We designed the study recruiting 63 workers, involved in different tasks within the same foundry facility.
We collected blood samples at the beginning of the work day 4, after three days of work.
Cantone et al, Environ Health Perspectiv 2011

46. Exposure concentrations in 11 work areas:
Exposure Assessment
Exposure concentrations in 11 work areas:
Exposure = area concentrations x time in the area
total time at work
Exposure during the study reflects usual exposure (high correlation [r2>0.90] in a subset of the study group)
Dynamic Reaction Cell
method with ammonia
Arsenic
Cadmium
Nickel
Lead
Manganese
Chromium
PM10
PM1
Total Quant method
Micro-gravimetric method on filter
In 11 work areas, identified as representative for the work positions of the study subjects, We measured air concentrations of arsenic, cadmium, nickel, lead, manganese, chromium, as well as of particulate matter with diameter lower that 10 and 1 microns;
Individual exposure was calculated as the average concentration of the work areas weighted by the time that each worker spent in the areas.
PM10 levels in each of the work areas have shown very little variability over time, as repeated measures over 1 year showed very high correlation between PM10 concentrations (r2 > 0.90)
(We only had access to environmental samplers ; Our environmental samplers had only filters for PM1, 5 PM and PM10)
Cantone et al, Environ Health Perspectiv 2011

47. 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

48. Exposure Levels Exposure (μg/m3) Mean SD Min Max Arsenic 0.18 0.21
0.005
0.500
Cadmium
0.007
0.001
0.03
Chromium
0.09
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
In this table I reported concentrations of PM and metals analyzed, with range from minimum to max and the mean, expressed in ug per cubic meter. Coarse particle concentration was defined as the difference between PM10 and PM1 ;
Metal components represented 15% of all pollutants (higher than what is usually found in ambient air [for example, in Milan is 5%]
Daily exposure to PM10, divided according to the exposure ,
PM10 exposure* , nr (%)
µg/m3 23 (36.5%)
µg/m3 24 (38.1%)
µg/m3 16 (25.4%)
Cantone et al, Environ Health Perspect 2011

49. Years of employment and histone modifications
H3K4 Dimethylation
H3K9 Acetylation
p-trend=0.04
p-trend=0.006
(n=19)
(n=17)
(n=20)
(n=19)
(n=17)
(n=20)
Cantone et al, Environ Health Perspect 2011

50. Exposures and histone modifications
H3K4 Dimethylation
H3K9 Acetylation
Exposure
β*†
(95% CI)* p* β
95% CI
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
(-0.15; 0.43)
0.346
0.510
Noemi, potresti formattare questa tabella?
*Adjusted by age, BMI, smoking, cigarettes/day, % granulocytes, and education
† Standardized regression coefficients
Cantone et al, Environ Health Perspect 2011

51. Joint effect of employment length and metal exposure on H3K4 dimethylation
Lead
years of employment
exposure
Arsenic
H3K4 dimethylation
H3K4 dimethylation
exposure
years of employment
Chromium
years of employment
exposure
Nickel
Job duration (yrs)
Lead
H3K4 dimethylation
H3K4 dimethylation
exposure
years of employment

52. Joint effect of employment length and metal exposure on H3K4 dimethylation
Manganese
years of employment
exposure
H3K9 acetylation
Chromium
H3K9 acetylation
exposure
years of employment

53. Exposure-related histone modifications:
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

54. ?
But wait… There’s More!
Online resources for epigenetic research

55. 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. The Human Epigenome Projects A public/private collaboration to catalogue Methylation Variable Positions (MVPs) in the human genome NAME21 A German National Initiative to analyze DNA methylation Patterns of Genes on Chromosome 21

56.
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57. http://www. sciencemag. org/feature/plus/sfg/resources/res_epigenetics
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58.
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59.
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60. Databases
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61.
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62. An atlas of human reference epigenomes and the results of their integrative and comparative analyses
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63. A searchable database for DNA methylation and environmental epigenetic effects
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64. An online repository of epigenetic data sets
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65. A searchable database of information from experimental data to facilitate understanding of histone modifications at a systematic level, with the current release incorporating 43 location-specific histone modifications in humans
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66.
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67.
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68. Scientific Journals
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69. Editor-in-Chief Manel Esteller Cancer Epigenetics and Biology Program
Barcelona, Spain
Impact Factor 4.78
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70. More focused on basic science
Impact Factor 5.33
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71. First issue in October 2009 Impact Factor 4.64
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72. Environmental Epigenetics
Opened submissions in August 2015
No impact factor yet
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73. Imprinting
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74.
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75.
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76. Lecture 3 Epigenetics: Environmental Instructions for the Genome
Next lecture
Lecture 3 Epigenetics: Environmental Instructions for the Genome