Epigenetics

1
Epigenetics

• ...is the study of heritable changes in gene
  expression that occur without a change in DNA
  sequence,
• DNA methylation,
• chromosome remodeling,
• RNA turnover.

2
Methylation

• Methylation Inhibits Transcription.

3
GC Islands

• Promoters in Eukaryotic cells are GC rich,
• The cytosines in these regions (and in the gene
  itself) can become methylated (5-methylcytosine),
• Specific proteins bind to methylated DNA, and in
  turn, inhibit transcription (RNA polymerase).

4
Stem Cells
Animal Cells

• Embryonic stem cells are undifferentiated,
• DNA is under-methylated,
• As the organism differentiates, genes become
  methylated, thus inactive,
• De-methylation can take place, however, incorrect
  de-methalation appears to be a critical feature
  in cloning regimes.

5
Chromosome Remodeling

• The XIST gene encodes a large molecule of RNA,
  and is encoded on the X chromosome,
• When two X chromosomes are present, the
  transcription, of one of them, predominates,
• XIST RNA accumulates along the X chromosome
  containing the active XIST gene and proceeds to
  inactivate all (or almost all) of the other
  hundreds of genes on that chromosome,
• XIST RNA does not travel over to any other X
  chromosome in the nucleus,
• Barr bodies are inactive X chromosomes compressed
  by XIST RNA.

6
Other Remodeling RNAs?

• ESTs (Expressed Sequence Tags) cDNA libraries
  are end sequenced,
• lots of non-protein-coding transcripts are
  found,
• Upwards of 60,000 RNA of these transcripts have
  been identified in the human genome,
• ignored until this year
• one active hypothesis is that they are involved
  in chromosome remodeling.

7
Epigenetics So Far

• ...epigenetic control of gene expression is now
  considered a component of normal development,
• stable repression of select genes in specific
  cell types.

8
Genome Surveillance

• ...epigenetic control of gene expression is now
  also considered a component of cellular defense,
• stable repression of select genes in specific
  cell types,
• defense against viruses,
• control of transposable elements,
• others?

9
Over Expression Studies

• Make a gene construct with,
• Structural Gene,
• Active promoter (often from a virus promoter),
• Marker gene to be able to determine
  transformation.

• Expect,
• Higher levels of protein,
• Gene-dosage phenotypes,
• Glorious publication.

Frequent Results no protein produced, scorn from
senior scientists.
10
Anti-Sense Studies

• Another good idea use a transgene with the
  coding sequence reversed...

11
Huh?
12
Expected Results

• Low, to no detectable transcript,
• Low, to no protein products,
• Glorious publication detailing gene function.

• Actual Results (Wacky)
• Phenotypes ranged from death to
  over-expression,
• Transcript levels were also extremely variable,
• Scorn from senior scientists.

13
Co-suppression(RNA turnover)

• T-DNA insertion can happen more than once per
  plant,
• Hard-headed, high self-esteem plant scientists
  published results indicating that...
• transgene expression often decreased as the copy
  number of transgene increased,
• Plants with more than one over-expression, or
  anti-sense construct, seemed to have the least
  transgene expression.

14
Modes

• ...Transcriptional Gene Silencing (TGS),
• RNA influences the methylation of promoters,
• ...Post-Translational Gene Silencing (PTGS),
• appears to involve the specific degradation of
  mRNA via a double-stranded RNA intermediate,
  dsRNA.

15
Co-Suppression as Reverse Genetics

• Plant scientists began using over-expression as a
  means to turn-off gene expression,
• Strangely, over-expressed genes were present on
  Northern blots as very small molecules,
• i.e. transcripts that should have been kbs in
  length, were present as 20-mers on Northerns.

16
RNAi(RNA interference)

• ...while attempting to do anti-sense KO of gene
  expression in C. elegans, Guo and Kemphues, Cell
  81, 611 (1995) observed that sense and anti-sense
  strands worked equally,
• in an anti-sense experiment, a gene is
  constructed so that it produces a complementary
  strand to an expressed transcript,
• the goal is to complement, thus inactivate the
  mRNA.
• ...following up, they found that dsRNA worked at
  least an order of magnitude better that either
  sense or anti-sense strands.

17
dsRNA
18
dsRNA Delivery

• ...dsRNA can move across cell boundaries,
• through the gut of C. elgans,
• injected,
• ingested, E. coli expressing a dsRNA,
• soaking,
• (through the vascular system of plants),
• ...or delivered as a heritable transgene.

19
amplification
20
(No Transcript)
21
It Works

• Arabidopsis

22
(No Transcript)
23
The Experiment

• Compare dsRNA transgenes with co-suppression and
  antisense constructs.

24
The Transgenes
1 transcript, 1 dsRNA molecule.
2 transcripts, 1 dsRNA molecule.
1 transcript, 1 antisense molecule.
1 transcript, 1 sense molecule.
25
The Transformation

• Agrobacteria mediated,
• electroporate modified T-DNA construct (with the
  transgene) into Agro,
• Suspend the Agro in a solution, dip the plants in
  the broth, and apply a vacuum,
• The Agro is pulled/pushed past the plant cuticle,
  and moves into location to form infections,
• Transformed plants are selected via a T-DNA
  specific marker.

26
T-DNA
marker gene(s)
active promoters transgene
virulence genes
Construct T-DNA
27
The Cast of Genes

• Agamous transcription factor, pistol is replaced
  by petals (organ identity)...

28
Clavata and Apetela(transcription factors)
Clavata (floral organ size) reduced internodes,
enlarged meristem
Apetela (floral meristem identilty) floral
organs replace petals.
29
Perianthia(transcription factor)
The perianthia mutant leads to flowers with 5
sepals, 5 petals, 5 stamens and 2 carpels. This
particular number and pattern of organs is the
predominant form of dicot flowers, but is not
found in the family Brassicaceae, of which
Arabidopsis is a member.
30
Results
31
RNA Levels?
Anti-sense probes...
Sense probes...
32
Protein Levels?
Western Blot
33
Is it Heritable?

• What do you expect?

34
Conclusions

• dsRNA does operate in plants,
• Provides reproducible, inheritable phenotypes,
• Results are highly penetrant, variable
  expressibility.

35
(No Transcript)
36
(No Transcript)
37
RNAi Range

• Yeast,
• Plants,
• Insects,
• Invertebrates,
• Vertebrates?

38
Vertebrates?
39
Mice?

• RNAi works in embryos, but as soon as the embryo
  is implanted in a womb, RNAi stops functioning.

40
Humans?

• All of the genes required to perform RNAi are
  present, and expressed,
• We dont know how they operate.

41
Next Week

• RNAi
• Genetics,
• Biochemistry.

42
Homologous Recombination Range

• Yes...
• Mice, many well characterized mammalian cells,
• bacteria, yeast,
• No (maybe)...
• C. elegans,
• Arabidopsis (done once, not repeated),
• Drosophila (shown in principle, not repeated),
• the rest?