Regulation of Gene Expression Overview

Regulation of Gene Expression

• Gene expression can be regulated at every step of the pathway including:
  • Transcription initiation
  • Transcript processing
  • mRNA degradation
  • Translation
  • Post-translational processing
  • Protein degradation

Transcription Initiation

• The most important regulated step in gene expression
• The human genome contains ~2600 genes encoding DNA-binding proteins, most are presumed to be transcriptional regulators (>10% of human genes are involved in regulating transcription)

Regulation of Transcriptional Initiation

• There are two main mechanisms
  • Interplay of trans-acting factors and cis-acting elements to control RNA polymerase recruitment.
    • All genes contain consensus sequences called "cis-acting elements" which regulate the gene's expression.
    • Cis-acting elements are bound by protein "trans-acting factors" which affect the recruitment of RNA polymerase.
    • These mechanisms are important in both prokaryotes and eukaryotes.
  • cis-acting elements: short discreet DNA sequences that play a role in regulating a gene on the same chromosome. They are found within larger regulatory regions such as promoters, enhancers and silencers.
  • trans-acting elements: regulation is conferred by a molecule different than that being regulated. They include enhancers and silencers.
    • Enhancers: activate transcription and can be located upstream, downstream, or within introns of genes. Rarely found in exons.
    • Silencers: repress transcription. They are usually location and orientation non-specific and can be > 106 bp from the gene.
    • They can regulate transcription initiation by DNA-folding, which brings the region into closer proximity of the promoter.

Eukaryotic Regulatory Elements

• Discrete DNA sequences of 6-12 bp
• Bind transcription factors
• Found in promoters, enhancers or silencers
• Response elements: mediate a response to a stimuli
• Insulator elements: prevent enhancers or silencers from regulating the inappropriate genes
• Barrier Sequences: prevent heterochromatin from spreading inappropriately
  • Heterchromatin can vary in size in different cells and may cause genes nearby the heterochromatin region to be silenced inappropriately.

Identifying Regulatory Elements

• Reporter Assays: mutational analysis of a regulatory region can be used to identify specific regulatory elements.

Specific Transcription Factors

• Thousands of factors that bind regulatory elements and control:
  • Strength of expression
  • Specificity of expression (cell type and temporal specificity)
  • Response to stimuli
• Only regulate specific gene(s)
• May regulate many different genes, sometimes having drastically different effects

Mediator Proteins

• Specific transcription factors affect transcriptional initiation indirectly.
• They make contacts with either:
  • General transcription factors, or
  • Mediator proteins
    • Act as "bridges” between the general transcription factors and don't bind to specific DNA sequences.
    • May have enzymatic activity (HATs, HDACs, histone modifications, chromatin remodelling).
    • They act as coactivators or corepressors.

Steroid Agonists vs. Antagonists

• Agonists: Bind to a receptor and stimulate gene expression.
  • Examples: most endogenous steroids, Dexamethasone (a synthetic glucocorticoid agonist).
• Antagonists: Block the activity of the endogenous steroid by competitive binding to the receptor and repress gene expression and/or block activation
  • Examples: Mifepristone (RU486) – a glucocorticoid and progesterone antagonist.

Treatment of Breast Cancer by Tamoxifen

• Growth of some breast cancers depends on estrogen signaling
• Tamoxifen:
  • An estrogen antagonist in the breast.
  • Estrogen Receptor bound to Tamoxifen recruits a corepressor instead of a coactivator, stopping cell division
  • An estrogen agonist in the endometrium.
  • Estrogen Receptor bound to Tamoxifen recruits a coactivator, increasing the risk of endometrial cancer.

Coordinated Gene Expression

• A single transcription factor can coordinate the expression of many genes.
• Example: Upon glucocorticoid signaling, the glucocorticoid receptor works with other factors to activate all genes.
• This is a mechanism for:
  • Coordinated expression of many genes
  • Cell-type specificity because the glucocorticoid receptor can activate different sets of genes in different cell types, depending on the transcription factors expressed in that cell type.

