Monday, November 11th Lecture Notes

  • In multicellular eukaryotes, the individual cells of the body differentiate by expressing different genes

  • Differential Gene Expression

    • Muscle Cells

      • Specialized for contraction

      • Express muscle-specific cytoskeletal proteins that allow the cell to rapidly shorten when it receives the appropriate signals

    • Nerve Cells

      • Specialized for the conduction and integration of electrical signals

      • Express nerve-specific synaptic proteins that allow them to communicate their signals to other cells

  • Helen Blau Study

    • Fused human liver cells to skeletal muscle cells of mice, and the hybrid cells began to stain with antibodies specific for human muscle proteins

      • Mouse muscle proteins activated transcription of human genes

  • Human Genome

    • Approximately 19,000 protein-coding genes

    • The majority of human genes are only expressed in one or a few cell types

  • In general, different cell types transcribe different genes because they contain different transcription factors

  • Pitx1 Gene

    • The Pitx1 gene of mice and humans is expressed in 4 different tissues

    • Although it has a single transcription unit, Pitx1 has 4 tissue-specific enhancers each of which responds to the combination of transcription factors found in 1 of these 4 tissues

    • The cis-regulatory region is said to have a modular organization:

      • A single gene with multiple enhancers, each of which is individually sufficient to activate transcription at the same core promoter

      • Each enhancer is activated by a different set of transcription factors, and therefore responds to different conditions

  • The molecular basis of differential gene expression has been extensively studied in developing embryos

    • The fertilized egg starts life with a single diploid genome; there is no transcription at this time

    • Gene expression and cell differentiation first appear as the embryo divides into multiple cells

  • Two different strategies that can bring out differential gene expression in embryos

    • Maternally derived transcription factors can be localized in different regions of the egg/embryo

    • Cells can communicate with intracellular signals that activate transcription factors asymmetrically

      • Some intracellular signals require cell contact; others are secreted molecules that diffuse between cells

  • Maternal Gene Products

    • In both animals and plants, fertilization involves the fusion of a very small sperm cell with a very large egg cell that contains huge quantities of

      • RNA

      • Protein

      • Nutrients

    • The RNA and protein contained within the egg were produced from the mother’s genes during the process of egg formation; hence, they are referred to as maternal gene products

  • Sea Squirt Embryo

    • Macho-1 encodes a zinc finger transcription factor that plays a critical role in formation of embryonic tail muscles

    • But it is not the embryo’s own Macho-1 gene that is used; rather, the mother squirt transcribes the Macho-1 genes during egg formation

    • Macho-1 mRNA is widely distributed at first

    • During egg formation, Macho=1 mRNA becomes concentrated at the bottom of the egg and is then moved to the posterior side

    • As the embryo divides, only 2 of 8 cells inherit this localized mRNA

    • All cells contain a set of muscle differentiation genes

    • But only the B4.1 cells inherit Macho-1 RNA and therefore synthesize Machho-1 protein

    • The Macho-1 TF selectively activates expression of muscle-specific proteins in this cell

  • How does an egg cell localize an mRNA to a particular region of its cytoplasm?

    • Some mRNAs have a localization signal sequence of their 3’-UTR

    • This nucleotide sequence can bind the RNA to a motor protein , which in turn uses the energy of ATP hydrolysis to walk along cytoskeletal protein fibers

    • As it moves, the motor protein drags the mRNA along with it

  • P2 and C. elegans

    • In the 6-cell C. elegans embryo, cell P2 secretes a protein that influences its neighbor cell MS, but not the more distant cell E

    • This signal alters gene expression in MS, but has no effect on E

  • All cell-to-cell interactions require a signal and a receptor:

    • Most receptors are cell-surface proteins embedded in the plasma membrane, but some are in the cytoplasm

    • The signals that pass between cells are generally chemicals or proteins, which are the ligands for their particular receptor

  • Once a cell-surface receptor detects its ligand molecule outside the cell, it activates a sequence of biochemical events inside the cell; the ensuing molecule events are known as signal transduction pathway

    • Many signal transduction pathways change the subset of genes that are expressed by the receiving end

  • Eukaryotic cells have thousands of different receptor proteins that can activate dozens of different signal transduction pathways

  • Jak/Stat Pathway

    • The JAK/STAT pathway is one example of a signal transduction pathway that can activate specific target genes within the receiving cell

    • The JAK-STAT pathway plays a critical role in the immune system, but like most signal transduction pathways it is also used in other ways by other cells

    • Process

      • The first step in this process is ligand binding

        • The ligand is a secreted protein called a cytokine

        • Ligand binding causes 2 subunits of the cytokine receptor to dimerize within the plane of the cell membrane

