Genomics and Behavior

DNA Protein BehaviorRNA

“gene expression”

“Central Dogma”

Outline

• Transcription

• Analyzing genes and gene expression– Early life experience and serotonin transporter

• Manipulating gene expression– Pair bonding behavior

• Epigenetics– Maternal behavior

Protein Synthesis

cytoplasm

nucleusDNA

mRNA

Transcription

ribosome

Translation

protein

Transcription (Gene expression)

• DNA double helix is unwound

• A strand of RNA is produced from the complementary DNA strand by RNA polymerase

ATG AAC TCCTAC TTG AGG

TAC TTG AGGA AAU UG C CC

gene

Translation

• mRNA migrates out of the nucleus to the ribosome

• Protein synthesis takes place based on the genetic code

• A three base codon codes for an amino acid

Mutation can Change Protein Structure

• A mutation in DNA can change physiology or behavior by changing properties in the resulting protein

• Siamese cats have a mutation that makes the tyrosinase enzyme heat sensitive

J. Hered. 21: 309-318, 1930.

Genetic Disorders I

• A few rare disorders are caused by single mutations that cause result in a defective enzyme– Huntington’s Disease

Gene Regulation

• Although mutations causing changes in protein can have important effects on behavior, most mutations are either silent or harmful

• Usually it is a difference in gene expression that influences a behavior

Transcription

• The production of messenger RNA (mRNA) from a sequence of DNA (gene)– Gene expression– “Turning on the gene”

Important DNA Sequences

• Promoter– Indicates where transcription should begin

• Coding region– Sequence that codes for a protein

• Terminator– Indicates where transcription should stop

promoter coding region terminator

Transcription machinery

• RNA Polymerase– Transcribes DNA into RNA

• Transcription factors– Help RNA polymerase bind to DNA– There are hundreds of different transcription factors– Steroid hormone receptors

RNA polymeraseTranscription factors

Initiation of Transcription

• Transcription factors bind to promoter region

Transcription factors

promoter

Initiation of Transcription

• Transcription factors allow RNA polymerase to bind to promoter region

RNA polymerase

promoter

Transcription

• RNA polymerase begins to move down the strand of DNA and transcribe it into RNA. It unwinds the DNA as it moves down the strand.

RNA polymerase

promoter

RNA

Transcription

RNA polymerase

promoter

RNA

Termination of Transcription

RNA polymerase

terminator

RNA

• When the RNA polymerase reaches the terminator sequence, it stops transcribing

Transcriptional Regulation

• Promoter sequence• Expression of

transcription factors• Splicing

Last point on transcription

• Gene regulation doesn’t mean much unless a change in protein occurs

• Translating the genetic code of RNA into a string of amino acids

Start codon

AUG

coding region Stop codon

UGA

UAA

UAG

The entire mRNA molecule is not translated

5’ untranslated

coding region 3’ untranslated

Somatostatin receptor: AY585720

aca gac aca cac gtg acg aag atg aac tcc tcc tcg…..

Start codon

…..aag acc ttc aca tcc tgc tga gag cct cct gct cct

Stop codon

Untranslated mRNA

• 5’ and 3’ untranslated mRNA can affect stability of the mRNA molecule and also how it is translated

Coding vs. Noncoding region

• Across species, usually it is the coding region of a gene that is more strongly conserved than the noncoding region

• Most mutations affecting the function of a protein are harmful, so they tend to be selected against

Measuring Gene Expression

PCR and Microarrays

Using a sequenced genome for behavioral research

• Most methods for measuring gene expression require knowing the sequence in advance

• It’s much easier to go to a database to retrieve the sequence that have to sequence it yourself

Sequenced Genomes

• Mammals: Human, mouse, rat, cow, pig

• Birds: Chicken, zebra finch

• Fish: Zebrafish, Fugu (pufferfish), tilapia

Polymerase Chain Reaction (PCR)

• Generally credited to Kary Mullis– 1993 Nobel Prize

• One of the most widely used techniques in molecular biology

PCR Steps

• Melting– Heat up sample so that double stranded DNA

unwinds

• Annealing– Cool down the sample so that gene specific

primers anneal to gene of interest

• Extension– Heat up sample so that DNA polymerase

synthesizes new strand of DNA

Materials for PCR

• Taq polymerase

• buffers

• dNTP: raw materials

• Specific primers: short sequences of DNA that flank the region of interest

• Template: a source of DNA– Can be genomic or cDNA

PCR animation

• Run PCR.exe file

Gel Electrophoresis

• Run gel animation

PCR Applications

• Amplifying specific sequences of DNA used for– Detecting genetic differences (mutations,

deletions, etc.)– Genetic fingerprinting and paternity testing– DNA sequencing– Manipulation of DNA (recombination)

