Lecture 5 BIO3147 (2024) PDF

Figure 3.6 Steps in the production of β-globin and hemoglobin Transcription of the βglobin gene creates a pre-mRNA containing exons and introns as well as the cap, tail, and 3′ and 5′ untranslated regions. Processing the premRNA into messenger RNA removes the introns. Translation on ribosomes uses t...

Figure 3.6 Steps in the production of β-globin and hemoglobin Transcription of the βglobin gene creates a pre-mRNA containing exons and introns as well as the cap, tail, and 3′ and 5′ untranslated regions. Processing the premRNA into messenger RNA removes the introns. Translation on ribosomes uses the mRNA to produce a protein. The β-globin protein is inactive until it is modified and complexed with αglobin and heme to form active hemoglobin. Figure 3.7 The bridge between enhancer and promoter can be made by transcription factors The example shown here is the mouse βglobin gene. (A) Transcription factors assemble on the enhancer, but the promoter is not used until the GATA1 transcription factor binds to the promoter. (B) GATA1 can recruit several other factors, including Ldb1, which forms a link uniting the enhancer-bound factors to the promoterbound factors. Figure 3.8 Enhancer region modularity Figure 3.8 Enhancer region modularity (Part 1) (i) The top diagram shows the exons, introns, promoter, and enhancers of a hypothetical gene A, but does not show how the two enhancers are involved in the expression of the gene (see ii and iii). In situ hybridization (left) shows that gene A is expressed in limb and brain cells. Figure 3.8 Enhancer region modularity (Part 2) (ii) In developing brain cells, brain specific transcription factors bind to the brain enhancer, causing it to bind to the Mediator, stabilize RNA polymerase II at the promoter, and modify the nucleosomes in the region of the promoter. The gene is transcribed in the brain cells only; the limb enhancer does not function. Figure 3.8 Enhancer region modularity (Part 3) (iii) An analogous process allows for transcription of the same gene in the cells of the limbs. The gene is not transcribed in any cell type whose transcription factors the enhancers cannot bind. Figure 3.8 Enhancer region modularity (Part 4) (B) The Pax6 protein is critical in the development of several widely different tissues. Enhancers direct Pax6 gene expression (yellow exons 1–7) differentially in the pancreas, the lens and cornea of the eye, the retina, and the neural tube. (C) A portion of the DNA sequence of the pancreas-specific enhancer element. This sequence has binding sites for the Pbx1 and Meis transcription factors; both must be present to activate Pax6 in the pancreas. Figure 3.8 Enhancer region modularity (Part 5) (D) When the lacZ reporter gene (which codes for βgalactosidase) is fused to the Pax6 enhancers for expression in the pancreas and lens/cornea, βgalactosidase enzyme activity (blue) is seen in those tissues. Figure 3.9 The genetic elements regulating tissue-specific transcription The genetic elements regulating tissue-specific transcription can be identified by fusing reporter genes to suspected enhancer regions of the genes expressed in particular cell types. Figure 3.9 The genetic elements regulating tissue-specific transcription (A) The GFP gene is fused to a zebrafish gene that is active only in certain cells of the retina. The result is expression of green fluorescent protein in the larval retina (below left), specifically in the cone cells (below right). Figure 3.9 The genetic elements regulating tissue-specific transcription (B) The enhancer region of the gene for the musclespecific protein Myf5 is fused to a lacZ reporter gene that codes for β-galactosidase. When stained for β-galactosidase activity (darkly stained region), the 13.5-day mouse embryo shows that the reporter gene is expressed in the muscles of the eye, face, neck, and forelimb and in the segmented myotomes (which give rise to the back musculature). Figure 3.10 A silencer represses gene transcription (A) Mouse embryo containing a transgene composed of the L1 promoter, which is a portion of the neuronspecific L1 gene, a lacZ gene and the L1 second exon, which contains the NRSE sequence. (B) Same-stage embryo with a similar transgene but lacking the NRSE sequence. Dark areas reveal the presence of βgalactosidase (the lacZ product). NRSE= neuro-restrictive silencing element Figure 3.11 Epigenetic regulation by histone modification Methyl groups condense nucleosomes more tightly, preventing access to promoter sites and thus preventing gene transcription. Acetylation loosens nucleosome packing, exposing the DNA to RNA polymerase II and transcription factors that will activate the genes Figure 3.16 Two DNA methyltransferases are critically important in modifying DNA The “de novo” methyltransferase Dnmt3 can place a methyl group on unmethylated cytosines. The “perpetuating” methyltransferase, Dnmt1, recognizes methylated Cs on one strand and methylates the C on the CG pair on the opposite strand. Figure 3.15 Modifying nucleosomes through methylated DNA (HDAC & HMT mediated-repression) MeCP2 recognizes the methylated cytosines of DNA. It binds to the DNA and is thereby able to recruit (A) histone deacetylases (which take acetyl groups off the histones) OR (B) histone methyltransferases (which add methyl groups to the histones). Both modifications promote the stability of the nucleosome and the tight packing of DNA, thereby repressing gene expression in these regions of DNA methylation. Table 3.1 Some major transcription factor families and subfamilies Figure 3.17 Three-dimensional model of the homodimeric transcription factor MITF (one protein shown in red, the other in blue) binding to a promoter element in DNA (white) The amino termini are located at the bottom of the figure and form the DNAbinding domains that recognize an 11base-pair sequence of DNA having the core sequence CATGTG. The proteinprotein interaction domain is located immediately above. MITF has the basic helix-loop-helix structure found in many transcription factors. The carboxylterminus of the molecule is thought to be the trans-activating domains that bind the p300/CBP transcription co-regulator. Figure 3.18 From differentiated fibroblast to induced pluripotent stem cell with four transcription factors If the “Yamanaka factors” (the Oct3/4, c-Myc, Sox2, and Klf4 [and nanog] transcription factor genes) are virally inserted into differentiated fibroblasts, these cells will dedifferentiate into induced pluripotent stem cells (iPSCs). Like embryonic stem cells, iPSCs can give rise to progeny of all three germ layers (mesoderm, ectoderm, and endoderm). Figure 3.22 Gene regulatory networks of endodermal lineages in the sea urchin embryo Sea urchin embryo across four developmental stages showing the progressive specification of endodermal cell fates (top) and the corresponding gene regulatory network of this specification from maternal contributions and signals to pioneer transcription factors leading to the final differentiation genes (bottom). Figure 3.23 Differential pre-mRNA processing Differential splicing can process the same pre-mRNA into different mRNAs by selectively using different exons Figure 3.24 Some examples of alternative pre-mRNA splicing Figure 3.25 The Dscam gene of Drosophila can produce 38,016 different types of proteins by alternative pre-mRNA splicing (B) Dscam is required for self-avoidance between dendrites that fosters a dispersed pattern of dendrites (left). Loss of Dscam in Drosophila, however, causes crossing and disrupted growth of dendrites from the same neuron (right; arrows). (C) Expression of alternatively spliced forms of Dscam (4.1, 4.2, 4.9, 4.12) in isolated populations of mushroom body neurons (white) in midpupal brains of the fly. Figure 3.26 Model of ribosomal heterogeneity in mice ((12th edition) (A) Ribosomes have slightly different proteins depending on the tissue in which they reside. Ribosomal protein Rpl38 (i.e., protein 38 of the large ribosomal subunit) is concentrated in those ribosomes found in the somites that give rise to the vertebrae. (B) A wild-type embryo (left) has normal vertebrae and normal Hox gene translation. Mice deficient in Rpl38 have reduced Hox gene translation and an extra pair of vertebrae. Figure 3.26 Maternal contributions to DNA replication in the zebrafish blastula (A) Wild-type blastulae show BrdUlabeled nuclei (blue) in all cells. (B) Although the correct number of cells is present in futile cycle mutants, they consistently show only 2 labeled nuclei, indicating that these mutants fail to undergo pronuclear fusion. Even in the absence of any zygotic DNA, early cleavages progress perfectly well due to the presence of maternal contributions. However, futile cycle mutant embryos arrest at the onset of gastrulation. Figure 3.28 Model for RNA interference from double-stranded RNA (dsRNA) and miRNA Double-stranded RNA (dsRNA) that is added to a cell or produced through transcription (miRNA). miRNAs are processed by the Drosha RNAase in the cell and then expoxted into the cytoplasm where it will interact with the RNA-induced silencing complex (RISC), made up primarily of Dicer and Argonaute, that prepares the RNA to be used as a guide for targeted mechanisms of interference. Figure 3.29 The role of miR430 during the maternal-to-zygotic transition in zebrafish (A) Numerous mRNAs derived from maternal contributions fuel development during the cleavage stages, but transitioning into the gastrula requires active transcription of the zygotic genome. miRNAs play a major role in clearing these maternally derived transcripts during this transition. (B) miR430 plays a major role in the interference of a majority of maternal transcripts in the zebrafish blastula as it transitions to zygotic control during gastrulation. In this graph, the different curves denote the reduction in three specific transcripts, two genes of which (purple and red) are differentially degraded by miR430 (green). Figure 3.30 Localization of mRNAs (A) Diffusion and local anchoring. nanos mRNA diffuses through the Drosophila egg and is bound (in part by the Oskar protein) at the posterior end of the oocyte. This anchoring allows the nanos mRNA to be translated. (B) Localized protection. The mRNA for Drosophila heat shock protein (Hsp83) will be degraded unless it binds to a protector protein (in this case, also at the posterior end of the oocyte). (C) Active