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Systematic Protein Location
Mapping Reveals Five
Principal Chromatin Types in
Drosophila Cells
Guillaume J. Filion, Joke G. van Bemmel, Ulrich Braunschweig, Wendy
Talhout, Jop Kind, Lucas D. Ward,
Wim Brugman, Ines J. de Castro, Ron M. Kerkhoven, Harmen J. Bussemaker,
and Bas van Steensel
Dristi Angdembey
Anton Arce
D145
Chromatin: structure and general
definition
http://www.nature.com/scitable/content/ne0000/ne0000/ne0000/ne0000/113158606/18847_6.jpg
Traditional classification
2 types of chromatin:
1. Heterochromatin
o repressed genes
o dark staining = tighter
packing
2. Euchromatin
o
An electron micrograph of a nucleus
http://www.histology.leeds.ac.uk/cell/nucleus.php
expressed genes
Distinct types of heterochromatin
discovered in Drosophila
1. PcG chromatin
o
o
Polycomb group (PcG) proteins + methylation of
lysine 27 of histone H3 (H3K27)
repressive type that regulates genes with
developmental functions
●
Sparmann and van Lohuizen, 2006
2. HP1 chromatin
Heterochromatin protein 1 (HP1) + associated
proteins + methylation of H3K9
o found around the centromere
o reporter genes integrated in or near likely repressed
o
●
Hediger and Gasser, 2006
Properties of distinct chromatin
types
2 important principles of chromatin
organization:
1. domains with distinct protein compositions
2. coverage of long stretches of DNA
Are there other types of
chromatin that follow these
principles?
Genome-wide location maps of 53
Chromatin Proteins
● Embryonic D. melanogaster cell line Kc167
used
● Representative cross-section of chromatin
proteome
● Used new technique DNA adenine
methyltransferase Identification
http://www.entu.cas.cz/ , https://www.broadinstitute.org/ , http://www.wikimedia.org/wikipedia/commons
DNA adenine methyltransferase
Identification (DamID)
● Fusion protein of E. coli
Dam and target chromatin
protein
● Methylates adenines of
nearby GATC sequences
● Uses microarray to ID
http://research.nki.nl/Vansteensellab/DamID_FAQ.htm
methylated A’s
● Sensitive to transient DNA
interactions
● Has 1 kbp resolution
● Goes over whole genome
300 bp at a time
Why not ChIP?
● Not great for large-scale
application
o paper probes for 53 proteins
● Dependent on quality
and specificity of
antibodies
● Dependent on
crosslinking reagents
o inability to bind to chromatin
proteins with short
residence times
http://www.usbweb.com/assets/78460_fig1.gif
Chromatin Binding Map
Chromatin Types by Arbitrary Color
● GREEN: HP 1 chromatin; classic
heterochromatin
corresponds to the
heterochromatin
● BLUE: PcG chromatin
discussed earlier
● BLACK: Half of genome, very repressive,
related to nuclear lamina, late replicating
● RED: Euchromatin-like, “hub” of cell
regulation, DBF hot spot, replication origins
● YELLOW: Euchromatin-like, constitutive
“housekeeping” genes, DNA repair
‘GREEN’ Chromatin
●
●
●
●
corresponds to classic heterochromatin, i.e. HP1 chromatin
marked by SU(VAR)3-9, HP1, and HP-1 interacting proteins
prominent in pericentric regions and on chromosone 4
H3K9me2, histone mark generated by SU(VAR)3-9, highly and specifically
enriched in GREEN chromatin
‘BLUE’ Chromatin
● corresponds to PcG chromatin
o extensive binding by the PcG proteins: PC, E(Z), PCL, SCE
● well-known PcG target loci such as the Hox gene clusters are localized in
BLUE domains
● H3K27me3, histone mark generated by E(Z), highly enriched in BLUE
chromatin
‘BLACK’ Chromatin
●
●
●
●
The most prevalent type of chromatin
Least genes per genome coverage
Most silent genes, with no mRNA for 66%
Genes are actively repressed
How did they figure out BLACK
Chromatin is repressive?
The question: “Does BLACK chromatin
actively repress transcription or merely forms
secondary to a lack of transcription?”
● if repressive, transgenes
inserted into BLACK chromatin
may exhibit reduced transcription
● if secondary, transgenes should
be unaffected
Developmental Regulation of Genes
in BLACK chromatin
● 33% of non-silent genes in BLACK chromatin
o tissue-specific expression
● suggests that BLACK chromatin domains can be remodeled
into a different chromatin type in some cell types
● rich in Highly Conserved Noncoding Elements (HCNEs)
o mediate gene regulation?
‘RED’ and ‘YELLOW’ Chromatin
● Most like classic euchromatin
o
o
o
High rates of transcription and polymerase activity
High gene density
Replicated first
● Share a set of proteins that make them like
euchromatin
○
○
○
Histone Deacetylases: remove acetylene, regulates
expression
ASH2: Methyltransferase, regulates expression
DF31: Chromatin decondensation
‘RED’ Chromatin
● Is replicated first and
has most origins of
replication
● Concentrates
nucleosome remodeling
● Contains key protein in
chromosome structure
● Genes are linked to
behavior and tissue
specific processes
‘YELLOW’ Chromatin
● Lots of histone
H3K36me3 with
chromodomain
modification
o
Leads to constitutive
activity
● Genes transcribed are
widely expressed and
include universal cell
processes
Predicting DNA binding from
Chromatin Types
● Genome has more binding motifs than DBFs
actually bind to
● Guided by chromatin type via interacting
chromatin proteins
● Specific to organism as chromatin does not
interact with foreign protein
Future directions
● How do these principal chromatin types
differ between cell types and species?
o
Do species with more “complex” genome have more
chromatin types?
 Drosophila: 4 chromosomes
 Humans: 23 chromosomes
● Do the chromatin types contribute to
epigenetic memory? Mechanism?
DamID- shortfalls/disadvantages
● Not standardized, mainly uses
microarrays not Nextgen
● Not perfect targeting leads to background
artifacts
o
Non-specific binding in more “open” chromatin
or high concentration of GATC
● High Dam concentrations leads to overmethylation
o
Needs weak induction because of high activity
Take this home guys
● DamID is a good option for large chromatin
protein sets with lots of coverage
● Chromatin is more diverse than euchromatin
and heterochromatin
o
o
Unique protein content leads to different expression
and repression of genes
Protein DNA binding is dependent on chromatin type
Acknowledgement
● Professor Blumberg
● Bassem Shoucri