Jan 30, 2016
DNA METHYLATION I(DNA METHYLATION AND ITS DETECTION)
EPIGENETIKA
Epigenetika se zabývá přenosem vlastností (informací), které nejsou uložené v sekvenci DNA.
Tyto informace jsou přenášené především v:
- struktuře a modifikacích chromatinu
- chemických modifikacích DNA
- RNA molekulách
Bacteria - protection against RE
Eukaryota - numerous effects
Bacteria - protection against RE
Bacteria - wide range of functions
Protista, Plantae, (Mammalia?)
See Ratel 2006
-CcGG- -GGcC-
all sites * Hemophilus parainfluenzae
-cCGG- -GGCc-
all sites *Moricardia sp.
-GaTC- -CTaG-
all sites (dam)Escherichia coli
-Cc(A/T)GG- -GG(T/A)cC-
all sites (dcm)Escherichia coli
-cG- -Gc-
some sites **mammals, some fungi (Neurospora) and plants
-cNG- -GNc-
some sites **plants
An example of a modified restriction enzyme recognition site. These sites are usually modified in organisms with the corresponding restriction activity.
** Fraction of CG dinucleotides or CNG trinucleotides varies with species and, to a lesser extent, with tissue.
Specific nucleotides are modified
DNA methylation
http://www.med.ufl.edu/biochem/keithr/research.html
5-methyl cytosine methylation
Mammalian DNA methyltransferases
Maintenance DNA methylation
De novo DNA methylation
Ignore
HW: Why?
Robertson 2002
Methods to study DNA methylation
Global methylation analysis
HPLC (complete hydrolysis, AP)
TLC (complete hydrolysis, AP, 32P labeled)
Sequence-specific methylation analysis
Methylation sensitive restriction enzymes - a number of methods
Bisulfite sequencing
MeDIP
For more details, see Oakeley, 1999
Bisulfite sequencing
From Oakeley, 1999
http://www.methods.info/Methods/DNA_methylation/Bisulphite_sequencing.html
dsDNA is resistant to conversion!
HW:Why isn’t 5mC
converted?
Classical protocol (bisulfite protocol.doc)
- starting material: cells or DNA up to 200 ng- extremely sensitive (100 DNA molecules)- based on agarose embedding
Drawbacks:- time consuming (approx. 10-11 hours)- low throughput (typically up to 8 samples/run)- low yield (200 ng max/reaction)
Epitect Bisulfite Sequencing Kit(Qiagen)
http://www1.qiagen.com/Products/Epigenetics/Epitect/EpitectBisulfiteKit.aspx?ShowInfo=1
Epitect
