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Genomics of Gene Regulation
ANSC 497B
Ross Hardison
Nov. 10, 2009
DNA sequences involved in regulation of
gene transcription
Protein-DNA interactions
Chromatin effects
Distinct classes of regulatory regions
Act in cis, affecting
expression of a gene
on the same
chromosome.
Cis-regulatory modules
(CRMs)
Maston G, Evans S and Green M (2006) Annu Rev Genomics Hum Genetics 7:29-59
General features of promoters
• A promoter is the DNA sequence required for correct initiation of
transcription
• It affects the amount of product from a gene, but does not affect the
structure of the product.
• Most promoters are at the 5’ end of the gene.
RNA polymerase II
Upstream regulatory
elements:
Regulate efficiency of
utilization of minimal
promoter
TATA box + Initiator:
Core or minimal
promoter. Site of
assembly of
preinitiation complex
Maston, Evans & Green (2006) Ann Rev Genomics & Human
Genetics, 7:29-59
Conventional view of eukaryotic gene promoters
Maston, Evans & Green (2006) Ann Rev Genomics & Human Genetics, 7:29-59
Most promoters in mammals are CpG islands
TATA, no CpG island
About 10% of promoters
CpG island, no TATA
About 90% of promoters
Carninci … Hayashizaki (2006)
Nature Genetics 38:626
Fraction of mRNAs
Differences in specificity of start sites for transcription
for TATA vs CpG island promoters
Carninci … Hayashizaki (2006)
Nature Genetics 38:626
Enhancers
• Cis-acting sequences that cause an increase in expression of a gene
• Act independently of position and orientation with respect to the
gene.
CRM
pr
luciferase
UCE
pr
lacZ
Tested UCE
Pennacchio et al.,
http://enhancer.lbl.
gov/
About half of the enhancers predicted by interspecies
alignments are validated in erythroid cells
Wang et al. (2006) Genome Research 16:1480- 1492
Over half of ultraconserved noncoding sequences are
developmental enhancers
Pennacchio et al. (2006) Nature 444:499-502
CRMs are clusters of specific binding sites for
transcription factors
Hardison (2002) on-line textbook Working with Molecular Genetics http://www.bx.psu.edu/~ross/
Enhancers can occur in a variety of positions
with respect to genes
Enhancer
Upstream
Enhancer
P Transcription unit
Adjacent
Downstream
Internal
Distal
Ex1
Ex2
Silencer
• Cis-acting sequences that cause a decrease in gene expression
• Similar to enhancer but has an opposite effect on gene expression
• Gene repression - inactive chromatin structure (heterochromatin)
•
•
SIR proteins (Silent Information Regulators)
Nucleates assembly of multi-protein complex
– hypoacetylated N-terminal tails of histones H3 and H4
– methylated N-terminal tail of H3 (Lys 9)
Insulators and boundaries
• A boundary in chromatin marks a transition from open to closed chromatin
• An insulator blocks activation of promoter by an enhancer
– Requires CTCF
• Example: HS4 from chick HBB complex has both functions
Pr
neoR
Insulator Enhancer
Neo-resistant colonies
% of maximum
10
Silencer
50
100
Repression by PcG proteins via chromatin
modification
Polycomb Group (PcG) Repressor Complex 2:
ESC, E(Z), NURF-55, and PcG repressor
SU(Z)12
Methylates K27 of Histone H3 via the SET
domain of E(Z)
me3
K27
H3 N-tail
OFF
trx group (trxG) proteins activate via chromatin
changes
• SWI/SNF nucleosome remodeling
• Histone H3 and H4 acetylation
• Methylation of K4 in histone H3
– Trx in Drosophila, MLL in humans
• http://www.igh.cnrs.fr/equip/cavalli/link.PolycombTeaching.html#Part_
3
Me1,2,3
K4
H3 N-tail
ON
Histone modifications modulate chromatin structure
H3K4me2, 3
http://www.imt.uni-marburg.de/bauer/images/fig2.jpg
H3K27me3
Uta-Maria Bauer
Repressed and active chromatin
Dustin Schones and Keiji Zhao (2008) Nature
Reviews Genetics 9: 179
Biochemical features of DNA in CRMs
Accessible to cleavage:
DNase hypersensitive site
Clusters of binding site motifs
Bound by specific transcription factors
Coactivators
Pol
Pol IIa
II
Associated with RNA polymerase
and general transcription factors
Nucleosomes with histone modifications:
Acetylation of H3 and H4
Methylation of H3K4
Lack of methylation at H3K27 or H3K9 …
Methods in Genomics of Gene
Regulation
Chromatin immunoprecipitation: Greatly enrich
for DNA occupied by a protein
Elaine Mardis (2007) Nature
Methods 4: 613-614
ChIP-chip: High throughput mapping of DNA
sequences occupied by protein
http://www.chiponchip.org
Bing Ren’s lab
Enrichment of sequence tags reveals function
Barbara Wold & Richard M Myers (2008) “Sequence Census Methods” Nature Methods 5:19-21
Illumina (Solexa) short read sequencing
- 8 lanes per run
- 10 M to 20 M reads of 36 nucleotides
(or longer) per run.
