Download Epigenetics an introduction

Epigenetics therapy
an introduction
Epigenetics refers to heritable changes in gene
expression that occur without alteration in DNA
sequence.
There are two primary and interconnected
epigenetic mechanisms - DNA methylation and
covalent modification of histones.
In addition, it is also becoming apparent that
RNA is intimately involved in the formation of a
repressive chromatin state.
Definition
• Epigenetics: all meiotically and mitotically
heritable changes in gene expression that are not
coded in the DNA sequence itself
• DNA methylation
• RNA associated silencing
• Histone modification
• Genotype and phenotype
Nature 2004;429:457-73
• DNA methylation
• Histone covalent modification
• Genetic and epigenetic interplay
towards cancer
• DNA methylation and cancer
• Histone modifications and cancer
• An epigenetic role for RNA
• Genes
• Chromatin
• Histone
• nonhistone
chromosomal proteins
• Nucleosome
• Chromosome
Initiation of DNA
transcription
Methods to repress genes
• Competitive DNA binding
• Masking the activation surface
• Direct interaction with the general
transcription factors
• Recruitment of repressive chromatin
remodeling complex
• Recruitment of histone deacetylases
Methods to repress genes
Methylation
Scaffold
protein
Histone
modification
Epigenetic diseases
Nature 2004;429:457-73
DNA methylation
DNA methylation
•At promoter, DNA
methylation suppresses
transcription
DNA methyltransferase
•With deamination, DNA
methylation induces C T
mutation
DNA methylation in cancer
Tumor suppressor
genes?
Nature Clin Pract Oncol
2005;2:S4-S11
Demethylation in cancer therapy
Nat Rev Drug Discov
2006;5:37-50
DNA methylation inhibitor, nucleoside
analogues and non-nucleoside analogues
Nat Rev Cancer 2006;5;37-50
Demetheylation effect of cytidine analogs in
fibroblast cell lines
Differentiation
Methylation
azacytidine
decitabine
Cell 1980;
20:85-93
Demethylation agent for MDS
Role of p15ink4b
p15 protein
Blood 2002;100:2957-64
Blood 2004;103:1635-40
Azacitidine improve response
rate* and time to leukemia
transformation in MDS but not
overall survival
JCO 2002;20:2429-40
Time to leukemia
transformation or death
P<0.0001
Overall survival p=0.10
Decitabine for MDS
Decitabine improves quality of life, decreases
RBC transfusion for MDS
Decitabine didn’t improve AML-free survival or
overall survival(14.0vs 14.9mo, p= 0.636)
Cancer 2006;106:1794-80
NPM-ALK and STAT5 methylation
•
•
•
•
NPM-ALK kinase, t(2;5) in ALCL
Suppression of STAT 5 in ALCL cell line
siRNA of NPM-ALK reactivate STAT5
NPM-ALK transfection results in methlyation of STAT5
promoter inactivate STAT5
Nat Med 2007;13:1341-8
AML1/ETO and microRNA-223
•
•
•
•
AML1/ETO = t(8;21) in AM2L
Low level of miR-223 in AM2L t(8;21)
AML1/ETO induces methylation of miR-223 promoter
Inhibition of AML1/ETO reverses miR-223 expression
AML1-ETO(-)
AML1-ETO(+)
Cancer cell 2007;12:457-66
Ras mediates methylation of
unrelated genes
The specificity of epigenetic change
Nature 2007;449:1073-78
Histone acetylation
(HDAC)
Role of histone
modification
• DNA transcription
• DNA repair
• DNA replication
Cell 2007;128:693-705
Histone modification
the histone code
•
•
•
•
•
Acetylation
Methylation
Phosphorylation
Ubiquitylation
sumoylation
Nature
2007;447:407-12
Nature
2007;447:407-12
Nat Rev Drug Disc
2006;5:769-84
HDAC inhibitors
Nat Rev Cancer 2006;5;37-50
SAHA (vorinostat) in cutaneous T-cell lymphoma
JCO 2007;25:3109-15
SAHA in ovarian cancer
Gynecologic Oncology 2008 online published
HDAC inhibitor suppress ERα activity in
estrogen dependent breast cancer cell line
trichostatin A
sensitizes estrogen
receptor α-negative
breast cancer cells
to tamoxifen
Oncogene
2004;23:1724-36
HDAC inhibitor Trichostatin A induces
global and gene-specific DNA demethylation
Biochem. Pharmacol 2007;73:1297-1307
T24
Problems in epigenetics
agents in cancer therapy
• Differentiation or cytotoxic agents?
• Specificity and Selectivity of the
treatment targets?
• Correction of epigenetic change and
underlying gene mutaion?
• No survival benefit till now!!
Interchromosome regulation of genes
Nature 2007;447:413-7
Scaffold protein
SATB1
• More higher
structure than
chromatin
• Play important role
in T-cell
development
Nat Genetics 2006;38:1229-30
SATB1
SATB1 reprogrammes gene
expression to promote breast
tumour growth and metastasis
Nature 2008;452:187-193
Summary
Nature 2007;447:433-40
Summary
• Methylation and HDAC
• control of transcription through electric
change or 3-D conformational change of
chromatin
• Control the phenotype
• Application of cancer therapy?
