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Lecture 15:

HW2 Feedback

Ultraconservation

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GGTGCCAGGGAAAGGGCAGGAGGTGAGTGCTGGGAGGCAGCTGAGGTCAACTTCTTTTGAACTTCCACGTGGTATTTACTCAGAGCAATTGGTGCCAGAGGCTCAGGGCCCTGGAGTATAAAGCAGAATGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCGAAAGACCTGTTGGAGGCTATGAATGCAATCAAGGTGACAGACAACTGGTGCAATGATGGTAGTGGAAATGGAGGAGAGGGGATTGATTCAAGATGCATTTAGGACCAAGAATCGGGAGCTTGTGAACGTGTGTATGAGTACTGTAGACGGAGTGGGTGTGTCATCAGAGAAGATCTGAGCATTTGGGCTTGCTCTCCTCAGAGGCCCTGCGAGTGGAGTTCAGCTTTTCCTCATGGGGCAAATCTCACTTTCGCTCCAGTTCCTGGGGCTCAGAGTCCCTGGCCCAGATGCCTCTTGCCATCTCATCTTCACCCTGCCTGGCTTCCCTTGCTTGTTCCAGGATTGTTTCATAAAGAGGGATGTGGTTGGTCTTTAACCCTATGAATGCTGGCTGAGGATGCCTGCGGAACCTGTAGTGAAGCTTTCAGGGGCTGCTCGGGTTCTGGCTGGTAGGTGAACACTGTCCATCTTGCCGGCTGGGACACAGTGACTCTGGGTAGTTGTGTAAGAGAGGGGCCCTTGGCAGACAAACAGGTTCTTCTCTGTTGGTGGGCCAGCCAGCAGGTCAGTGGGAAGGTTAAAGGTCATGGGGTTTGGGAGAACTGGGTGAGGAGTTCAGCCCCATCCCCCGTAAAGCTCCTGGGAAGCACTTCTCTACTGGGGCAGCCCCTGATACCAGGGCACTCATTAACCCTCTGGGTGCCAGGGAAAGGGCAGGAGGTGAGTGCTGGGAGGCAGCTGAGGTCAACTTCTTTTGAACTTCCACGTGGTATTTACTCAGAGCAATTGGTGCCAGAGGCTCAGGGCCCTGGAGTATAAAGCAGAATGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAGGAAAGACCTGTTGGAGGCTATGAATGCAATCAAGGTGACAGACAACTGGTGCAATGATGGTAGTGGAAATGGAGGAGAGGGGATTGATTCAAGATGCATTTAGGACCAAGAATCGGGAGCTTGTGAACGTGTGTATGAGTACTGTAGACGGAGTGGGTGTGTCATCAGAGAAGATCTGAGCATTTGGGCTTGCTCTCCTCAGAGGCCCTGCGAGTGGAGTTCAGCTTTTCCTCATGGGGCAAATCTCACTTTCGCTCCAGTTCCTGGGGCTCAGAGTCCCTGGCCCAGATGCCTCTTGCCATCTCATCTTCACCCTGCCTGGCTTCCCTTGCTTGTTCCAGGATTGTTTCATAAAGAGGGATGTGGTTGGTCTTTAACCCTATGAATGCTGGCTGAGGATGCCTGCGGAACCTGTAGTGAAGCTTTCAGGGGCTGCTCGGGTTCTGGCTGGTAGGTGAACACTGTCCATCTTGCCGGCTGGGACACAGTGACTCTGGGTAGTTGTGTAAGAGAGGGGCCCTTGGCAGACAAACAGGTTCTTCTCTGTTGGTGGGCCAGCCAGCAGGTCAGTGGGAAGGTTAAAGGTCATGGGGTTTGGGAGAAACTGGGTGAGGAGTTCAGCCCCATCCCCCGTAAAGCTCCTGGGAAGCACTTCTCTACTGGGGCAGCCCCTGATACCAGGGCACTCATTAACCCTCTGGGTGCCAGGGAAAGGGCAGGAGGTGAGTGCTGGGAGGCAGCTGAGGTCAACTTCTTTTGAACTTCCACGTGGTATTTACTCAGAGCAATTGGTGCCAGAGGCTCAGGGCCCTGGAGTATAAAGCAGAATGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCGAAAGACCTGTTGGAGGCTATGAATGCAATCAAGGTGACAGACAACTGGTGCAATGATGGTAGTGGAAATGGAGGAGAGGGGATTGATTCAAGATGCATTTAGGACCAAGAATCGGGAGCTTGTGAACGTGTGTATGAGTACTGTAGACGGAGTGGGTGTGTCATCAGAGAAGATCTGAGCATTTGGGCTTGCTCTCCTCAGAGGCCCTGCGAGTGGAGTTCAGCTTTTCCTCATGGGGCAAATCTCACTTTCGCTCCAGTTCCTGGGGCTCAGAGTCCCTGGCCCAGATGCCTCTTGCCATCTCATCTTCACCCTGCCTGGCTTCCCTTGCTTGTTCCAGGATTGTTTCATAAAGAGGGATGTGGTTGGTCTTTAACCCTATGAATGCTGGCTGAGGATGCCTGCGGAACCTGTAGTGAAGCTTTCAGGGGCTGCTCGGGTTCTGGCTGGTAGGTGAACACTGTCCATCTTGCCGGCTGGGACACAGTGACTCTGGGTAGTTGTGTAAGAGAGGGGCCCTTGGCAGACAAACAGGTTCTTCTCTGTTGGTGGGCCAGCCAGCAGGTCAGTGGGAAGGTTAAAGGTCATGGGGTTTGGGAGAACTGGGTGAGGAGTTCAGCCCCATCCCCCGTAAAGCTCCTGGGAAGCACTTCTCTACTGGGGCAGCCCCTGATACCAGGGCACTCATTAACCCTCTGGGTGCCAGGGAAAGGGCAGGAGGTGAGTGCTGGGAGGCAGCTGAGGTCAACTTCTTTTGAACTTCCACGTGGTATTTACTCAGAGCAATTGGTGCCAGAGGCTCAGGGCCCTGGAGTATAAAGCAGAATGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAGACGTGAGCAGGTGAGCAGCTGGGGCTGTCTGCTCTCTGTGCCCAG

