Lecture 8 (Kaufmann) Flashcards
Eukaryotic TF's
Four-winged fly experiment
Nobel prize for physiology/medicine
Four-winged fly experiment
- Maternal morphogen gradients, like nanos and bicoid, establish the anterior-posterior axis and provide positional information for development.
- Gap genes, such as Krüppel, are activated by maternal gradients and define broad embryonic regions like the head, thorax, and abdomen.
- Pair-rule genes, including even-skipped, create a striped pattern across the embryo, dividing it into segments.
- Segment polarity genes, like engrailed and wingless, refine segment boundaries and establish segment polarity.
- Hox genes, such as Ultrabithorax (UBX) and abdominal A, determine the identity and specialization of segments, guiding the formation of structures like the thorax and abdomen.
- The research revealed a hierarchical genetic cascade that regulates segmentation and specialization, providing key insights into genetics, evolution, and developmental biology.
HIF transcription factor
Nobel prize for physiology/medicine
HIF transcription factor
- Discovery of how cells sense and adapt to oxygen availability, focusing on the HIF transcription factor.
- Under normoxia (normal oxygen levels), HIF-1α is hydroxylated by prolyl hydroxylase enzymes, marking it for degradation by the proteasome through binding with the VHL protein.
- Under hypoxia (low oxygen levels), hydroxylation is inhibited, preventing HIF-1α degradation.
- Stabilized HIF-1α translocates to the nucleus, where it dimerizes with ARNT and binds to HRE (Hypoxia-Responsive Elements) in the DNA.
- This activates transcription of genes that enable the cell to adapt to low oxygen, such as those involved in erythropoiesis, angiogenesis, and metabolism.
- The research revealed how oxygen sensing directly controls gene expression, with implications for understanding diseases like cancer and anemia.
Stages of Gene Expression Control
Stages of Gene Expression Control
- Local Structure of the Gene (Hetero- & Euchromatin)
- mRNA modification & processing (e.g. splicing)
- mRNA stability (e.g. miRNA)
- mRNA export from the nucleus (mRNP)
- Translation & Posttranslational mechanisms
Organization of Transcription Factors
Organization of Transcription Factors
- Modular organization (composed of distinct, independent domains)
- DNA-binding activity and transcription activation are carried out by independent domains of an activator
- Specificity for activation is determined by the DNA-binding domain
Tat (HIV)
An RNA-binding protein that acts like a transcription factor
Tat (HIV)
- Tat binds the TAR RNA: While the RNA is still being transcribed, Tat binds to the TAR (Trans-Activation Response) element, a structured sequence in the nascent (newly made) mRNA at the 5’ end.
- Recruits P-TEFb: Tat recruits the cellular factor P-TEFb, which contains CDK9 (a kinase). This kinase phosphorylates the CTD (C-terminal domain) of RNA Polymerase II.
- Overcomes Pausing: Without Tat, RNA Polymerase II often stalls or pauses after initiating transcription, resulting in incomplete or aborted transcripts. Tat and P-TEFb enable the transition of RNA Polymerase II from pausing into productive elongation, allowing the full-length viral transcript to be made.
Transcriptional Activation Domains
Transcriptional Activation Domains
Acidic Activation Domains
- Contain acidic amino acids like glutamate and aspartate.
- Interact with coactivators (e.g., CBP/p300) to acetylate histones and open chromatin.
- Recruit Mediator and chromatin remodelers to stabilize RNA Polymerase II.
Glutamine-Rich or Proline-Rich Domains
- Enriched in glutamine (e.g., Sp1) or proline (e.g., AP-1).
- Stabilize transcription machinery by interacting with general transcription factors.
- Facilitate PIC assembly and maintain open chromatin.
How can many different transcription factors act with the same
coactivator complexes such as Mediator or p300?
IDRs
- Transcription factors interact with coactivators like Mediator or p300 through intrinsically disordered regions (IDRs), which lack a fixed structure and adapt upon interaction with partners.
- IDRs are versatile, classified by their amino acid composition (e.g., acidic, proline-rich) or shapes (e.g., acid blobs, negative noodles).
- Coactivators are modular and flexible, allowing simultaneous interaction with multiple transcription factors.
- Mediator bridges transcription factors and RNA Polymerase II, while p300 modifies chromatin through histone acetylation, making DNA accessible.
Fuzzy Complex Hypothesis
Fuzzy Complex Hypothesis
- Fuzzy complexes involve proteins with flexible, unstructured regions that remain dynamic even after binding.
- These unstructured regions are called intrinsically disordered regions (IDRs) and are found in transcription factors and coactivators.
- Fuzzy complexes allow multiple modes of interaction, making them adaptable to different binding partners.
