Everything you wanted to know about ENCODE

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Everything you wanted to know about ENCODE

• But were afraid to ask

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Top 5 Reasons Biologists Go Into Bioinformatics

• 5 - Microscopes and biochemistry are so 20th
  century.

3
Top 5 Reasons Biologists Go Into Bioinformatics

• 5 - Microscopes and biochemistry are so 20th
  century.
• 4 - Got started purifying proteins, but it turns
  out the cold room is really COLD.

4
Top 5 Reasons Biologists Go Into Bioinformatics

• 5 - Microscopes and biochemistry are so 20th
  century.
• 4 - Got started purifying proteins, but it turns
  out the cold room is really COLD.
• 3 - After 23 years of school wanted to make MORE
  than 23,000/year as a postdoc.

5
Top 5 Reasons Biologists Go Into Bioinformatics

• 5 - Microscopes and biochemistry are so 20th
  century.
• 4 - Got started purifying proteins, but it turns
  out the cold room is really COLD.
• 3 - After 23 years of school wanted to make MORE
  than 23,000/year as a postdoc.
• 2 - Like to swear, _at_ttracted to _ Perl !!

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Top 5 Reasons Biologists Go Into Bioinformatics

• 5 - Microscopes and biochemistry are so 20th
  century.
• 4 - Got started purifying proteins, but it turns
  out the cold room is really COLD.
• 3 - After 23 years of school wanted to make MORE
  than 23,000/year as a postdoc.
• 2 - Like to swear, _at_ttracted to _ Perl !!
• 1 - Getting carpel tunnel from pipetting

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Top 5 Reasons Computer People go into
Bioinformatics

• 5 - Bio courses actually have some females.

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Top 5 Reasons Computer People go into
Bioinformatics

• 5 - Bio courses actually have some females.
• 4 - Human genome more stable than Windows XP

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Top 5 Reasons Computer People go into
Bioinformatics

• 5 - Bio courses actually have some females.
• 4 - Human genome more stable than Windows XP
• 3 - Having mastered binary trees, quad trees, and
  parse trees ready for phylogenic trees.

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Top 5 Reasons Computer People go into
Bioinformatics

• 5 - Bio courses actually have some females.
• 4 - Human genome more stable than Windows XP
• 3 - Having mastered binary trees, quad trees, and
  parse trees ready for phylogenic trees.
• 2 - Missing heady froth of the internet bubble.

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Top 5 Reasons Computer People go into
Bioinformatics

• 5 - Bio courses actually have some females.
• 4 - Human genome more stable than Windows XP
• 3 - Having mastered binary trees, quad trees, and
  parse trees ready for phylogenic trees.
• 2 - Missing heady froth of the internet bubble.
• 1 - Must augment humanity to defeat evil
  artificial intelligent robots.

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The Paradox of Genomics
How does a long, static, one dimensional string
of DNA turn into the remarkably complex, dynamic,
and three dimensional human body?
GTTTGCCATCTTTTGCTGCTCTAGGGAATCCAGCAGCTGTCACCATG
TAAACAAGCCCAGGCTAGACCAGTTACCCTCATCATCTTAGCTGATA
GCCAGCCAGCCACCACAGGCATGAGT
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Looks like code not enough, must study actual
cells DNA
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How DNA is Used by the Cell
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Promoter Tells Where to Begin
Different promoters activate different genes
in different parts of the body.
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A Computer in Soup
Idealized promoter for a gene involved in making
hair. Proteins that bind to specific DNA
sequences in the promoter region together turn a
gene on or off. These proteins are themselves
regulated by their own promoters leading to a
gene regulatory network with many of the same
properties as a neural network.
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Regulation By Txn Factor Binding
When I-KB is removed from by phosphorylation,
NF-KB complex binds to dna.
Note that you would need Both NF-KB p65 and NF-KB
p50 Subunits to be expressed in same cell For
this transcription activation Pathway to work.
Selective, combinatorical expression of txn
factors is very important In defining different
types of cells.
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The Decisions of a Cell

• When to reproduce?
• When to migrate and where?
• What to differentiate into?
• When to secrete something?
• When to make an electrical signal?

The more rapid decisions usually are via the cell
membrane and 2nd messengers. The longer acting
decisions are usually made in the nucleus.
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Nucleus Used to Appear Simple

• Cheek cells stained with basic dyes. Nuclei are
  readily visible.

