What are stem cells

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Biol/Chem 473
Schulze lecture 10 Stem cells and chromatin
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What are stem cells?

• Non-specialized cells that have the capacity to
  divide in culture and differentiate into more
  mature cells with specialized functions.
• Can be used for both reproductive and therapeutic
  cloning.

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Reproductive and therapeutic cloning begin the
same way
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A decade of reproductive cloning
2000
2001
1996
1998
2003
RETRACTED
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Stem cell chromatin

• Lots of dramatic DNA methylation changes
• Changes in chromatin accessibility at key
  developmental loci (homeotic gene clusters)
• Key PcG genes are essential for development ES
  lines cant be established without them

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Two important histone methyltransferasesEnhancer
of Zeste (PcG) trithorax (trxG)
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HCNEs highly conserved non-coding elements in
vertebrates
Fugu rubripes
Homo sapiens
Compare non-coding sequence
Filter out transposons etc and matches less than
100bp
Map sequences on human genome
Functional in vivo assay in zebrafish

• These HCNEs map in clusters (i.e., non-randomly
  distributed)
• 93 of these clusters are located within 50kb or
  one or more genes important in transcriptional
  regulation or development

Woolfe et al. (2005) PLOS Biology 3(1) 0116
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HCNEs cluster near developmentally important
genes in vertebrates
Woolfe et al. (2005) PLOS Biology 3(1) 0116
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A bivalent chromatin structure marks key
developmental genes in embryonic stem cells

• Bernstein, B.E. Angelina Jolie, Brad Pitt,
  Jennifer Aniston et al. (2006)
• Cell 125 315-326

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Hypothesis

• HCNEs represent conserved regulatory sequences
  that control vertebrate developmental genetic
  expression

Prediction

• The regulation of developmental gene expression
  programs will correlate with specific epigenetic
  markers on the HCNE control regions

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Strategy

• Map histone methylation patterns in mouse
  embryonic stem (ES) cells across specific
  regions of the genome
• Use ChiP (chromatin immunoprecipitation) on a
  genomic Chip (tiling genomic oligonucleotide
  arrays)
• Focus on arrays that represent HOX and HCNE
  sequences

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Bivalent domains contain BOTH repressive AND
activating histone modifications

• Confirmed high concordance of H3K4me and
  transcriptional start sites (TSS)
• H3K4me domains relatively small
• H3K27Me domains much larger
• Three quarters of the H3K27Me domains contained
  H3K4me domains within them
• These are termed bivalent domains as they
  harbour both activating and repressive marks

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Bivalent domains repressive AND activating
histone modifications
H3K4me
H3K27me
A higher than expected incidence of bivalents
occur in HCNEs
Fig 1A
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Hypothesis

• Genes that encode proteins that establish cell
  identity are enriched for bivalent domains
• These bivalent domains are responsible for
  maintaining developmentally important genes in a
  poised state that resolve one way or the other
  through differentiation

Prediction

• Differentiated cells will contain few, if any,
  bivalent domains

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Strategy

• Look at chromatin marks on same regions in
  differentiated cell types
• Mouse embryonic fibroblasts (MEFs)
• Mouse primary lung fibroblasts (MLFs)
• C2C12 myoblasts
• Neuro2a neuroblastoma cells

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Fig 2
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Bivalent domains in ES cells are not bivalent in
differentiated cells

• Bivalent domains on TSSs (transcriptional start
  sites) of ES cells are monovalent in
  differentiated cells

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Validation?

• This is a novel chromatin mark
• Nobody will believe us
• Also, maybe these two states (H3K27Me and H3K4Me)
  exist separately in different subpopulations of
  chromatin pulled out of the stem cells in ChIP
• Test coincidence of H3K4Me and H3K27Me with
  sequential ChIP
• Test fold enrichment quantitatively with real
  time quantitative (Q)PCR

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QPCR (Real Time quantitative PCR)

• Amplification products are labeled by a DNA
  binding dye or probe chemistry that emit
  fluorescent signal when excited
• The signal strength of the emitted light is
  directly proportional to the amount of PCR
  product in the reaction
• The fluorescence intensity is detected and
  recorded every cycle
• DNA amplification is monitored as the reaction
  occurs (hence, real time)
• Reverse transcriptase PCR and real time PCR are
  not necessarily the same things always check
  context!

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QPCR, like regular PCR, occurs in stages
PCR just getting started amount of product not
proportional to amount of starting material
(cant measure it anyway)
End of reaction and PCR components depleted
amount of product not proportional to amount of
starting material
Linear phase amount of product is proportional
to amount of starting material
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Real time measurement during log phase of PCR
correlates with starting concentration
http//www.dorak.info/genetics/realtime.html
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Fig 3
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Bivalents keep genes silent, but poised for
later expression
H3K27Me is epistatic to H3K4Me
Fig 4
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Poised state to resolved state differentiation
in cell culture
Reverse transcriptase PCR ie., starting
template is mRNA population (not the same as QPCR)
All genes associated with bivalent domains
Bivalent marks resolve into monovalent K4Me or
K27Me, depending on the transcriptional state
after differentiation
Fig 5
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Findings and significance

• Bivalent domains hold developmentally important
  genes in a poised state in stem cells
• This poised state is fundamentally repressive,
  but contains within it the potential for
  activation upon differentiation
• The poised state can also resolve into a
  continued repressed state upon differentiation
• The resolved monovalent domains are much larger
  than the bivalent domains
• This may create a larger pool of modified
  histones with which to perpetuate the epigenetic
  mark

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How is are the bivalent domains established?

• DNA sequence features???
• H3K4Me in ES cells positively correlates with CpG
  islands (a marker for promoters)
• H3K4Me is a mark made by trxG proteins
• trxG proteins associate with CpG-rich DNA
• H3K27Me in ES cells positively correlates with
  transposon-poor sequences (as do the HCNEs)
• Transposon rich sequences acquire different
  repressive marks that may interfere with bivalent
  structure
• What is the mark in this region that attracts the
  PcG proteins that methylate K27? Dont know
• These correlations between sequence features and
  methylation states breaks down in differentiated
  cells because lineage-specific transcriptional
  programs result in transfer to a greater degree
  of epigenetic control

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Questions

• HCNEs do they define targets for creating
  specific chromatin conformations and/or nuclear
  localizations that affect the establishment of
  bivalent domains?
• Do bivalents that persist in differentiated cell
  types correspond with genes that have the
  potential for further, later induction?