- starting material: DNA 1 ng - 2 g- sensititvity OK for most applications- faster (cca 6 hours), throughput OK
Whatever …
The critical component are primers!!
AAAn
ORF1 ORF25’ UTREN RT
~6 kb
- active, autonomous, non-LTR class
- retrotransposition in cis
- the most abundant retrotransposon in mammalian genomes
- 4-500 000 insertion in the human genome
- ~100 full length intact elements
- typically silenced in somatic cells (hypermethylation)
Model case:L1 promoter methylation analysis
>L1 5’ UTRGGGGGGAGGAGCCAAGATGGCCGAATAGGAACAGCTCCGGTCTACAGCTCCCAGCGTGAGCGACGCAGAAGACGGTGATTTCTGCATTTCCATCTGAGGTACCGGGTTCATCTCACTAGGGAGTGCCAGACAGTGGGCGCAGGCCAGTGTGTGTGCGCACCGTGCGCGAGCCGAAGCAGGGCGAGGCATTGCCTCACCTGGGAAGCGCAAGGGGTCAGGGAGTTCCCTTTCCGAGTCAAAGAAAGGGGTGACGGACGCACCTGGAAAATCGGGTCACTCCCACCCGAATATTGCGCTTTTCAGACCGGCTTAAGAAACGGCGCACCACGAGACTATATCCCACACCTGGCTCGGAGGGTCCTACGCCCACGGAATCTCGCTGATTGCTAGCACAGCAGTCTGAGATCAAACTGCAAGGCGGCAACGAGGCTGGGGGAGGGGCGCCCGCCATTGCCCAGGCTTGCTTAGGTAAACAAAGCAGCAGGGAAGCTCGAACTGGGTGGAGCCCACCACAGCTCAAGGAGGCCTGCCTGCCTCTGTAGGCTCCACCTCTGGGGGCAGGGCACAGACAAACAAAAAGACAGCAGTAACCTCTGCAGACTTAAGTGTCCCTGTCTGACAGCTTTGAAGAGAGCAGTGGTTCTCCCAGCACGCAGCTGGAGATCTGAGAACGGGCAGACTGCCTCCTCAAGTGGGTCCCTGACCCCTGACCCCCGAGCAGCCTAACTGGGAGGCACCCCCCAGCAGGGGCACACTGACACCTCACACGGCAGGGTATTCCAACAGACCTGCAGCTGAGGGTCCTGTCTGTTAGAAGGAAAACTAACAACCAGAAAGGACATCTACACCGAAAACCCATCTGTACATCACCATCATCAAAGACCAAAAGTAGATAAAACCACAAAG
1) Find your sequence – NCBI Genbank and Pubmed
BISULFITE SEQUENCING STEP BY STEP
BISULFITE SEQUENCING STEP BY STEP
>L1 5’ UTRGGGGGGAGGAGCCAAGATGGCCGAATAGGAACAGCTCCGGTCTACAGCTCCCAGCGTGAGCGACGCAGAAGACGGTGATTTCTGCATTTCCATCTGAGGTACCGGGTTCATCTCACTAGGGAGTGCCAGACAGTGGGCGCAGGCCAGTGTGTGTGCGCACCGTGCGCGAGCCGAAGCAGGGCGAGGCATTGCCTCACCTGGGAAGCGCAAGGGGTCAGGGAGTTCCCTTTCCGAGTCAAAGAAAGGGGTGACGGACGCACCTGGAAAATCGGGTCACTCCCACCCGAATATTGCGCTTTTCAGACCGGCTTAAGAAACGGCGCACCACGAGACTATATCCCACACCTGGCTCGGAGGGTCCTACGCCCACGGAATCTCGCTGATTGCTAGCACAGCAGTCTGAGATCAAACTGCAAGGCGGCAACGAGGCTGGGGGAGGGGCGCCCGCCATTGCCCAGGCTTGCTTAGGTAAACAAAGCAGCAGGGAAGCTCGAACTGGGTGGAGCCCACCACAGCTCAAGGAGGCCTGCCTGCCTCTGTAGGCTCCACCTCTGGGGGCAGGGCACAGACAAACAAAAAGACAGCAGTAACCTCTGCAGACTTAAGTGTCCCTGTCTGACAGCTTTGAAGAGAGCAGTGGTTCTCCCAGCACGCAGCTGGAGATCTGAGAACGGGCAGACTGCCTCCTCAAGTGGGTCCCTGACCCCTGACCCCCGAGCAGCCTAACTGGGAGGCACCCCCCAGCAGGGGCACACTGACACCTCACACGGCAGGGTATTCCAACAGACCTGCAGCTGAGGGTCCTGTCTGTTAGAAGGAAAACTAACAACCAGAAAGGACATCTACACCGAAAACCCATCTGTACATCACCATCATCAAAGACCAAAAGTAGATAAAACCACAAAG
