MCB104 - Lecture 13 - Epigenetics and Genome Sequencing

What is epigenetics?

“Stuff on top of genetics”

• DNA sequence in genes is not the only carrier of genetic information

• Epigenetic phenomena: heritable self perpetuating changes in gene expression not caused by base pair sequence changes

• Usually involves modified cytosine residues, modified histone tails in chromatin, small RNAs

• Epigenetic factors that can determine whether a gene is “on” or “off” can change

How can 2 mice have the same genotype for coat color?

• Epigenetics can change phenotype

• Yellow mouse expresses yellow coat color but grey mouse has coat color gene silenced form epigenetic modifications

• Epigenetic information can therefore determine if gene is “on” or “off”

What is DNA methylation?

• A methyl group is added to the cytosine base in a 5’ CpG 3’ dinucleotide by DNA methyl transferases (DNMTs)

• DNA methylation maintained through cell division

• Special DNMT at the replication fork methylates the newly synthesized DNA strand

What is the ROLE of DNA methylation?

• CpG methylation turns OFF gene expression

• Methyl-CpG-binding proteins (MeCPs) bind to methylated CpG islands and close the chromatin structure

  • These are transcriptional repressors

What are CpG Islands?

• Deamination of methylated Cytosine results in Thymine

• This means that frequently methylated CpGs tend to be lost

• As active promoters tend to be unmethylated, CpG islands tend to be found in their vicinity

What are Histone Tail Modifications?

• Modifying histone tails alter affinity to DNA

• Methylation of Lysine decreased negative charge, increasing affinity to DNA

• Acetylation of Lysine increases negative charge, decreasing affinity to DNA

• (Remember, DNA is negatively charged)

• TIghter coiling limits access for transcription factors

What does methylation of lysine do?

• Methylation of Lysine decreased negative charge, increasing affinity to DNA

What does acetylation of Lysine do?

• Acetylation of Lysine increases negative charge, decreasing affinity to DNA

What is Genomic Imprinting?

• Genomic Imprinting: Phenomenon in which expression of an allele depends on the parent that transmits it

• Methylation at imprinting control regions (ICRs) affects gene expression

• RNA-Seq has identified about 100 imprinted genes

  • Paternally imprinted: paternal allele not transcribed

  • Maternally imprinted: material allele isn’t transcribed

  • Imprinted = silenced

If I silence a paternal or maternal allele, what happens?

• RNA-Seq has identified about 100 imprinted genes

  • Paternally imprinted: paternal allele not transcribed

  • Maternally imprinted: material allele isn’t transcribed

  • Imprinted = silenced

How can imprinting in pedigrees look?

• Alleles from the non-imprinting parent are dominant in offspring

How was imprinting discovered?

• Scientists predicted existence of imprinting prior to understanding the mechanism

• In the 1980s, pedigrees were observed where the sex of the parent carrying the disease allele determined if the child had the disease

• Deletion of the paternally imprinted gene doesn’t affect a father’s child if the wild type allele from the mother is expressed

• Deletion of a maternally imprinted gene doesn’t affect a mother’s child if the wild type allele from the father is expressed

How can deleting paternally imprinted gene affect a child?

• Deletion of the paternally imprinted gene doesn’t affect a father’s child if the wild type allele from the mother is expressed

How can deleting maternally imprinted gene affect a child?

• Deletion of a maternally imprinted gene doesn’t affect a mother’s child if the wild type allele from the father is expressed

Describe Imprinting Mechanism: INSULATORS

• EX: ICR is an insulator that controls transcription of Igf2 gene

• Nonmethylated maternal insulator is functional,; binds CTCF and transcription is silenced

• Methylated paternal insulator is nonfunctional and transcription is active

Describe Imprinting Mechanism: ncRNA MECHANISM

• ICR contains a noncoding RNA whose transcription is controlled by CpG islands

• Nonmethylated paternal CpG island allows production of Air ncRNA; transcription of lgfr2 is silenced

• Methylated maternal CpG island prevents production of Air; transcription of lgfr2 is active

How does Resetting of Genomic Imprints work?