        • Dimerization brings together the cytoplasmic tails of the receptors, each of which is bound to a molecule of a second protein called Janus kinase (JAK)

        • Once the receptor has undergone autophosphorylation, the phosphorylated tails now serve as binding sites for the cytoplasmic protein STAT

        • JAK phosphorylates STAT as well

        • Once phosphorylated, STAT can function as a transcription factor

        • The phosphorylated STAT dimerizes is actively transported into the nucleus where the STAT dimers bind to specific enhancers

          • In this way, the JAK-STAT pathway activates transcription of its target genes

      • Kinases are enzymes that can transfer a phosphate from ATP to another molecule

      • Protein kinase is a kinase that phosphorylates another protein

      • Most kinases have a high degree of specificity

      • Although JAK has a kinase domain, it is unable to phosphorylate itself or the receptor molecule its bound to

      • But when the cytokine receptor dimerizes, each molecule of JAK protein can phosphorylate the other JAK and other receptor subunit

        • Receptor auto-phosphorylation

  • MAPK Signaling Pathway

    • The MAP kinase (MAPK) pathway is a second signal transduction involving different ligands, different receptors, and different cytoplasmic signaling proteins

      • Activation of the MAP Kinase pathway also involves:

        • Ligand binding

        • Receptor dimerization

        • Receptor autophosphorylation

    • Process

      • Once activated the MAP kinase signal transduction pathway involves a series of kinases, each of which phosphorylates the next protein in the sequence ultimately resulting in the phosphorylation/activation of one or more transcription factors

      • Amplification: At each step in this pathway a single molecule of kinase enzyme can phosphorylate thousands of molecules of its substrates

    • Signaling between cells, activation of signal transduction pathways, and alternations in expression are widespread features of animal development

      • But sometimes these signaling pathways don’t work properly, and the result is cancer or abnormal development

    • Achondroplasia

      • Caused by a mutated allele for Fibroblast Growth Factor Receptor 3 (FGFR3)

      • Results in the arms and legs failing to elongate during late pregnancy and childhood

      • Greater than 99% of achondroplasia results from dominant alleles of this one gene, and greater than 97% of those alleles involve the exact same missense mutation

      • The G380R mutation results in a constitutively active receptor protein

        • The receptor dimerizes and auto phosphorylates even when FGF is absent

      • Normally, FGFR3 receives signals that tell a few cells at a time to burn into bone

        • This process continues throughout adulthood

      • But cartilage cells with the G380R mutant receptor all convert to bone at once leaving no cartilage for future bone elongation

  • Mini Study Guide

    • Different cell types express different genes

    • Cell-specific gene expression is usually regulated by the different transcription factors in those cells

    • Tissue-specific enhancers can independently regulate the same gene in different cell types

    • Name and describe two ways in which a developing embryo can start turning on different genes in different cells; give one example of each

    • Understand JAK/STAT signaling

    • Understand MAPK signaling

    • Explain one example of how mis regulation of signaling can lead to aberrant development

  • I-Clicker Questions

    • Pitx1 is expressed in the developing thymus and the hindlimb buds. How is this transcriptional regulation achieved?

      • Pitx1 has several modular enhancers that respond to different transcription factor that suppresses pitx1 expression everywhere else

    • What is a cis-regulatory module?

      • An enhancer that, when bound, is sufficient for activation of transcription of the gene that it regulates, independently of binding at other enhancers

    • As an embryo grows, eventually cells must diverge in fate to make all the different tissues in the body. How are these differences achieved?

      • Changes in gene expression within each cell

    • Where do the earliest proteins translated in an embryo come from?

      • Maternally transcribed mRNAs deposited in the egg

    • The macho1 mRNA is localized to one place within the sea squirt egg. How is this mRNA identified as needing to be localized?

      • There is a signal sequence in the 3’ untranslated region

    • How are mRNAs with a 3’ signal sequence moved within the cell?

      • By motor proteins along the cytoskeleton

    • Cytokines act as a ligand for what signal transduction pathway?

      • Jak/STAT pathway

    • During autophosphorylation, a receptor/kinase phosphorylates itself

      • False

    • What do SH2 domains bind to?

      • Phosphorylated tyrosine’s

    • What needs to happen to STAT before it can regulate transcription?

      • Translocation into the nucleus

      • Dimerizaion

      • Phosphorylation by Jak

      • Binding to the cytokine receptor

    • Which molecule in the MAPK pathway likely contains an SH2 domain?

      • Grb2

    • One important feature attributed to signal transduction pathways is that they amplify the signal as it moves through the cell. What does this mean?

      • The number of molecules representing the signal increases at successive steps in the pathway.

    • What is the best way to describe the effects of the mutation in FGFR3 that causes achondroplasia?

      • The receptor is activated even in the absence of ligand