1) Find your sequence
HW:look at CpG density of: EGFP, Bluescript, Actin promoter and transcribed region, IAP LTR, L1 ORF2
BISULFITE SEQUENCING STEP BY STEP
>L1 5’ UTRGGGGGGAGGAGCCAAGATGGCXYAATAGGAACAGCTCXYGTCTACAGCTCCCAGXYTGAGXYAXYCAGAAGAXYGTGATTTCTGCATTTCCATCTGAGGTACXYGGTTCATCTCACTAGGGAGTGCCAGACAGTGGGXYCAGGCCAGTGTGTGTGXYCACXYTGXYXYAGCXYAAGCAGGGXYAGGCATTGCCTCACCTGGGAAGXYCAAGGGGTCAGGGAGTTCCCTTTCXYAGTCAAAGAAAGGGGTGAXYGAXYCACCTGGAAAATXYGGTCACTCCCACCXYAATATTGXYCTTTTCAGACXYGCTTAAGAAAXYGXYCACCAXYAGACTATATCCCACACCTGGCTXYGAGGGTCCTAXYCCCAXYGAATCTXYCTGATTGCTAGCACAGCAGTCTGAGATCAAACTGCAAGGXYGCAAXYAGGCTGGGGGAGGGGXYCCXYCCATTGCCCAGGCTTGCTTAGGTAAACAAAGCAGCAGGGAAGCTXYAACTGGGTGGAGCCCACCACAGCTCAAGGAGGCCTGCCTGCCTCTGTAGGCTCCACCTCTGGGGGCAGGGCACAGACAAACAAAAAGACAGCAGTAACCTCTGCAGACTTAAGTGTCCCTGTCTGACAGCTTTGAAGAGAGCAGTGGTTCTCCCAGCAXYCAGCTGGAGATCTGAGAAXYGGCAGACTGCCTCCTCAAGTGGGTCCCTGACCCCTGACCCCXYAGCAGCCTAACTGGGAGGCACCCCCCAGCAGGGGCACACTGACACCTCACAXYGCAGGGTATTCCAACAGACCTGCAGCTGAGGGTCCTGTCTGTTAGAAGGAAAACTAACAACCAGAAAGGACATCTACACXYAAAACCCATCTGTACATCACCATCATCAAAGACCAAAAGTAGATAAAACCACAAAG
2) Word -> REPLACE CG WITH XY
BISULFITE SEQUENCING STEP BY STEP
>L1 5’ UTRGGGGGGAGGAGTTAAGATGGTXYAATAGGAATAGTTTXYGTTTATAGTTTTTAGXYTGAGXYAXYTAGAAGAXYGTGATTTTTGTATTTTTATTTGAGGTATXYGGTTTATTTTATTAGGGAGTGTTAGATAGTGGGXYTAGGTTAGTGTGTGTGXYTATXYTGXYXYAGTXYAAGTAGGGXYAGGTATTGTTTTATTTGGGAAGXYTAAGGGGTTAGGGAGTTTTTTTTTXYAGTTAAAGAAAGGGGTGAXYGAXYTATTTGGAAAATXYGGTTATTTTTATTXYAATATTGXYTTTTTTAGATXYGTTTAAGAAAXYGXYTATTAXYAGATTATATTTTATATTTGGTTXYGAGGGTTTTAXYTTTAXYGAATTTXYTTGATTGTTAGTATAGTAGTTTGAGATTAAATTGTAAGGXYGTAAXYAGGTTGGGGGAGGGGXYTTXYTTATTGTTTAGGTTTGTTTAGGTAAATAAAGTAGTAGGGAAGTTXYAATTGGGTGGAGTTTATTATAGTTTAAGGAGGTTTGTTTGTTTTTGTAGGTTTTATTTTTGGGGGTAGGGTATAGATAAATAAAAAGATAGTAGTAATTTTTGTAGATTTAAGTGTTTTTGTTTGATAGTTTTGAAGAGAGTAGTGGTTTTTTTAGTAXYTAGTTGGAGATTTGAGAAXYGGTAGATTGTTTTTTTAAGTGGGTTTTTGATTTTTGATTTTXYAGTAGTTTAATTGGGAGGTATTTTTTAGTAGGGGTATATTGATATTTTATAXYGTAGGGTATTTTAATAGATTTGTAGTTGAGGGTTTTGTTTGTTAGAAGGAAAATTAATAATTAGAAAGGATATTTATATXYAAAATTTATTTGTATATTATTATTATTAAAGATTAAAAGTAGATAAAATTATAAAG
