Epigenomics and Microbiome

Michael Snyder

October 19, 2015

Slides from: Joe Ecker, Anne Brunet, Joanna Wysocka, Jason Reuter Disclosures: Personalis, GenapSys, AxioMx

A Symphonic Example

DNA Phenotype

Image: mooogmonster

Epigenetic changes

It is likely that factors outside of DNA sequence may also contribute to human diseases and possibly evolution

Definition: Epigenomics The study of the complete set of epigenetic modifications on the genome that can modify DNA instructions, turning on and off genes, or control the production of proteins. These modifications occur when chemical compounds or proteins bind to DNA and “mark” the genome. They do not alter the sequence itself. Methods for marking the genome: • DNA methylation • Histone modification

The Epigenome – DNA Methylome

Often occurs at CpG >28 million CpG sites in the human genome. Human genome consists of vast oceans of DNA sequence containing few but heavily methylated CpG dinucleotides. Punctuated by short regions with unmethylated CpGs occurring at higher density, forming “CpG islands” in the genome, often at gene promoters. HydroxyC methylation (not discussed)

The Human Epigenome

Maleszewska & Kaminska, Cancers, 2013

Methylation in promoters: Gene expression turned off

Inactive A

ctive Chrom

atin

Ac: Acetylation

The Human Epigenome

Maleszewska & Kaminska, Cancers, 2013

Methylation in promoters: Gene expression turned off

Inactive A

ctive Chrom

atin Ac: Acetylation

Dnmt: DNA methyltransferase TET: “Demethylation” HMT: Histone MethylTransferase KDM: Histone Demethylase HAT: Histone AcetylTransferase HDAC: Histone DeACetylase

Modifying enzymes

DNA Methylation Associated with Many Diseases and Traits

Asthma and Allergies (Nadeau)

Aging Nutrition

Cancer Exercise

Horvath Genome Biology 2013 14:R115 doi:10.1186/gb-2013-14-10-r115

DNA Methylation Correlates With Age

Measure Methylation at 353 CpG Sites “Clock CpGs” Correlates Well with Age Across Many Tissues

Nutrition and DNA Methylation Honey Bees Extensive differential methylation workers and queen “Royal Jelly”silences Dnmt3 DNA Methyl transferase

Nutrition and DNA Methylation

Agouti Mouse • Paracrine signalling molecule

that affect coat color • Yellow, obese, diabetic

unmethylated • Brown healthy methylated • Transposon

Nutrition and DNA Methylation

BPA exposure

Agouti Mouse • Affected by Nutrition and Environment • Methyl rich diet by Mom Suppresses yellow • BPA – (bisphenol A) polycarbonate plastic precursor

Human DNA methylation at putative MEs is influenced by season of conception in the Gambia

Waterland et al., PLoS Genet 2010

Gambia – rainy season: hungry, dry season: lots of food

DNA from peripheral blood leukocytes

Exercise and DNA Methylation

Lindholm et al. Epigenetics. 2014 Dec 2;9(12):1557-69. doi: 10.4161/15592294.2014.982445.

• Many studies have linked exercise and DNA methylation differences are specific genes

Recent study Lindholm et al 23 people. Bike use one leg 4X/Week 45’ for 3 months; other leg is control Measure gene expression and DNA methylation (arrays) beginning and end of training on each leg (muscle biopsy) 4919 sites differed in trained leg (5% FDR)

Father

Mother

T C Inactivated by

mutation

Inactivated by DNA

Methylation M M M

Methylated CpGs

Few RNAs

Lots of RNA

PDE4 DIP Gene

Gene Inactivation by Mutation and Methylation: PDE4 involved in eosinophilia

electron micrograph of chromatin

nucleosome

DNA

histones K. Luger

Epigenetics and Chromatin

Chromatin undergoes reversible chemical modifications, which have regulatory properties

Chromatin modification plays a regulatory role

Covalent modifications of histone proteins

acetylation lysine methylation

arginine methylation

phosphor- ylation

Me

K Me Me K Ac

R Me Me

S/T P Ub

K Sumo

K

ubiquitin- ylation

sumo- ylation

Non-covalent mechanisms: ATP-dependent chromatin remodeling

Mind-boggling array of histone modifications

Writers, readers and erasers of histone modifications

writer eraser reader

acetylation

methylation

acetyltransferases deacetylases

methyltransferases demethylases

kinases phosphatases phosphoryl.

