Not IN Our Genes - A Different Kind of Inheritance.! Christopher Phiel, Ph.D. University of Colorado Denver Mini-STEM School February 4, 2014

Transcription

1 Not IN Our Genes - A Different Kind of Inheritance! Christopher Phiel, Ph.D. University of Colorado Denver Mini-STEM School February 4, 2014

2 Epigenetics in Mainstream Media

3 Epigenetics *Current definition: The study of mitotically and meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence *This definition is being actively debated within the scientific community. What does this mean? And why should we care?

4 Regulation of gene expression in eukaryotic cells Transcription factor Marmorstein, Nature Reviews Mol Cell Bio (2001) Is this version of gene regulation over-simplified?

5 Direct cell reprogramming Vierbuchen and Wernig (2012)

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7 Ramen Noodles and Chromatin? 4 x 5 x 1

8 Ramen Noodles and Chromatin? 4 x 5 x of noodles!

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11 Histones can be modified, and these modifications can affect gene expression

12 A Histone Code

13 Regulation of gene expression - transcription factors + epigenetics Berger, Nature (2000)

14 Epigenetics Nova ScienceNow, July 2007 Histone modifications Acetylation, methylation, phosphorylation, arginylation, ubiquitination DNA methylation

15 Epigenetics Nova ScienceNow, July 2007 Histone modifications Acetylation, methylation, phosphorylation, arginylation, ubiquitination DNA methylation

16 How are these epigenetic modifications established? There are a host of proteins whose function is to write, erase, or read epigenetic modifications HATs - histone acetyltransferases HDACs - histone deacetylases HMTs - histone methyltransferases DNMTs - DNA methyltransferases

17 How do are cells know which regions should have open or closed chromatin? And how are these regions marked?

18 ChIP experimental workflow PCR Band or no band?

19 Human Genome Sequencing Using traditional Sanger sequencing, it took an international team 11 years and $3 billion to sequence the human genome (about $1 per base pair) Advances in sequencing technology have dramatically reduced the time and cost - currently about $5000 and can be completed in a week - expected to drop to below $1000 in the next months

20 NGS is not just for whole genome sequencing! ChIP-seq - chromatin immunoprecipitation + seq Methyl-seq - DNA methylation sequencing RNA-seq - capture RNA and sequence (vs. microarray) Small RNA-seq - focus on mirnas, snorna, trnas, etc. RIP-seq - RNA immunoprecipitation + seq (to identify RNAs associated with specific proteins) A common thread throughout all of these strategies is to first use another technique to enrich the samples for a given type of analysis, followed by NGS

21 Illumina Next-Generation Sequencing Metzker, Nat Rev Genetics (2010) Allows tens of millions of sequencing reactions to be performed simultaneously Also referred to as massively parallel sequencing

22 Illumina Next-Generation Sequencing Metzker, Nat Rev Genetics (2010) 51x --> 51 bp reads

23 How does next-generation sequencing work? Another name for next-generation sequencing is massively parallel sequencing Illumina

24 Analyzing NGS data requires the assistance of bioinformatics (topic of future Mini-STEM lecture) Mortazavi, et al, Nature Methods (2008)

25 ChIP-seq experimental workflow

26 Mapping the precise locations of histone modifications throughout the entire genome Specific histone modifications have previously had functions ascribed to them, such as facilitating gene expression (H3K4me3) or suppressing gene expression (H3K27me3) H3K4me3 = trimethylation of lysine 4 on histone H3 H3K27me3 = trimethylation of lysine 27 on histone H3

27 ENCODE Encyclopedia of DNA Elements Project that aims to delineate all of the functional elements encoded in the human genome Identification and quantification of RNA species in whole cells and sub cellular compartments Analysis of RNA binding proteins Mapping of noncoding and proteincoding genes Delineation of chromatin and DNA accessibility Mapping of histone modifications and transcription factor-binding sites by ChIP Measurement of DNA methylation Performed 1,640 sequencing studies on 147 different human cell types Examination of long-range chromatin interactions