- 1 lane can produce enough reads to
map locations of a transcription factor in a
mammalian genome.
Example of ChIP-seq
ChIP vs NRSF = neuron-restrictive silencing factor
Jurkat human lymphoblast line
NPAS4 encodes neuronal PAS domain protein 4
Johnson DS, Mortazavi A, Myers RM, Wold B. (2007) Genome-Wide Mapping of in Vivo Protein-DNA Interactions.
Science 316:1497-1502.
ChIP-seq for chromatin modifications
Dustin Schones and Keiji Zhao (2008)
Nature Reviews Genetics 9: 179
Histone modifications around HBB locus
Known CRMs
UCSC genes
trithorax
Polycomb
Transcription
associated mark
DNase hypersensitive sites
Distributions at all GenCode TSSs
Symmetrical distribution of:
- H3K4me3, H3K4me2
- H3Ac, H4Ac, DHS
- E2F1, E2F4, Myc, Pol II
Birney et al. (2007) Nature 477: 799-816
Distribution of
histone
modifications and
factor binding
around regulatory
regions
• Promoters
– H3K4me3, H3K4me2
– E2F1, E2F4, Myc, Pol II
• Distal HSs
– H3K4me1: enhancers
– CTCF: insulators
Birney et al. (2007) Nature, 447:799-816
Enhancers predicted from chromatin
signatures
(2009) Nature 459: 108-112
Enhancer
predictions in
human cells
Characteristics and validation of predicted
enhancers
Data Resources for Genomics of Gene
Regulation
UCSC Genome Browser
•
Visualize data described in publications, e.g.
– Expression data
•
–
Regulation
•
•
•
•
•
•
Affymetrix gene arrays, GNF, Su et al. 2004
Kim et al. 2005, PICs (TAF1)
Kim et al., 2008, CTCF
Boyle et al., 2008, DNase hypersensitive sites
Heintzman et al., 2009, Enhancers predicted by H3K4me1
Mikkelsen et al., 2007, Chromatin modifications in pluripotent and lineage-committed cells
ENCODE project, Production phase
– Expression
•
•
–
Affy high density tiling arrays
RNA-seq from several sources (CSHL, Helicos)
Regulation
•
•
•
•
•
•
•
Broad histone modifications
HAIB DNA methylation
Open Chromatin
UW DNase HS
HAIB TFBS
Yale TFBS
SUNY RBP
Factor
occupancy
and
DNase
hypersensitivity
ENCODE Tracks:
Broad histone
modifications,
Open chromatin,
UW DHS, Yale
TFBSs
HS5
Locus control region
4
3
2
1
Collated sets of published regulatory regions
• http://www.bx.psu.edu/~ross/dataset/Reguldata.html
• Noncoding DNA segments with high regulatory potential
• PRPs: Intersection of the High RP segments and the PReMods
(clusters of conserved transcription factor binding site motifs)
• Most constrained DNA segments, phastCons
• DNase hypersensitive sites in CD4+ T cells
• DNA segments occupied by CTCF in primary fibroblasts
• Preinitiation complexes (TAF1) in IMR90 cells
• Predicted erythroid cis-regulatory modules
GeneTrack
• Genomic data analysis and integration
– Istvan Albert, Frank Pugh, et al., PSU
– http://genetrack.bx.psu.edu/
• Install on your system
• Gallery of data for visualization
– Yeast H2AZ nucleosome predictions, 454 sequencing
– Drosophila H2AZ nucleosome predictions, 454
sequencing
Yeast
nucleosome
map
HIS3:
nucleosomefree region
modENCODE
http://www.modencode.org/
Worm and Fly
Gene annotations
Expression
Chromatin modifications
TFBs in vivo, etc.
