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Biol/Chem 473. Schulze lecture 10: Stem cells and chromatin. 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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slide1

Biol/Chem 473

Schulze lecture 10:

Stem cells and chromatin

what are stem cells
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.
a decade of reproductive cloning

2004

2005

2005

A decade of reproductive cloning

2000

2001

1996

1998

2003

RETRACTED

slide6

Many (all?) fates possible

Limited set of fates possible

can’t culture

can culture

stem cell chromatin
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 can’t be established without them
two important histone methyltransferases enhancer of zeste pcg trithorax trxg

E(z) and orthologs recognize and methylate H3K27 (forms part of repressor complex PRC2 that maintains repression of homeotic and other genes in development)

Trithorax and orthologs recognize and methylate H3K4 (forms part of an activating complex that maintains activation of homeotic and other genes in development)

Two important histone methyltransferasesEnhancer of Zeste (PcG) & trithorax (trxG)
hcne s highly conserved non coding elements in vertebrates
HCNE’s: highly conserved non-coding elements in vertebrates

Fugu rubripes

Homo sapiens

Compare non-coding sequence

Filter out transposons etc and matches less than 100bp

  • These HCNE’s 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

Map sequences on human genome

Functional in vivo assay in zebrafish

Woolfe et al. (2005) PLOS Biology 3(1): 0116

hcnes cluster near developmentally important genes in vertebrates
HCNEs cluster near developmentally important genes in vertebrates

Woolfe et al. (2005) PLOS Biology 3(1): 0116

a bivalent chromatin structure marks key developmental genes in embryonic stem cells

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

hypothesis
Hypothesis
  • HCNE’s 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
strategy
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
bivalent domains contain both repressive and activating histone modifications
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
bivalent domains repressive and activating histone modifications
Bivalent domains: repressive AND activating histone modifications

H3K4me

H3K27me

A higher than expected incidence of bivalents occur in HCNE’s

Fig 1A

hypothesis16
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
strategy17
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
bivalent domains in es cells are not bivalent in differentiated cells
Bivalent domains in ES cells are not bivalent in differentiated cells
  • Bivalent domains on TSS’s (transcriptional start sites) of ES cells are monovalent in differentiated cells
validation
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
qpcr real time quantitative pcr
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!
qpcr like regular pcr occurs in stages

Logarithmic

phase

Initial phase

Plateau

phase

QPCR, like regular PCR, occurs in stages

PCR just getting started: amount of product not proportional to amount of starting material (can’t 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

real time measurement during log phase of pcr correlates with starting concentration
Real time measurement during log phase of PCR correlates with starting concentration

http://www.dorak.info/genetics/realtime.html

slide24

Differentiated cells

H3K27Me only

Bivalent

H3K4Me only

Fig 3

bivalents keep genes silent but poised for later expression
Bivalents keep genes silent, but “poised” for later expression

H3K27Me is epistatic to H3K4Me

Fig 4

poised state to resolved state differentiation in cell culture
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

findings and significance
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
how is are the bivalent domains established
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 HCNE’s)
  • 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? Don’t 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
questions
Questions
  • HCNE’s – 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?