Transcription Factors Notes

Transcription Factors

Overview

• This lecture expands on prokaryotic gene expression, the necessity of cell signaling for multicellularity, and eukaryotic gene expression.
• It uses two examples of signals coupled to a transcriptional response to facilitate understanding different signaling pathways.
• A workshop on Endpoint and RT-qPCR for measuring mRNA is mentioned.

Learning Objectives

• LO1: Describe the organization of a typical protein coding gene.
• LO2: Describe the nature of trans-acting factors and cis-regulatory elements.
• LO3: Describe the relationship between general transcriptional factors and RNA polymerase(II).
• LO4: Describe the roles of specific transcription factors that bind enhancer/silencer regions.
• LO5: Describe the features of the Wnt Signaling Pathway and experimental evidence.
• LO6: Describe the features of Hypoxia Sensing.

Organization of a Typical Protein Coding Gene (LO1)

• Protein-coding genes consist of more than just the protein-encoding sequence.
• Genes include regulatory regions located upstream or downstream.
• Eukaryotic genes include:
  • Exons: protein-encoding parts.
  • Introns: parts that are removed.
  • Regulatory sequences: sequences that alter gene expression.

Trans-acting Factors and Cis-regulatory Elements (LO2)

• Cis-regulatory elements: DNA regions involved in gene regulation, dictating where, when, how, and at what level a gene should be expressed. These include promoters, enhancers, and silencers.
• Trans-acting factors: usually proteins, bind to cis-regulatory elements to regulate gene transcription.
• Transcription factors are trans-acting factors that bind directly to DNA or associate with protein complexes bound to DNA, altering the activity of cis-regulatory elements.

Structural Motifs of Transcription Factors (LO2)

• Examples of transcription factor structural motifs include:
  • Three Zinc Fingers
  • Leucine Zipper
  • Helix-turn-helix motif

Function of Transcription Factors (LO3)

• Transcription of a protein-coding gene requires RNA polymerase II.
• RNA polymerase is essential for transcribing DNA into mRNA.
• RNA polymerase forms a complex with general transcription factors to initiate transcription.
• Not all general transcription factors bind directly to DNA; many bind indirectly via other proteins in the complex.
• General transcription factors form a complex at the TATA box.
• The TATA box is a DNA region around 30 base-pairs upstream of the transcription start site and is part of the promoter, with the conserved sequence T-A-T-A.
• Different general transcription factors have different functions, including TATA box recognition, RNA polymerase II recruitment, and unwinding the DNA helix.

TATA Box and TBP (LO2, LO3)

• The TATA box is recognized by TBP (TATA binding protein), which bends the DNA 80 degrees to separate the strands for RNA polymerase II.
• Lower number of hydrogen bonds between T-A base pairs compared to G-C base pairs.
• Ease separation (melting) of DNA strands needed for transcription initiation.

General Transcription Factors (LO3)

• General transcription factors aid RNA polymerase II in its function.
• They position RNA polymerase II correctly at the promoter.
• They aid in pulling apart the two strands of DNA to allow transcription to begin.
• They release RNA polymerase from the promoter to start its elongation mode.
• They are general because they are needed at nearly all promoters used by RNA polymerase II.
• They consist of interacting proteins denoted as TFIIA to TFIID (which contains TBP), etc.
• TFII stands for ‘transcription factor for polymerase II’.
• They function similarly to bacterial σ factors.
• Portions of TFIIF even have the same three-dimensional structure as the equivalent portions of σ.

Overview of General Transcription Factor Function (LO3)

• A: The promoter contains a TATA box 25 nucleotides away from the transcription site.
• B: TFIID, via TBP, binds the TATA box.
• C: TFIID with TBP bends the DNA and facilitates TFIIB binding.
• D: Other general transcription factors and RNA polymerase gather at the promoter.
• E: TFIIH uses ATP to unwind DNA, exposing the template strand, and phosphorylates RNA polymerase II, allowing it to begin transcription elongation and release of the general transcription factors.

Roles of General Transcription Factors in Transition Initiation (LO3)

• Note: No need to memorize!.

Specific Transcription Factors (LO4)

• Specific transcription factors usually bind to enhancer and silencer regions.
• They influence the activity of the transcription initiation complex.
• Enhancers bind proteins that activate transcription, while silencers bind repressor proteins that inhibit transcription.
• Cells can alter which genes are active by controlling when and where these specific transcription factors are active.

Binding Motifs (LO4)

• Specific transcription factors typically recognize regions of 6-12 bases long.
• The length of the recognition region (the binding motif) is important: short motifs have a higher chance of appearing by chance.