3) Word -> REPLACE C WITH T
BISULFITE SEQUENCING STEP BY STEP
>L1 5’ UTRGGGGGGAGGAGTTAAGATGGTCGAATAGGAATAGTTTCGGTTTATAGTTTTTAGCGTGAGCGACGTAGAAGACGGTGATTTTTGTATTTTTATTTGAGGTATCGGGTTTATTTTATTAGGGAGTGTTAGATAGTGGGCGTAGGTTAGTGTGTGTGCGTATCGTGCGCGAGTCGAAGTAGGGCGAGGTATTGTTTTATTTGGGAAGCGTAAGGGGTTAGGGAGTTTTTTTTTCGAGTTAAAGAAAGGGGTGACGGACGTATTTGGAAAATCGGGTTATTTTTATTCGAATATTGCGTTTTTTAGATCGGTTTAAGAAACGGCGTATTACGAGATTATATTTTATATTTGGTTCGGAGGGTTTTACGTTTACGGAATTTCGTTGATTGTTAGTATAGTAGTTTGAGATTAAATTGTAAGGCGGTAACGAGGTTGGGGGAGGGGCGTTCGTTATTGTTTAGGTTTGTTTAGGTAAATAAAGTAGTAGGGAAGTTCGAATTGGGTGGAGTTTATTATAGTTTAAGGAGGTTTGTTTGTTTTTGTAGGTTTTATTTTTGGGGGTAGGGTATAGATAAATAAAAAGATAGTAGTAATTTTTGTAGATTTAAGTGTTTTTGTTTGATAGTTTTGAAGAGAGTAGTGGTTTTTTTAGTACGTAGTTGGAGATTTGAGAACGGGTAGATTGTTTTTTTAAGTGGGTTTTTGATTTTTGATTTTCGAGTAGTTTAATTGGGAGGTATTTTTTAGTAGGGGTATATTGATATTTTATACGGTAGGGTATTTTAATAGATTTGTAGTTGAGGGTTTTGTTTGTTAGAAGGAAAATTAATAATTAGAAAGGATATTTATATCGAAAATTTATTTGTATATTATTATTATTAAAGATTAAAAGTAGATAAAATTATAAAG
4) Word -> REPLACE XY WITH CG
BISULFITE SEQUENCING STEP BY STEP
>L1 5’ UTRGGGGGGAGGAGTTAAGATGGTCGAATAGGAATAGTTTCGGTTTATAGTTTTTAGCGTGAGCGACGTAGAAGACGGTGATTTTTGTATTTTTATTTGAGGTATCGGGTTTATTTTATTAGGGAGTGTTAGATAGTGGGCGTAGGTTAGTGTGTGTGCGTATCGTGCGCGAGTCGAAGTAGGGCGAGGTATTGTTTTATTTGGGAAGCGTAAGGGGTTAGGGAGTTTTTTTTTCGAGTTAAAGAAAGGGGTGACGGACGTATTTGGAAAATCGGGTTATTTTTATTCGAATATTGCGTTTTTTAGATCGGTTTAAGAAACGGCGTATTACGAGATTATATTTTATATTTGGTTCGGAGGGTTTTACGTTTACGGAATTTCGTTGATTGTTAGTATAGTAGTTTGAGATTAAATTGTAAGGCGGTAACGAGGTTGGGGGAGGGGCGTTCGTTATTGTTTAGGTTTGTTTAGGTAAATAAAGTAGTAGGGAAGTTCGAATTGGGTGGAGTTTATTATAGTTTAAGGAGGTTTGTTTGTTTTTGTAGGTTTTATTTTTGGGGGTAGGGTATAGATAAATAAAAAGATAGTAGTAATTTTTGTAGATTTAAGTGTTTTTGTTTGATAGTTTTGAAGAGAGTAGTGGTTTTTTTAGTACGTAGTTGGAGATTTGAGAACGGGTAGATTGTTTTTTTAAGTGGGTTTTTGATTTTTGATTTTCGAGTAGTTTAATTGGGAGGTATTTTTTAGTAGGGGTATATTGATATTTTATACGGTAGGGTATTTTAATAGATTTGTAGTTGAGGGTTTTGTTTGTTAGAAGGAAAATTAATAATTAGAAAGGATATTTATATCGAAAATTTATTTGTATATTATTATTATTAAAGATTAAAAGTAGATAAAATTATAAAG
5) Design primers- size typically up to 500 bp, ideally around 300 bp
- avoid low complexity sequences- Tm around 55oC - select the right region!