• Epigenetic imprints remain throughout the lifespan of mammal

• During meiosis, imprints are erased and new ones are set based on the sex of organism

What is EXAMPLE of Resetting of Genomic Imprints?

• Two syndromes associated with small deletions of Chromosome 15, q11-13 region

  • At least 2 genes within region are differently imprinted

  • Prader-Willi syndrome occurs when deletion is inherited from father

  • Angelman syndrome occurs when the deletion is inherited from the mother

  • Affected individuals have mental/development disorders

SEE IF INCORRECT DOUBLE CHECK

What are reasons for imprinting?

• In placental mammals, most imprinted genes control prenatal growth

• Parental conflict hypothesis: mother’s best interest for her baby to be small; father’s interest for his baby to be large and robust

  • Viewed as overly simplisitic, other theories considered

  • Most control of imprinting is maternal, so why would mother cede control to paternal allele

  • Alternate hypothesis is to encourage rapid adaptation by increasing selective pressure on developmental genes

What is parental conflict hypothesis?

• Parental conflict hypothesis: mother’s best interest for her baby to be small; father’s interest for his baby to be large and robust

What is cell fate?

• Imprinting is RARE

• Many epigenetics marks are only transmitted through mitosis

• All cells in multicellular organsims have the same genes

• Cell types are different bc of gene expression differences

• Master regulator, often a transcription factor, determines gene expression and potential fates

• Cells “remember” their fate from cell generation to cell generation in part by copying histone modifications at replication fork

  • Nucleosomes on both daughter strands are composites of new and old histones

Example of master regulators?

HOX GENES

Describe Hox Genes

• Drosophila Hox (homeobox) genes encode transcription factors that set up animals segmented body plan

• Cells in each fly segment get information about which segment they are in due to Hox proteins made there

• Hox genes are repressed in specific groups of cells

• HIGHLY CONSERVED

• If epigenetic markers affected - likely passed down (asked in class by me)

How can Hox Genes Repression work?

• PRE (Polycomb response element): Cis-acting DNA sequence that binds repressors that recruit PRC2 co-repressor complex

• PRC2 contains a histone methyltransferase that methylates lysine27 (K27) in the tail of histone H3

• H3K27me mark is propagated to adjacent nucleosomes and Hox genes is repressed

• PRC2 is recruited to H3K27me

How can Hox repression be maintained?

• Once established H3K27me and closed chromatin are recapitulated at the Hox gene in all of the descendants of a cell

• PRC2 recruitment to PRE maintains robust silencing

  • H3K27me marks are self perpetuating but not indefinitely

How does Constitutive Heterochromatin work?

• Constitutive Heterochromatin: highly compacted chromatin found in all cells

• Found at centromeres, which are enriched in transposable elements; important to prevent TE mobilization

• Centromeric heterochromatin formation often involves methylation of K9 amino acid on the tail of histone H3 (H3K9me)

How does Heterochromatin formation work in plants?

• ncRNA transcribed from centromeres of plant chromosomes intiates heterochromatin formation

• ncRNA is converted to siRNAs which bind to Argonaute (ago) proteins

• Ago recruits histone methyltransferases (HMTs) that methylate H3K9 and DNMTs that methylate cytosine in DNA; heterochromatin forms

How can plant heterochromatin be maintained?

• Plant heterochromatin be re-formed after cell division

• H3K9me marks remaining on some nucleosomes recruit a DNMT that methylates cytosines

• Methylated cytosines attract an HMT in feedback loop

What is X-inactivation?

• Cells in early female mammalian embryos inactivate a random X chromosome, making it a Barr Body

• Xist IncRNA binds to X chromosome that expresses it and recruits histone modifying enzymes including PRC1 and 2

• Chromatin marked by H3K9me, H3K27me, methylated CpG islands among others

How does X-inactivation spread?

