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Přírodovědecká fakulta UK
EPIGENETIKA MB150P85
Petr Svoboda
mail:
tel:
[email protected]
241063147
A few comments on the course:
- the goal of this course is to present you the latest original information on
epigenetics, to give you some idea on how is such information obtained and to
make you a better scientist.
- this course is designed for advanced students, particularly for those who
consider career in science. The course is modeled after advanced Msc./PhD.
courses at the University of Pennsylvania and is likely very different from
anything you have experienced at the university, so far.
- this course requires active participation and is quite is demanding. You will
have to read the original literature, make a few homeworks, take an exam and
write a scientific text. You earn your credits.
- the course is taught in English now. Mp3 records in Czech from 2007 Spring
semester are available per request.
- there are no stupid question. It is stupid not to ask. Exception: don’t ask if this
or that is going to be included in the exam because the answer is always YES.
- take the course as a challenge. Don’t take it if you don’t like to be challenged.
Resources
Suggested reading:
Allis et al., Epigenetics
Alberts et al. Molecular Biology of the Cell
Passarge E., Color Atlas of Genetics
Tost, Epigenetics
Original articles a reviews will be provided
during the course (as .pdf )
Resources
Suggested reading for minimalists:
NCBI - literature (Pubmed, OMIM),
sequences (Genbank) a BLAST
Ensembl - anotated sequences, data mining
BioGPS – atlas of gene expression
BCM Search Launcher – sequence analysis (old and decaying)
Google and Wikipedia work very well too
Additional information
Office hours:
- no specific time (unless you insist), you can come anytime
- to make sure I’ll have time, please, write me or call me in advance
Course requirements and the final exam
- “take-home” two week exam
- the code of academic integrity will be strictly enforced
EPIGENETIKA B150P85
Introduction
-overview of the course, basic concepts of epigenetic marks, diversity of epigenetic mechanisms and
effects
Lecture 1
24.2. 2011
Histones I
- concept of chromatin structure. Heterochromatin and euchromatin. Core histones, linker histones,
replacement histones, protamines. Methods for studying chromatin.
Histones II
- histone modifications, polycomb proteins, acetylation, fosforylation and histone methylations, effects on
gene expression.
DNA methylation I
Lecture 2
10.3. 2011
- molecular basis of DNA methylation. CpG and non-CpG methylation. Adenosin methylation. Metods for
studying DNA methylation. Bisulfite sequencing.
DNA methylation II
- effects of DNA methylation on gene expression, Methyl-binding proteins and mechanisms of inhibition of
gene expression, distribution of DNA methylation within genes and mammalian genomes.
RNA silencing I – molecular machines for RNA silencing
Lecture 3
24.3. 2011
-“historical” introduction into RNA silencing. Post-transcriptional effects. Roles and effects of dsRNA.
Proteins and complexes in RNA silencing.
RNA silencing II - RNAi technology
- experimental and therapeutic use. Design of RNAi experiments
RNA silencing III – roles of RNA silencing pathways
-miRNA pathway, chromatin connection.
Imprinting
Lecture 4
7.4. 2011
- concept of imprinting, mammalian imprinting. Molecular mechanisms of imprinting. Role of imprinting,
Battle of the sexes.
X-inactivation
- principles and different strategies for dosage compensation. Control of X-inactivation in mammals.
Epigenetic reprogramming in mammalian life-cycle
Lecture 5
21.4. 2011
-integration of epigenetic modification in the mammalian life cycle. Reprogramming of gene expression
during development, artificial reprogramming – the traditional view.
Chromatin in transcribed regions - journal club
Integrated view of epigenetic regulation of gene expression
Lecture 6
5.5. 2011
- establishment of pluripotency in ES cells and embryos
Course overview, feedback session
EPIGENETICS
Epigenetics deals with heritable information
which is not encoded in the DNA sequence
Such information can be encoded in:
• structure and chromatin modifications
• DNA modifications
• RNA molecules
HISTONES I
(CHROMATIN STRUCTURE AND FUNCTION)
Regulation of complex genomes is a problem
TF binding site length?
Core promoter length?
