Lecture 7: Enhancers and Insulators

Holoprosencephaly

Cyclopia often caused by mutations in the SHH gene

cyclopamine, smoothened, hedgehog

Corn lily creates a compound called _____ that inhibits the _____ gene which is normally activated by the ____ gene

Heidelberg Screen

A large-scale mutagenesis screening that isolated mutations affecting the pattern or structure of the Drosophila melanogaster larval cuticle— identified the hedgehog mutation

Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh)

Most vertebrates have 3 hedgehog genes:

Shh

Gene is vertebrates that plays a crucial role in the development of the nervous system, limbs, and other tissues

MFCS1

Enhancer of the Shh gene promoter

preaxial polydactyly

Point mutation in the human Shh enhancer causes…

FISH, 3C/4C

____ and ____ methods can be used for any long-distance DNA-DNA interactions

Fluorescent In Situ Hybridization

A technique involving fluorescently labelled DNA probes that identify and map specific DNA sequences on chromosomes

Direct Labelling

In FISH, refers to probes containing fluorophores directly attached to the nucleotide

Indirect Labelling

In classic ISH, refers to probes containing a bulky side chain directly attached to the nucleotide

hapten

The bulky side chain often used for indirect FISH labelling is called…

Digoxigenin (DIG)

A steroid hapten isolated from Digitalis spp.

alkaline phosphatase, horseradish peroxidase, coloured products

Anti-DIG antibodies can be coupled with compounds such as _____ or ____ which create _____ that label adjacent structures

Primary Antibody

Antibody that specifically binds the protein of interest

Secondary Antibody

Antibody that recognises the primary antibody and carries a fluorophore; are typically species-specific

in different species

Primary antibodies and secondary antibodies must be produced…

MFCS1, within the Lmbr1 gene

The Shh enhancer, _____, is found…

Chromosome Conformation Capture

Relies on “capturing” the moment when an enhancer is physically close to its corresponding promoter

crosslinking to connect promoter and enhancer— proximity ligation to fuse the promoter and enhancer DNA— PCR amplification or sequencing

The steps of 3C are:

mediator, cohesin

Interaction between ____ and ____ enables chromatin looping

Insulator

DNA sequences that act as barriers to prevent inappropriate interactions between enhancers and promoters

use of insulators and promoter competition

The two mechanisms of regulating enhancer-promoter specificity are:

Scr

Gene in Drosophila that encodes a homeodomain transcription factor which functions in cephalic and thoracic development

Ftz

Gene in Drosophila that encodes a homeodomain transcription factor which functions in establishing a segmented pattern

TATA, DPE, TATA, promoter competition

The AE-1 enhancer works with ____ and ____ promoters, but preferentially activates ____ promoters— this refers to _____

is it placed physically between an enhancer and promoter

An insulator only works if…

looping out of the intervening chromatin thus bringing them into close spatial proximity to facilitate gene expression

The looping enhancer model proposes that enhancers and promoters interact by…

creating a protein scaffold between them which facilitates enhancer-promoter interaction and gene regulation

The linking enhancer model proposes that enhancers and promoters interact by…

enhancer-binding proteins bind the enhancer and recruit RNAP before physically moving towards the promoter

The tracking enhancer model proposes that enhancers and promoters interact by…

looping facilitated by an enhancer-binding protein complex that moves down the DNA by dragging DNA behind as it moves (creating a bigger loop)

The facilitated tracking enhancer model proposes that enhancers and promoters interact by…

decondensed, chromosome territories

Interphase chromosomes are _____ and are orderly structures into ______

Transcription Factories

Area where active genes from several chromosomes may be localised to create areas of high transcriptional activity

insulator, Mod(mdg4), hold chromatin fibres to the nuclear lamina

Su(Hw), an _______, requires the addition of _____ which tethers proteins to the nuclear membrane to _______

Gypsy

A retro-transposon found in the Drosophila 5’ UTR that contains the Su(Hw) binding site

Su(Hw)

A C₂H₂ zinc finger protein that binds to 26 base pair sequences; binds Gypsy and endogenous sites

Mod(mdg4)

Protein that binds Su(Hw) which has a protein-protein interaction domain (BTB/POZ) found in many transcription factors

Su(Hw), Mod(mdg4)

Gypsy is tethered by ___ and ____ to the nuclear membrane

movement of chromatin fibres, tethering chromatin to the nuclear membrane

Insulators restrict ______ via ______ thus the movement of the enhancer is restricted and the enhancer can not access the promoter

finding nutrients, avoiding threats, and finding a mate

The roles of olfaction/chemosensing are:

MOE

Main olfactory epithelium— surface contains many finger-like projections to maximise surface contact with the air

MOB

Main olfactory bulb

VNO

Vomeronasal organ

AOB

Accessory olfactory bulb

a single odorant receptor, 1000+ odorant receptor genes in the genome, the sensory neuron identity

Each olfactory sensory neuron expresses ________ despite _____, as determined by _____

Anosmia

Loss of the sense of smell

ACE2 and TMPRSS2

The two key proteins required for Sars-CoV-2 entry are:

ACE2

Main receptor for Sars-CoV-2 that the spike proteins attach to

TMPRSS2

Secondary receptor for Sars-CoV-2 that leads to viral activation

Sustentacular Cells

Cells adjacent to the olfactory nerves which act as support for the neurons— if containing a high viral load such as in Sars-CoV-2 infection, the sense of smell may be affected

G-protein coupled receptors, largest group of membrane receptors, seven-transmembrane

Odorant receptors are ______ and are the ______— they have _____ domain receptors

M71

A mammalian odorant receptor protein

MAPK

Mitogen-activated protein kinase

CREB

cAMP-responsive element-binding protein

MAPK, CREB, a special G protein, adenylyl cyclase

___ and ___ affect GPCR transcription, however for olfaction ___ activates _____ to produce an action potential

ephedrine binds the odorant receptor which activates the G protein and stimulates adenylyl cyclase to form cAMP— cAMP activates PKA by binding to the regulatory subunit, thus releasing catalytic subunit— the catalytic subunit stimulates phosphorylase kinase whcih activates glycogen phosphorylase that cleaves glycogen to G1P— cAMP actiavtes CNG channel which causes influx of Na+ and Ca2+ which in turn activates chloride channel for Cl- efflux— depolarization occurs and triggers an action potential

Odorant GCPR activation steps:

Cyclic Nucleotide-Gated Channels

Ion channels that function in response to the binding of cyclic nucleotides

Chromosome Kissing

Transient physical interactions between chromosome territories within the nucleus often facilitated by chromatin looping

H enhancer, M50, MOR23

_____ DNA colocalizes with odorant receptor RNA (___ and ___)

H and M71, 3C, chromosome kissing, PCR with H and OR-specific primers, inverse PCR with M71 primers, or inverse PCR with H primers

Interchromosomal interactions (for example between _______) can be characterised using ______ by crosslinking _______ events, ligating, and conducting _______, ______, or _____