Molecular Genetics 2: Control of Gene Expression (PDF)

MG2_5 Moleculaire Genetica 2 Control of gene expression (DNA rearrangements, Feedback loops, Imprinting) Molecular genetic mechanisms that create and maintain special...

MG2_5 Moleculaire Genetica 2 Control of gene expression (DNA rearrangements, Feedback loops, Imprinting) Molecular genetic mechanisms that create and maintain specialized cell types Chapter 7: p 423-445 (Albert) + Lecture Discover the world at Leiden University Alia Matysik 1  A neuron and a liver cell share the same genome Transcription regulators Cell memory (permanent changes in gene expression) decide when and where a Other transient changes (temporary) gene can be transcribed Discover the world at Leiden University Alia Matysik 2  The non-uniform distribution of transcription regulators in an early Drosophila embryo Eve gene → important for embryo development. It can read the concentrations of transcription regulators at each position. Figure 7–26 The nonuniform distribution of transcription regulators in an early Drosophila embryo. At this stage, the embryo is a syncytium; that is, multiple nuclei are contained in a common cytoplasm. Although not shown in these drawings, all of these proteins are concentrated in the nuclei. How such differences are established is discussed in Chapter 21. Discover the world at Leiden University Alia Matysik 3 The seven stripes of the protein encoded by the Eve gene in a developing Drosophila embryo Figure 7–27 Two and one-half hours after fertilization, the egg was fixed and stained with antibodies that recognize the Eve protein (green) and antibodies that recognize the Giant protein (red). Where Eve and Giant proteins are both present, the staining appears yellow. At this stage in development, the egg contains approximately 4000 nuclei. The Eve and Giant proteins are both located in the nuclei, and the Eve stripes are about four nuclei wide. Evenskipped (Eve) How does it work?. Lets see the regulatory region of the Eve gene Discover the world at Leiden University Alia Matysik 4 Experiment demonstrating the modular construction of the Eve gene regulatory region Figure 7–28 Experiment demonstrating the modular construction of the Eve gene regulatory region. (A) A 480-nucleotidepair section of the Eve regulatory region was removed and (B) inserted upstream of a test promoter that directs the synthesis of the enzyme β-galactosidase (the product of the E. coli LacZ gene). (C, D) When this artificial construct was reintroduced into the genome of Drosophila embryos, the embryos (D) expressed β-galactosidase (detectable by histochemical staining) precisely in the position of the second of the seven Eve stripes (C). 20 different transcription regulator bind in 7 different combinations to Eve gene (one combination for each strip) Discover the world at Leiden University Alia Matysik 5 The Eve gene control region of stripe 2 contains cis regulatory sequences for four transcription regulators Four regulatory proteins are responsible for the proper expression of Eve in stripe 2. Flies that are deficient in Bicoid and Hunchback fail to efficiently express Eve in stripe 2. Flies deficient in either of the two gene repressors, Giant and Krüppel, stripe 2 expands and covers an abnormally broad region of the embryo. In some cases the binding sites overlap, and the proteins can compete to bind the DNA. Discover the world at Leiden University Alia Matysik 6 Distribution of the transcription regulators for ensuring that Eve is expressed in stripe 2 The expression of Eve in stripe 2 occurs only at the position where the two activators (Bicoid and Hunchback) are present and the two repressors (Giant and Krüppel) are absent. Discover the world at Leiden University Alia Matysik 7  Eve gene control the expression of other genes Eve gene itself encode a transcriptional regulator which is setup in seven strips and control the expression of other genes which eventually give rise to different body parts of the adult fly. Discover the world at Leiden University Alia Matysik 8  Eve gene control the expression of other genes multiple inputs at a promoter Discover the world at Leiden University Alia Matysik 9  The integration of multiple inputs at a promoter Figure 7–31 The integration of multiple inputs at a promoter. Multiple sets of transcription regulators, coactivators, and co- repressors can work together to influence transcription initiation at a promoter, as they do in the Eve stripe 2 module illustrated in Figure 7–29. It is not yet understood in detail how the cell achieves integration of multiple inputs, but it is likely that the final transcriptional activity of the gene results from a competition between activators and repressors. Final transcriptional activity of the gene results