bromodomains

chromodomains, PHD fingers, MBT

14-3-3, BRCT

MODIFICATION:

From J. Wysoka

Identification of proteins recognizing histone lysine acetylation and methylation

SPECIFICITY DOMAIN PROTEIN YEAR NOTES REFERENCES

Acetyl lysine Bromodomain Gcn5 1999 Not lysine specific

Dhalluin et al., 1999

Methyl lysine Chromodomain HP1 2001 H3K9me specific

Bannister et al., 2001; Jacobs, Khorasanizadeh, 2002; Lachner et al., 2001

PHD finger BPTF, MLL etc etc

2006 Specific for histone, lysine & me’n state

Wysocka et al., 2006; Li et al., 2006; Pena et al., 2006;

Tudor, MBT etc 2007 Reviewed by (Taverna et al., 2007)

Multiple histone-binding modules often co-exist within the same polypeptide

Ruthenburg et al., Nat. Rev. MCB, 2007

24

Histone Marks Representing a Variety of Functional Elements

Active Genes

Repressed Genes

Chromatin variation across individuals

SAN

CEU

YRI

Asian

Enhancer related marks are more variable than RNA

Kasowski et al 2013 Science

Epigenetics and Cancer

Cancer is a genetic disease Cancer is also an epigenetic disease

The methylome and major changes that occur in cancer

The cancer methylome is characterized by widespread Hypo-methylation & focal CpG-island Hyper-methylation, often in promoters of tumor suppressor genes. Stirzaker et al., Trends in Genetics, 2014

Epigenetic modifiers are frequent targets for mutation in cancer

Recent sequencing studies show that mutations in the three classes of epigenetic modifiers are frequently observed in various types of cancers, highlighting the crosstalk between genetics and epigenetics: - DNMT3A mutations in AML

(next slide) - IDH1/2 mutations in

glioblastoma - EZH2 mutations in breast,

colon, pancreas, melanoma,…

You & Jones, Cancer Cell, 2012

DNMT3A mutations in leukemia Mutations in DNMT3A, an enzyme concerned with de novo methylation of CpG dinucleotides, are among the most common somatic mutations, occurring in 25% of acute myeloid leukemia (AML). DNMT3A mutations have also been found in myelodysplastic syndromes (MDS). Indicates that aberrant epigenetic modulation of the genome has a pathological role in leukemogenesis.

Location of mutations in DNMT3A in AML

Methylation-based classification of colorectal cancer correlates with cancer

gene mutation status

Hinoue, T et al. (2012). Genome research, 22(2), 271–82

BRCA DNA Methylation and Mutation in Ovarian Cancer

Genes can be inactivated by either Mutation or DNA Methylation

Cancer Genome-Epigenome Interaction: Gene body methylation contributes to cancer-causing mutations

• Methylation of cytosine strongly increases the rate of C→T transition mutations.

• Due to “spontaneous deamination” of cytosine, which forms thymine.

• In somatic cells, gene body methylation is a major

cause of cancer gene mutations in tumor suppressor genes, such as TP53.

The epigenome as a therapeutic target

Abnormal hyper-methylation often silences the expression of tumor suppressor genes. Drugs, such as Dacogen and Vidaza (5-azacytidine), may reverse tumor-associated gene silencing through their ability to disrupt DNA methylation.

Hamm and Costa, Pharmacology & Therapeutics, 2015

The histone deacetylase inhibitors Zolinza and Istodax may reverse tumor-associated gene silencing through their ability to disrupt aberrant posttranslational histone alterations. (Subcutanteous T-cell lymphoma; CTCL)

The reversible nature of epigenetic modifications provides an exciting opportunity for the development of therapeutics.

Methylation of certain genes can influence sensitivity to chemotherapeutic drugs

Toyota et al.,

Cancer Science, 2009

Best known example for promoter methylation influencing treatment: MGMT

• The O(6)-methylguanine-DNA methyltransferase (MGMT) encodes a DNA repair enzyme that can abrogate the effects of alkylating chemotherapy such as temozolamide.

• Alkylating chemotherapy damages DNA and kills tumor cells.

• However, if the MGMT gene is active (unmethylated promoter), the damage is rapidly repaired.

• Malignant gliomas may have the MGMT gene inactivated due to methylation of its promoter region.

• Thus, in gliomas, MGMT promoter methylation is a favorable prognostic marker in the setting of either radiation or chemotherapy.

DNA methylation of specific marker genes can be used as a biomarker for cancer detection and diagnosis

Summers et al., Journal of Cancer, 2013 http://www.epiprocolon.com

History of cancer epigenetics

Hattori et al., Biochem. Biophys. Res. Commun., 2014

Conclusions

1. Epigenomic regulation is widespread 2. Occurs by multiple mechanisms 3. Is involved in many important biological processes Cancer, Aging, Nutrition

Microbiome

What is the microbiome? The collection of microbiomes that exist in a given area, tissue or organism. It refers to all microbes including bacteria, fungi and viruses.

The Human Microbiome The human microbiome is the collection of microbes that live on and in us

30 trillion human cells

300 trillion microbial cells

>10 million genes (compared to 20K for humans)

Enormous biochemical diversity What do the microbes do??

The Role of the Microbiome It breaks down our food into constituents we absorb.

It synthesizes essential vitamins. E.g. Vitamin B12, Biotin, Folic Acid

Body ecology Different Kingdoms Bacteria Eukaryota (e.g fungi) Archaea Viruses Different Phyla/Classes Typically 5-10 major types 100s – 1000s of species Macro-ecology: Plants, Animals, Fungi, Protists

Different niches Aerobic, anaerobic Dry, moist Oily, nutrient-rich/poor Exposed, protected from environment

Where did our microbiome come from?

Microbes have been on earth for 3 billion years We evolved in the midst of microbes We evolved organs to house our microbes

Each body site has its own microbial community Adaptive immunity allowed richer microbiome?

Image: Darryl Leja, NHGRI

When does a person’s microbiome form?

At birth a baby’s gut microbiome is similar to the mother’s vaginal microbiome It forms at lot during the first year as baby ingest food Microbiome from breast fed microbiome has similarities to its Mother’s breast/milk

Non breast fed babies have a different microbiome.

Dominguez-Bello et al. (2010) PNAS 107:119

Sampling of the microbiota 5-60 minutes after birth

• Acinotobactor " Baclllales • Conobactotineae • Corynebacterineae • Haomopnnus • Lactobacd us

• Mlc:roooconeae • NGI$$8t14

Pasteur..aceae • Prevotella • Propion1bacteMeae Sneathia

Staphylococcus Slroptococcus Other

Dominguez-Belloeta!. (2010) PNAS 107:11971x

Vaginal (Baby l ike Vaginal) Caesarian (baby looks l ike skin)

Approaches to metagenomic study of the microbiome

Microbial community is very diverse Bacteria, viruses, eukaryotes 100s-1000s of taxa A few are >80% of total MANY minor taxa 99% of microbes cannot be cultured! Two approaches Targeted sequencing Shotgun sequencing

Targeted sequencing

16S rRNA sequences/sample Average 5000 sequences

Must have >1000 sequences Sample 100s of taxa

Describe communities and genus abundance in many samples –

“Average” community and variations

Cost < $50/sample 16S rRNA = bar code identifier of species

Liu et al. Nucleic Acid. Res. 2007 35. e120

Primers to variable parts of 16S rRNA

Shotgun sequencing Sample every gene in the community by high throughput DNA sequencing

Analyze for: Bacterial community structure, Viruses, Fungi Metabolic pathways, other genes of interest (antibiotic resistance; virulence)

Slide courtesy of Ami Bhatt

Studying communities – shotgun sequencing

Assemble

Illumina shotgun of sample

community DNA

10 Gb/sample 100 million reads 100s of samples

Align

Catalog of 2000 reference genomes

Blast vs DBs (GenBank eg)

Analyze each genome for breadth and depth of read coverage,

SNVs

Gene predictions

Growth of Archae/Bacteria genome sequences

Nikos Kyrpides, Genomes Online Database

1 genome/day added to GenBank

Bacterial sequencing dominates

Major subgroups of the stool biome type

women men

St. Louis Houston

BMI>=30

BMI<25

NA

not hispanic/latino/spanish Hispanic/latino/spanish

25 <= BMI <30

Lots of diversity of individual level: People Have Their Own “Personal Microbiome”

Each row a different sample

Histograms of genera in each sample

Bacteroides

Prevotella

Ruminococcus

Yanjiao Zhou & George Weinstock

Diversity at the organismal level but wide array of shared microbial genes comprising a “core microbiome” at the gene level

Turnbaugh PJ, et al. Nature. 2009

The microbiome differs with different organ sites

Dirk Gevers (Broad Institute) + HMP Consortium

Other cool facts

Niels O. Verhulst PLoS One 2011

HA = Highly Attractive PA = Poorly attractive

Does microbiome affect attraction between humans?

