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Understanding the function of conserved non-coding regions in the
human genome
Sofie Salama – Haussler lab
CS273A, November 17, 2008
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Haussler Lab
• Dry lab – comparative genomics research• Browser staff – UCSC genome browser, ENCODE data
coordination center, 1000 genomes• Wet lab - Experimental analysis of interesting human
genomic regions
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• Origin of conserved non-coding regions and co-regulated gene networks
• Function of ultraconserved elements
• Discovery of novel non-coding RNA genes
• Detailed analysis of Human Accelerated Regions (HAR’s)
Understanding the function of conserved non-coding regions in the
human genome
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How are we different from chimps?
• Brain anatomy– 3X larger, especially cortex– More later developing
neurons of the upper cortical layers projecting within the cortex
– functional asymmetries
• What are the genotypic differences responsible for these phenotypic differences?
Hill, R. S. & Walsh, C. A. Nature 437, 64–67 (2005)
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Clues from comparative genomics
• Human vs. chimpanzee genome– Genomes are almost identical– BUT, almost 29 million differences – What are the important
differences???
• Multiple mammalian genomes sequenced – Conservation used to identify functional
elements – only 1/3 of conserved regions are protein
coding
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The HAR screen • Identify previously conserved regions
– ≥100 bp 96% identical between the chimpananzee, mouse and rat genomes
– ~35,000 mammalian conserved regions
• Compare to human sequence to identify Human Accelerated Regions– Look for orthologous segments with a large
number of changes– Develop statistical methods to rank and
evaluate each HAR
• Identified 49 regions with a significant increased substitution rate in humans (genome wide FDR<5%)
Katie Pollard
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Wet lab HAR projects
• HAR population resequencing
• Analysis of HAR1
• Characterization of HAR2 knockout and knockin mice
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Why resequence the HARs?
• Positive selection– Beneficial mutation enters
population– Spreads. Nearby (neutral)
alleles from mutated chromosome hitchhike towards fixation – a selective sweep
– Skew DAF spectrum towards both ends
• Confounding factor: time – Neutral drift removes variation
in 4Neff generations (~1 MYr in human)
• Human/chimp ancestor 5-7 MYA
Stringer Nature 2003
Noonan et al. Science 2006
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Resequence HARs 1 to 49
• 40kb around each HAR (~2.5Mb total with 13 control regions)
• 24 samples (48 chromosomes) YRI hapmap samples (panel P2 Seattle SNPs)
• Enough to do population genetic analysis on a HAR-by-HAR basis (not like our paper on ultras in the average)
• High throughput sequencing technology enables cost effective investigation.
Sol Katzman
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“Next-Gen” Sequencing• ABI SOLiD (fluoro seq by repeated ligation)
– 35bp reads (fragment, not mate-pair)– $3-4K per run– 2 slides per run– multiple samples per slide
• barcoded samples• Isolated drops on a slide
– 50 to 100 Million reads per slide• Total 2.5Gb of reads• 50% mapped? 50% enriched?• 250X coverage of 2.5Mb target regions?• Divide by number of samples in run for sample coverage
– From 1000 Genomes project:• Need 11X to get both alleles @ 99% prob• Need 27X average to get 11X @ 99% prob
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Project Overview (part 1 of 2)
to Part 2 Sol Katzman
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Project Overview (part 2 of 2)from Part 1
Sol Katzman
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Wet lab HAR projects
• HAR population resequencing
• Analysis of HAR1
• Characterization of HAR2 knockout and knockin mice
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and the winner is….HAR1!