BISULFITE SEQUENCING STEP BY STEP
>L1 5’ UTRGGGGGGAGGAGTTAAGATGGTCGAATAGGAATAGTTTCGGTTTATAGTTTTTAGCGTGAGCGACGTAGAAGACGGTGATTTTTGTATTTTTATTTGAGGTATCGGGTTTATTTTATTAGGGAGTGTTAGATAGTGGGCGTAGGTTAGTGTGTGTGCGTATCGTGCGCGAGTCGAAGTAGGGCGAGGTATTGTTTTATTTGGGAAGCGTAAGGGGTTAGGGAGTTTTTTTTTCGAGTTAAAGAAAGGGGTGACGGACGTATTTGGAAAATCGGGTTATTTTTATTCGAATATTGCGTTTTTTAGATCGGTTTAAGAAACGGCGTATTACGAGATTATATTTTATATTTGGTTCGGAGGGTTTTACGTTTACGGAATTTCGTTGATTGTTAGTATAGTAGTTTGAGATTAAATTGTAAGGCGGTAACGAGGTTGGGGGAGGGGCGTTCGTTATTGTTTAGGTTTGTTTAGGTAAATAAAGTAGTAGGGAAGTTCGAATTGGGTGGAGTTTATTATAGTTTAAGGAGGTTTGTTTGTTTTTGTAGGTTTTATTTTTGGGGGTAGGGTATAGATAAATAAAAAGATAGTAGTAATTTTTGTAGATTTAAGTGTTTTTGTTTGATAGTTTTGAAGAGAGTAGTGGTTTTTTTAGTACGTAGTTGGAGATTTGAGAACGGGTAGATTGTTTTTTTAAGTGGGTTTTTGATTTTTGATTTTCGAGTAGTTTAATTGGGAGGTATTTTTTAGTAGGGGTATATTGATATTTTATACGGTAGGGTATTTTAATAGATTTGTAGTTGAGGGTTTTGTTTGTTAGAAGGAAAATTAATAATTAGAAAGGATATTTATATCGAAAATTTATTTGTATATTATTATTATTAAAGATTAAAAGTAGATAAAATTATAAAG
5) Design primers- size typically up to 500 bp, ideally around 300 bp
- avoid low complexity sequences- Tm around 55oC - select the right region!
TTTT doesn’t matter
3’ End dG should be above -13.0(calculated from the last 7 nucleotides)
- ignore all dimers with positive dG- only primers with negative dG <-1.5 should concern you- combination of low negative dG and perfect basepairing at the 3’ end is the worst combination
Duplex analysis
Tips how to improve amplification
HOT START PCR
-add Taq pol only after denaturation step- use hot start Taq pol, e.g. Amplitaq GOLD from Perkin Elmer/ABI
- Amplitaq GOLD requires the intital denaturation step for 10-15 min at 95 oC
TOUCHDOWN PCR
94 oC for 15 min
94 oC for 30 sec62->55 oC for 30 sec72 oC for 1 min
94 oC for 30 sec55 oC for 30 sec72 oC for 1 min
72 oC for 15-20 min
14 cycles0.5 oC down/cycle
36 cycles
EXTREMELY SENSITIVE TO
CONTAMINATIONS !!!
BISULFITE SEQUENCING STEP BY STEP
5) Run PCR
6) cut 7) Gel Extraction(Qiagen)
8) TOPO TA II cloning(Invitrogen)
9) Miniprep(Qiagen)
10) Sequencing with SP6 primer
Restriction digest- cheap and fast- less information (up to a few CpGs)- not good for polymorphic sequences
Pyrosequencing http://www.pyrosequencing.com/
- short read (30nt)- quantitative ratio of polymorphic nucleotides- good for one sequence analyzed from many samples
Direction of sequencing is important!
-GT-rich regions are more difficult to sequence- cause problems especially with products >300 bp
400
700
The same PCR product (amount, purity …) but different strands sequenced
BioEdit –it’s good and it’s free!http://www.mbio.ncsu.edu/BioEdit/bioedit.html
Vector NTI is good but f*cking expensive
52 58 61 70
analysis of individual elements
single-locus analysis
AAAn
ORF1 ORF25’ UTREN RT
L1 p
ool
L1 6
p21
L1 6
p22
L1 6
q16
L1 8
q24
L1 X
p22
L1 X
q22
? ? ?
femaleundifferentiated
hES sample
How would you explain it?How would you test your explanation?
