1. Op, excuse me, your DNA is showing
Jordan Roselli
Graduate Student
San Francisco State University, California
CIRM Scholar

2. Overview Background Information Previous Research
Epigenetics, cell cycles, pluripotency
Previous Research
“Chromatin signatures of pluripotent cell lines” – Véronique Azuara et al.
Current Research

3. Background: Epigenetic Modifications
Epigenetics:
chemical and structural modifications made to the genome
do not alter the DNA sequence
are passed along as cells divide
can influence how genes are expressed.

4. Background: chomatin, nucelosomes, and histones
modifications
1. Chromatin
remodeling
3. DNA
modifications
Chromosome Image 1: The O’Sullivan Lab at the UPCI, ( Pittsburg )
Nucleosome Image: Nature Reviews, (Histone deacetylases and cancer: causes and therapies. Paul A Marks et al.)

5. Background: The cell cycles
Video from:

6. Background: Stem cells
endoderm
mesoderm
ectoderm
Embryonic stem cells
Image from:
Hematopoietic stem cells

7. Previous Research
Oct 2002
Transcriptional profiling of stem cells
The idea of a ‘signature’ for stem cells
Attempted to define a general gene expression profile for the stem cell ‘state’.

8. Previous Research
Looking at the gene expressions common to humans, mice, and non hematopoietic SCs.

9. Previous Research
Oct 2002
Genome-wide DNA replication profile for Drosophila melanogaster: a link between transcription and replication timing.
Schubeler, D. et al. Nature Genet. 32, 438–442 (2002).

10. Previous Research
Nov 2003
2003 Refined method to assess replication timing
Positive correlation between replication timing and transcriptional activity

11. Previous Research: Replication timing of the human genome
Genes represented on the 1Mb array of Lymphoblastoid cells in A.
Genes in Chromosome 22 in C.
Replication timing ratio in BLUE
Expression level in RED
“However, we were unable to find a significant correlation with absolute levels of expression. An example replication timing and expression level profile for chromosome 2 is shown Figure 7B, illustrating this lack of correlation.”
Still unsure about what is causing replication timing

12. Previous Research
The adjustable nucleosome: an epigenetic signaling module Bryan M. Turner
1997 X-ray crystallography of nucleosome
Nucleosome picture: Crystal structure of the nucleosome core particle at 2.8 Å resolution
Karolin Luger1, Armin W. Mäder1, Robin K. Richmond1, David F. Sargent1 and Timothy J. Richmond1

13. Previous Research
April 2006

14. A bivalent chromatin structure marks key developmental genes in embryonic stem cells
Previous Research:
ES – Embryonic stem cells
C2C12 – Myoblasts
Neuro2a – Neuroblastoma cells
MEF – mouse embryonic fibroblasts
MLF – mouse lung fibroblasts
YELLOW: Lys27 methylation only
GREEN: Lys4 methylation only
RED: Bivalent domain
BLUE: Transcriptional Start Sites

15. May 2006
Published May 2006

16. The question
Do embryonic stem cells have epigenetic features that distinguish them from stem cells with more restricted potential?

17. Methods Cell Types ES HSC T lymphocytes
CCE, OSG, and OS25
HSC
A4 and Pax5-/-
T lymphocytes
BrdU labeling – Thymidine analog
PI staining
CCE, OSG, and OS25: Mouse (Murine) embryonic stem cells
LABELED with BrdU – Bromodeoxyuridine. It has a Br instead of a CH3 like Thymidine (thymidine is a nucleoside thymine + deoxyribose sugar)
STAINED with PI – propidium Iodide. An intercalating agent between nucleic acid bases.
SEPARATED into 6 fractions according to DNA content.