• Xist serves as a scaffold for various RNA binding proteins (RBPs)

• SAF-A links Xist to the chromosome, while Lamin-B receptor links it to the nuclear lamina

• Causes X chromosome to clump together and facilitates spread of Xist

What is the heritability of X inactivation?

• Not really understood

  • Transfer of histone marks/chromatin methylation is important but not fully explanatory

• Possible that Barr body is compartmentalized enuff to maintain repressive environment during replication

What is transgenerational epigenetic inheritance?

• Transgenerational epigenetic inheritance: when an environmentally induced trait (not caused by base pair mutation) appears in an individual descendants whose DNA was not directly exposed to environmental trigger

• Some examples:

  • RNA directed DNA methylation

  • piRNA memories of Transposable Element (TE) invasion

Describe Toadflax Flower specification?

• Wild type, bilateral, vs. Peloria, radial, flower symmetry specified by a recessive nontranscribed allele of a Lcyc gene

• Same DNA sequence, but Peloria Lcyc allele is extensively methylated and silenced

  • Epialleles: alleles that derive from epigenetic marks instead of sequence changes

• Cause is methylation of upstream TE extending into lcyc

• Heritable bc DNA methylation marks aren’t erased in plant gametogenesis

What is piRNAs?

• Organisms try to limit TE mobilization to prevent mutations/chromosomal rearrangements

• In Drosophila, small non-coding RNAs called piRNAs (Piwi-interacting RNAs) suppress TE movement

• piRNAs are made in female germ-line cells and transmitted to progeny in oocytes

• Mechanism to preserve memories of TE invasions and help protect against future TE mobilization

What is piRNA mechanism?

• TE invading Drosophila genome hops into piRNA cluster, which contains inactive remnants of different TEs

• Cluster is transcribed to make piRNA precursors

• piRNAs promote destruction of TE mRNAs blocking expression of TE genes and immobilizing TEs

How does piRNA inheritance work?

• piRNAs can regenerate themselves in the female germ line

• piRNAs guide the Argonaute protein piwi back to the same piRNA cluster by basepairing with piRNA transcripts as they are being transcribed

• Piwi recruits histone modifying enzymes

• HP1 binds to histone and closes chromatin, but the HP1 at piRNA clusters brings RNA polII to clusters, leading to transcription

• Self-sustaining germline production of piRNAs

What is Hybrid Dysgenesis?

• STERILITY of hybrid progeny

• Outside flies crossed to lab Drosophila had STERILE progeny

  • But only when outside males were crossed with lab females and not reverse

• Genomes of outside flies contained P elements; lab fly genomes had none

• Oocytes of females w/out P element don’t have piRNAs to press P element mobilization

  • Leads to so many mutations in gametes that progeny are sterile

• Oocytes of females with P elements have piRNAs that inactivate P elements in germ line of progeny

What is Genomics?

Human Genome Project: an accurate sequence of the human genome was initiated in 1990 and completed (97% converage) in 2003

We’ve now seqeucned thousands of eukaryotic and hundreds of thousands of prokaryotic species

General ideas behind genome sequencing are simple:

• Fragmenting the genome

• Cloning DNA fragments

• Sequencing DNA fragments

• Reconstructing genome from fragments

What are Restriction Enzymes?

• Restriction Enzymes fragment the genome at specific sites by recognizing a specific sequence of bases

  • Cuts sugar-phosphate backbones of both strands

  • Restriction fragments are generated by digestion of DNA with restriction enzymes

  • Hundreds of restriction enzymes now available

• Recognition sites for restriction enzymes are usually 4 to 8 bp of double strand DNA

  • Often palindromic - base sequences of each strand are identical when read 5’ to 3’

  • Each enzymes cuts at the same place relative to its specific recognition sequence

What are some commonly used restriction enzymes?

What are Blunt vs. Sticky ends?

Blunt ends: cuts are straight through both DNA strands at the line of symmetry

Sticky ends: cuts are displaced equally on either side of line of symmetry

• Ends have either 5’ overhangs or 3’ overhands

Different restriction enzymes produce different fragment length - calculate?

What is an example of a restirciton map?