AAAn
ORF1 ORF25’ UTREN RT
8q24
Xp22
HOMEWORK
Oct-4 promoter analysis-PCR cloned in pCR II-sequenced with SP6
MeDIP
DNA METHYLATION II(EFFECTS OF DNA METHYLATION)
EUMETAZOA
PSEUDOCOELOMATES
COELOMATESPROTOSTOMES
DEUTEROSTOMESECHINODERMATA
CHORDATA
AMPHIBIA
NEMATODA
PISCES
MAMMALIA
ARTHROPODA
>350 MYA
>400 MYA
>600 MYA
Mus
Xenopus
Danio
Strongylocentrotus
Drosophila
Caenorhabditis
- a number of conserved genes - common basic cell types - highly variable DNA methylation - different development - conserved histone modifications- different sex determination- different epigenetic mechanisms
DNA METHYLATION ACROSS PHYLA
Jabbari 2004
-genomes carrying CpG methylation show lower frequencies of CpG
EXTENSIVE DNA METHYLATION LEAVES TRACES ….
http://www.chemsoc.org/chembytes/ezine/2001/pufulete_mar01.htm
C-T CONVERSION
0.1-0.2% of the cytosines methylated
Drosophila
DNA methylation in the zebrafish
DNA METHYLATION DISTRIBUTION- trends and exceptions
repetitive sequences
- noncoding tandem repeats (satellites) hypermethylated- coding tandem repeats - rDNA variable- interspersed elements - L1, IAP, Alu hypermethylated- telomeric repeats hypermethylated
unique sequences
- promoter - active genes +/- unmethylated- inactive genes variable
- “gene body” methylated
upstream of the UCE UCE core p.
dim-H3K9(inactive)
H4Ac(active)
HEK-293total DNA
rDNA
- pol I transcription- tandemly arrayed on five pairs of human acrocentric chromosomes- ~400 copies per haploid genome- typically half active, half inactive (all active in the oocyte)
Methylationhypo hyper
transformed cell lines 50% 50%primary human cells 100% 0%primary mouse cells 100% 0%undiff. ES cells 100% 0%diff. ES cells 100% 0%
130 bp repeats UCE Core
Annotation of UCE and CORE sequences based on Heix and Grummt , 1995
400 bp
human
mouse
- different CpG density between closely related species- gene activity does not correlate with methylation in both species- only inactive genes in transformed lines acquire methylation- sometimes, methylation is not very informative
Kim
et
al.
BM
C C
an
cer
20
06
6:1
80
CpG islands
CpG island is a region least 200 bp long and with a GC
percentage that is greater than 50% and with CpG frequency
that is greater than 6% (genome average is 1%).
Found in and near approximately 40% of
promoters of mammalian genes (about 70% in human promoters). A “typical” CpG island is 300-3000 bp long. The CpG sites in the CpG islands of promoters are typically unmethylated if
genes are expressed. This observation led to the
speculation that methylation of CpG sites in the promoter of a
gene may inhibit the expression of a gene.
PROMOTERS OF INACTIVE GENES
- hundreds of papers with contradictory data- methylation correlates with inactivity … but that’s it ...
CpG poor promoters - hypermethylated regardless of activitystrong CpG island promoters - hypomethylated regardless of activityweak CpG island promoters - distinct …testis-specific promoters - methylated in somatic cells
Mammalian DNA methyltransferases
Maintenance methylation - DNMT1
substrate: hemimethylated DNAfunction: restoration of DNA methylation after replication
De novo methylation - DNMT3a, 3b (3l)
substrate: unmethylated DNAfunction: establishemnt of new DNA methylation patterns
Li 2002
Mammalian DNA methyltransferases
Maintenance DNA methylation
De novo DNA methylation
Ignore
HW: Why?
Robertson 2002
Setting up and interpreting the mark …
Klose 2006
Setting up and interpreting the mark …
Klose 2006
DNA Methyl Binding Proteins MBDs
http://www.wcb.ed.ac.uk/bird.htm
http://homepages.ed.ac.uk/dmac/Bird_Lab/birdlab.html
MBDs
Klose 2006
Hendrich 2003
MBD1
MBD2
MBD3
MBD4
MECP2
KO MUTATIONSCHARACTERIZATION
-transcriptional repressor- X-linked-able to bind a single methyl-CpG-binds tightly to chromosomes, pericentromeric heterochromatin- associates with Sin3A HDAC complex and BRM complex- expression correlates with maturation of the neural system
-mutant ES cells grow and can differentiate (Tate et al., 1996)-essential for embryonic development (Tate et al., 1996)-not essential for embryonic development (Chen et al. 2001)
Rett syndrome- various mutations- only in females (lethal in m.)