transport on the cytoskeleton, causing the accumulation of mRNA at a particular site. Here, bicoid mRNA is transported along microtubules by the motor protein dynein to the anterior of the oocyte. Meanwhile, oskar mRNA is brought to the posterior pole by the motor protein kinesin along microtubules. Figure 3.31 In situ hybridization (A) Whole mount in situ hybridization for odd-skipped mRNA (blue) in a stage-9 Drosophila embryo. (B) Antisense RNA probe with uridine triphosphate conjugated to digoxigenin (DIG). (C) Illustration of two cells at the border of the odd-skipped expression pattern seen in the box in (A). The cell on the left is not expressing odd-skipped, whereas the cell on the right is. The antisense DIGlabeled RNA probe with complementarity to the oddskipped gene becomes hybridized to any cell expressing oddskipped transcripts. Figure 3.31 In situ hybridization Following probe hybridization, samples are treated with anti-DIG antibodies conjugated to the enzyme alkaline phosphatase. When nitroblue tetrazolium chloride (NBT) and 5-Bromo-4-chloro-3indolyl-phosphate (BICIP) are then added to the sample, alkaline phosphatase converts them to a blue precipitate. Only those cells expressing odd-skipped turn blue. Figure 3.32 Chromatin immunoprecipitation-sequencing (ChIP-Seq) Chromatin is isolated from the cell nuclei. The chromatin proteins are crosslinked to their DNA-binding sites, and the DNA, bound to its proteins, is fragmented into small pieces. Antibodies bind to specific chromatin proteins, and the antibodies—with whatever is bound to them—are precipitated out of solution. The DNA fragments associated with the precipitated complexes are purified from the proteins and sequenced. These sequences can be compared with the genome maps to discover the precise locations of the genes these proteins may be regulating. Figure 3.33 RNA sequencing: RNA-Seq (Part 1) Begin with specific tissues, often comparing different conditions, such as embryos of different ages (chick embryos, as shown here), isolated tissues (such as the eye; boxed regions) or even single cells, and samples from different genotypes or experimental manipulations Figure 3.33 RNA sequencing: RNA-Seq (Part 2) (1) RNA is isolated to obtain only those genes that are actively expressed. (2) These transcripts are then fragmented into smaller stretches and used to create cDNA with reverse transcriptase. (3) Specialized adaptors are ligated to the cDNA ends to enable PCR amplification and immobilization for: (4) Subsequent sequencing. Figure 3.34 CRISPR/Cas9-mediated gene editing The CRISPR/Cas9 system is used to cause targeted indel formation or insertional mutagenesis within a gene of interest. A gene-specific guide RNA (gRNA) is designed and introduced into cells together with the nuclease Cas9, for instance by co-injection into a newly fertilized zygote. The gRNA will bind to the genome with complementarity and will recruit Cas9 to this same location to induce a double-stranded break. Figure 3.34 CRISPR/Cas9-mediated gene editing Non-homologous end joining (NHEJ) is the cell’s DNA repair mechanism that often results in small insertions or deletions (approximately 2–30 base pairs; a 2 base-pair insertion is shown here), which can cause the establishment of a premature stop codon and potential loss of the protein’s function. In addition, plasmids carrying insertions with homology to regions surrounding the gRNA target sites are used to insert known sequences at the double-stranded break. Such methods are being explored as a way to repair mutations. Figure 3.35 Targeted expression of the Pax6 gene in a Drosophila non-eye imaginal disc A strain of Drosophila was constructed wherein the gene for the yeast GAL4 transcription factor was placed downstream from an enhancer sequence that normally stimulates gene expression in the imaginal discs for mouthparts. If crossed to a strain that contains a transgene that places GAL4-binding sites upstream of the Pax6 gene, in the progeny, the Pax6 gene will be expressed in whichever imaginal disc the GAL4 protein is made. (B) Drosophila ommatidia (compound eyes) emerging from the mouthparts of a fruit fly in which the Pax6 gene was expressed in the labial (mouthpart) discs. Figure 3.36 The Cre-lox technique for conditional mutagenesis, by which gene mutations can be generated in specific cells only Mice wild-type alleles (in this case, the genes encoding the Hnf4α transcription factor) have been replaced by alleles in which the second exon is flanked by loxP sequences. These mice are mated with mice having the gene for Cre-recombinase fused to a promoter that is active only in particular cells. In this case, the promoter is that of an albumin gene that functions early in liver development. Figure 3.36 The Cre-lox technique for conditional mutagenesis, by which gene mutations can be generated in specific cells only In mice with both of these altered alleles, Cre-recombinase is made only in the cells where that promoter is activated (i.e., in these cells synthesizing albumin). The Cre-recombinase binds to the loxP sequences flanking exon 2 and removes that exon. Thus, in the case depicted here, only the developing liver cells lack a functional Hnf4α gene.