18. Methods: supplemental data

19. Do ES cells replicate transcriptional regulatory genes at different times than other stem cells?

20. Figure 1: Replication Timing Profiles of ES, HSC, and T cells
Embryonic genes
Embryonic development
Haematopoietic genes
Neural Specific Genes
FINDINGS:” Implying that these epigenetic changes may reflect differences in transcriptional ‘competence’ and lineage affiliation, rather than overt changes in gene transcription”
Myogenic genes
Neuronal genes

21. Does replication-timing distinguish different embryonic stem cells?

22. Figure 2: Replication-timing Profiles of ES, EC and NP cells
EC: Embryonic Carcinoma cell lines F9 and P19
NP: Neural Progenitors
Investigating whether replication-timing profiles distinguish different stem cells, a subset of genes in two embryonic carcinoma cell lines, F9 and P19, which share the expression of numerous markers with ES cells but have a much narrower range of lineage potential.
F9 cells have a propensity to differentiate towards endoderm lineages (i.e. LUNG and PANCREAS)
P19 cells preferentially differentiate towards neuronal lineages.
FINDINGS: “The replication profiles observed reflect the different potentials of these embryonic carcinoma cell lines”

23. Are there underlying differences in the chromatin structure leading to the late replication of neural-specific genes in T-cells compared to ES cells?

24. Figure 3: A comparison of histone tales of ES and T cells
AcetylH3K9
MethylH3K4
Myf5 (early replication) and Hoxa1 (late replication) are controls.
“THE ABUNDANCE OF MODIFIED HISTONES AT THE PROMOTER REGION OF EACH GENE WAS ASSESSED AND EXPRESSED AS RATIO OF TOTAL H3 DETECTED (RELATIVE ABUNDANCE).”
FINDINGS: “Trimethylated H3K27 was absent from the promoters of expressed Oct4 and Sox2 genes, suggesting that this modification was selectively marking non-expressed genes.”
MethylH3K9
MethylH3K27

25. Is the trimethylation of H3K27 on the promoters of non-expressed genes selectively marking non-expressed genes?

26. Figure 4: The effect of Eed on gene expression in ES cells
Eed deficient ES cells
Cell lines: G8.1 and B1.3
Eed is required for the Ezh2 histone methylation function and H3K27 methylation
NO Eed means
NO H3K27 methylation
Eed is required for repressing neural-specific gene expression in ES cells
RT-PCR results
To verify that this increased expression wasn’t because of the Eed loss rather than the reflection of the different genetic backgrounds, they created Eed heterozygous cell lines Eed+/- ES cells “2.2”
The consistently lower expression levels in Eed heterzygous cells confirms that the loss of Eed (and H2K27 methylation) results in the upregulation of several neural genes in ES cells.

27. Does the loss of methylated H3K27 in Eed-deficient ES cells result in the global changes in chromatin accessibility?

28. Figure 5: The effect of Eed on replication timing in ES cells
Heterochromatin
Neural-specific genes
Rex1 is a locus that has been previously shown to switch from early to late replication when ES cells commit to neural progenitor fate and six neighboring loci.
Replication patterns are the same in ES cells as they are in Eed-/- deficient cells. γ-satellite and X141 are regions of DNA that are known to in Heterochromatin and should replicate LATE.
“The loss of H3K27 methylation resulted in the premature expression of several neural-specific genes in ES cells, but had modest effect on the timing of locus replication.”

29. Findings
Epigenetic changes may reflect differences in transcriptional ‘competence’ and lineage affiliation, rather than overt changes in gene transcription.
Trimethylated H3K27 marking non-expressed genes
Trimethylated H3K27 was absent from the promoters of expressed Oct4 and Sox2 genes, suggesting that this modification was selectively marking non-expressed genes

30. Findings
Several inactive, neural-specific genes replicate earlier in ES cells than in HSC and lymphocytes
Those same genes are enriched for acetylated H3K9 and methylated H3K4
Unexpectedly, these genes were trimethylated for H3K27
PR2C preventing the inappropriate expression of some neural-specific genes in ES cells?

31. Findings
Genes showing high levels of acetylated H3K9 replicated early in S phase regardless of whether they were expressed or not.
Low levels of acetylated H3K9 were associated with late replication.
Replication profiles useful for detecting cellular differentiation stage and lineage preference in EC cell lines.

32. Current Research
The adjustable nucleosome: an epigenetic signaling module Bryan M. Turner
Histones are either the star player or say very little

33. Thank you