Control of Gene Expression - Focus on Eukaryotes

Eukaryotic transcription regulators + regulatory sequences

• In eukaryotes, transcription regulators can act FAR from the promoter

  • This is because enhancers, the eukaryotic DNA regulatory sequences, can be over 1000 base pairs away from the core promoter site

• Such transcription regulators that bind to the DNA also ENHANCE transcription

  • Which is why the DNA regulatory sequences are called ENHANCERS

Activators vs. Repressors

• Activators

  • A specific type of transcription regulator that functions to promote the formation of the transcription initiation complex OR pre-initiation complex

    • Such as promoting greater binding of general transcription factors and RNA Polymerase II to the core promoter

  • They can also increase the ability of the mediator to bind to the core promoter

• Repressors

  • A specific type of transcription regulator that can:

    • Physically block activators or GTF/RNA Pol II by binding DNA

    • Bind to activators to prevent their DNA binding

    • And as a result, in some way prevent initiation

Big Idea about Enhancers and Proximal Promoters

• Both refer to specific DNA sequences that act as binding sites for transcription regulators

• They work together to control when, where, and how much of a gene is expressed in eukaryotic cells

• Proximal Promoters

  • Are located immediately upstream of a gene’s transcription start site

• Enhancers

  • Can be located thousands of base pairs away

Analogy - Relationship between core and proximal promoters, and enhancers

• The core promoter provides the physical landing dock for the transcription machinery to land

  • Think of it as the keyhole that a key must be inserted into in a car or else the car won’t start

• The proximal promoter is right next to the core promoter (upstream) and acts as the decision maker that decides the immediate conditions under which the transcription machinery can start

  • Think of it as in a car, even if you turn the key in the keyhole, the engine won’t fire unless the correct “ready” signals are ready

• The enhancer, again these specific DNA regulatory sequences that can be located far away, can dramatically amplify the cell’s ability to produce proteins when they are needed

  • Think of it as the turbo booster in a car that causes the car to go from 2 mph to 100 mph

Mediators

• A mediator complex is a multi-subunit protein assembly

  • It acts as a master regulatory hub for gene expression

    • Specifically, it can relay signs from DNA-binding transcription factors (activators/repressors) to the core transcription machinery

    • It also provides extensive surface area for protein-protein interactions → Allowing for different subunits to work together

Stem Cells

• Characteristics

  • Unspecialized

  • Potent (contain the ability to become different cell types)

  • Self-renew

• Types

  • Embryonic

  • Adult

  • Induced Pluripotent (iPSC)

Waddington’s Landscape

• This is a model that describes cell potential (specifically the levels of potency and how they differ)

• From highest to lowest potency

  • Zygote

    • Totipotent: Having the highest potency of all cell types

    • This is because at the very beginning of development, absolutely nothing has been set in stone yet and the cell contains the genetic information to become anything without restrictions

  • Embryonic Stem Cells (ES)

    • Pluripotent: Most cell types but not all

    • This is because there has been some development that has made some cells specialized (ex. for the placenta, etc.)

  • Adult Stem Cells

    • Multipotent: Some cell types but limited

    • This is because a lot of the cells have become specialized for a lot of the functions that an adult living organism needs, but there can exist some cells that provide a “blank slate” to use

  • Differentiated Cell Types

    • Unipotent: Lowest potency, single cell type

    • This is because once cells become differentiated and therefore specialized, it is unlikely that this will be reversed as the reasons these specialized cells exist is to support functions that a living organism needs

Master Regulators

• Certain transcription factors (or regulators!) that drive the expression of MANY genes

  • They’re sort of like the top of a domino effect → knocking one domino over causes a cascading effect to happen

• Master regulators can bind to the enhancers of hundred of genes OR they can bind to the promoters of other transcription factors

• Master regulators often use POSITIVE FEEDBACK LOOPS

  • Meaning they can turn on and drive the expression of many genes that eventually code for proteins like other transcription factors/regulators that support the functions of the master regulators itself and vice versa

    • Which eliminates the need to rely on an external signal to sustain the activity of master regulators

Combinatorial Control

• BIG IDEA: Cells will express various combinations of transcription regulators to drive expression of genes required for that cell’s function

  • Specifically, groups of transcription regulators can work together to ensure the proper set of genes are turned ON (or OFF) → altering cell fate and function

  • AS A RESULT, from a limited number of transcription regulators, you can get many different cell types

• To do so, combinatorial control exists by integration of information through AND, OR, NOT commands

  • Meaning that for a gene to be active, it has to meet a combination of requirements

    • For example, for a gene to be transcribed, it might need Regulator 1 AND Regulator 2

    • Or a gene might need Regulator 1 AND Regulator 2 NOT Regulator 3 (meaning that all these statements need to be met before the expression of the particular gene is turned on)