Comparative Genomics: 5HTT

• Individual variation in the length of the serotonin transporter promoter observed in rhesus monkeys

Serotonin Transporter

Activity

• The rhesus monkey short allele has reduced transcriptional activity compared to the rhesus monkey long allele

Serotonin function

• Researchers examined a serotonin metabolite (5HIAA) in cerebral-spinal fluid (CSF)

• Examined monkeys raised in two different environments– Peer-reared (nursery)– Parent-reared (mother)

Serotonin metabolites

• In monkeys raised in nursery, heterozygotes had reduced 5HIAA

• In monkeys raised by mothers, there was no effect of genotype

Serotonin and Behavior

• Individuals with the lowest CSF 5-HIAA levels are more likely to show aggressive behavior and engage in risk taking behavior (making extreme jumps, also drinking excessive amounts of alcohol)

• What’s missing from the story???

Real-time PCR

• A method for measuring the relative amount of gene expression in tissue samples

• Extremely sensitive, often referred to as quantitative PCR or qPCR

• All methods depend on increasing fluorescent signal as target gene is amplified

Why is it called real time?

• After each cycle, the instrument measures the amount of fluorescence in the reaction

Cycle Threshold (Ct)

• Ct: the number of cycles needed to reach a certain level of fluorescence

• The more copies of an mRNA present, the lower the Ct

Cycle thresholds

• Every ten-fold increase in mRNA should lead to a 3.33 increase in cycle threshold

• A standard curve can be used to determine how many copies of mRNA are present in a sample

0

2

4

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8

10

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1 10 1001,000

10,000

100,000

Copies

Cyc

le th

resh

old

Hormone Receptors in Cichlid Fish

• Cichlid fish Astatotilapia can have two phenotypes

• Dominant males are more aggressive than subordinate males

Measuring hormone receptor mRNA

• Dominant males had more androgen receptor mRNA in forebrain

• Suggested that dominant males may be more sensitive to androgens

Burmeister et al. 2007, Horm. Behav: 51 164-170.

Real time PCR +s and -s

• Advantages– Relatively easy– Very sensitive– Gold standard of measuring gene expression

quantitatively– Often used to confirm microarray results

• Disadvantages– Usually poor spatial resolution– Gene expression does not always reflect protein

expression

Microarrays

• Allow for the measurement of tens of thousands of genes simultaneously

• Generally requires that the species’ genome sequenced– Sometimes can use a closely related species– Human, mouse, rat, zebrafish commercially

available– Zebra finch, honey bee, and cichlid fish also

exist

Microarray data

• Can be overwhelming

• Many microarrays have probes from more than 10,000 genes

• Data are usually more useful if the scientist has an idea of what to look for

Honey Bee Behavior

• Younger bees stay inside the hive and take care of brood (nurses)

• Older bees forage for nectar and pollen outside the hive

Age vs. Behavior Confound

• In a normal colony age is confounded with behavior

• To address this confound, researchers created colonies in which all the bees were the same age– In this environment, some young bees will

become foragers

Behavioral Genomics

• Abbreviations– Young nurse (YN)– Young forager (YF)– Old nurse (ON)– Old foragers (OF)

• Yellow = increased gene expression

• Blue = decreased gene expression

Different Behavior, Different Genes

When bees switch from nursing to foraging

These genes turn off

These genes turn on

Gene Expression Signatures

• Changes in gene expression were so reliable that researchers could predict behavioral status based on gene expression (95% accurate)

Specific Genes

Microarrays +s and -s

• Advantages– Can generate a mind blowing amount of data– Can identify gene networks– Can help identify unexpected new directions for

research

• Disadvantages– Frequently yield false positives and false negatives– Large datasets can be overwhelming, sometimes

hard to make sense of it all– Expensive

Measuring Gene Expression

• Measuring gene expression is an important component of behavioral genetics research

• Like any correlational approach, its main weakness is that it cannot establish cause and effect relationships

Manipulating Gene Expression

Knockout mice

• Breeding of knock-out mice is becoming routine. Many universities have facilities that will create lines of mice for a fee

• Key point: traditional knockout mice lose function of the gene of interest through development and as adults

Transgenic Mice: “Knock-ins”

• Similar procedure as creating knock-out mice.