from a competition between activators and repressors How do different cells get different levels of activation of transcriptional regulators? Discover the world at Leiden University Alia Matysik 10 Some ways in which the activity of transcription regulators is controlled inside eukaryotic cells Figure 7–32 Some ways in which the activity of transcription regulators is controlled inside eukaryotic cells. (A) The protein is synthesized only when needed and is rapidly degraded by proteolysis so that it does not accumulate. (B) Activation by ligand binding. (C) Activation by covalent modification. Phosphorylation is shown here, but many other modifications are possible (see Table 3–3, p. 165). (D) Formation of a complex between a DNA-binding protein and a separate protein with a transcription-activating domain. (E) Unmasking of an activation domain by the phosphorylation of an inhibitor protein. (F) Stimulation of nuclear entry by removal of an inhibitory protein that otherwise keeps the regulatory protein from entering the nucleus. (G) Release of a transcription regulator from a membrane bilayer by regulated proteolysis. Discover the world at Leiden University Alia Matysik 11 A “decision” to make different transcription regulators is made after each cell division It is self-perpetuating once initiated. In this way, through cell memory, the final combinatorial specification is built up step by step. Discover the world at Leiden University Alia Matysik 12 “Critical gene regulatory proteins” act as a “Master switches” How can one or few transcription regulators trigger activation or inactivation of a whole set of genes? “Critical gene regulatory proteins” act as a “Master switches” that can be quickly turn on or off Example Changing liver cell to neuronal cell Eye formation in leg of Drosophila Discover the world at Leiden University Alia Matysik 13  Changing liver cell to neuronal cell A small set of transcription regulators can convert one differentiated cell type into another Figure 7–34 In this experiment, (A) liver cells grown in culture were converted into (B) neuronal cells via the artificial expression of three nerve-specific transcription regulators. Both types of cells express an artificial red fluorescent protein, which is used to visualize them. This conversion involves the activation of many nerve-specific genes as well as the repression of many liver-specific genes. Discover the world at Leiden University Alia Matysik 14  Eye formation in leg of Drosophila Expression of the Drosophila Eyeless gene in precursor cells of the leg triggers the development of an eye on the leg Discover the world at Leiden University Alia Matysik 15 Expression of “master transcription regulators” is sufficient to trigger a change in cell identity Figure 7–36 A combination of transcription regulators can induce a differentiated cell to de-differentiate into a pluripotent cell. The artificial expression of a set of three genes, each of which encodes a transcription regulator, can reprogram a fibroblast into a pluripotent cell with embryonic stem (ES)-cell-like properties. Like ES cells, such induced pluripotent stem (iPS) cells can proliferate indefinitely in culture and can be stimulated by appropriate extracellular signal molecules to differentiate into almost any cell type found in the body. Transcription regulators such as Oct4, Sox2, and Klf4 are often called master transcription regulators because their expression is sufficient to trigger a change in cell identity. Discover the world at Leiden University Alia Matysik 16 Single transcription regulator can coordinate the expression of many different genes Figure 7–38 A single transcription regulator can coordinate the expression of many different genes. The action of the glucocorticoid receptor is illustrated schematically. On the left is a series of genes, each of which has various transcription regulators bound to its regulatory region. However, these bound proteins are not sufficient on their own to fully activate transcription. On the right is shown the effect of adding an additional transcription regulator—the glucocorticoid receptor in a complex with glucocorticoid hormone—that has a cis regulatory sequence in the control region of each gene. The glucocorticoid receptor completes the combination of transcription regulators required for maximal initiation of transcription, and the genes are now switched on as a set. When the hormone is no longer present, the glucocorticoid receptor dissociates from DNA and the genes return to their pre-stimulated levels Discover the world at Leiden University Alia Matysik 17 Differentiated cells maintain their identity by positive feedback loop Protein A is a regulatory protein Figure 7–39 A positive feedback loop can create cell memory. Protein