• Site of 3 restirction enzymes in 200 kb region of human chromosome 11

• Names and locations of genes in this region are shown below restriction sites

• Rsal 4bp, EcoRI 6bp, Notl: 8bp

What is Mechanical Fragmentation?

• Some experiments require random cutting of DNA, not guided cuts with restriction enzymes

• Mechanical forces can break phosphodiester bonds

  • Passing DNA through thin needle at high pressure

  • Sonication (ultrasound energy)

• Ends can be blunt, may have protruding single-strand regions

How to do Gel Electrophoresis?

• DNA analysis via electrophoresis, movement of charged molecules (ex: DNA fragments) in electric field

• Steps:

  • 1) Pour heated molten agarose into acrylic plate to which comb has been attached allow to cool and harden

  • 2) Remove comb, place gel in buffered aqueous sol, load DNA samples into wells in gel

  • 3) Apply electric current

  • 4) DNA has negative charge, so moves toward postitive charge

  • 5) Remove gel from tank after electrophoresis

  • 6) Visualize DNA fragments by staining gel with fluorescent dye, photograph gel under UV light

    • With linear DNA fragments migration distance through gel depends on size

    • Determine size of unkwown fragments by comparison of migration to DNA markers of known size

How can DNA fragments be cloned?

• Genomes of animals, plants, and microorganisms are too large to analyze all at once

• Molecular cloning purifies a specific DNA fragment away from all other fragments and makes identical copies of fragment

• 2 basic steps:

  • Insert DNA fragments into cloning vectors to specialized chromosome-like carriers that ensure transport, replication, and purification of DNA inserts

  • Transport recombination DNA into living cells to be copied

• Group of replicated DNA molecules = DNA clone

What as Plasmid Vectors?

• Plasmid cloning vectors have 3 main features:

  • Origin of replication

  • A selectable marker gene (ex: antibiotic resistance)

  • Insertion site

    • Often a synthetic polylinker, DNA sequence containing multiple restriction enzyme sites

• Different vectors carry different size insertions

  • Plasmid ~20 kb

  • Bacterial artificial chromsomes (BAC) ~~ 300 kb

  • Yeast artificial chromosomes (YAC) ~~ 2000 kb

How can recombinant vectors be created?

• Digest the vector and target genomic DNA with a restriction enzyme that results in complementary sticky ends

• DNA Ligase is used to seal the phosphodiester backbones between vectors and insertion

Each genomic DNA fragment can form _____?

DIFFERENT RECOMBINANT MOLECULE

• Pieces being inserted are random

• Result is many different recombinant vectors

What can you do with recombinant DNA to transform Host cells?

• Transformation: process by which cell or organism takes up foreign DNA

• In E.coli only 0.1% of cells will be transformed with plasmid

• Only cells with plasmid will grow on media with ampicillin

• Each cell with plasmid will produce a colony on agar plate, millions of identical plasmids in colony are a DNA clone

What is a Genomic Library?

• Genomic library: long lived collection of cellular clones that contains copies of every sequence in the whole genome inserted into a suitable vector

• Each colony contains a different recombinant plasmid, each with a part of the genome

• A perfect genomic library has one copy of every sequence in the entire genome

  • Genomic Equivalent: Minimum # of clones in a perfect library = length of genome/length of insert

• Impossible to obtain perfect library

  • Usually libraries are made that have 4-5 genomic equivalents

  • Gives an average of 4-5 clones for each locus (95% prob that each locus is present at least once)

What is Sanger Sequencing?

• Uses DNA polymerase to synthesize new DNA

• DNA polymerase requires:

  • TEMPLATE: single strand of DNA to copy

  • Deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP), basic building blocks for new DNA

  • Primer: short single stranded DNA molecule that is complementary to part of template, includes free 3’ end to which DNA polymerase can attach new nucleotides

What are Sanger Sequencing Steps?

