ch 19

Gene Regulation in Eukaryotes (Chapter 19)

§19.1 Overview of Eukaryotic Gene Regulation

• Gene expression control occurs through various mechanisms affecting gene products.

• Key Steps Regulated in Gene Expression:
    - Transcription initiation
    - Transcript processing
    - Export from nucleus
    - Translation of mRNA
    - Protein localization
    - Protein modification

§19.2 Control of Transcription Initiation Through Enhancers

Three Types of RNA Polymerases in Eukaryotes

• RNA polymerase I (RNA pol I):
    - Transcribes rRNA genes.

• RNA polymerase II (RNA pol II):
    - Transcribes all protein-coding genes (mRNAs) and micro-RNAs.

• RNA polymerase III (RNA pol III):
    - Transcribes tRNA genes and some small regulatory RNAs.

Major Cis-acting Regulatory Elements

• Promoters:
    - DNA sequences adjacent to the gene.
    - Bind RNA polymerase and have a TATA box.
    - Establish the basal level of transcription.

Major Cis-acting Regulatory Elements: Enhancers

• Enhancers:
    - DNA sequences that can be located far from the promoter.
    - Enhance transcription levels (10-to 1000 fold) when proteins bind to them.
    - Can be 5′ or 3′ of the transcription start site; some located in introns.
    - Function independently of orientation and can maintain activity even when repositioned.

§19.3 Regulation After Transcription

Proteins Acting in Trans to Control Transcription Initiation

• Transcription factors:
    - Sequence-specific DNA-binding proteins that interact with promoters and enhancers.
    - Recruit additional proteins affecting transcription.
    - Types of Transcription Factors:
      - Basal factors: Help in the basic transcription process.
      - Activators: Enhance transcription levels.
      - Repressors: Suppress transcription initiation.

Regulation of RNA Polymerase II Activity

• TFIIs: General transcription factors for RNA polymerase II; assist in binding to promoters.

• Key Components:
    - TFIID: Composed of TBP (TATA box-binding protein) and TAFs (TBP-associated factors).
    - RNA polymerase II holoenzyme: Contains the core RNA polymerase II and C-terminal domain (CTD).
    - Mediator: A multiprotein complex facilitating RNA polymerase II's response to transcriptional activators.

Role of Activators in Transcription

• Activators bind to enhancers, influencing variation in transcription levels across genes.

• Mechanisms of Activator Action:
    - Stimulating recruitment of basal factors and RNA polymerase II to promoters.
    - Recruiting co-activators to modify chromatin structure.

Domains within Activators

• Activators possess at least two functional domains:
    - DNA Binding Domain: Binds to specific enhancer.
    - Activation Domain: Interacts with neighboring proteins like basal factors or coactivators.

• Examples of DNA binding domains include:
    - Helix-turn-helix
    - Zinc finger

Repressor Proteins and Their Functions

• Repressors: Suppress transcription by recruiting corepressors.

• Corepressors' Functions:
    - Prevent RNA polymerase II complex from binding to the promoter.
    - Modify histones leading to closed chromatin structure.

Cell-type Specific Transcription and Allosteric Regulation

• Mechanism of Regulation: Transcription factors' activities can vary through allosteric interactions with effectors.

• Example: Steroid hormone receptors;
    - Hormone binding induces a shape change, increasing affinity for DNA-binding domains to target enhancers.

• Post-translational Modifications (PTMs): Alter transcription factor activity via covalent modifications.

Multifunctionality of Transcription Factors

• A single transcription factor can function differently in distinct cell types.

• Example: Drosophila Dorsal protein acts as an activator for one gene and as a repressor for another by recruiting corepressor Groucho.

Enhancer Functions

• Enhancers determine tissue-specific and temporal-specific gene expression.

• In humans, approximately 2,000 genes encode regulatory proteins, each capable of acting on multiple genes.

Reporter Genes in Enhancer Identification

• Enhancers can be identified by constructing recombinant DNA molecules with a putative enhancer sequence fused to a reporter gene (e.g., GFP).

• Generation of transgenic organisms allows visualization of enhancer activity.

Insulators: Mechanism and Function

• Insulators: DNA elements between a promoter and an enhancer that block enhancer activation of that promoter.

• Organize DNA into loops; enhancers activate transcription from promoters within the same loop.

• Interaction of CTCF proteins forms DNA loops, also referred to as topologically associating domains (TADs).

• An enhancer can only interact with a promoter if both are in the same TAD.

§19.4 A Comprehensive Example: Sex Determination in Drosophila

Comparison with Human Sex Determination

• In humans, the presence of the SRY gene on the Y chromosome determines maleness; absent results in femaleness.

• In Drosophila, maleness is the default state, with two X chromosomes activating transcription of the Sxl gene essential for female development.

Sxl Gene and its Cascade Effects in Drosophila

• Transcription of Sxl in XX (females) initiates various pathways:
    1. Determine external appearance and behavior as male or female.
    2. Guide germ cell development into eggs or sperm.
    3. Produce dosage compensation by increasing transcription of X-linked genes in males.

Understanding Sex-specific Traits

• Drosophila males and females exhibit distinct sex-specific traits biologically and behaviorally.

• Phenotypes identified as specific to males (blue), females (red), or shared variations (green).

Gene Control in Sexual Behavior

• The fruitless (fru) gene codes for male-specific behavior in courting.

• Mutant males display aberrant courting behavior linked to fru gene dysfunction.

Drosophila Mutations and Their Impacts

Expression of Sxl mRNA During Early Development

• The Sxl gene is crucial for female-specific development.

• In XX cells, Sxl transcription from the establishment promoter (Pe) depends on X-linked activators (Scute, Runt, SisA, Upd), requiring two X chromosomes for sufficient activator concentration.

The Role of Sxl in Alternative Splicing

• Post-embryogenesis, Sxl transcription occurs from the maintenance promoter (Pm).

• Male transcripts include a stop codon in exon 3 whereas female transcripts undergo alternative splicing to produce functional protein.

Lethality in Sxl Loss-of-Function Males vs. Females

• Sxl absence does not affect males due to lack of expression, while females rely on Sxl for development, leading to lethality.

• Aberrant expression of dosage compensation genes, specifically Msl-2, in Sxl mutants triggers female lethality after incorrect synthesis of male proteins.

Sxl Protein Functions in Splicing Cascade

• Sxl protein plays a central role in regulating the splicing of transformer (tra) and doublesex (dsx) mRNAs.

Alternative Splicing Outcomes of Dsx

• Two splicing products:
    - Dsx-F (female): Activated by Sxl and tra products when two X chromosomes are present.
    - Dsx-M (male): Produced in the absence of Sxl product, influencing male-specific development.

Fru Proteins and Their Regulation

• Alternative splicing yields Fru-M (male-specific) and Fru-F (female-specific).

• Different splicing leads to varied Fru protein expression in Drosophila based on sex.

Eukaryotic Transcription Summary

• Key Points:
    - Multiple RNA polymerases present (particularly RNA Pol II).
    - Essential gene components include promoters and TATA boxes.
    - Enhancers can regulate at a distance and exhibit bidirectional activity.
    - Insulators play a role in organizing gene expression.
    - Transcription Factors (TFs) have complex interactions, including allosteric regulation and dual functionality.
    - Analysis of enhancers can be performed through reporter assays.
    - Drosophila's sex determination pathway serves as a prime example of regulatory cascades in gene expression by analyzing specific gene interactions.