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Mammalian Nuclei Stained in Various Ways
Image from Tom Misteli lab
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Artists rendition of nucleus
Image from nuclear protein database
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Chromatin
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Turning on a gene

• Getting DNA into the right compartment of the
  nucleus (may involve very diffuse signals in DNA
  over very long distances)
• Loosening up chromatin structure (this involves
  enhancers and repressors which can act over
  relatively long distances)
• Attracting RNA Polymerase II to the transcription
  start site (these involve relatively close
  factors both upstream and downstream of
  transcription start).

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HISTONE MODIFICATIONS
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Modification
Effect
H3K4me3
H3K4me2
H3K4me1
H3acK9/14
H4acK5/8/12/16
Slide adapted from Christoph Kock, Sanger
Institute
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(No Transcript)
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Methods for Studying Transcription

• Traditional
• Genetics in model organisms
• Promoters/enhancers hooked to reporter genes
• Gel shifts and DNAse footprinting.
• ENCODE/High Throughput
• Phylogenic footprinting
• Motif searches in clusters of coregulated genes.
• Chromatin Immunoprecipitation CHIP/CHIP
• DNAse hypersensitivity

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Drosophila Genetics
antennapediamutant
normal
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Reporter Gene Constructs
promoter to study
easily seen gene
Drosophila embryo transfected with ftz promoter
hookedup to lacz reporter gene, creating stripes
where ftz promoteris active.
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Biochemical Footprinting Assays
Gel showing selective protection of DNA from
nuclease digestion where transcription factor is
bound.
Txn factorfootprint
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Comparative Genomics
Webb Miller
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Comparative Genomics at BMP10
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Conservation of Gene Features

• Conservation pattern across 3165 mappings of
  human RefSeq mRNAs to the genome. A program
  sampled 200 evenly spaced bases across 500 bases
  upstream of transcription, the 5 UTR, the first
  coding exon, introns, middle coding exons,
  introns, the 3 UTR and 500 bases after
  polyadenylatoin. There are peaks of conservation
  at the transition from one region to another.

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Normalized eScores
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Conservation Levels of Regulatory Regions in
Human/Mouse Alignments
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Dnase I Hypersensitivity, CHIP/CHIP,
transcription data on ENR333
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CHromatin ImmunoPrecipitation

• Crosslink cells with formaldehyde.
• Sonicate to shear DNA
• Add antibody to a protein involved in
  transcription.
• Precipitate antibody and and everything attached
• Heat to release DNA.
• Analyse DNA with PCR or microarrays
• CHIP on microarray CHIP/CHIP

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CHIP/CHIP in ENCODE

• groups Sanger, Yale, Affy, UCSD, Stanford, GIS
  (more?)
• proteins RNA Pol II, TAF1, histones in various
  states of acylation/methylation
• cells various cell lines treated various ways.

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CHIP/CHIP Groups

• Sanger - sequencing center in UK that does a lot
  of annotation as well.
• UCSD/Ludwig Institute - where Bing Ren, a pioneer
  of CHIP lives
• GIS - Genome Institute Singapore - using
  paired-end ditag CHIP.
• Stanford, YALE, Affy you all know.

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CHIP/CHIP Targets

• RNA Polymerase II, converts DNA-gtRNA for protein
  coding genes.
• Antibody targets form in initiation complex
  (start of gene)
• TAF1 - A basal transcription factor. Involved in
  recruiting Pol II to initiation site
• Histones 34 - the balls DNA winds around
• Antibodies against various acylated and
  methylated forms, most of which are associated
  with chromatin opening

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Cell Types

• HELA - cervical epithelial carcinoma
• HCT116 - colon epithelial carcinoma
• IMR90 - lung fibroblast
• THP1 - blood monocyte leukemia
• GMO6990 - lymphoblastoid
• HL-60 - promyelocytic leukemia cell line
• Many others in Stanford promoter track.

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DNAse hypersensitivity

• Very old technique being adapted to high
  throughput.
• DNA cutting enzymes can access open chromatin
  faster than closed chromatin
• Other things may also influence how susceptible a
  particular piece of DNA is to DNAse cutting.
• What is hypersensitive in a particular cell line
  is quite reproducible.
• There are various techniques for seeing where cut
  is sequencing cut ends, PCR around cut site,
  etc.

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Dnase I Hypersensitivity, CHIP/CHIP,
transcription data on ENR333
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Dnase I Hypersensitivity, CHIP/CHIP,
transcription data on ENR333
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Close up of same region
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The END
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How is a gene turned on?

• Pioneering transcription factors bind to DNA
  and tag it for chromatin opening
• Histones are acylated/methylated which opens
  chromatin.
• More transcription factors bind newly exposed
  sites in DNA.
• RNA Polymerase II attracted to txn factors
• Yet more txn factors phosphorylate tail of Pol
  II, allowing it to start transcription.