Conserved Region Analysis of Oncogenic Human Papillomavirus Genome
Understanding the function of conserved non-coding regions in the human genome
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Transcript of Understanding the function of conserved non-coding regions in the human genome
Understanding the function of conserved non-coding regions in the
human genome
Sofie Salama – Haussler labCS273A, November 17, 2008
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
• 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
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)
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
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
Wet lab HAR projects
• HAR population resequencing
• Analysis of HAR1
• Characterization of HAR2 knockout and knockin mice
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
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
“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
Project Overview (part 1 of 2)
to Part 2 Sol Katzman
Project Overview (part 2 of 2)from Part 1
Sol Katzman
Wet lab HAR projects
• HAR population resequencing
• Analysis of HAR1
• Characterization of HAR2 knockout and knockin mice
and the winner is….HAR1!
• 118 bp segment with 18 changes between the human and chimp sequences
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
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
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
HAR1F is expressed in the in the neocortex
Nelle Lambert, Marie-Alexandra Lambot, Sandra Coppens, Pierre Vanderhaeghen
500µm 250µm
Reelin and cortical development
Amadio, JP & Walsh, CA, Cell 126:1033-1035 (2006)
HAR1F is expressed in the marginal zone and the cortical plate
Nelle Lambert, Marie-Alexandra Lambot, Sandra Coppens, Pierre Vanderhaeghen
125 µm
Expression of HAR1F in the neocortex continues though 19 GW
Nelle Lambert, Marie-Alexandra Lambot, Sandra Coppens, Pierre Vanderhaeghen
250 µm
1000 µm
Co-expression of Reelin and HAR1F in Cajal-Retzius neurons
Nelle Lambert, Marie-Alexandra Lambot, Sandra Coppens, Pierre Vanderhaeghen
250 µm
250 µm
Expression of HAR1F elsewhere in the brain at later embryonic stages
Nelle Lambert, Marie-Alexandra Lambot, Sandra Coppens, Pierre Vanderhaeghen
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
HAR1F is predicted to form a stable RNA structure
Jakob Pederson
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
Human HAR1F differs from the ancestral RNA stucture
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
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
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?
Wet lab HAR projects
• HAR population resequencing
• Analysis of HAR1
• Characterization of HAR2 knockout and knockin mice
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
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
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)?
HAR2 is a neural-specific enhancer
Bryan King and Armen Shamamian
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
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
AcknowledgementsHaussler Lab
Jeff Long, Ting Wang, Danielle Gomez
Manny AresHaller Igel
Harry NollerDavid FeldheimJena Yamada
Nader Pourmand
UCSC Collaborators
FundingHHMI, NIDA
Pierre Vanderhaegen – Univ. of BrusselsKatie Pollard – UCDUCSF/GladstoneAndy Kern - Dartmouth