Homo sapiens
E. coli
42= 16
43= 64
44= 256
45= 1024
46= 4096
47= 16384
48= 65356
chromatin represents a
structural solution for
maintaining and accessing
complex genomes
Higher order structure of genetic information in Eucaryots
HETEROCHROMATIN vs. EUCHROMATIN
Dyes, like carminic acetic acid or orceine can be used to
stain certain domains of a chromosome. The resulting
pattern is characteristic for the respective chromosome of a
species. During interphase, the chromosomal structure is
usually resolved. The intensity of the nuclear staining
becomes feebler and less uniform than that of the
chromosomes. The stainable substance has been called
chromatin by E. HEITZ (formerly at the Botanical Institute of
the University of Hamburg, 1927, 1929). He distinguished
between heterochromatin and euchromatin.
Heterochromatin are all the intensely stained domains,
euchromatin the diffuse ones. Heterochromatin is usually
spread over the whole nucleus and has a granular
appearance. It is known today that the heterochromatic
domains are those where the DNA is tightly packed (strongly
condensed) which is the reason for their more intense
staining. The euchromatic domains are less tightly packed.
http://www.biologie.uni-hamburg.de/b-online/e11/11c.htm#05
CHROMOSOME BANDING TECHNIQUES
Prior to 1960, when Moorehead and
Nowell described the use of Giemsa in
their chromosome preparations,
conventional cytologic stains such as
acetoorcein, acetocarmine, gentian violet,
hematoxylin, Leishman's, Wright's, and
Feulgen stains were used to stain
chromosomes. The Romanovsky dyes
(which include Giemsa, Leishman's, and
Wright's stain) are now recommended for
conventional staining, because the slides
can be easily destained and banded by
most banding procedures. Orcein-stained
chromosomes cannot be destained and
banded; therefore, orcein is generally not
used in routine chromosome staining.
Giemsa stain is now the most popular
stain for chromosome analysis
(Gustashaw, 1991).
Banding protocols
http://homepage.mac.com/wildlifeweb/cyto/text/Banding.html
http://www-biology.ucsd.edu/classes/bimm110.SP06/lectures_WEB/L08.05_Cytogenetics.htm
http://www-biology.ucsd.edu/classes/bimm110.SP06/lectures_WEB/L08.05_Cytogenetics.htm
metaphase and prometaphase G-banded human chromosome 1 and the standard
nomenclature for labeling the bands;short arm: p (petite); long arm: q; 1 - 4 regions for
each arm labeled from centromere towards telomere each region has several bands,
again numbered away from the centromere
http://fig.cox.miami.edu/~cmallery/150/proceuc/chromosome.jpg
Evolution of chromatin structure models
Molecular Biology of the Cell
1994
Molecular Biology of the Cell
2002
Molecular Biology of the Cell
2007
Things to remember …
Nucleosome
H2A, H2B, H3, H4 – core histones
H1 – linker histone
http://en.wikipedia.org/wiki/File:Nucleosome.JPG
Things to remember …
Closed
http://sgi.bls.umkc.edu/waterborg/chromat/chroma09.html
Open
Things to remember …
apparent global chromatin patterns
are underlied by repetitive sequences
Martens 2005
NUCLEOSOME AND CORE HISTONES
H2A, H2B, H3, H4 – core histones
H1 – linker histone
Replication-dependent core histones
- localized in large clusters (common chromatin domains? RNA processing?)
