Lecture #13.1 | Oncogenes as Transcription Factors: Fos/Jun

Nucleosomes

made of histone octamers (2x H2A, H2B, H3, H4) with Lys-rich tails at the N-terminal domain (NTD)

• DNA wraps itself around and the tails protrude (n-terminal domains)

• diameter of 10 nm

• H1 histone is an open segment then there is a linker segment until another histone

What does it mean the tails stick out?

tails are modified by acetylation, methylation, and phosphorylation on Lys/Arg residues that changes the charge

• Lys/Arg: Positively charged

Histone code hypothesis

different modifications determine if the chromatin is open (active) or closed (inactive).

Acetylation of lys

leads to open chromatin because it decreases the positive charge on the Lys in the histone tails.

• Methylation

generally leads to repressed transcription, with the exception of H3K4me3, which is a mark for active transcription.

Promoters

• contain specific DNA sequences (response elements, RE) to which TFs bind.

• Promoters are immediately upstream of the transcription start site (+1, ~5 kb).

• RE = TF binding sites (TFBS), typically 6-15 bp.

Enhancers

• (~500-1000 bp) contain multiple TFBS.

  • They can be farther from +1 (up to 100 kb) and can be upstream (e.g., -10 kb) or downstream of +1 (e.g., +10 kb).

Pioneering TF

bind the histone-bound DNA, opening up the chromatin and making it easier for other TFs to bind.

• first thing that binds

Co-activators

• recruit histone acetyltransferases (HATs).

• HAT activity leads to acetylation of histone tails, resulting in open chromatin.

• Acetylation decreases the positive charge on the Lys in the histone tails.

Co-repressors

• recruit histone deacetylases (HDACs), leading to deacetylation of histone tails.

• Non-acetylated histone tails result in closed chromatin.

Mediator Complex

• After TFs bind enhancers and recruit co-activators, the Mediator complex is recruited to coordinate with the General Transcription Factors (GTFs).

• GTFs orient RNA polymerase in the correct direction for transcription.

• DNA bending brings enhancers close to promoters.

• RNA polymerase straddles +1, the start site of transcription.

• GTFs (TFII proteins, including TATA-binding protein) bind the core promoter (-60 bp to +60 bp) containing the TATA box.

• RNA Pol II transcribes protein-coding genes.

• The Mediator complex coordinates with the GTFs.

Summary of transcription initiation

1. Activators bind DNA in a sequence-specific fashion in promoters/enhancers.

2. Activators recruit co-activators, which modify the nucleosomes (nucleosome remodeling complexes move nucleosomes out of the way, requiring ATP).

3. Activators and co-activators recruit the General Transcription Factors (GTFs) – Mediator, TFIID, RNA Pol II.

4. Transcription is initiated once the C-TD of RNA Pol II is phosphorylated by TFHII (one of the 9 subunits is a kinase).

5. Promoters often stay “marked” for transcription even after RNA Pol II leaves the promoter.

   1. Primer promoter for reactivation

Oncogenes function as transcription factors (TFs).

• They possess a modular structure with a DNA-binding domain (DBD), a dimerization domain, and an activation domain.

• They bind specific DNA response elements (RE) in the regulatory regions of genes, typically as dimers.

  • REs often consist of half-sites.

• TFs are often members of families of related proteins that form homodimers and heterodimers, increasing the complexity of gene regulation.

• Binding of dimers increases the stability of the TF bound to DNA.

• The activation domain interacts with co-activators, the Mediator complex, and GTFs (including RNA Pol II), and sometimes co-repressors.

What is meant by the modular nature of TFs?

Well defined activation domain and DNA binding domain

• forms a homodimer with a combination of 2 factors

  • complexity is created by dimerization of half sites

    

• small changes in the motif or spacing lead to different transcriptional outcomes

AGGTCA

core recognition motif/half-site for a class of transcription factors, especially nuclear receptors

How does this relate to the ST Pathway

• TFs are phosphorylated in response to the signal transduction (ST) pathway (e.g., MAPK, JNK, PKC, etc.).

• Phosphorylation may activate or inhibit the activity of a TF.

• Phosphorylation may affect:

  • Nuclear localization

  • Protein dimerization

  • DNA binding

  • Co-activator/co-repressor recruitment

  • Protein stability

C-fos activation

MAPK phosphorylates TFs TCF and SRF, which bind the SRE in the c-Fos promoter

• initiates transcription

What is c-fos?

• proto-oncogene and a transcription factor.

  • Originally identified as a viral oncogene, v-Fos.

  • It is an immediate early response gene activated by growth factors in serum.

    • Does not need to wait to increase its activity, its an immediate response

  • c-Fos is phosphorylated in response to growth factors (immediate), and its RNA/protein levels are increased (early).

  • bZIP protein like c-Jun

c-Jun

• another bZIP protein, is also directly activated by the ST pathway and is phosphorylated by MAPK and JNK protein kinase.

• Ras→ Raf → Mek → Mapk → Elk1 and Jun

Ap1

Heterodimer between Fos + Jun that work as a TF

• AP1 turns on genes involved in cell proliferation, such as Cyclin D1.

bZIP Proteins

• Basic region (“b”):

  • Rich in positively charged amino acids (e.g., lysine, arginine).

  • Binds to specific DNA sequences, often with a consensus like TGACGTCA (the CRE motif).

  • has a Leucine Zipper region

    • A stretch of amino acids where leucine appears every 7 residues, forming a coiled-coil structure.

    • Allows dimerization (either homodimers or heterodimers).

    • Acts like a molecular Velcro, zipping two proteins together.

      

JNK

• stress-activated pathway is named after c-Jun.

c-Jun stabilization

• by phosphorylation, increasing its concentration in the nucleus.

• Phosphorylation also increases its transcriptional activity.

c-Fos concentration

• increased by the action of TCF/SRF on its promoter.

• High levels of both Jun and Fos allow for the formation of the Jun-Fos heterodimer, AP-1.

• The Fos/Jun heterodimer is the most stable and has the highest affinity for DNA, higher than Fos or Jun homodimers.

Fos/Jun heterodimer

most stable and has the highest affinity for DNA, higher than Fos or Jun homodimers.

• Ras/MAPK - Jun/Fos Connection

• Ras activates MAPK and JNK in response to GFs and other signals.

• Both phosphorylate c-Jun; MAPK phosphorylates c-Fos.

• Phosphorylation of c-Jun stabilizes it, increasing the concentration of c-Jun.

• MAPK phosphorylates Elk-1, which turns on the expression of the c-Fos gene.

• c-Jun heterodimerizes with c-Fos, forming the AP1 complex.

• AP1 turns on genes involved in cell proliferation, such as Cyclin D1.

  • Fibroblasts lacking c-Jun cannot be transformed by Ras, indicating that c-Jun is required for the transformation of cells by Ras!

Grb2 and SOS

Right next to RTK

• Growth factor receptor-bound protein 2 (adaptor protein) which is an adaptor protein (has no enzymatic activity).

  • ensures specificity

• SOS: Son of Sevenless (named from Drosophila genetics) which is a GEF (Guanine nucleotide Exchange Factor) for Ras.

Cyclin D1

increases after exposure of serum-starved cells to a mitogen.

• cells grown in a medium without serum cannot get mitogenic cells

• so no cyclin D1

• if a mitogen is added → initiates proliferation