The skin microbiome affects our attractiveness to mosquitos!

The Role of the Microbiome in Health and Disease

Ecological diseases from our microbiome

Not infectious diseases: causative agents are part of the normal flora - e.g. Candida albicans - Disease results from disturbance to the environment, altering microbial balance Antibiotic treatment - Clostridum difficile no longer held in check by excess beneficial organisms - Reduced protection against deleterious organisms Dental caries - Streptococcus mutans overgrowth due to high carbohydrate utilization - Increased amounts of deleterious substances, eg lactic acid Toxic Shock Syndrome - Staphylococcus aureus emergence due to abnormally dry environment - Change in the environment Many more - to

be discovered

Disease metagenomics projects Illness of unknown origin

Necrotizing enterocolitis

IBD/Crohn’s disease

Urethritis

Nasal microbiome after vaccination and flu infection

Acne

Antibiotic effects on physiology

Cystic fibrosis

Periodontitis

Lung microbiome and HIV

Kawasaki disease

Bacterial vaginitis

Obesity

Diabetes

Sepsis

Trachoma and sequelae

Autism

Myocardialinfarction (heart attack)

http://guardianlv.com/2013/10/flu-vaccine-may-cut-heart-attack-risk/

Carnitine from meat is converted to TMAO using gut microbiome - Antibiotics suppress TMAO levels in humans - Germ free mice, no TMAO CUT genes responsible Possible solutions: 1) Change diet to reduce relevant bacteria 2) Produce inhibitors of the CUT gene product. 3) Genetic engineering

High levels of TMAO (trimethylamine-N-oxide) are associated with MI

Some examples: Obesity and the microbiome

Our food affects our microbiome High fat vs low fat diet.

Typically American diet Soy

Vervet 2009

Less gut bacterial diversity in obese vs lean people

Turnbaugh PJ, et al. Nature. 2009

Obesity and the microbiome Differences between obese mouse and lean mouse

Ley et al PNAS 2005

Leptin mutant (ob/ob)

Normal mouse (+/+)

TLR5 (toll like-receptor deficient) mice fight bacteria differently from normal mice.

Vijay-Kumar et al Science 2010

Their transmissible microbiome makes them eat more

Vijay-Kumar et al Science 2010

What about humans?

Ley et al Host Cell & Microbe 2006

Pilot study in humanized mice

Smith et al Science 2013

Gut microbes studies in discordant twins Implant microbiome into Mice

Kawshiokar = malnurished

Diabetes and the microbiome

Diabetics have a different microbiome than nondiabetics in humans and mouse models. Inject bacteria from healthy mouse into mouse prior to inducing diabetes. Reduces diabetes formation.

Wang et al Cell 2012

Inflammatory bowel disease (IBD)

Different microbiome in Chrohn’s disease and IBD In some cases fecal transplants seemed to help (In case you were wondering – Administered through Ingestion or the rectum)

Infections: Organ transplantation Microbial infections often appear after organ transplantation Example: Hematopoetic cell transplantation with umbilical cord cells at Harvard 11 people infected after tranplant e.g. 30 yr old woman with ALL. Pursued cord tranplant. Developed coiltis

WBC 135K/uL, 53% blasts

Acute Leukemia (ALL)

Bhatt et al, NEJM 2012

Organ transplantation

Extract DNA from fixed tissue Shotgun sequencing

Many reads did not map to known bacterial genomes!

Lots!

Bhatt et al, NEJM 2012

Organ transplantation Assemble unmapped reads from two patients into new sequence: Bradyrhizobium enterica

Slide courtesy of Ami Bhatt

Classification of the CCS-associated bacterium

Phylogenetic analysis using the draft genome to classify the organism

Slide courtesy of Ami Bhatt

Febrile children study

Plasma samples positive for HHV6 or Enterovirus 6 8 1

PCR Seq

Greg Storch

Conclusions 1) The microbiome is vast and essential 2) It is associated with many, and possibly all, human diseases. 3) It is not clear what is cause and what is effect. 4) It may provide novel avenues for treating human disease.