Thank you
for
your attention
Decitabine induces ER gene
expression in ER(-) breast cancer
cell line(MDA-MB-231ELuc.6 cells)
Cancer Res
1995;55:2279-83
HDAC inhibitor and azacitidine together
induce ER expression in ER(-) breast
cancer line (MDA MB-231)
Cancer Res 2001;61:7025-29
Nature 2004;429:457-73
Cell 2007;128:655-68
Proteins that associate
preferentially with modified versions
of histone H3 and histone H4.
Crosstalk between histone modification
Cell 2007;128:693-705
Epigenetics
The genomes of several plants have been sequenced, and those
of many others are under way.
But genetic information alone cannot fully address the
fundamental question of
how genes are differentially expressed during cell
differentiation and plant development, as the DNA sequences in
all cells in a plant are essentially the
same.
Several important mechanisms regulate transcription by
affecting the structural properties of the chromatin:
- DNA cytosine methylation,
- covalent modifications of histones, and
- certain aspects of RNA interference (RNAi),
They are referred to as “epigenetic” because they direct “the
structural adaptation of chromosomal regions so as to register,
signal or perpetuate altered activity states”.
DNA methylation
Three distinct DNA methylation pathways with overlapping
functions have been characterized in Arabidopsis.
1 The mammalian DNMT1 homolog METHYLTRANSFERASE 1
(MET1) primarily maintains DNA methylation at CG sites (CG
methylation).
2 The plant-specific CHROMOMETHYLASE3 (CMT3) interacts
with the H3
Lys9 dimethylation (H3K9me2) pathway to maintain DNA
methylation at CHG sites (CHG methylation, H = A, C, or T).
3 The DNMT3a/3b homologs DOMAINS REARRANGED
METHYLASE 1 and 2 (DRM1/2) maintain DNA methylation at CHH
sites (CHH methylation), which
requires the active targeting of small interfering RNAs (siRNAs).
DNA methylation
Methylated and unmethylated DNA can be distinguished by
three major types
of experimental approaches:
(1) sodium bisulfite treatment that converts cytosine (but not
methylcytosine) to uracil,
(2) enzymatic digestion (using methylation-specific
endonucleases or methylation sensitive isoschizomers), and
(3) affinity purification or immunoprecipitation (with methylcytosine binding proteins and antibodies to methyl-cytosine,
respectively).
The methylated fraction of the genome is then visualized by
hybridizing treated
DNA to microarrays.
DNA methylation
Results from these microarray studies were largely consistent:
1
~20% of the Arabidopsis genome is methylated.
2 Transposons and other repeats comprise the largest fraction,
whereas the promoters of endogenous genes are rarely
methylated.
3 Surprisingly, methylation in the transcribed regions of
endogenous genes is unexpectedly rampant (dt. ungezügelt).
4 More than one-third of all genes contain methylation (called
“body methylation”) that is enriched in the 3′ half of the
transcribed regions and primarily occurs at CG sites.
DNA methylation
DNA methylation is critically important in silencing transposons
and regulating plant development.
Severe loss of methylation results in a genome-wide massive
transcriptional reactivation of transposons, and quadruple
mutations in drm1 drm2 cmt3 met1 cause embryo lethality.
Interestingly, the role of DNA methylation in regulating
transcription appears to
depend on the position of methylation relative to genes:
- Methylation in promoters appears to repress transcription.
- Paradoxically, however, body-methylated genes are usually
transcribed at moderate to high levels and are transcribed less
tissue-specifically relative to unmethylated genes.
DNA methylation: new paper
Recently, Cokus et al. combined sodium bisulfite treatment of
genomic DNA with ultrahigh-throughput sequencing (>20× genome
coverage) to generate the first DNA methylation map for any
organism at single-base resolution.
This “BS-Seq” method has several advantages over microarray-based
methods :
1 it can detect methylation in important genomic regions that are not
covered by any microarray platform (such as telomeres, ribosomal DNA,
etc.).
2 it reveals the sequence contexts of DNA methylation (i.e., CG,
CHG, and CHH) and therefore provides important information regarding
the epigenetic pathways that function at any given locus. E.g. all three
types of methylation colocalize to transposons, but gene body methylation
occurs exclusively exclusively at CG sites.
3 BS-Seq is more effective in detecting light methylation and subtle
changes (e.g., in mutants).
4 the theoretically unlimited sequencing depth makes it possible to
quantitatively measure the percentage of cells in which any particular
cytosine is methylated, thereby offering important clues regarding
potential cell-specific DNA methylation.