A is a master transcription regulator that activates the transcription of its own gene—as well as other cell-type-specific genes (not shown). All of the descendants of the original cell will therefore “remember” that the progenitor cell had experienced a transient signal that initiated the production of protein A. Discover the world at Leiden University Alia Matysik 18 Direct and indirect feedback loop strengthen cell memory Transcription circuits allow the cell to carry out logic operations Figure 7–40 Common types of network motifs in transcription circuits. A and B represent transcription regulators, arrows indicate positive transcription control, while lines with bars depict negative transcription control. In the feed-forward loop, A and B represent transcription regulators that both activate the transcription of target gene Z Positive feedback → for keeping stable memory Negative feedback → to keep gene expression close to standard level during fluctuating conditions Discover the world at Leiden University Alia Matysik 19 How cell ignore rapid fluctuations of the input signal and respond only to persistent levels Figure 7–41 (A) In this theoretical example, transcription regulators A and B are both required for transcription of Z, and A becomes active only when an input signal is present. (B) If the input signal to A is brief, A does not stay active long enough for B to accumulate, and the Z gene is not transcribed. (C) If the signal to A persists, B accumulates, A remains active, and Z is transcribed. This arrangement allows the cell to ignore rapid fluctuations of the input signal and respond only to persistent levels. This strategy could be used, for example, to distinguish between random noise and a true signal. The behaviour shown here was computed for one particular set of parameter values describing the quantitative properties of A, B, and the product of Z, along with their syntheses. With different values of these parameters, feed-forward loops can in principle perform other types of “calculations.” Many feedforward loops have been discovered in cells, and theoretical analysis helps researchers to discern—and subsequently test—the different ways in which they may function Discover the world at Leiden University Alia Matysik 20 The exceedingly complex gene circuit that specifies a portion of the developing sea urchin embryo Figure 7–42. Each colored small box represents a different gene. Those in yellow code for transcription regulators and those in green and blue code for proteins that give cells of the mesoderm and endoderm, respectively, their specialized characteristics. Genes depicted in gray are largely active in the mother and provide the egg with cues needed for proper development. Arrows depict instances in which a transcription regulator activates the transcription of another gene. Lines ending in bars indicate examples of gene repression. Discover the world at Leiden University Alia Matysik 21  Summary Each type of cell in a higher eukaryotic organism contains a specific set of transcription regulators that ensures the expression of only those genes appropriate to that type of cell. The transcription of any particular gene is generally controlled by a combination of transcription regulators. A given transcription regulator may be active in a variety of circumstances and is typically involved in the regulation of many different genes. Since specialized animal cells can maintain their identity, the gene regulatory mechanisms involved in creating them must be stable once established and heritable when the cell divides. Direct or indirect positive feedback loops, enable transcription regulators to perpetuate their own synthesis, thus provide stability mechanism for cell memory. Discover the world at Leiden University Alia Matysik 22 Mechanisms that reinforce cell memory in plants and animals Patterns of DNA methylation can be inherited when vertebrate cells divide CG-rich islands are associated with many genes in mammals Genomic imprinting is based on DNA methylation Chromosome-wide alterations in chromatin structure can be inherited Epigenetic mechanisms ensure that stable patterns of gene expression can be transmitted to daughter cells Discover the world at Leiden University Alia Matysik 23 Patterns of DNA methylation can be inherited when vertebrate cells divide Figure 7–43 Formation of 5- methyl cytosine occurs by methylation of a cytosine base in the DNA double helix. In vertebrates, this event is largely confined to selected cytosine (C) nucleotides located in the sequence CG. Discover the world at Leiden University Alia Matysik 24 Patterns of DNA methylation can be inherited when vertebrate cells divide How DNA methylation patterns are faithfully inherited? Figure 7–44 How DNA methylation patterns