Both environment and genotype affect phenotype

 Yellow ---> brown (Agouti)

 Fed the mother mice a compound called genistein, a normally occuring compound found
in soy products & tends to promote DNA methylation
 Doesn't change DNA sequence
 A^vy is affected by the methylation state of DNA
 Decreased methylation: yellow
 High methylation: brown
 When a mouse's agouti gene is completely unmethylated, it's yellow and obese and prone
to diabetes and cancer.
 When agouti is methylated (as in normal mice), the coat color is brown and the mouse has
a low disease risk
 Epi allele: the expression phenotype of being yellow or brown depends on the
methylation state of DNA (cytosines are methylated)
 A yellow coat correlates with a hypomethylated and active epiallele
 CpG islands: sections of DNA that are repeated CG clusters and the cytosine can receive a
methyl group
 The increase in methylation increases the condensation of DNA so transcription factors
have a hard time accessing DNA
 Histones: 4 different types, they bind together as dimers and the dimers bind together to
form a histone octomer which is what the DNA winds around
 Ex: 22 11
33 44
 As the DNA is wrapped around the histone octomer it forms the nucleosome
 The regions of DNA bound to that histone is blocked from access
 DNA methylation promotes that interaction with the histones and other proteins
and it recruits factors that will modify the histones themselves. Modified histones
bind DNA more tightly and in some cases assemble into multi nucleosome
complexes that bind the DNA further
 Diseases affected by epigenetic factors: ex diabetes
 Ex:EGCG, genistein, royal jelly (bees become queens)
Modification of histones
 Histone tail: sequence of amino acids hanging off the histone, series of lysines (K) and
arginines (R )
 You can have methylation of these amino acids
 Dimethylation or methylation at other lysines can be repressing.
 Not methylating the DNA here, methylating the protein that's associated with
the DNA
 Acetylation: activation
 These modifications also effect the likelihood of another lysine in the histone to be
modified
 These are all controlled epigenetically, environmental signals
 There are many dietary ingredients with epigenetic and chromatin remodeling properties
 Sulforaphanes from brassica- inhibitors of histone deacetylases
 Lunasin from soy: prevents histone acetylation
 Garlic inhibits HDAC
 Teratogens: Prevent proteins from functioning properly in the development of the
individual
  TNF alpha important for angiogenesis
 Thalidomide- no effect on DNA, inhibits TNF alpha
Norm of the reaction
 To determine the genetic component of the phenotype one needs to examine and
quantify the phenotype of interest in a controlled environment
 Genetically identical individuals (sibling crosses)
 Constant reproducible conditions
 Penetrance : % of genetically identical individuals that show the trait or mutant phenotype
of interest
 Ex: purple eggs at top, 5/9
 Expressivity: the degree to which an individual with a specific genotype displays the
mutant phenotype
 Ex: second row, differences in strength of phenotype
 Both penetrance and expressivity are affected by the environment
 Polydactyly
 Affects # of digits in both hand and feet
 Dominantly inherited trait
 Controlled epigenetically
 Sonic hedgehog gene mutations affects the enhancer element that makes it more or
less sensitive to DNA methylation
 Agouti vy: specific allele of agouti, a neomorphic mutation that results in an epi allele of
agouti
 An insertion of a particle IAP, intercisternal A particle, a piece of a retrovirus, inserts
upstream of normal promoter of agouti and takes over agouti expression
 Wt: brown coloration, yellow middle section… turns on and turns off at different
stages in the hair cycle
 This retrovirus has the ability to drive transcription independent of the normal
promoter. It drives transcription of the agouti locus, when it's active the mice will be
yellow because it will never turn off
 New rogue promoter: shuts it off based on methylation
 This IAP has a region of CpG sites, 9 CpG islands that can be methylated. As
these are methylated, this then has decreased ability to drive expression of
the agouti transcript and it starts to shut down. As methylation increases you
get pseudo-agouti, normal promoter is able to act bc the new rogue promoter
is shut off by methylation.
 Shut off the promoter by increasing the amount of genestein you get pseudo agouti
mice
Forward genetics in model systems
 What does dominance tell you about the function of the wild-type allele?
 Recessive mutations that caused an increase in trichome branching. Likely to be loss of
function.
 The normal function of the gene is to inhibit trichome branching.
 Trichome forms from epidermal cell at the surface of the cell. Some of the cells randomly
endoreduplicate, synthesize their DNA, duplicate their genome as if they're going to divide
but then the cells don't divide
 2n --> 4n (Can go to 8n, to 16n)
 During the process of trichome branching, you go through several rounds of
endoreduplication
 Increase in Dna content somehow lead to increasing in branching
 The normal function is to inhibit endoreduplication
Ex: Lof mutation
 Ovaries that have mutation in foxl2, part of a class of fox gene tf
 Ovaries started to look like sertoli cells (testes), and germ cells started to look like sperm
instead
 The default state is male if we loose foxl2
 In the ovary, the foxl2 function is to suppress male germ line character
 Homozygous for that loss of function, you get that development
Genes involved in trichome formation
 Four different genes responsible for preventing 4-5-6 trichome
 Ex: polychome, kaktus, tryptochome, rastafari
  To figure out how many different genes were involved, they had to figure out if these
mutations were really recessive or if they could be dominantly inherited
 Complementation testing
 Complementation tells us: different gene
 No complementation: same gene, different alleles of the same gene
 Can only do complementation testing for recessive mutations
Test for dominance
Complementation testing
 C & D cross --> wildtype, different genes, complementation
 In wt: moderate level of endoreduplication
 In sfi: more individuals with higher level of endoreduplication
 Pym: very high levels of endoreduplication
 Function of the wt gene for rfi and pym is to inhibit endoreduplication
 When those genes are knocked out, increase in endoreduplication
 RFI could promote pym which inhibits endoredpulciation
 Or pym could upregulate RFI which inhibits endoreduplication
 Knock out RFI and pym together, the phenotype of the double mutant should be the
about the same amount as the single mutants bc they work together
 But rfi and pym could independently inhibit endoreduplication
 If we knock out rfi, we still could have pym to reign it back a little bit
 Knock out both, if they were an independent pathway, the phenotype should be more
severe
Drosophila
 Homozygous for eye-gone gene
 Homozygous for eyeless
 Both have very similar phenotypes
 Did experiments to show that phenotypes were recessive
 Then did crosses to eyesgone to eyeless, F1 were wildtype, which tells us that they're
mutations in two different genes
 2 different genes in drosophila are required to make eyes
 In the F2, 9:3:3 ratio to wt:eyesgoen: eyeless
 Homozygous for eyesgone and eyeless, embryonic lethal
 Both eyesgone and eyeless are part of a pathway essential
 Synthetic lethality: two different pathways that are converging upon an essential process
 Pathway B or pathway A are enough to keep the organism alive

Recessive Lethality
 Agouti yellow is not an example of recessive lethality in real life
 Manx mutation: manx allele is a loss of funciton allele and wt cats are homozygous for an
allele, heterozygous make half the amount of functional product, and homozgyous for the
mutation don't make the product at all
 Semidominant mutation
 Little m is semidominant to the wildtype in the heterozygous
 Recessive lethality: homozygosity in a single locus instead of being a double mutant
 Different from synthetic lethality which involves two different genes
 Manx phenotype is similar to what we see in snap dragon (red flower, pink flower, white
flower), same idea of semidominance