• Introduce a construct containing the gene of interest to mouse embryonic stem cells

• The construct should include a strong promoter, to ensure the gene will be expressed. This technique can also be used to determine if a given promoter is, in fact, strong

Vasopressin

• Vasopressin

• Behavioral effects– Aggression– Parental behavior– Pair bonding/affiliation

• Vasopressin receptors– V1a– V1b

Prairie vole (Microtus ochrogaster)

Vasopressin Receptor 1a (V1a) Gene

• Prairie vole (Microtus ochrogaster) and meadow vole (M. montanus) genes are 99% identical

• Biggest difference lies in the promoter region (more extensive in M. ocrhogaster)

Does the Prairie Vole Promoter Affect Behavior?

Regulation of the transcription of the V1a RNA can influence the amount of V1a

protein

Prairie Vole V1A Promoter in Mice

• Transgenic mice expressing the prairie vole V1a gene show patterns of receptor expression like prairie voles– White: wt mice– Striped: transgenics– Black: voles

Behavioral Effect of Prairie Vole Transgene

• Increased expression of the receptor alone had no effect on affiliative behavior

• Injection of vasopressin (AVP) in transgenic mice increased sniffing and grooming of a female

Complicating Issues

• The prairie vole promoter gene is present in many species of voles that are not monogamous

Fink et al. 2007, PNAS 103:10956

Partner Preference vs. Monogamy

• Partner preference behavior has been examined in only a few species of voles

• It may be that many non-monogamous species show some form of partner preference that is affected by vasopressin

• The effects of the vasopressin promoter may depend on the expression of other genes

Epigenetics

Glossary

• Epigenetic: modifications to genes that do not involve changing the DNA sequence itself and can be transmitted across generations

Levels of Organization

• Chromosome–Histone

• Gene–DNA

coarse

fine

Packaging of DNA

DNA Must be Unpackaged Before it is Read

• Unwrapping DNA from around histones allow polymerases to read DNA for either transcription or replication

Box closed with tapeShipping crate

Methylated DNA Non-Methylated DNA

A specific kind of chemical change that can affect how easily

DNA is unpacked from the histone

Methylation

DNA Methylation

• Methylated DNA has a small methyl group attached to some of its bases

• Usually methyl groups attach to cytosine (C) only

Non-Methylated DNAMethylated DNA

Maternal Care, Methylation, and “Anxiety”

• Previous studies showed that pups raised by high licking and grooming dams showed– Increased exploratory behavior– Reduced physiological responses to

stress (namely corticosterone secretion)

Methylation of Glucocorticoid Receptor Promoter

• Cross-fostering experiment showed that rat pupss raised by low licking/grooming dams had increased methylation of the glucocorticoid receptor promoter

Effect of Methylation on Stress Response

• Experimentally blocking methylation with a drug blocks the effect of low licking/grooming on corticosterone secretion during stress

Methylation of Estrogen Receptor

• Low parental care rats have low estrogen receptor expression

• Estrogen receptor promotes parental care

• Maternal behavior reduces methylation of estrogen receptor

Cross Generational Effects

• Pups raised by low licking/grooming dams grow up to lick their own pups at a low rate

• Behavioral patterns are transmitted across generations

Cross fostered

Question

• Is this inheritance of an acquired characteristic?

• Some people think so, others are not convinced

• What do you think?

“The Modern Synthesis”

• Refers to the integration of ideas on genetics with theories of natural and sexual selection– Hardy-Weinberg Theorem– Genetical Theory of Natural Selection

• Continuous variation has same method of inheritance (quantitative) as discontinuous variation (discrete)

“The Modern Synthesis”

• Populations contain genetic variation that arises randomly (mutation, recombination)

• Evolution as changes in gene frequency

• Most adaptive genetic variants have small effects on phenotype (evolution is slow)

• Acquired traits are not inherited

• Until Ronald Fisher’s “Genetical Theory of Natural Selection”, many evolutionary biologists held views on inheritance that are generally not accepted today. One prominent theory was the “inheritance of acquired characteristics”, in which traits acquired by parents could be passed on to successive generations. Although usually attributed to Jean Baptiste Lamarck, it was a commonly accepted method of inheritance in the late 19th and early 20th centuries. The rise of the “modern synthesis” of evolution rejects this mode of inheritance, but recent findings in epigenetics raise some interesting issues. Do recent findings showing behavioral modification of DNA methylation constitute a challenge to the “modern synthesis” or do these results conform with existing ideas from the modern synthesis?