- the major human cluster - 6p21(mouse chr. 13)
- smaller clusters on 1p21 (mouse chr. 3) and 1q42 (mouse chr. 11)
- the major cluster tends to colocalize with Cajal bodies (functional link isn‘t well understood)
histone type
cluster
gene nomenclature
HIST1H2AG
family member
older nomenclature and synonyms can be clarified at the GNF Symatlas and NCBI webpages
Marzluff 2002
Expression of core histones
cell-cycle dependent
http://www.unc.edu/depts/marzluff/research.html
specific 3‘ end processing
CPSF-73
http://www.reactome.org/cgi-bin/eventbrowser?DB=gk_current&ID=77588&
Mammalian core histone variants
H2A.X
- estimated to make 10% of nuclear H2A in mammals
- rapidly phosphorylated in a response to DNA damage
CENP-A (variant of histone 3, Cid in Drosophila)
- found at centromeric regions
macroH2A
- enriched on the inactive X chromosome
H2A.Z
- possibly involved in initial steps of gene activation in euchromatin
H3.3
- deposited within chromatin independent on DNA replication
- enriched at sites of transcription
- accumulates in non-cycling cells
H3.1
- synthesized and deposited during S-phase
H2A.Bbd
- excluded from the inactive X chromosome
- H2A.Bbd histone octamer organizes only approximately 130 bp of DNA
Protamines - non-histone replacement in sperms
Braun 2001
Methods to study chromatin – Immunofluorescence I
- Small resolution on mammalian chromosomes
- useful of analysis of large domains (centomeres, rDNA arrays …) and global protein distribution
-IF and FISH combination - colokalization
A
B
349 CENP-A
349
350
HA
UBF
UBF
349
Merge
Merge
Merge
HEK293
C
Methodsto study chromatin – Immunofluorescence I
- Higher resolution in polytene chromosomes in
Drosophila
Polytene chromosomes (blue)
stained for Hairy (green) and
Groucho (red) binding
Methods to study chromatin – Chromatin IP
• good resolution (typically 0.5 - 1.0 kb)
• useful for analysis of individual genes, promoters
• genome-scale analysis nowadays possible
• relatively expensive
tips and tricks
Detection:
•qPCR
•promoter/tiling microarray = ChIP-Chip
•deep sequencing = ChIP-Seq
Methods to study chromatin – Chromatin IP
human rDNA repeat
A
5´ETS
10kb
5kb
0kb
18S 5.8S
1kb 3kb
6kb
15kb
20kb
25kb
30kb
35kb
40kb
43kb
28S
13kb
20kb
29kb
38kb
42kb
B
14
12
349
% of input
10
unspecific antibody
8
6
4
2
0
GAPDH
1kb
3kb
6kb
13kb
20kb
29kb
38kb
42kb
HISTONES II
(MODIFICATIONS AND THEIR INTERPRETATION)
http://biology.plosjournals.org/perlserv?request=get-document&doi=10.1371/journal.pbio.0020136
http://sgi.bls.umkc.edu/waterborg/chromat/chroma09.html
Proc Natl Acad Sci U S A. 1964 May; 51(5): 786–794.
http://biology.plosjournals.org/perlserv?request=get-document&doi=10.1371/journal.pbio.0020136
Kuo 1998
Histone acetylation - deacetylation
Kuo 1998
Histone acetylation - deacetylation
Kuo 1998
Histone acetylation - deacetylation
Histone acetylation
Deposition-related (B HATs)
Transcription-related (A HATs)
Annemieke 2003
Histone deacetylases
Trichostatin A is an
inhibitor of histone
deacetylases.
+ Sir2 family of deacetylases - target nonhistone proteins
Histone methylation - lysine residues
SET
domain
HMTs
http://www.imt.uni-marburg.de/bauer/research.html
Bannister 2002
specific
structure
modification
specific
locus
specific
effect
must be maintained!
(memory)
specific methylation level
specific residue
specific lysine residue
specific
effect
specific sequence
specific complex
specific
HMT
specific
protein
Histone methylation - lysine residues
Bannister 2002
histone code concept
Shi 2007
Histone
methylation
is
reversible!
JmjC domain
(JumanjiC)
Histone methylation - arginine residues
http://www.imt.uni-marburg.de/bauer/research.html
Abcam = common source of Abs and information
http://www.abcam.com/
Bannister 2005
Shi 2007
mono
di
tri
and
back
Arney 2007
… BACK TO HETEROCHROMATIN vs. EUCHROMATIN
Martens 2005
… BACK TO HETEROCHROMATIN vs. EUCHROMATIN
DISTINCT REGIONS WITHIN CHROMATIN
Histone code and its interpretation
Bannister 2002
Histone code and its interpretation
Complexes, complexes, complexes …
Bannister 2002
Li 2002
Schwartz 2007
Polycombs
Schwartz 2007
Polycombs
H3K27
Histone phosphorylation
It has been estimated that a
typical human cell must repair
over 10,000 DNA lesions per day
(Lindahl, T. Nature, 1993).
Histone phosphorylation