Specific Transcription Factors - DNA Base Symbolism (LO4)

• A = Adenine, C = Cytosine, G = Guanine, T = Thymine
• Stacked letters mean that the binding site may sometimes have a different DNA base at that location.
• The height of the letter is how often that base appears in that position relative to the other bases.

Protein Recruitment (LO4)

• Most transcription factors contain:
  • A DNA binding domain
  • An activation or repressor domain
  • A domain for interactions with other proteins
• Many specific transcription factors recruit other proteins.

Enhancers and Silencers (LO4)

• Enhancers and silencers can be several thousand base pairs from the promoters.
• Specific transcription factors can influence transcription over these long distances by forming loops.
• Multiple enhancers and silencers can influence transcription.
• These interact with the Transcription Initiation Complex via the Mediator Complex.
• The effects of enhancers and silencers provide the organization of cells within a multicellular organism.
• Different cells express different specific transcription factors, activating different genes despite having the same genome.

Gene Regulatory Hierarchies (LO4)

• Transcription factors regulate the expression of multiple downstream targets, including other specific transcription factors.
• The result is transcription factors regulate cascades of gene transcription.

Wnt Signaling Pathway (LO5)

• The Wnt pathway is critical for embryo development and tissue regeneration in adult bone marrow, skin, and intestines.
• Wnt is the messenger that activates the Frizzled receptor on the cell membrane.
• The message is carried through a series of steps to stop the degradation of β-catenin.
• Wnt activates Frizzled, which in turn activates Dishevelled.
• Dishevelled inhibits the β-Catenin Destruction Complex, which contains Axin, APC, and GSK-3β.
• This leads to the stabilization of β-Catenin, which can then activate the transcription factor TCF.

Wnt Signaling and Cancer (LO5)

• In nearly all colorectal cancers, the Wnt signaling pathway is mutated, often resulting in the deactivation of APC (Adenomatous Polyposis Coli).
• APC has a critical role in attenuating Wnt-signaling, and these mutations result in the hyperactivation of the Wnt pathway and the formation of tumors.
• APC is a tumor suppressor because it ensures that cells grow and proliferate at the right time and place.

Measuring Transcription Factor Binding (LO4, LO5)

• One way to measure the binding of a transcription factor (e.g., β-catenin) to DNA is ChIP-qPCR.
• Process:
  • Cross-link DNA to the transcription factors using formaldehyde.
  • Shear the DNA using ultrasound.
  • Purify the DNA using antibodies for the transcription factor (e.g., β-catenin).
  • Measure how much DNA is present by amplifying the region where the TF is thought to be bound.
• If the TF isn’t bound, no DNA will amplify; if it is, more DNA will be obtained.

ChIP-qPCR Results (LO4, LO5)

• Comparing the binding of β-catenin at a control site to its binding at the promoter of the gene MYC, it was found that more DNA is immuno-precipitated from the MYC gene than from the control region.
• β-catenin therefore appears to bind at the MYC gene.

ChIP-Seq (LO4, LO5)

• If the DNA is sequenced instead of performing qPCR for a single region, the DNA sequences obtained can be aligned to the genome; this is called ChIP-Seq.
• ChIP-Seq allows to see where β-catenin binds near the promoter of MYC and to see possible enhancers.
• ChIP-Seq is a valuable technology as it allows the assessment of binding sites across the genome.

Hypoxia Sensing (LO6)

• Multicellular organisms can buffer the environment, which presents challenges.
• As depth increases of tissues within the human body, oxygen becomes less accessible to the cells.
• The body has developed the ability to sense hypoxia (lack of oxygen) and stimulate the growth of blood vessels.

HIF1A and Hypoxia (LO6)

• The protein HIF1A (Hypoxia-inducible factor) is a transcription factor.
• In normoxic conditions (i.e., when oxygen levels are fine), HIF1A is modified by a proline hydroxylase.
• This marks HIF1A in a way that is recognized by pVHL (Von Hippel–Lindau tumor suppressor), which targets HIF1A for degradation.
• Once degraded, HIF1A is unable to activate genes.
• When oxygen is low, the proline hydroxylase is unable to mark HIF1A, and HIF1A is not degraded because the proline hydroxylase needs oxygen to mark the HIF1A protein.
• Now that the HIF1A is stable, it binds DNA near genes like VEGF and activates them.
• VEGF is of particular interest as it is a signaling molecule that causes the development of blood vessels.