Ultraconserved Elements in theHuman Genome: The Hip & The Hype

Dept. of Developmental BiologyDept. of Computer Science

Stanford University

Gill Bejerano

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Sequence Conservation implies Function

(but whichwhich function/s?...)

human

mouse

mammalianancestor

...CTTTGCGA-TGAGTAGCATCTACTATTT...

...ACGTGGGACTGACTA-CATCGACTACGA...

functional region!

Comparative Genomics of related species highlights:

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HumanGenome:

3*109 letters

Human Genome full of Conserved Non-Coding Elements

1.5%known

function >50%junk

3x more functional DNA than known!

compare to other species

>5% human genome functional

~106 genomic loci do not code for protein

What do they do then?

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Conserved elements in the Human Genome

all human-mouse alignmentshuman-mouse ancestral repeats alignment

Difference: 5% of

Human Genome

[Mouse consortium, Nature 2002]

election

human-mouse ancestral repeats alignment

85%id on average

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Conserved elements in the Human Genome

all human-mouse alignmentshuman-mouse ancestral repeats alignment

Difference: 5% of

Human Genome

election

human-mouse ancestral repeats alignment

85%id on average

UltraconservationUltraconservation

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Typical DNA Conservation levels

Conserved elements between human and mouse are on average 85% identical. [mouse consortium, 2002]

(dot = base identical to human)

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Ultraconserved Elements

[Bejerano et al., Science 2004]

fish

481 elements perfectly conserved (100%id) over

200bp or more between human, mouse and rat.

using2 vs. 3species

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Contamination

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What exactly is an Ultraconserved Element?

Aha!!

using3 vs. 43species

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Ultraconservation as a Phenomenon

Few species More and more species

Hmmm….

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Ultraconserved Elements: Why?

Hundreds of long genomic regions identical between amniotes they must have rejected many different changes.

But... all functions we understand in our genome are encoded using redundant codes.

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CDS ncRNA TFBS

seq.

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Conserved elements in the Human Genome

all human-mouse alignmentshuman-mouse ancestral repeats alignment

Difference: 5% of

Human Genome

election

human-mouse ancestral repeats alignment

85%id on average

UltraconservationUltraconservation

Why did I Why did I look at the tail?look at the tail?

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...ACGTACGACTGACTAGCATCGACTACGA........TCTGACTAGCATCGACTACGA...

DNA Replication is Imperfect

It’s imperfect on all scales: small, medium and large.

In particular it begets novel functional entities:

...ACGTACGACTGACTAGCATCGACTACGA...

...ACGTACGACTGACTAGCATCGACTACGA........TCTGACTAGCATCGACTACGA...

functionaljunk

functionalfunctional

functional’’ functional’

regionalduplication

functionaldivergence

Protein & RNA gene families come to life this way. What else does?