Experimental Tests in the Genomics
of Gene Regulation
GATA-1 is required for erythroid maturation
Common
myeloid
progenitor
MEP
Hematopoietic
stem cell
G1E cells
GATA-1
Myeloblast
Common
lymphoid
progenitor
G1E-ER4 cells
Basophil
Eosinophil
Neutrophil
Aria Rad, 2007 http://commons.wikimedia.org/wiki/Image:Hematopoiesis_(human)_diagram.png
Monocyte,
macrophage
GATA1-induced changes in gene expression and
occupancy genome-wide
Genes induced or
repressed after restoration
of GATA1
Occupancy by TFs and histone modifications
along a 60 Mb region
High sensitivity and specificity of high
throughput occupancy data
High
throughput
occupancy
matches
known
CRMs at
Hbb locus
Confirmed and novel regulatory regions for Gypa
Known CRMs
Gypa gene
Response
DHSs
GATA1
TAL1
Trx: H3K4me1
Trx: H3K4me3
PcG: H3K27me3
Input DNA
Induced genes have GATA1 occupied segments close
to their TSS
DNA segments occupied by GATA-1 were tested for
enhancer activity on transfected plasmids
Occupied
segments
Some of the DNA segments occupied by GATA1 are active as enhancers
Cheng et al. (2008) Genome Research 18:1896-1905
Binding site motifs in occupied DNA segments
can be deeply preserved during evolution
Consensus binding site motif for GATA-1: WGATAR or YTATCW
5997
constrained
7308
not constrained
2055
no motif
All GATA1-occupied segments active as
enhancers are also occupied by SCL and LDB1
Genetic Determinants of Variation in
Gene Expression
Variation of gene expression among individuals
• Levels of expression of many genes vary in humans (and other
species)
• Variation in expression is heritable
• Determinants of variability map to discrete genomic intervals
• Often multiple determinants
• This variation indicates an abundance of cis-regulatory variation in
the human genome
• For example:
– Microarray expression analyses of 3554 genes in 14 families
• Morley M … Cheung VG (2004) Nature 430:743-747
- Expression analysis of about 16 HapMap individuals
• Storey et al. (2007) AJHG 80: 502-509
– Expression analysis of all 270 individuals genotypes in HapMap
• Stranger BE … Dermitzakis E (2007) Nature Genetics 39:1217-1224
Variation in expression between populations
Figure 5.Allele-specific qPCR analysis of SH2B3. a, Log2-fold change of SH2B3 expression for all CEU and YRI
individuals, relative to the average expression level in the YRI sample obtained from allele-specific qPCR. The
distribution of SH2B3 expression is significantly different between samples (t-test, P= .0157), which confirms the
microarray results.
b, Allele-specific qPCR of a coding polymorphism (rs1107853), which demonstrates that the log2-fold change of
the G allele relative to the A allele is significantly different between heterozygous DNA (Het DNA) and
heterozygous cDNA (Het cDNA) samples (t-test, P= .00118).
Storey et al., 2007, AJHG 80:502-509
Mapping determinants of expression variation
•
•
Stranger et al., 2007, Nature Genetics 39:1217-1224
Expression analysis of EBV-transformed lymphoblastoid cells from all 270
individuals genotypes in HapMap
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–
–
–
•
30 Caucasian trios (90) of European descent in Utah (CEU)
30 Yoruba trios (90) from Ibadan, Nigeria (YRI)
45 unrelated Chinese individuals from Beijing Univ (CHB)
45 unrelated Japanese individuals from Tokyo (JPT)
Measure levels of expression of 47,294 probes (about 24,000 genes) in each
individual
– Focus on 13,643 genes “selected on criteria of variance and population
differentiation”
•
•
Already know genotypes at about 2.2 million SNPs for each individual
(HapMap)
Test for significant association of variation at each SNP with variation in
expression of each gene
– Linear regression model
– Spearman rank correlation test
•
Evaluate significance of regression P values by 10,000 permutations of the
data, focus on those associations above the 0.001 permutation threshold
Association of SNPs with expression
• Significant
association between
expression and cisSNPs (within 1 Mb)
• 831 genes in at least
one population
• 310 genes in at least
2 populations
• 62 genes in all 4
populations
• Also find associated
SNPs in trans:
perhaps regulatory
proteins
Stranger et al., 2007, Nature Genetics 39:1217-1224
Location of expression-associated SNPs
• Most are “close” to
transcription start site (TSS)
• Symmetrical arrangement
(similar to biochemical
features of promoters)
• Three of the SNPs have been
shown to affect promoter
activity in transfection assays
(Hoogendoorn et al. (2004)
Human Mutation 24: 35-42
Figure 4 Properties of significant cis associations
as a function of SNP distance from the
transcription start site.
Stranger et al., 2007, Nature Genetics 39:1217-1224
Relevance to human health
• "We predict that variants in regulatory regions make a
greater contribution to complex disease than do variants
that affect protein sequence”
– Manolis Dermitzakis, ScienceDaily
Risk loci in noncoding regions
(2007) Science 316: 1336-1341
Biochemical features of DNA in CRMs
Accessible to cleavage:
DNase hypersensitive site
Clusters of binding site motifs
Bound by specific transcription factors
Coactivators
Pol
Pol IIa
II
Associated with RNA polymerase
and general transcription factors
Nucleosomes with histone modifications:
Acetylation of H3 and H4
Methylation of H3K4
Candidate functions in T2D SNP intervals
Overlap of SNP rs564398 with DHS suggests a role in transcriptional regulation,
but overlap with an exon of a noncoding RNA suggests a role in post-transcriptional
regulation. Different hypotheses to test in future work.