are faithfully inherited. In vertebrate DNA, a large fraction of the cytosine nucleotides in the sequence CG is methylated. Because of the existence of a methyl-directed methylating enzyme (the maintenance methyl transferase), once a pattern of DNA methylation is established, that pattern of methylation is inherited in the progeny DNA, as shown. “maintenance methyl transferase“ Discover the world at Leiden University Alia Matysik 25  Patterns of DNA methylation can be inherited when vertebrate cells divide Role of DNA methylation? It contribute to stable gene repression Unexpressed genes are less leaky Figure 7–45 In this schematic example, histone reader and writer proteins, under the direction of transcription regulators, establish a repressive form of chromatin. A de novo DNA methylase is attracted by the histone reader and methylates nearby cytosines in DNA, which are, in turn, bound by DNA methyl-binding proteins. During DNA replication, some of the modified (blue dot) histones will be inherited by one daughter chromosome, some by the other, and in each daughter they can induce reconstruction of the same pattern of chromatin modifications. At the same time, the mechanism shown in Figure 7–44 will cause both daughter chromosomes to inherit the same methylation pattern. In these cases where DNA methylation stimulates the activity of the histone writer, the two inheritance mechanisms will be mutually reinforcing. This scheme can account for the inheritance by daughter cells of both the histone and the DNA modifications. It can also explain the tendency of some chromatin modifications to spread along a chromosome. The rate at which vertebrate gene is transcribed can vary 10 6 fold between one tissue than other. But in bacteria difference between expressed & unexpressed gene is 1000x Discover the world at Leiden University Alia Matysik 26 CG-rich islands are associated with many genes in mammals More than ¾ CGs have been lost during evolution 20,000 CG islands in human genome 60% coding gene have promotor embedded in CG islands Figure 7–46 The CG islands surrounding the promoter in three mammalian housekeeping genes. The yellow boxes show the extent of each island. (CG island: each ~1000 nucleotide pair long) Discover the world at Leiden University Alia Matysik 27 Why CG-rich islands survived during evolution Because they remained unmethylated in germ line C → U (repair occur) 5-methyl C → T Figure 7–47 White lines mark the location of CG dinucleotides in the DNA sequences, while red circles indicate the presence of a methyl group on the CG dinucleotide. CG sequences that lie in regulatory sequences of genes that are transcribed in germ cells are unmethylated and therefore tend to be retained in evolution. Methylated CG sequences, on the other hand, tend to be lost through deamination of 5-methyl C to T, unless the CG sequence is critical for survival. Discover the world at Leiden University Alia Matysik 28 Genomic imprinting Expression of certain genes depends on whether they are inherited from mother or father Genomic imprinting is based on DNA methylation Imprinting provides an important exception to classical genetic behavior, and several hundred mouse genes are thought to be affected in this way. If the two alleles of gene A are distinct, these different imprinting patterns can cause phenotypic differences, even though they carry exactly the same DNA sequences of the two A gene alleles. Discover the world at Leiden University Alia Matysik 29 Mechanism of imprinting Figure 7–49 (A) On chromosomes inherited from the female, a protein called CTCF binds to an insulator, blocking communication between cis-regulatory sequences (green) and the Igf2 gene (orange). Igf2 is therefore not expressed from the maternally inherited chromosome. Because of imprinting, the insulator on the male-derived chromosome is methylated (red circles); this inactivates the insulator by blocking the binding of the CTCF protein, and allows the cis-regulatory sequences to activate transcription of the Igf2 gene. (B) Imprinting of the mouse Kcnq1 gene. On the maternally derived chromosome, synthesis of the lncRNA is blocked by methylation of the DNA (red circles), and the Kcnq1 gene is expressed. On the paternally derived chromosome, the lncRNA is synthesized, remains in place, and lnc= long non-coding RNA by directing alterations in chromatin Kcnq1 encode voltage gated Ca 2+ channel structure blocks expression of the Kcnq1 gene. Although shown as directly binding to lncRNA, the histone-modifying enzymes are likely to be recruited indirectly, through additional proteins. Discover the world at Leiden University Alia Matysik 30 A striking example of imprinting inheritence Entire X-chromosome inactivation in female XX ( ) XY ( ) X (>1000 genes) Y (