Shiri FreilichJanet Thornton’s group, EBICambridge University

Relating the evolution of gene content to tissue specialization

“...And when he was 93, Grandpa decided that the time had come for a man-to-man conversation.

(I was 36 at the time, I have been married for 15 years).

‘All my life I am all the time looking at woman, looking and learning. Nu, and what I learned, I want to learn to you now also.’

‘Woman, in some ways is just like us exactly the same. But in some other ways a woman is entirely different.’

‘But you know what? In which ways a woman is just like us and in which ways she is very different –

nu, on this I am still working’.”

(Amos Oz, A Tale of Love and Darkness)

The full genome sequence from various species can highlight the common versus unique

What’s new?

The transition from unicellularity to multicellularity as an interior

design challenge

- gene

Overview

• Part 1: Expression pattern of ‘old’ and ‘new’ proteins in mouse tissues

• Part 2: Expression pattern of singleton and duplicate proteins in mouse tissues

• Part 3: The evolution of tissue-specific metabolic pathways in mammals

Part 1:

Relating age and function of a protein to its expression pattern in mouse tissues

A collaboration with Tom Freeman’s group

(MRC RFCGR)Freilich et al, Genome Biol. 2005;6(7):R56.

Processing mouse expression and sequence data

Classification of mouse proteins into phylogenetic groups

Tissues have a similar composition of functional classes

Tissue type

Frac

tion

enzymes

transcription regul.signal transductiontransporters

Tissues have a similar composition of phylogenetic classes

Tissue type

Frac

tion

universaleukaryote sp.metazoan sp.mammalian sp.

Expression distribution of proteins from different

categories• Tissues have almost identical functional/

phylogenetic composition• Tissue diversity must be achieved through

differences in the protein composition within each category

• Do proteins from different categories duffer in their expression pattern?

Number of tissues where protein is expressed

Frac

tion

Regulatory proteins are more specifically expressed

Number of tissues where is expressed

Frac

tion

~1/3

~1/10

Mouse proteinsclassified into functional groupsRegulatory

proteins

Metabolic proteins

enzymes

transcription regul.signal transductiontransporters

Metazoan-specific proteins are more specifically expressed

Present in a unicellular ancestor of metazoa

Number of tissues where protein is expressed

Frac

tion

universaleukaryote sp.metazoan sp.mammalian sp.

Mouse proteinsclassified into phyletic groupsSpecific to

metzoa

Functional categories overlap with phylogenetic categories

• Most of the pre-metazoan proteins are metabolic proteins (transporters and enzymes)

• Most of the metazoan-specific proteins are regulatory proteins (signal transduction and transcription regulation)

Identifying the dominant influence: function or age

• Obvious differences between ‘old’ and ‘new’ proteins, within the metabolic functional group

• Yet, less than 1/3 of the pre-metazoa proteins are expressed in all tissues

Number of tissues where protein is expressed

Frac

tion

Still, some of the pre-metazoa proteins are tissue specific

• Functions occurring in the unicellular cell become tissue-specific in multicellular species (Ldh example)

• Universal genes that have been duplicated become specific to a tissue whilst a second copy maintains its original expression pattern (Pgk-2 example)

Part 2:

Relating duplication events to expression pattern in mouse tissuesFreilich et al, Genome Biol. 2006;7.

“…duplication events had contributed greatly to the attainment of the complex body organisation in metazoa, where cells having identical genetic material can differentiate … due to the presence of duplicated genes in their genomes” Ohno S. (1970). Evolution by gene duplication.

The subfunctionalization model

Lynch M & Force A, Genetics. 154 (2000):The division of expression of an ancestor gene between its daughter duplicates promotes the retention of a gene in the genome

Microarray expression data provide support to the

subfunctionalization model• Gu et al: expression divergence between

duplicate genes increases with evolutionary time (differentiation modes in yeast). Trends Genet. 2002;18: 609-13.

• Makova et al: spatial expression divergence between duplicate genes increases with evolutionary time (human tissues). Genome Res. 2003;13:1638-45 .

• Huminiecki and Wolfe: a general trend for increased tissue-specificity of expression as family size increase was observed for mammalian genes. Genome Res. 2004;14:1870-79 .

Project Overview:The relationship between gene

duplication and breadth of expression

Protein’s perspective: 1. Does duplication event lead to an increase in tissue

specificity?