Transcription Factors and Disease

• Mutations in transcription factors are associated with many diseases.
• Cancer
  • Many proto-oncogenes are transcription factors (c-myc, c-jun, c-fos)
• Autoimmune
  • Polyendocrinopathy Syndrome (AIRE)
• Developmental
  • Nail-patella syndrome (LMX1B)
  • Cleidocranial dysplasia (RUNX2)
  • Bicuspid aortic valve (GATA4)
  • Synpolydactyly (HOXD13)
• Diabetes
  • Maturity Onset Diabetes of the Young (MODY) (HNF1α, HNF1β, HNF4α, PDX1, NEUROD1)

Prokaryotic Regulation

• Promoters: the only regulatory region
• Few regulatory elements / consensus sequences
• Sigma factors: specificity and transcriptional initiation
• Co-repressors & Inducers: ligands that act via a repressor
• Co-Activators & Repressors: ligands that act via an activator
• Operons: polycistronic clusters of genes regulated by a single promoter
  • Coordinated regulation
• Lac Operon: active when Lactose is available and Glucose is not
  • Allolactose: the inducer that causes repressors to dissociate from the operator
  • cAMP: the co-activator that causes CAP proteins to bind to the CAP site

Eukaryotic Regulation: Chromatin Structure

• DNA Methylation: silencing, permanent
• Histone Acetylation: activating, dynamic
• Histone Methylation: variable, dynamic
• ATP-Dependent Chromatin Remodeling (SWI/SNF): removes/modifies nucleosomes.

Summary

• Regulatory Regions:
  • Promoters: immediately 5' of every gene, initiate transcription via the TATA Box, Inr Sequence (bind Basal/General Transcription Factors), and control specificity via other regulatory elements that bind Specific Transcription Factors.
  • Enhancers, Silencers: can be located upstream, downstream, in introns of some genes, and control specificity via other regulatory elements that bind Specific Transcription Factors. Enhancers activate, Silencers repress.
• Regulatory Elements: Response elements mediate a response to a stimuli
• Reporter Assays: used to identify positive and negative regulatory elements
• Transcription Factors:
  • General: Assemble around a TATA or Inr Sequence on ALL genes, directly recruit RNA polymerase.
  • Specific: Bind at other regulatory elements only on specific genes, indirectly recruit RNA polymerase and may act synergistically. They contact DNA through DNA-binding domains, which form sequence-specific bonds with bases.
• Mediator Proteins: “Bridges” that may have enzymatic activity and act as coactivators or corepressors.

Gene Expression Regulation

• Gene expression can be regulated at any stage of the gene expression pathway
• The most important regulated step is transcriptional initiation
• The human genome contains around 2600 genes that encode DNA-binding proteins, many of which are presumed to be transcriptional regulators

Transcriptional Initiation

• Two major mechanisms regulate transcriptional initiation:
  • Interplay between trans-acting factors and cis-acting elements to control RNA polymerase recruitment.
  • Alteration of chromatin structures to control accessibility of the gene to transcription factors and RNA polymerase.

Cis-Acting Elements

• Cis-acting elements are short discreet DNA sequences
• They regulate a gene on the same chromosome
• They are found in regulatory regions such as promoters, enhancers and silencers
• They bind to trans-acting factors which are proteins that regulate gene expression

Trans-Acting Factors

• These are proteins that regulate gene expression on a different molecule, often a DNA strand within a chromosome.
• They include transcription factors, activators and repressors.
• The activity of these proteins may be controlled by other molecules.

Measuring Gene Expression

• Gene expression can be measured by quantifying mRNA levels.
• Common methods include: Northern Blot, RT-PCR, gene expression microarray and Next-generation sequencing (RNA-seq).

Prokaryotic Gene Expression

• Prokaryotic promoters are regulatory regions found upstream of the transcription start site
• They are the only regulatory regions used in prokaryotes.
• Promoter sequences influence the rate of transcription.
• They contain binding sites for transcription factors.

Lac Operon

• The Lac operon is a group of genes related to lactose metabolism in bacteria.
• It contains a promoter, an operator, three structural genes and one regulatory gene (Lac I) encoding a repressor.
• The Lac repressor binds to the operator, preventing transcription.
• The presence of lactose induces the expression of the Lac operon via the inducer allolactose.
• Allolactose binds to the repressor and causes it to dissociate from the operator, allowing transcription to proceed.

Apolipoprotein B (ApoB)

• The APOB gene encodes apolipoprotein B (ApoB), a protein component of lipoproteins.
• ApoB is produced in the liver and the intestine.
• The liver produces ApoB100, a full-length protein.
• The intestine produces ApoB48, a truncated protein that is only 48% the size of the liver protein.
• ApoB48 is produced due to the editing of the APOB mRNA in intestinal cells.
• This editing involves deamination of a specific cytosine in the APOB mRNA, resulting in a premature stop codon, causing the creation of a truncated protein.