28 Summary of ENCODE findings The vast majority of the human genome (80.4%) participates in at least one biochemical RNA- and/or chromatin-associated event in at least one cell type 95% of the genome lies within 8 kb of a DNA-protein interaction, and 99% is within 1.7 kb of at least one biochemical event Classifying the genome into seven chromatin states indicates 399,124 regions with enhancer-like features and 70,292 regions with promoter-like features It is possible to correlate quantitatively RNA production and processing with both chromatin marks and transcription factor binding at promoters, indicating that promoter functionality can explain most of the variation in RNA expression Many non-coding variants in individual genome sequences lie in ENCODE-annotated functional regions; this number is at least as large as those that lie in protein-coding regions Single-nucleotide polymorphisms (SNPs) associated with disease by GWAS are enriched within non-coding functional elements, with a majority residing in or near ENCODE-defined regions that are outside of protein coding genes. In many cases, the disease phenotypes can be associated with a specific cell type or transcription factor.

29 Green, et al, Nature (2011)

30 Correlating ChIP-seq data with GWAS SNP studies GWAS = genome-wide association studies SNP = single nucleotide polymorphisms ENCODE Consortium, Nature (2012)

31 Drugs that modify epigenetic modifications DeWoskin and Million, Nature Reviews Drug Discovery (2013)

32 Lithium and valproic acid are both use to treat bipolar disorder - do they work the same way?

33 VPA is an HDAC inhibitor VPA = valproic acid Because of our discovery, VPA has been successfully used clinically in the treatment of different leukemias

34 DNA Methylation Simple modification of cytosine bases, mediated by enzymes called DNA methyltransferases! Approximately 5% of the genome is methylated! The pattern of DNA methylation can overlap, but is distinct for each cell type in our bodies

35 Glycogen Synthase Kinase-3 (Gsk-3) Applies the Brakes to Many Signaling Pathways Cohen, Nature Rev Mol Cell Bio (2006) Barker and Clevers, Nature Rev Drug Discovery (2006)

36 Gsk-3-Deficient Mouse Embryonic Stem Cells WT ESCs Gsk-3 DKO ESCs

37 Whole-Genome Expression Analysis via Microarray 1. Isolate RNA samples. Synthesize DNA copies. RNA population or probes Clinical sample 4. Analyze data and correlate with histoclinical data 2. Hybridize labeled probe with DNA microarray on a chip 8x4x2 2x4x8 3. Scan the chip We recently found that cells deficient in Gsk-3 also have reduced DNA methylation at specific regions of the genome

38 Genome-wide DNA Methylation Sequencing Tony D Ippolito Gavin Meredith, Ph.D. The Ohio State University Major - Molecular Genetics! *Currently - Duke University Ph.D. Program in Genetics Life Technologies R&D Manager

39 Genome-wide DNA Methylation Sequencing Tony D Ippolito Gavin Meredith, Ph.D. The Ohio State University Major - Molecular Genetics! *Currently - Duke University Ph.D. Program in Genetics Life Technologies R&D Manager Which genomic regions in Gsk-3 DKO ESCs are hypomethylated compared to WT ESCs?

40 Genome-wide DNA Methylation Sequencing Tony D Ippolito Gavin Meredith, Ph.D. The Ohio State University Major - Molecular Genetics! *Currently - Duke University Ph.D. Program in Genetics Life Technologies R&D Manager Which genomic regions in Gsk-3 DKO ESCs are hypomethylated compared to WT ESCs?