- They may help proteins form biomolecular condensates, which organize transcription machinery.
Pioneer TF’s
Pioneer transcription factors (TFs)
- bind DNA even in compacted chromatin, locating target sites buried in nucleosomes.
- They open chromatin by displacing nucleosomes with the help of co-factors or chromatin remodelers.
- This process makes chromatin accessible for other TFs, coactivators, and transcription machinery, enabling gene activation.
- Examples like Oct4, Sox2, and Klf4 adapt to nucleosome surfaces and facilitate cooperative binding with other TFs, such as c-Myc.
- NF-Y is a pioneer transcription factor that binds the CCAAT box in compacted chromatin, resembling histones structurally, and facilitates chromatin remodeling and early gene activation.
Transcriptional Repression
Transcriptional Repression
- Repressor proteins block activator TFs or general transcription machinery from binding DNA.
- impacts chromatin structure, reducing accessibility for transcriptional machinery.
- Repression condensates are membraneless compartments formed through liquid–liquid phase separation, concentrating corepressors like GROUCHO and chromatin-modifying enzymes (e.g., HDACs, HMTs) to compact chromatin and silence gene expression.
Corepressors recruit enzymes like:
- HMTs: Add H3K9Me, H3K27Me to silence genes.
- HDACs: Remove acetyl groups (H3Ac, H4Ac) to tighten chromatin.
- KDMs: Demethylate H3K4Me, reducing activation marks.
- ATPases: Remodel chromatin to restrict access.
Transcriptional Repression
Example
GROUCHO-type co-repressors
- interact with repressor transcription factors to mediate transcriptional repression.
- They lack enzymatic activity but recruit HDACs (Histone Deacetylases) to remove acetylation marks, tightening chromatin.
- Bind to specific repressor domains, such as WRPW motifs, on target transcription factors.
- Involved in chromatin remodeling, ensuring transcription machinery cannot access DNA.
- Play a role in regulating developmental processes by repressing genes critical for differentiation or pattern formation.
How are TF’s activated?
How are TF’s activated?
- Protein Synthesis: Some TFs are activated upon synthesis, such as homeoproteins.
- Phosphorylation: Inactive TFs can be activated via phosphorylation, e.g., Heat Shock Transcription Factor (HSTF).
- Dephosphorylation: Certain TFs require dephosphorylation for activation.
- Ligand Binding: Steroid receptors are activated when they bind specific ligands.
- Inhibitor Release: NF-κB is activated when released from an inhibitor.
- Partner Exchange: TF activation can occur via a change of binding partners, such as HLH proteins (MyoD/ID).
- Proteolytic Cleavage: Membrane-bound proteins can be activated through cleavage that releases an active TF, as in sterol response.
Cys 2/His 2 Zinc Finger Domain
Example for a transcription factor domain
Cys 2/His 2 Zinc Finger Domain: Example for a transcription factor domain
- Zinc finger domains are structural motifs stabilized by zinc ions, formed by His and Cys amino acids coordinating the zinc ion.
- Each zinc finger typically contains 23 amino acids and forms an α-helix that binds one turn of the major groove of DNA.
- Zinc finger proteins usually have multiple zinc fingers, allowing them to interact with longer DNA sequences.
- They can bind DNA, RNA, or both, depending on the specific protein and its function.
- Zinc fingers can be engineered to target specific nucleotide sequences for synthetic biology and gene-editing purposes.
Zinc Fingers of Steroid Receptors
Example for a transcription factor domain
Zinc fingers in steroid receptors
- Unique because they contain Cysteine (Cys) residues but lack Histidine (His) residues.
Steroid receptors, such as glucocorticoid and estrogen receptors, have two zinc fingers:
- One zinc finger is responsible for DNA binding.
- The second zinc finger helps maintain proper spacing between the binding sites.
Steroid receptors bind DNA as dimers:
- Homodimers (two identical receptors) bind to palindromic sequences in DNA.
- Heterodimers (two different receptors) bind to direct repeat sequences in DNA.
- These mechanisms allow precise DNA recognition and regulation of gene expression by steroid hormones
bHLH (basic Helix-loop-Helix)
Example for a transcription factor domain
bHLH (basic Helix-loop-Helix)
- Composed of 40-50 amino acids.
- Contain two amphipathic α-helices separated by a basic loop region.
- The basic region interacts with DNA, typically binding to E-box sequences (CANNTG).
- The α-helices mediate dimerization, enabling the formation of homo- or heterodimers.
- Function in regulating cell differentiation, proliferation, and other critical processes.
- Note: Flanking sequences modulate TF binding by altering DNA shape, emphasizing that binding specificity isn’t determined solely by the core binding sequence but also by the structural context of the DNA.