Angelmann syndrome?Autism?
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM
-transcriptional repressor from the MeCP1 complex- also described as demethylase - but nobody reproduced that!-MBD2/ NuRD complex, HDACs-MBD2 recruiting the NURD complex to methylated DNA?
-glycosylase domain, DNA repair- interacts with mismatch repair protein MLH
primary microsatellite-instability (MSI) tumors
-Mbd4 -/- mice have a 3-fold increase in the frequency of C-to-T transitions at CpG sites (Millar et al. 2002)
-transcriptional repressor from the MeCP1 complex-requires more than a single methyl-CpG-in a complex with HDAC3-interacts with the Suv39h1-HP1 heterochromatic complex
-does not bind methylated DNA-MBD3/ NuRD complex (distinct from MBD2 NURD)-MBD3 an integral part of the NURD complex?
-Mbd3 -/- ES cells are viable but fail to form a stable NuRD complex, differentiation defect, LIF-independent self-renewal. - Mbd3-/- mice die during early embryogenesis
Kaiso -lacks MBD domain-recognizes 5mC in CGCG through zinc-finger domains- transcriptional repressor
Mbd-/- mice have deficits in adult neurogenesis and hippocampal function (Zhao et al., 2003)
Autism?
Mbd2-/- mice are viable and fertile! Maternal behavior of Mbd2-/- mice is defective (Hendrich et al. 2001)
Kaiso -/-t mice show resistance to intestinal cancer
Klose 2006
Klose 2006
- when it is associated with transcriptional repression, there are usually also chromatin modifications consistent with the pattern
- information about DNA sequence methylation without information about location (promoter, intergenic, intronic, exonic …) and expression has low information content
- methylated promoter does not automatically belong to an inactive gene while unmethylated promoter does not automatically belong to an active gene
- it’s OK to say that DNA methylation is typically associated with transcriptional repression
- be open minded and expect the unexpected ...
A few final notes regarding DNA methylation
- inhibition of DNMT1, - - replication dependent
DNA DEMETHYLATION
PASSIVE
- replication independent- clearly demonstrated in some cases- unknown mechanism, unknown demethylase. DNA repair/glycosylase?
ACTIVE
- DNA methylation is stable but reversible
CpG methylation during development
Adapted from Mayer et al. (2000). Nature 403(6769):501-2
1-cell 6h 1-cell 8h 2-cell 22h 4-cell 45h
Adapted from Rougier et al. (1998). Genes Dev 12(14):2108 -13
4-cell asymmetrical staining 8-cell weak labeling, rare asymmetrical chr.
NOBODY EVER REPRODUCED THESE DATATHIS PAPER WAS NEVER RETRACTED
HW: What’s the closest mammalian homologue of ROS1?
Introduction and Background:What hypotheses are being tested in this paper?
What information induced the authors to perform the experiments?What new methods or insights brought to bear on the problem?
Methods:What are the critical methods of the paper?
What enabling technologies are used?What are the weaknesses of the methods used?
Are there other or better approaches that could be used?If this is a genetics approach, what would be a biochemical or molecular approach?
If this is a biochemical or molecular approach, what genetics methods could be used?
Results and DiscussionWhat are the primary conclusions of the paper?
Did the authors prove their hypotheses?What novel information or directions come from this work?
What control experiments were performed?What assumptions still remain in the work?How could these assumptions be tested?
What other explanations for the observations are still possible?
Reading the original literature ...
Journal Club ofAbysmal Disasters
• success stories of poor science• bad but Nature papers!• autopsies of crappy papers• examples one shouldn’t follow• retractions and bad reputations• learning from mistakes, which cost careers
… in English ...… once a month ...
… seminar room, first floor …… watch out of further announcements ...
The Institute of Molecular Genetics, PragueDepartment of Epigenetic Regulations
organizes:
Bad papers also deserve attention … therefore: