L14 Gene Regulation in Eukaryotes III

Eukaryotic Gene Expression III

Control of Transcription Factors

Main Routes of Control

  • Unmasking of activation domain: The activation domain of transcription factors is often masked by other domains or proteins and can be unmasked to activate transcription.

  • Transport into and out of nucleus: The localization of transcription factors is crucial; they need to be transported into the nucleus to activate transcription and can also be exported back to the cytoplasm.

  • Combinatorial control: The combined actions of multiple transcription factors can lead to differential gene expression, where multiple pathways and signals converge.

Gene Regulation in Eukaryotes

Flow of Genetic Information

  1. Gene

  2. Transcription

    • hnRNA (heteronuclear RNA)

    • mRNA (messenger RNA)

  3. RNA Processing

  4. RNA Export

  5. Translation

  6. Post-Translation

  7. RNA Degradation

    • Nuclear

    • Cytoplasmic

Transcriptional Silencing

Position Effect

  • Genes may become “silenced” depending on their location on the chromosome, particularly near telomeres and centromeres, which are transcriptionally inactive.

  • Silencing can extend over broad regions, leading to the repression of multiple genes simultaneously.

  • This process is linked with the formation of heterochromatin, which is influenced by histone modifications and DNA methylation in higher eukaryotes.

Silencing at Yeast Telomeres

  • Telomeres are structured densely, inhibiting transcription within the final 1-5 kb.

  • The “silencing complex” is formed by DNA binding proteins such as Rap1, which recognizes telomere DNA repeats and recruits silencing proteins:

    • Sir2: A histone deacetylase.

    • Sir3 and Sir4: Proteins that bind to deacetylated histones.

DNA Methylation and Gene Silencing

Mammalian Cells

  • In mammals, DNA methylation is crucial for silencing. Histone deacetylases and methylases are recruited to methylated DNA.

  • This process plays an essential role in genomic imprinting, where one allele (either paternal or maternal) is active and the other is silenced; for example, the Isf2 (insulin-like growth factor) gene is expressed only from the paternal allele.

  • An illustration shows the role of an insulator in maintaining gene expression states, where methylation inhibits binding.

Maintenance Methylase

  • A specific enzyme known as maintenance methylase targets hemimethylated DNA to preserve the methylation pattern across cell divisions, which is essential for epigenetic gene regulation.

Alternative Splicing

Mechanism

  • Alternative splicing allows a single gene to produce multiple protein variants. Various sequences can be used to produce different mRNAs:

    • Exon skipping

    • Alternative 3' exon usage

    • Alternative poly(A) sites

  • Splicing is modulated by specific activators and repressors that impact splice sites and sequences involved.

Biological Example

  • A case study related to the dsx gene in fruit flies illustrates how alternative splicing determines sex:

    • Male development is associated with splicing that skips certain exons, producing male-specific Dsx protein (150 amino acids) versus 30 amino acids for female-specific protein.

mRNA Regulation and Localization

  • For mRNA to be transported from the nucleus to the cytoplasm, RNA processing steps must occur. Blocking processing can prohibit export, thereby regulating expression levels.

  • RNA localization refers to the transport of RNA to specific cellular compartments, typically directed by sequences within the 3' untranslated region (UTR) and can occur via direct transfer or random diffusion followed by trapping.

Small RNA and Their Role in Gene Expression

Types of Small RNAs

  • Micro RNAs (miRNAs): Endogenously expressed as precursors, involved in post-transcriptional regulation of target mRNAs.

  • Small interfering RNAs (siRNAs): Derived from double-stranded RNA (dsRNA) and play a key role in RNA interference (RNAi).

  • Both miRNAs and siRNAs typically range from 19-25 nucleotides and participate in the RNA-induced silencing complex (RISC), which targets mRNA for degradation or inhibits its translation.

Mechanism of miRNA Processing

  • MiRNA undergo various processing steps:

    1. Cropping by Drosha: In the nucleus, primary miRNA is processed into pre-miRNA.

    2. Dicing by Dicer: In the cytosol, pre-miRNA is further processed into mature miRNA.

  • RISC then binds and regulates target mRNAs based on the complementarity of miRNA and mRNA sequences.

Summary of Key Points

  • Transcription initiation is a primary control point in gene regulation.

  • DNA and chromatin modifications can silence gene regions.

  • Gene regulation includes levels beyond transcription, such as RNA processing, stability, export, and localization.

  • Small RNAs have a significant regulatory role, operating through various mechanisms including mRNA degradation, translation inhibition, and histone modification.

  • The overarching goal of gene regulation studies encompasses understanding gene expression levels and interactions between proteins and nucleic acids.

Definitions Related to Gene Regulation

  • Genome: The complete set of genes of an organism or its organelles.

  • Transcriptome: The complete set of mRNA molecules present in a cell, tissue, or organ, which is context-dependent, as expression levels change with physiological or pathological conditions.

  • Proteome: The complete set of protein molecules present in a cell, tissue, or organ, also subject to variation based on context.

Methods of Analysis in Gene Regulation

Transcriptomic Analysis

  • Tools utilized include:

    • Northern analysis

    • Reporter genes

    • RT-PCR (Reverse Transcription Polymerase Chain Reaction)

    • SAGE (Serial Analysis of Gene Expression)

    • Microarrays

    • RNA-seq

Proteomic Analysis

  • Techniques employed:

    • Western blotting

    • 2D-electrophoresis

    • Mass fingerprinting

    • LC-MS (Liquid Chromatography-Mass Spectrometry)

    • Protein arrays

Protein-DNA Interaction Studies

  • Involved methods:

    • Gel shift assays (EMSA)

    • DNase I footprinting

    • ChIP (Chromatin Immunoprecipitation)

Protein-Protein Interactions

  • Methods such as:

    • Yeast two-hybrid

    • Co-immunoprecipitation

    • Tandem affinity purification

Techniques for Transcript Profiling

Northern Blots

  • Detects the presence and quantity of specific mRNA transcripts, useful for studying gene expression patterns under varied conditions.

  • Workflow incorporates gel electrophoresis followed by hybridization with a labeled probe for detection.

Microarrays

  • Utilize a solid support with DNA spots corresponding to genes. mRNA is isolated, converted into labeled cDNA, and hybridized to the array.

  • Signal intensity quantification assesses levels of gene expression.

RNA-Seq

  • Involves generating large datasets of RNA sequences that are aligned to reference genomes, facilitating various studies such as differential gene expression, transcript discovery, and RNA editing analysis.