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Computational Approach I

Group them into paralog families of human functional regions of common origins: • Annotated members induce function on all. • Examine core, substitutions in family. • Test for “guilt by association”. [Bejerano et al., ISMB 2004]

.....ACGTGCATGACTGACTAGCATCAGACGACTAC..GATAATACGCTACGACTAGCTAC.....human DNA

...TGACTAGCATCGACTAC..GATAATACGAC... ...CATCGACTAC..GATAATACGACGGTTGGT...AC T

~400bp

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Functional Annotation by Families

[Bejerano et al., ISMB 2004]

Puzzling News:96% of the 700,000appear unique(!)

Good News:We still find12,027 families

novel putative ncRNAs, cis-regulatory elements, etc.

After removing from top 5% Human all annotated regions, and more:

700,000 elements, covering 3.5% Human Genome

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human

mouse

rat

related genesrelated elements(75%id over 200bp)

same element96%id over 200bp

same element95%id over 200bp

Computational Approach II

Classical Biological approach: experiment to understand these regions

Computational approach: how many regions like this or “better” are there?

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Out popped the Ultraconserved Elements

Puzzling News:96% of the 700,000

conserved non-codingelements appear

unique(!)

Same with Ultras

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What could ultras be doing?

•exonic•non•possibly

Associating distal peaks in a gene-based context is statistically inappropriate

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Gene transcription start site

Ultraconserved Element

Ontology term (e.g. ‘development’)

http://cs273a.stanford.edu [Bejerano Fall10/11]

N = 8 genes in genome

K = 3 genes annotated with

n = 3 genes selected by proximal peaks

k = 2 selected gene annotated with

P = Pr(k ≥1 | n=2, K =3, N=8)

1.Set gene regulatory domain.

2.Associate Ultras with genes.

3.Per ontology term, count annotated genes selected.

4.Rank terms by enrichment hypergeometric p-value.

Evolved into

http://great.stanford.edu/

Enrichment Association of Ultraconserved Elements

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Exo

nic

Ultr

asN

on

-exo

nic

Ultr

as

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Ultras are Functional

Back in 2004 we hypothesized:

481 ultraconserved elements

exonic subset –

post transcriptional regulation

[Ni et al., Genes Dev.; Lareau et al., Nature, 2007]

“nonexonic” subset –

transcriptional regulators

[Pennacchio et al., Nature, 2006]

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Ultraconserved Non-coding RNA

[Calin et al, Cancer Cell, 2007]miRNA complementarity

About 1/3 of all ultras are expressed.

Some are predicted to provide microRNA targets.

A few are anti-correlated with miRNAexpression levels.

A few even act as oncogenes.

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Ultras are Under Strong Human Selection

Ultra DAF NonSyn DAF

[Katzman et al, Science ,2007]

Mutational cold spots? NO. Rare (new) mutations are introduced to the population.

Fierce purifying selection? YES. Very few of these get anywhere near fixation.

chimpA

humans

G AAA

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Touch an Ultra And You - DIY

[Ahituv et al., PLoS Biology, 2007]

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What can’t we measure in the lab?

sN

s

e ee

esN 21

1),|fixationPr(

Ne is population size, s selective dis/advantage.Both of which are VERY wrong in the lab.

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So it can happen – but does it FIX?

tDNA element

mouse

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Count Fraction Lost, Binned by %id

t human

macaque

dog

mouse

rat

100bp

sliding

window

count_all

count_hole

bin

by

%id

humandog rat mouse

macaque

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Quite Some Time Later

[McLean & Bejerano, Genome Res., 2008]

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Ultras are Fiercely Retained through Evolution

Ultras are

>300 fold

more

persistent

than

neutral DNA(25% deleted)

the genomic deletiongenomic deletion is

100%id primates-dog: 1,691,090bp

rodents deleted: 1,447bp (0.086%)

sN

s

e ee

esN 21

1),|fixationPr(

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How special are the Ultras?

election

UltraconservationUltraconservation

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Ultraconservation as a Phenomenon

Few species More and more species

Hmmm….

We do not see a bump in the curve

Ultraconserved Elements: What do we know?

• Excessive sequence conservation exists.• Set is heterogeneous from a functional perspective.• Four can be KO-ed with no clear phenotype.• Yet, the set is under extreme selection in natural

populations, both for mutations and deletions.• Most ultras have deep orthology, and no paralogy.• One ultra comes from a mobile element co-option events.• Others may have come from similar events.• Ultras appear the tip of a continuum, not a unique peak.

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Ultraconserved Elements: What we don’t

• What maintains so much conservation?

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