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EH298 – Lecture 2 Histone Modifications
Andrea Baccarelli, MD, PhD, MPH Exposure, Epidemiology & Risk Program Department of Environmental Health EH298 – Lecture 2 Histone Modifications
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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
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Definition & Mechanisms
Lecture 2 Outline 1 Definition & Mechanisms 2 Methods & Analysis 3 Case Study Applying Epigenetics to Human Studies
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Histone Modifications: Definition and Mechanisms
1 Histone Modifications: Definition and Mechanisms Delving into the histone code
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Published in PNAS EH298 Environmental Epigenetics Day 1
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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 EH298 Environmental Epigenetics Day 1
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Chromatin Euchromatin – Heterochromatin – Partially decondensed
Transcribed genes Heterochromatin – Hypercondensed in interphase Transcriptionally inert Formation of chromosomal structures Centromeres, telomeres 7
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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
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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
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Electron Micrographs of “chromatin preparations”
Beads on a string 30-nm fibers 10
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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
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Histone modifications
Biochimie Aug 4 Histone modifications
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Epigenetic Marks Berger, Nature 2007
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Something is writing this code…
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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
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The newly deposited nucleosomes adopt the modification pattern
of the preexisting nucleosomes This allows the stable inheritance of particular chromatin organizations EH298 Environmental Epigenetics Day 1
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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
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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
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Histone acetylation
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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
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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
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Histone phosphorylation
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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
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Ubiquitin
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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
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Something is reading this code…
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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
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Histone code hypothesis
Proteins that bind the modified histones
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All together now! EH298 Environmental Epigenetics Day 1
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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 EH298 Environmental Epigenetics Day 1
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Methods for Histone Modification Analysis
2 Methods for Histone Modification Analysis Deciphering the histone code
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Histone Modification Analysis
Step 1: Histone Purification & Isolation Step 2: Histone Analsyis (Several Methods) ELISA (Enzyme-Linked ImmunoSorbent Assay) ChIP (Chromatin ImmunoPrecipitation) EH298 Environmental Epigenetics Day2
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Histone Purification and Isolation
Hypertonic Solution Any kind of analysis Shechter et al. Nature Protocols 2, (2007) EH298 Environmental Epigenetics Day2
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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 EH298 Environmental Epigenetics Day2
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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 EH298 Environmental Epigenetics Day2
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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 EH298 Environmental Epigenetics Day2
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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 EH298 Environmental Epigenetics Day2
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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 EH298 Environmental Epigenetics Day2
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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? But wait… There’s More! Online resources for epigenetic research
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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
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http://www. sciencemag. org/feature/plus/sfg/resources/res_epigenetics
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Databases EH298 Environmental Epigenetics Day 1
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An atlas of human reference epigenomes and the results of their integrative and comparative analyses
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A searchable database for DNA methylation and environmental epigenetic effects
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An online repository of epigenetic data sets
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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 EH298 Environmental Epigenetics Day2
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Scientific Journals EH298 Environmental Epigenetics Day 1
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Editor-in-Chief Manel Esteller Cancer Epigenetics and Biology Program
Barcelona, Spain Impact Factor 4.78 EH298 Environmental Epigenetics Day 1
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More focused on basic science
Impact Factor 5.33 EH298 Environmental Epigenetics Day 1
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First issue in October 2009 Impact Factor 4.64
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Environmental Epigenetics
Opened submissions in August 2015 No impact factor yet EH298 Environmental Epigenetics Day 1
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Imprinting EH298 Environmental Epigenetics Day 1
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Lecture 3 Epigenetics: Environmental Instructions for the Genome
Next lecture Lecture 3 Epigenetics: Environmental Instructions for the Genome
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