Time of duplication perspective:2. Does the date of duplication event matters? (i.e., do

duplication events occurring in the ancestral unicellular lead to an increase in tissue specificity)

Protein-family perspective: 3. does a protein family maintain a non-specific expression

pattern? (i.e., is a specific expression of proteins from big families complementary)

(Freilich et al, Genome Biol. 2006;7(10):R89)

Singleton proteins are more globally expressed

Number of tissues in which protein is expressed

Frac

tion

Singletons (570) Duplicate proteins (1886) Proteins with many close homologues (417)

Negative correlation between expression breadth and number

of homologues

Groups of proteins, ordered by their number of homologues

Mea

n nu

mbe

r of

ti

ssue

s

SingletonsDuplicate proteins

Large variation

Number of homologues proteins

Num

ber

of t

issu

es

Correlation -0.20P-value 1.5e-55

SingletonsDuplicate proteins

Mean number of expressed tissues

Correlation -0.20 P-value 1.5e-55

Does the date of duplication event matter?

(i.e., do duplication events occurring in the ancestral unicellular lead to an increase in tissue specificity)

?

Increase in tissue specificity

Global expression

Identifying ‘old’ and ‘new’ duplications

Only post-multicelullarity duplication events lead to

expression specificity

The protein-family perspective: Is the specific expression of

family members complementary?

Proteins from big families tend to be more specifically expressed.

Does a protein family maintain a non specific expression pattern?

?

Complementary expression

Overlapping expression

Calculating the cumulative tissue distribution of protein

families

Cumulative tissue distribution of protein families is not

correlated with family size

Complementary expression pattern in protein families:

While a duplication event leads to a tissue specialisation of one or

both copy, the total tissue-distribution of the protein family remains

constant.

Protein families, ordered by size

Aver

age

tiss

ue-

cove

rage

of

prot

ein

fam

ilies

Singletons

Families with any expression information

Families with >=75%expression information

The findings support the subfunctionalization model

Protein’s perspective: 1. Does duplication event lead to an increase in

tissue specificity? YesTime of duplication perspective:2. Does the date of duplication event maters?

only duplication events that that place in a multicellular species lead to a specific expression-> suggests that expression divergence, following gene duplication, promotes the retention of a gene in the genome

Protein-family perspective: 3. Does a protein family maintain a non-specific

expression pattern? Yes-> suggests the division of expression between family members

Part 3:The evolution of the mammalian

metabolic pathways

Some of the tissue-specific pathways are specific to mammals.

Can we understand how tissue-differentiation of animals’ metabolism reflects their evolution?

Freilich et al, BMC evolutionary biology 2008, 8:247.

STEROID HORMONE METABOLISM

Why studying metabolic networks?

• Metabolic networks’ structure and composition are well defined

• Available metabolic databases

• Genotype is highly related to phenotype

The structure of the KEGG database

STEROID HORMONE METABOLISM

The reactions within a pathway can be absent/present in a

species

Arabidopsis Thaliana

Homo Sapiens

Project overview:

• Identification of pathways absent/present in a species

• Classification of human pathways according to their phyletic origin

• Characterization of lineage-specific metabolic pathways

Phylogenetic classification of human pathways

All pathways in human (metabolic)Universal pathways

Eukaryota-specific pathways

Metazoan-specificpathways

Mammalian-specific pathways

Components of theEukaryotic membrane(sphingolipids, glycosaminoglycan)

Phylogenetic classification of human pathways

33 Universal pathways

8 Eukaryota-specific pathways

10 Metazoan-specific pathways

14 Mammalian-spc. pathways

Metabolic skeleton: sugars, nucleotides, some amino-acids, energy

Tissue specific activities:Neuronal guidance, hormonal activity, digestion Tissue specific activities and intracellular signaling(blood cell recognition)

The pathways can be linked to form a network

The network structure of the metabolic pathways

Universal pathwaysEukaryota-spc. pathwaysMetazoan-spc. pathwaysMammalian-spc. pathways

The integration of the steroid biosynthesis pathway into the sterol biosynthesis pathway

sterol

cholesterol

steroid hormonebile acid

UniversalEukaryotaHuman

From manually selected examples to a computational approach

Creating a list of adjacent reactions:

2.7.4.2 -> 4.1.1.334.1.1.33->2.5.1.1..2.5.1.1->2.5.1.212.5.1.21-

>1.14.99.7

Using the adjacency list for a large-scale characterization

of the metabolic network

What’s new (metabolic pathways)?

Summary

• ‘New’ genes tend to be more tissue-specific, ‘ancient’ genes tend to be globally expressed

• Despite this trend, many metazoan genes are ubiquitous and many universal proteins are tissue specific

• ‘New’ duplications of ‘old’ and ‘new’ proteins lead to a more specific expression, and therefore can facilitate the evolution of new, tissue-specific, functions

• The core of metabolic-pathways, inherited from a uniclellular ancestor, provides a platform for the evolution of mammalian-specific, tissue-specific pathways

Thanks

Thornton GroupJanet ThorntonTim MassinghamEric BlancExpression data:Tom Freeman

Sumit Bhattacharyya