• Cloned recombinant DNA is denatured (heat breaks H bonds btw strands)

• Strands are mixed with oligonucleotide primer (made in DNA synthesizer) approximately 20 bp complementary to template strand

• As temperature is lowered, primers and template strand anneal (hybridize)

• Hybridized template and primers are mixed with DNA polymerase, dNTPs, and small amounts of dideoxyribonucleotide triphosphates, ddNTPs

  • ddNTPs lack a 3’-OH, halting polymerization

  • Modtern method has each with unique florescent tag

• Result is a set of nested fragments, with different 3’ ends

How can you readout the Sanger Sequencing result?

• Special gel seperates DNA fragments that can differ in size by only one nucleotide

  • Smaller DNA fragments migrate faster

• Each lane displays the sequence obtained form a separate DNA sample and primer

• Each fragment terminates with a specific ddnTP labeled with unique florescent dye

  • DNA fragments are electrophoresed based on size, color of terminal ddNTP is recorded to identify nucleotide code

How can sequencing reads be automated?

• Fragments flow past a laser beam and the color of the terminal base is digitally recorded

• Computer reads of sequence complementary to the template strand

• Sequence is read from left to right (5’ to 3’ synthesis from primer)

Genome comparisons?

How can genomes be assembled (Describe Human Genome Project Method)

• Human Genome Project

  • 1) Construct BAC genomic library

  • 2) Identify sets of overlapping BAC clones

  • 3) Shear DNA from each BAC seperarely to make smaller clones

  • 4) Sequence DNA

  • 5) Assemble sequences based on overlap

• Strategy was successful but labor intensive, inefficient and expensive

How does Shotgun sequencing work?

• Celera developed the whole genome shotgun strategy for genome sequencing

  • Create genomic library of overlapping fragments in plasmid vectors

  • Sequence DNA inserts of randomly chosen library plasmids (“shotgun”)

  • Assembly sequences based on overlap of sequences into contigs - continuous base pair sequences

• The whole-genome shotgun approach can be highly automated

Can repeats disrupt or help shotguns sequencing?

DISRUPT

• MAny sequences repeat throughout genome

• With only short, single reads these sequences cannot be distinguished from each other

• Can lead to misassembly

How does Paired End Reads work?

• Paired end sequencing, sequencing a BAC clone library rather than smaller inserts of plasma clones

• Each BAC clone insert gave 3 pieces of into:

  • 2 approximately 1000 bp sequence reads

  • Knowledge that 2 sequences were approx 200-300 kb apart

• Can give information on flanking sequences of repeat

What is Next Generation Sequencing?

• Sequencing an entire genome (high coverage, missing some small regions) now costs about $1000

• Whole-exome sequencing (limited to expressed parts of genome) is less expensive

  • Sequence only fragments corresponding to exons

• High-throughout, or massively parallel, sequencing is like Sanger sequencing with a few modifications

  • Individual DNA molecules are anchored in place

  • Each base is identified before next one added

  • Increased sensitivity eliminates need for cloning or PCR

What is ILLUMINA SEQUENCING: LIGATION

• Cloning into vectors is time consuming so Illumina sequencing skips it

• Instead of ligating into vectors, ligates with adaptors that have

  • Sequencing and amplification primer sites

  • Complementation to DNA molecules anchored to flow cells

  • Sometimes a barcode (unique seq. identifier) to ID source of sequence

What is ILLUMINA SEQUENCING: AMPLIFICATION

• Anchorign primers used to synthesize new strands

• After denaturing the other end is bound by reverse primers also anchored to flow cell

• Synthesizes new strand

• Repeat process

• Cleave 1 strand type

• Result is clusters of identical sequences

What is ILLUMINA SEQUENCING: SEQUENCING

• Add sequencing primers

• Add Fluorescently tagged bases with blocked 3’OH and DNA polymerase

• Read Fluorescent label at each cluster

• Remove fluorophore and blocker

• Repeat

• Reads 50-200bp

What is Illumina Use Cases?

• Illumina sequencing is best used if reference sequence available

  • Not ideal for de novo whole genome sequencing

• This way the 50-200 bp fragments can be aligned to known sequences

• Good for sequencing whole genomes of humans