A high-resolution map of human evolutionary constraints using 29 mammals Kerstin Lindblad-Toh et al Presentation by Robert Lewis and Kaylee Wells

What is Evolutionary Constraint? Restrictions that conserve non- deleterious alleles! Explains why something didn’t (or doesn’t) evolve. Aspect of an organism that has not changed over time

Phylogeny and constrained elements from the 29 genome sequences Compared with the HMRD Looked at 100 bp sites 4.2 substitutions per site vs 0.68 HMRD Low probability of a non purifying sequence remaining fixed with 29 species! Therefore, better constraint detection

Shotgun Sequencing

Method for Detecting Constraint Generated 2X coverage Shotgun Sequence Contigs were 2.8kb Scaffolds were 51.8kb Depth (Coverage) = N x (L/G) N = # Reads G = Genome Length L = Read Length

Sequencing Assembly and Alignment! With 29 mammalian species they were able to find: 3.6 million elements spanning 4.2% of the human genome Length of elements significantly smaller 36 bp vs 123 bp in the HMRD comparison

PhastCons – How well individual bases are conserved SiPhy – Indicates bases under selection HMRD vs 29 Mammals HMRD  1 Element 29 Mammals  4 elements for NRSF binding

~1.5% of the genome is Protein coding 5% undergoing purifying selection Of the 5%, 3.5% are regulatory elements Genome Wide Association Studies Rely on Non-Coding Sequences

Exons and protein coding regions 3,788 candidate exons. (2% increase) Stop codon read through to subsequent stop codon in 4 genes (regulatory) >10,000 synonymous constrained elements in 25% of genes Regions with very low synonymous substitution rate (No change in AA) HIGHLY CONSTRAINED

HoxA2 (2 sites with SCE) Synonymous rate = base change but not AA change PhyloP = Nucleotide Conservation Scale from -14 to 3 (+) = More Conserved (-) = Faster Evolution (changing) dN/dS indicates selective pressure X > 1 = Change in phenotype These sites are known enhancers and drive expression in other Hox

RNA structures and structural elements! Look at RNA sequences  Determine secondary structure Found 37,381 possible elements Important b/c structure indicates function! (Look at structure and find likely target)

Promoters! Again, Structure = Function! Organized into 3 categories High Constraint Development Intermittent constrain Basic Cell Functions Low Constraint Immunity & Reproduction

Regulatory Motifs HMRD already created catalog of motifs conserved across genome  Not good for finding new motifs! 29 Mammals revealed 688 regulatory motifs associated with 345 transcription factors 2.7 million conserved instances  form regulatory network 375 motif targets with 21 regulators per target gene

Chromatin Signatures Indicate possible functions for 37.5% of unexplained conserved elements Functions of elements outside coding regions, UTRs, proximal promoters.

Accounting for constrained elements ~30% constrained elements overlap were associated with protein-coding transcripts ~27% overlap specific enriched chromatin states ~1.5% novel RNA structures ~3% conserved regulatory motifs ~60% of constrained elements overlap with any of those features

Implications for interpreting disease associated variants SNPs associated with human disease are 1.37-fold enriched for constrained regions. Only a small portion of SNPs are likely to be causative. HOXB1 and HOXB2 associated with tooth development phenotypes

Implications for Disease associated variance Look at SNPs for HOXB1 Helps resolve which SNPs disrupt function Rs disrupts Forkhead-family motif in an enhancer

Codon specific selection Looked at 6.05 million codons 84.2% Purifying (Negative) selection sites 2.4% Positive selection sites 4,431 Proteins with 15,383 positive selections sites Distributed positively selected sites for: immune response, taste perception, meiotic chromosome segregation and transcription regulation Localized positive selection sites for: microtubule based movement, topological change, telomere maintenance

Exaptation of mobile elements Elements can move and be retained where advantageous in the genome 280,000 mobile elements exaptations common to mammalian genomes Of the ~1.1 million constrained elements from 90 million years of divergence between marsupials and eutherians we can trace 19% to mobile elements 11% of mobile elements constrained

Accelerated evolution in the primate linage 564 human-accelerated regions (HARs) Previously 202 known 577 primate-accelerated regions (PARs) In these regions constrained elements for brain and limb development Influence genes harboring or neighboring are enriched for extracellular signaling, receptor activity, immunity, axon guidance, cartilage development, and embryonic pattern formation Why are we different from our primate linage?

Main Points and Key Techniques Analysis of 29 mammalian genomes showed a map of >3.5 million constrained elements. ~4% of the human genome The function of ~60% of these constrained sequences can be identified. Protein coding sequences RNA structures Promoters and transcriptional regulators Chromatin signatures This article shows the importance of constrained elements in the evolution of the mammalian lineage as well as their role in diseases.

Acknowledgments Lindblad-Toh et al., (2011) A high-resolution map of human evolutionary constraint using 29 mammals. Nature 478,476–482. Bejerano, G. et al. Ultraconserved elements in the human genome. Science 304,1321–1325 (2004).