• Combinatorial Control is a very efficient technique

  • Mix-and-match ability makes it so that thousands of regulators are not needed to generates thousands of different cell types

    • And because the transcription regulators are all shared, the combinations of them are different which is why differentiated cells are possible

Induced Pluripotent Stem Cells (iPSCs)

• These cells allow for adult stem cells to be pushed back in developmental time to an embryonic-like cell state (stem-like cells)

• Expression of three transcription factors could allow for this reprogramming of adult cells

  • Oct4, Sox2, Klf4 (Yamanaka factors)

• The expression of master regulators can revert an adult cell back to a stem-cell like fate

  • Because the master regulators can support the self-sustaining loop of the Yamanaka factors and keep them going/expressed even after scientists stopped manually providing them to the adult cells through a POSITIVE FEEDBACK loop

Epigenetics

• Means “Above” the Genetic Code

  • Meaning there is a layer of control for the genome that sits on top of your DNA sequence (beyond the letters that make it up)

  • ANALOGY: While your DNA is the “instructional manual” for creating different proteins and molecules, epigenetics determines which pages (which genes) are bookmarked, highlighted, or glued shut

• How it works

  • Epigenetics involves modifications to DNA-associated proteins and DNA itself that alter how the DNA is “READ” rather than the alteration of the genetic code itself

  • Epigenetic changes can occur during development and in response to the environment

    • Especially in the earlier stages of development, epigenetic markers allow for cells to be “set up” for various different functions because of how all cells contain the capability to be associated with specific functions

    • Responses to the environment like added stress, additional nutritional incorporations, and more can also influence such changes because they can determine whether the epigenetic markers are used

• As cells become specialized, they have their own unique epigenetic profile

  • They can contain specific patterns of chemical tags attached to your DNA that dictate which genes are turned “on” or “off”

• Changes in the epigenome can be inherited upon cell division

• Reversibility in epigenetic changes allows organisms to respond to their environment

• Types of Modifications

  • DNA Methylation

  • Histone Acetylation (and Methylation)

  • Chromatin remodeling factors

DNA Methylation

• The EPIGENETIC mechanism when methyl groups are added to certain cytosine bases

  • Usually found in CpG pairs

• DNA Methylation tends to turn OFF genes in the area by recruiting proteins that BLOCK transcription

Heterochromatin vs. Euchromatin

• Heterochromatin

  • DNA is CONDENSED and INACCESSIBLE to transcription machinery

  • Genes in the regions are OFF → Can be referred to a “silenced” region of the chromosome

• Euchromatin

  • DNA is DECONDENSED and OPEN

  • Genes in the region can be ON

    • But cells also need appropriate transcription regulators to be expressed

Histone Modifications - Broadly

• DNA wraps around the nucleosome

  • Of which consists of a complex of eight histone proteins that wind around a histone octamer

    • All four of the histones used have a high proportion of positively charged amino acids (lysine and arginine) that allow for tight binding to the negatively charged sugar-phosphate DNA backbone

• The amino acids in the “tail” of the histone proteins can be modified

• IMPORTANT: For methylation and phosphorylation, the most important thing to take away is that these modifications to the histone “tail” are possible

• Cells can use a “histone code” to regulate gene expression

  • Meaning combinations of methylation and acetylation can be “read” by the cell to determine if the region should be open or closed

    • Which then alters accessibility to promoter elements like the TATA Box

      • Where tightly wrapped nucleosomes can block TATA box and other DNA binding sites, and vice versa

Histone Acetylation

• The process of adding acetyl groups to the lysine amino acids on the histone “tails”

  • Is done by histone acetyltransferase (HATs)

• Addition of an acetyl group neutralizes the original slight positive charge that histone tails have from lysine amino acids

  • As a result, it causes the histone complex to loosen its hold on DNA

• The loosened hold the histone complex has on DNA allows for DNA regulator proteins to have improved access

• Acetylated proteins can also bind other proteins like chromatin remodeling factors to promote transcription

• Deacetylation

  • Removal of acetyl groups by histone deacetylase (HDACs) has the opposite effect → Making DNA less accessible

Chromatin remodeling complex

• DNA is tightly packed into chromatin

  • As a result, the structure of chromatin often needs to be changed to allow access to genes in response to cellular needs

• Chromatin remodeling complexes use energy from ATP hydrolysis to loosen the DNA and push it along the histone octamer

  • However, this can also make DNA less accessible sometimes

p53 Transcription Factor

• Is a regulatory protein that is normally supposed to turn on genes in response to DNA damage

  • However, cancer often arises because of how the p53 transcription factor is mutated → unable to turn on gene expression to fix certain types of DNA damage