41 Understanding How DNA Methylation Differences Are Determined via NGS Peaks are built up by aligning sequences to reference genome WT Each box represents a 51 bp fragment of DNA DKO

42 Methyl-Seq Data in Genome Viewer WT DKO

43 Genomic Imprinting, DNA Methylation, and Dnmt3a2 Mammals inherit one copy of each gene from both parents However, not all genes are equally expressed Imprinted genes are those in which one inherited copy is silenced by DNA methylation There have been approximately 100 imprinted genes identified in mice IMPORTANT - these are parent-of-origin effects, not gender effects

44 Genomic Imprinting, DNA Methylation, and Dnmt3a2 Mammals inherit one copy of each gene from both parents However, not all genes are equally expressed Imprinted genes are those in which one inherited copy is silenced by DNA methylation There have been approximately 100 imprinted genes identified in mice IMPORTANT - these are parent-of-origin effects, not gender effects Igf2 - Insulin-like growth factor 2 = methylation

45 Genomic Imprinting, DNA Methylation, and Dnmt3a2 Mammals inherit one copy of each gene from both parents However, not all genes are equally expressed Imprinted genes are those in which one inherited copy is silenced by DNA methylation There have been approximately 100 imprinted genes identified in mice IMPORTANT - these are parent-of-origin effects, not gender effects Igf2 - Insulin-like growth factor 2 = methylation

46 Genomic Imprinting, DNA Methylation, and Dnmt3a2 Mammals inherit one copy of each gene from both parents However, not all genes are equally expressed Imprinted genes are those in which one inherited copy is silenced by DNA methylation There have been approximately 100 imprinted genes identified in mice IMPORTANT - these are parent-of-origin effects, not gender effects Igf2 - Insulin-like growth factor 2 = methylation Human imprinting disorders Beckwith-Wiedemann syndrome/wilm s tumor, Prader-Willi syndrome/angelman syndrome

47 Genomic Imprinting, DNA Methylation, and Dnmt3a2 Mammals inherit one copy of each gene from both parents However, not all genes are equally expressed Imprinted genes are those in which one inherited copy is silenced by DNA methylation There have been approximately 100 imprinted genes identified in mice IMPORTANT - these are parent-of-origin effects, not gender effects Igf2 - Insulin-like growth factor 2 = methylation Human imprinting disorders Beckwith-Wiedemann syndrome/wilm s tumor, Prader-Willi syndrome/angelman syndrome **Dnmt3a/3a2 is the DNA methyltransferase that is responsible for methylating imprinted loci**

48 Genomic Imprinting, DNA Methylation, and Dnmt3a2 Methylated loci Methylation re-established Most of genome demethylated

49 Example of an Imprinted Locus H19/Igf2 Differentially methylated region (DMR)

50 DNA Methylation at the H19 DMR is Reduced in Gsk-3 DKO ESCs 303 bp fragment with 15 CpGs WT % Sequence of 13 clones from WT ESCs DKO % Sequence of 11 clones from DKO ESCs = Unmethylated CpG = Methylated CpG

51 Model for the Regulation of Igf2/H19 Gene Expression in Gsk-3 DKO ESCs

52 Constitutive Activation of Insulin Signaling in Wild-Type Embryonic Stem Cells WT p110* 100 kd 75 kd 75 kd 50 kd 75 kd 50 kd 50 kd PI3K p-akt Akt p-gsk-3 50 kd 50 kd GSK-3 Tubulin Western blots (protein) CST antibodies Cohen, Nature Rev Mol Cell Bio (2006) Phosphorylation of Gsk-3α and Gsk-3β by Akt results in inhibition of Gsk-3 activity

53 Increasing DNA methylation in pregnant Agouti mice results in reduced obesity in the offspring Jirtle and Skinner, Nature Reviews Genetics (2007)

54 The fungicide viclozolin has been reported to cause transgenerational effects in rats Kaiser, Science (2014)

55 Activation of Insulin Signaling via Activated PI3K Results in Reduced DNA Methylation Igf2/H19 DMD WT 81.3% p110* 52.9%

56 Connecting Food Availability to Epigenetics and Disease

57 Connecting Food Availability to Epigenetics and Disease Pembrey, et al, Eur J Med Gen (2006)

58 Could altered insulin signaling be the mechanism behind this effect?? (Insulin signaling & Gsk-3 inhibition)