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.

Reproductive and therapeutic cloning begin the same way

A decade of reproductive cloning

1996 1998 2000 2001 2003

200520052004

RETRACTED

can’t culture

can culture

Many (all?) fates possible Limited set of fates possible

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 methyltransferasesEnhancer 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)

HCNE’s: highly conserved non-coding elements in vertebrates

• 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

Compare non-coding sequence

Filter out transposons etc and matches less than 100bp

Map sequences on human genome

Functional in vivo assay in zebrafish

Fugu rubripes Homo sapiens

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

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

HCNEs cluster near developmentally important genes

in vertebrates

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• 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• 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

• 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

H3K27me H3K4me

Fig 1A

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

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

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

Most bivalents in ES cells are either H3K27Me OR H3K4Me in differentiated cells

Fig 2

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?• 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)

• 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

Initial phasePlateauphase

Logarithmic phase

PCR just getting started: amount of product not

proportional to amount of starting material (can’t

measure it anyway)

Linear phase: amount of product is proportional to amount of starting

material

End of reaction and PCR components

depleted: amount of product not

proportional to amount of starting

material

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

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

H3K27Me only

H3K4Me only

Bivalent

Differentiated cells

Fig 3

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

H3K27Me is epistatic to H3K4MeFig 4

Poised state to resolved state: differentiation in cell culture

All genes associated with bivalent domains

Fig 5

Bivalent marks resolve into monovalent K4Me or K27Me, depending on the transcriptional state after differentiation

Reverse transcriptase PCR – ie., starting template is mRNA population (not the same as QPCR)

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?

• 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

• 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?