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Přírodovědecká fakulta UK. EPIGENETIKA MB150P85. Petr Svoboda. mail : svobodap@img.cas.cz tel: 24106 3147. A few comments on the course :.

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slide1

Přírodovědecká fakulta UK

EPIGENETIKA MB150P85

Petr Svoboda

mail: svobodap@img.cas.cz

tel: 241063147

slide2

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.

slide3

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 )

slide4

Resources

Suggested reading for minimalists:

slide5

NCBI - literature (Pubmed, OMIM),

sequences (Genbank) a BLAST

BioGPS – atlas of gene expression

Ensembl - anotated sequences, data mining

BCM Search Launcher – sequence analysis (old and decaying)

Google and Wikipedia work very well too

slide6

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
slide7

EPIGENETIKA B150P85

  • Introduction
  • overview of the course, basic concepts of epigenetic marks, diversity of epigenetic mechanisms and effects
  • 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
  • - 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.

Lecture 1

24.2. 2011

Lecture 2

10.3. 2011

slide8

RNA silencing I – molecular machines for RNA silencing

  • “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
  • - 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
  • 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
  • establishment of pluripotency in ES cells and embryos
  • Course overview, feedback session

Lecture 3

24.3. 2011

Lecture 4

7.4. 2011

Lecture 5

21.4. 2011

Lecture 6

5.5. 2011

epigenetics
EPIGENETICS

Epigeneticsdeals withheritable information which is not encoded in the DNA sequence

  • Such information can be encoded in:
    • structure and chromatin modifications
    • DNA modifications
    • RNA molecules
regulation of complex genomes is a problem

42= 16

43= 64

44= 256

45= 1024

46= 4096

47= 16384

48= 65356

Regulation of complex genomes is a problem

E. coli

TF binding site length?

Core promoter length?

Homo sapiens

chromatin represents a structural solution for maintaining and accessing complex genomes

slide13

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

slide14

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

slide15

http://www-biology.ucsd.edu/classes/bimm110.SP06/lectures_WEB/L08.05_Cytogenetics.htmhttp://www-biology.ucsd.edu/classes/bimm110.SP06/lectures_WEB/L08.05_Cytogenetics.htm

slide16

http://www-biology.ucsd.edu/classes/bimm110.SP06/lectures_WEB/L08.05_Cytogenetics.htmhttp://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 telomereeach region has several bands, again numbered away from the centromere

slide17

http://fig.cox.miami.edu/~cmallery/150/proceuc/chromosome.jpghttp://fig.cox.miami.edu/~cmallery/150/proceuc/chromosome.jpg

slide18

Evolution of chromatin structure models

Molecular Biology of the Cell

1994

Molecular Biology of the Cell

2002

Molecular Biology of the Cell

2007

nucleosome

Things to remember …

Nucleosome

H2A, H2B, H3, H4 – core histones

H1 – linker histone

http://en.wikipedia.org/wiki/File:Nucleosome.JPG

slide20

Things to remember …

Closed

Open

http://sgi.bls.umkc.edu/waterborg/chromat/chroma09.html

slide21

Things to remember …

apparent global chromatin patterns are underlied by repetitive sequences

Martens 2005

nucleosome and core histones
NUCLEOSOME AND CORE HISTONES

H2A, H2B, H3, H4 – core histones

H1 – linker histone

slide23

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

family member

HIST1H2AG

older nomenclature and synonyms can be clarified at the GNF Symatlas and NCBI webpages

Marzluff 2002

slide24

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&

slide25

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

slide27

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

B

C

A

349

350

HA

349 CENP-A

UBF

UBF

349

Merge

Merge

Merge

HEK293

slide28

Methodsto study chromatin – Immunofluorescence I

- Higher resolution in polytene chromosomes in Drosophila

Polytene chromosomes (blue) stained for Hairy (green) and Groucho (red) binding

slide29

Methods to study chromatin – Chromatin IP

  • good resolution (typically 0.5 - 1.0 kb)
  • useful for analysisof individual genes, promoters
  • genome-scale analysis nowadays possible
  • relatively expensive

tips and tricks

  • Detection:
  • qPCR
  • promoter/tiling microarray = ChIP-Chip
  • deep sequencing = ChIP-Seq
slide30

Methods to study chromatin – Chromatin IP

human rDNA repeat

A

10kb

5kb

15kb

20kb

0kb

25kb

30kb

35kb

40kb

43kb

5´ETS

18S

5.8S

28S

1kb

3kb

6kb

13kb

20kb

29kb

38kb

42kb

B

14

12

349

10

unspecific antibody

8

% of input

6

4

2

0

GAPDH

1kb

3kb

6kb

13kb

20kb

29kb

38kb

42kb

slide32

http://biology.plosjournals.org/perlserv?request=get-document&doi=10.1371/journal.pbio.0020136http://biology.plosjournals.org/perlserv?request=get-document&doi=10.1371/journal.pbio.0020136

slide36

http://biology.plosjournals.org/perlserv?request=get-document&doi=10.1371/journal.pbio.0020136http://biology.plosjournals.org/perlserv?request=get-document&doi=10.1371/journal.pbio.0020136

slide38

Kuo 1998

Histone acetylation - deacetylation

slide39

Kuo 1998

Histone acetylation - deacetylation

slide40

Kuo 1998

Histone acetylation - deacetylation

Histone acetylation

Deposition-related (B HATs)

Transcription-related (A HATs)

slide41

Annemieke 2003

Histone deacetylases

Trichostatin A is an inhibitor of histone deacetylases.

+ Sir2 family of deacetylases - target nonhistone proteins

slide42

http://www.imt.uni-marburg.de/bauer/research.html

Histone methylation - lysine residues

SET

domain

HMTs

Bannister 2002

slide43

specific methylation level

specific residue

specific lysine residue

specific

effect

specific sequence

specific complex

specific

HMT

specific

protein

specific structure modification

specific

effect

specific

locus

must be maintained!

(memory)

slide46

Shi 2007

Histone methylation is reversible!

JmjC domain

(JumanjiC)

slide47

Histone methylation - arginine residues

http://www.imt.uni-marburg.de/bauer/research.html

slide50

Shi 2007

mono

di

tri

and back

slide51

Arney 2007

… BACK TO HETEROCHROMATIN vs. EUCHROMATIN

slide52

Martens 2005

… BACK TO HETEROCHROMATIN vs. EUCHROMATIN

DISTINCT REGIONS WITHIN CHROMATIN

slide54

Histone code and its interpretation

Complexes, complexes, complexes …

Bannister 2002

slide56

Schwartz 2007

Polycombs

slide57

Schwartz 2007

Polycombs

H3K27

slide58

Histone phosphorylation

It has been estimated that a typical human cell must repair over 10,000 DNA lesions per day (Lindahl, T. Nature, 1993).