RNA-directed DNA methylation
Putative pathway for RNA directed DNA
methylation in A. thaliana. Target loci (in
this case tandemly repeated sequences;
coloured arrows) recruit an RNA
polymerase IV complex consisting of
NRPD1A and NRPD2 through an unknown
mechanism, and this results in the
generation of a single-stranded RNA
(ssRNA) species. This ssRNA is converted
to double-stranded RNA (dsRNA) by the
RNA-dependent RNA polymerase RDR2.
The dsRNA is then processed into 24-nucleotide siRNAs by DCL3. The
siRNAs are subsequently loaded into the protein AGO4, which associates
with another form of the RNA polymerase IV complex, NRPD1B–NRPD2.
AGO4 that is ‘programmed’ with siRNAs can then locate homologous
genomic sequences and guide the protein DRM2, which has de novo
cytosine methyltransferase activity. Targeting of DRM2 to DNA sequences
also involves the chromatin remodelling protein DRD1.
DNA methyltransferase structure and function
Plant and mammalian
genomes encode
homologous cytosine
methyltransferases,
of which there are
three classes in
plants and two in
mammals.
A. thaliana MET1 and Homo sapiens DNMT1 both function to
maintain CG methylation after DNA replication, through a
preference for hemi methylated substrates, and both have aminoterminal BAH domains of unknown function.
De novo DNA methylation is carried out by the homologous
proteins DRM2 (in A. thaliana) and DNMT3A and DNMT3B (both in
H. sapiens). Despite their homology, these proteins have distinct Nterminal domains, and the catalytic motifs present in the cytosine
methyltransferase domain are ordered differently in DRM2 and the
DNMT3
proteins.
Plants
also
have
another
class
of
methyltransferase, which is not found in mammals. CMT3 functions
together with DRM2 to maintain nonassociated-CG methylation.
PWWP, Pro-Trp-Trp-Pro motif; UBA, ubiquitin.
Small RNAs
Four major endogenous RNAi pathways have been described in
Arabidopsis.
Functioning at at the posttranscriptional level through mRNA
degradation and/or translation inhibition are
the microRNA (miRNA),
transacting siRNA (ta-siRNA), and
natural-antisense siRNA (nat-siRNA) pathways.
In contrast, the siRNA pathway is involved in gene silencing both
transcriptionally by directing DNA methylation and
posttranscriptionally by guiding mRNA cleavage.
Function of small RNAs
MicroRNAs (miRNAs) and transacting siRNAs (tasiRNAs) are
primarily involved in regulating gene expression and plant
development,
siRNAs play a major role in defending the genome against the
proliferation of invading viruses and endogenous transposable
elements.
The function of the fourth type of sRNAs, natural-antisense
siRNAs (nat-siRNAs), is not entirely clear but is likely related to
plant stress responses
Small RNAs
Millions of 21- to 24-nucleotide (nt) siRNAs have been cloned and
sequenced from wild-type Arabidopsis plants and siRNA pathway
mutants.
Most of these studies generated not only sequence information
necessary to map the siRNAs back to their originating genomic
loci, but also the length information of siRNAs that is indicative
of the processing enzymes involved (e.g., DICER-LIKE enzymes,
DCLs).
Small
RNAs
The majority of the siRNAs (>90%) are produced from doublestranded RNA (dsRNA) precursors generated by RNA polymerase
IV isoform a (Pol IVa) and
RNA-dependent RNA polymerase 2 (RDR2).
RNAP IV is a recently identified class of RNAP that is specific to
plant genomes. Unlike RNAP I, II, and III, RNAP IV appears to be
specialized in siRNA metabolism.
These dsRNA precursors are then processed by DCL3 to 24-nt
siRNAs (with partially redundant contributions from DCL2 and
DCL4) and become
preferentially associated with ARGONAUTE4, which then interacts
withPol IVb to direct DRM1/2- mediated CHH methylation.
Most of these siRNAs are derived from genomic loci corresponding
to transposons with high levels of CHH DNA methylation, and very
few arefound in protein-coding genes
Conclusions
Two major fractions of the Arabidopsis genome are associated with
and regulated by different epigenetic mechanisms:
(1) Genes are regulated by pathways such as H3K27me3, H3K4me2,
and miRNAs/ta-siRNAs/nat-siRNAs, whereas
(2) transposons and other repeats are silenced by DNA methylation,
H3K9me2, and siRNAs.
Such a functional distinction, however, is blurred when the two
genetic fractions overlap, which occurs much more frequently in
larger and more complex genomes.
Conclusions
Although increasingly comprehensive, such an epigenomic picture
remains static. Relatively little is known about how the plant
epigenome changes in response to developmental or environmental
cues.
A particularly interesting question may be how mechanisms that
evolved to stably silence transposons could offer the flexibility
required for the developmental regulation of endogenous genes.
In addition, we do not yet have a clear understanding of the nature
and the maintenance of the boundaries separating epigenetically
distinct chromatin compartments.
In some cases, genetic landmarks (such as the transcription unit)
may serve as borders; in other cases, the balancing acts of opposing
epigenetic mechanisms may help to stably maintain the epigenetic
landscape of plant genomes.