• 118 bp segment with 18 changes between the human and chimp sequences
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HAR1 genomic landscape
• Browser gazing suggested the HAR1 element may be expressed in both orientations
• rt-PCR on human tissue RNA preps suggested brain specific expression of the HAR1 element
• Used RACE to clone both forward and reverse transcripts from cortical and cerebellar RNA
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HAR1 is transcribed
• HAR1F expressed in brain (cerebellum, forebrain structures), ovary and testes (~1/10 of brain expression)
• HAR1R expressed in brain (1/10 of HAR1F) and testes• Outside HAR1 element, little conservation beyond primates
HAR1
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RNA in situ hybridization
• Fix tissue (whole embryo or sections)• Synthesize digoxygenin labelled probe anti-
sense to desired target• Hybridize, wash, visualize using enzyme linked
anti-DIG anitbody
superfly.ucsd.edu
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HAR1F is expressed in the in the neocortex
Nelle Lambert, Marie-Alexandra Lambot, Sandra Coppens, Pierre Vanderhaeghen
500µm 250µm
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Reelin and cortical development
Amadio, JP & Walsh, CA, Cell 126:1033-1035 (2006)
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HAR1F is expressed in the marginal zone and the cortical plate
Nelle Lambert, Marie-Alexandra Lambot, Sandra Coppens, Pierre Vanderhaeghen
125 µm
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Expression of HAR1F in the neocortex continues though 19 GW
Nelle Lambert, Marie-Alexandra Lambot, Sandra Coppens, Pierre Vanderhaeghen
250 µm
1000 µm
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Co-expression of Reelin and HAR1F in Cajal-Retzius neurons
Nelle Lambert, Marie-Alexandra Lambot, Sandra Coppens, Pierre Vanderhaeghen
250 µm
250 µm
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Expression of HAR1F elsewhere in the brain at later embryonic stages
Nelle Lambert, Marie-Alexandra Lambot, Sandra Coppens, Pierre Vanderhaeghen
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The HAR1F neocortical expression pattern is found in macaque
• Expression pattern conserved since the divergence of hominoids and old world monkeys 25 MYA
Colette Dehay, Pierre Vanderhaeghen
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HAR1F is predicted to form a stable
RNA structure
Jakob Pederson
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Human
Chimp
Human Chimp
- 40
- 60
- 50
- 70
U G C A - 0 10 30 U G C A - 0 10 30DMS DMS
Haller Igel, Manny Ares
Structure probing reveals differences in the human and chimp structures
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Human HAR1F differs from the ancestral RNA stucture
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Resequencing/population genetics
• Samples– 24 member human diversity panel (HAR1 element)– 70 Caucasian and African American (6.5 kb region)– Other primates (gorilla, orangutan, macaque)
• Findings– human-specific changes fixed in the populations
(NO SNPs!)– Changes happened at least 1 MYA, no evidence of a
recent selective sweep– Large number of human changes extends throughout
HAR1F 1st exon
Sol Katzman, Bryan King, Andy Kern
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Summary• HAR1 is the most extreme of a set of genomic regions
showing increased substitutions specifically in the human lineage
• HAR1 overlaps 2 divergent ncRNA genes, HAR1F and HAR1R
• HAR1F is expressed in the neocortex in reelin producing Cajal-Retzius neurons which are critical for creating the architecture of the human cortex and also in other structures patterned by the reelin pathway
• HAR1F forms a stable RNA structure and the human substitutions appear to alter this structure
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What does HAR1 do???
• What is the cellular role of HAR1 ncRNAs?
• Where are they localize?
• Who do they interact with?
• What is their role in neural development?
• How do human HAR1 ncRNAs differ from other mammalian HAR1 ncRNAs?
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Wet lab HAR projects
• HAR population resequencing
• Analysis of HAR1
• Characterization of HAR2 knockout and knockin mice
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HAR2
• 12 human substitutions in a 119 bp segment• highly conserved in amiotes, present in frog• Not in a mature transcript, no RNA secondary
structure
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HAR2 Genomic Neighborhood
• HAR2 located in an intron of Centaurin-gamma 2• Closest neighbor is Gastrulation and brain-specific
homeobox protein 2 • CENTG2-HAR2-GBX2 relationship conserved back to
frog-human ancestor
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Transgenic assay for enhancer activity
LacZMinimal PromoterHAR2
Harvest at embryonic timepoints. Stain to visualize lacZ activity.
How does LacZ expression compare with that of nearby genes (centg2 and gbx2)?
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HAR2 is a neural-specific enhancer
Bryan King and Armen Shamamian
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HAR2 is a limb specific enhancer
• Human HAR2 shows significant activity in the limb buds
• Human HAR2 is stronger and shows a broader pattern of expression
• Making the human substitutions in the chimp construct is sufficient for increased limb bud staining
Prabhakar et al. (2008) Science
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HAR2 targeted mutants
• HAR2 knockout – marked allele is made, breeding with constitutive cre mouse to remove vector/marker sequences
• HAR2 knockin human HAR2 – Have ES cell line, no chimeras yet
• HAR2 knockin mouse HAR2 – Have construct
Robert Sellers, Armen Shamamian
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AcknowledgementsHaussler Lab
Jeff Long, Ting Wang, Danielle Gomez
Manny AresHaller Igel
Harry NollerDavid FeldheimJena Yamada
Nader Pourmand
UCSC Collaborators
Funding
HHMI, NIDA
Pierre Vanderhaegen – Univ. of BrusselsKatie Pollard – UCDUCSF/GladstoneAndy Kern - Dartmouth
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