Biol 456/656
Molecular Epigenetics
Lecture #5
Wed. Sept 2, 2015
Marmostein, Nature Reviews Molecular Cell Biology, 2001
http://www.nature.com/nrm/journal/v2/n6/fig_tab/nrm0601_422a_F1.html
-Chromatin consists of nucleosomes
-145-147 bp of DNA around octomeric core
-core: 2 molecules each of H2A, H2B, H3, H4
-NH2-terminal is basic charged histone tail region
NH2
Note (mistake from last class):
N terminal is end of protruding tail. Number starts at N-terminus
Turner, NSMB, 2005
Charting histone modifications
• Chromatin organization differs between cell
types
• enables differential access to regulatory ciselements
• Chromatin structure influenced by
transcription factors and transcription itself
Chromatin Immunoprecipitation
(ChIP)
http://joe.endocrinologyjournals.org/content/201/1/1/F1.large.jpg
ChIP-seq
http://www.bnl.gov/bnlweb/pubaf/pr/photos/2011/11/chip_seq_illustration_final-hr.jpg
ChIP
• Chromatin Immunoprecipitation
• Steps
1- Cross-link proteins to DNA with formaldehyde
2- Sonicate DNA to shear to ~500 bp fragments
3- Immunoprecipitate with antibody targeting epitope of
interest (histone modification, transcription factor, etc).
4- wash the bead-antibody-protein-DNA complex to
remove non-specific chromatin
5-reverse cross-links
6- remove proteins with proteinase K
7-isolate DNA
• With isolated DNA, can proceed with
– 1. PCR/qPCR (ChIP-PCR)
– 2. microarray (ChIP-chip)
– 3. deep sequencing (ChIP-seq)
• The focus has shifted from single genes to a
genome wide of where proteins bind DNA
• Generalities (universal rules) can be
extrapolated from experiments on individual
genes
What do histone modifications mark?
• Genome-wide studies show correlation of
marks with
– Promoters: high GC content vs low GC content (high vs low CpG)
– Enhancers: positively influence transcription at distal promoters
– Insulators: block enhancer activity
– Transcribed regions
– Repressed regions
H3K4me3
H3K36me3
H3K79me2
Transcribed gene
H3K79me2, H3K36me3
associated
with transcribed genes
H3K4me3 marks
promoters of transcribed
genes
Not-transcribed
Insulator binding protein
H3K9me3
H3K27me3
H3K9me3, H3K27me3
associated with nontranscribed regions
Zhou et al., Nature Reviews Genetics, 2011
Dashboard of histone modifications
@ Promoters:
Histone modifications contribute
To fine tuning of gene expression
@ Gene bodies:
Active vs Inactive conformations
Mammalian Promoter Regions
• Can be high or low GC content
– High CpG content promoter- HCP
– Low CpG content promoter- LCP
(the p is for phosphate!)
-open/active by default
-Applies to
housekeeping genes
-Applies to
developmental regulator
gene promoters in
Embryonic Stem (ES)
cells
-Inaccessible
to RNA Pol II.
Not Transcribed
Active
-selective activation
by transcription factors
Inactive
No histone methylation
DNA methylation
-Inactive by default
Low CpG promoters tend to be DNA methylated. High CpG promoters tend to not.
Heterochromatin
• Tightly packed DNA (vs euchromatin)
• Important for genome organization/stability, gene
regulation
• Lots of repetitive sequence
• H3K9me3 enriched
• Can use H3K9me3 to identify heterochromatin
domains
• Despite the repressive environment, some
expressed genes reside in heterochromatic
regions of the genome
H3K9me3 marks heterochromatin
size
Gray bars are unmappable regions
Ho et al., Supp Fig 33
Ho et al., Supp Fig 33
What do chromatin marks look like on
individual genes?
H3K9me3 marks heterochromatin
Ho et al., supp fig 37
H3K36me3 marks transcribed genes
H3K27me3 marks suppressed genes
Ho et al., supp fig 37
H3K4me3 marks TSS
(Isn’t so clear for this example, right??)
-remember, genome-wide trends won’t be observable for each gene example!
Ho et al., Supp Fig 36
Ho et al., Supp Fig 36
From Human to fly to worm, Histone methylation mark
(H3K9me3) “tracks” the same
Ho et al., Supp Fig 36
Ho et al., Supp Fig 36
Differentiated cells have more H3K9me3 regions than embryonic cells
LAD: Lamina associated domain
c
See Ho et. al paper. Or go to link:
http://www.nature.com/nature/journal/v512/n7515/full/nature13415.html#extended-data
Homework: Explain the important findings from this figure in words.
You can paraphrase what is in the text or give your own interpretation.
Send by email to pmiura@unr.edu before Friday.
Summary
• Histone modifications indicate:
– Functional genomic elements (promoters, enhancers)
– Expressed vs silenced genes
“epigenetics” does not appear in the text!!!
Supplemental data section has 42 figures
Chromatin composition and organization
– Worm, fly, human
Techniques:
– ChIP-seq: Chromatin IP, deep sequencing
– ChIP-chip: Chromatin IP, microarray
Profiles:
– core histones, histone variants, histone
modifications, chromatin associated proteins
Paper Summary: Marks profiled
• Histone modifications
– H3K27me3, H3K4me3
• Non-histones
– CHD3 (part of NuRD complex which deacetylates
histones)
– EZH2 (Histone methyltransferase)
– KDM4A (Lysine demethylase)
– RNA Pol II
• DNAse I hypersensitive sites (DHS)
• Nascent transcript sequencing (GRO-seq)
DHS
DHS: DNAse I hypersensitivity site
-susceptible to cleavage by DNAse I enzyme
https://en.wikipedia.org/wiki/DNase_I_hypersensitive_site
GRO-seq
https://en.wikipedia.org/wiki/Nuclear_run-on#/media/File:GRO-Seq.png
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2833333/
Transcription start site
Transcription start site
Transcription start site
Genome Architecture at promoters
fly/worm vs human
• Very similar to human cells
– Nucleosome density
– Methylation marks at TSS
• E.g. H3K4me3, H3K27ac
• Very different from human cells
– GC content
– Antisense nascent transcription
Ext Data Fig 1B- Fly
Heterochromatin
• Tightly packed DNA (vs euchromatin)
• Important for genome organization/stability, gene
regulation
• Lots of repetitive sequence
• H3K9me3 enriched
• Can use H3K9me3 to identify heterochromatin
domains
• Despite the repressive environment, some
expressed genes reside in heterochromatic
regions of the genome
c
See Ho et. al paper. Or go to link:
http://www.nature.com/nature/journal/v512/n7515/full/nature13415.html#extended-data
Homework: Explain the important findings from this figure in words.
You can paraphrase what is in the text or give your own interpretation.
Send by email to pmiura@unr.edu before Monday.