Molecular Biology of the Cell - Volume 2 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1993

Control of Gene Expression
Strategies of Genetic Control

The Morphology and function of various Cell types that make up a higher Organism often differ quite significantly. For example, a mammalian neuron and a lymphocyte look so little alike (see Fig. 13-29) that it is hard to imagine them harboring the exact same genome. Such vast differences between Cells led to the hypothesis that genes might be selectively lost during Cell Differentiation. However, it is now known that this is not the case; cell differentiation is determined by changes in Gene Expression, not by gene loss.

10.1.1. Different cell types of a multicellular organism contain the same DNA [1]

The diverse cell types in a multicellular organism differ from one another because they synthesize and accumulate distinct sets of RNA and Protein molecules. These processes occur without irreversible Changes in the DNA. The best evidence that The Genome is conserved during cell differentiation comes from classic experiments performed on frogs. If The Nucleus of a fully differentiated frog cell is injected into an unfertilized oocyte whose own nucleus has been removed, the injected "donor" nucleus directs The Development of a normal tadpole from the recipient egg. Since the tadpole possesses A wide variety of differentiated cells that derived their DNA sequences from the Nucleus of the original donor cell, we can conclude that the differentiated donor cell has not lost any essential DNA sequences. A similar Conclusion follows from experiments conducted on various plants. In these studies, pieces of differentiated tissue were cultured on an artificial medium and then dissociated into individual cells. It turned out that such an isolated cell is capable of regenerating an entire adult plant (Fig. 10-1).

Class="center">

Fig. 10-1. Diagram of an experiment that can be performed with various plants. Many differentiated plant cells retain the capacity for "dedifferentiation": under appropriate conditions, they give rise to a clone of cells that can develop into an entire organism (see Chapter 20).

Another piece of evidence indicating that large blocks of DNA are neither lost nor rearranged during vertebrate development is the following observation: mitotic Chromosomes from different cell types, when subjected to special staining techniques, exhibit the exact same transverse banding pattern (see Fig. 9-40). Based on this criterion, one must conclude that the chromosome Complement in differentiated cells of The Human Body is identical. Furthermore, when the genomes of various cells are compared using recombinant DNA Methods, it turns out that the changes in gene expression accompanying the development of Multicellular Organisms are generally not accompanied by alterations in the DNA sequences of the corresponding genes (some important exceptions are discussed in Section 18.4.2).

10.1.2. Different cell types synthesize different sets of Proteins [2]

To understand The Mechanism of cell differentiation, one must first know just how different various cell types really are. A complete answer to this fundamental question is not yet available, but certain Conclusions have already been reached.

1. There are numerous processes common to all cells, and consequently, All cells share many identical proteins. Some of these proteins are present in large amounts and are easily analyzed. Among them are the major structural Proteins of the Cytoskeleton and chromosomes, certain proteins belonging to the Endoplasmic reticulum and Golgi apparatus, ribosomal proteins, and so forth. Many less abundant proteins, such as various Enzymes involved in core metabolic reactions, are also present in all cell types.

2. Certain proteins are found in large quantities only in specialized cells, whereas in other cell types they cannot be detected even by the most sensitive methods. For example, Hemoglobin is found exclusively in red Blood Cells.

3. When roughly 2,000 of the most prevalent proteins (i.e., those present at about 50,000 molecules per cell) are compared among different cell types using two-dimensional Polyacrylamide gel Electrophoresis, relatively few differences are revealed. Whether the comparison is made between two cultured cell lines (e.g., Muscle and Nerve Cells) or between cells from two differentiated rodent Tissues (e.g., Liver and lung), the result is similar. The vast majority of proteins are synthesized in all examined cell types, with synthesis rates varying by no more than fivefold across cell types. Only a few percent of proteins fail to follow this pattern.

Fig. 10-2. Five levels of control of GENE EXPRESSION IN eukaryotes. Following Protein Synthesis, protein activity can be controlled through regulated degradation (G), reversible modifications (such as phosphorylation), and the targeted Intracellular Localization of the protein molecule.

Judging by the number of different mRNA sequences present, a typical higher Introduction/5.html">Eukaryotic Cell synthesizes between 10,000 and 20,000 different proteins (see Table 9-2), many of which are present in concentrations so low that they escape detection even by two-dimensional gel electrophoresis. If these minor proteins vary across different cells to the same extent as the more abundant classes, one must conclude that relatively modest changes in THE SPECTRUM OF proteins synthesized by a cell can profoundly alter its entire behavior and Structure.

10-3

10.1.3. Gene expression can be regulated at each step of the pathway from DNA to RNA and to protein [3]

If the differences between various cell types stem from which proteins are expressed within them, It is important to know at what level Protein synthesis is controlled. Along the pathway leading from DNA to protein, this control can be exerted at virtually any stage (Fig. 10-2). The principal "control points" can be: 1) the timing and frequency of METABOLISM/31.html">Transcription of a given gene (transcriptional control), 2) the Processing of the primary RNA transcript (RNA Processing control), 3) the Selection of mature mRNAs in the nucleus for export to the Cytoplasm (RNA transport control), 4) the selection of mRNAs in the cytoplasm for Translation on Ribosomes (translational control), 5) the selective destabilization of specific types of mRNA in the cytoplasm (mRNA degradation control), and 6) the selective activation, inactivation, or compartmentalization of protein molecules after their synthesis (protein activity control).

For most genes, transcriptional control is the most important. The earliest insights into the role that transcriptional control plays in vertebrate development came from comparisons of liver mRNA with Brain mRNA. Some of these RNAs were present exclusively in liver cells. Using cDNA probes, it was demonstrated that brain cells lack not only the corresponding mRNAs but also the primary transcripts from which they are formed. To verify this, nuclei were isolated from liver and brain cells and then incubated with highly radioactive RNA precursors (ribonucleoside triphosphates). As a result, all RNA transcripts synthesized during this incubation became radioactively labeled. This process, known as "nuclear run-on transcription," is illustrated schematically in Fig. 10-3. The labeled RNA molecules were subsequently subjected to rigorous analysis (Figs. 10-4 and 10-5). It was conclusively shown that brain heterogeneous nuclear RNA (hnRNA) contains no sequences homologous to any of the 11 different liver-specific mRNAs. In these experiments, liver hnRNA served as a positive control because it was known to contain sequences that hybridize with liver cDNA. The absence of liver-specific mRNAs (and consequently the proteins they encode) in the cytoplasm of brain cells can be attributed primarily to the transcriptional silencing of the corresponding genes in these cells. In other words, data on randomly selected liver-specific mRNAs indicate that the differences between liver and brain cells are controlled predominantly at the transcriptional level.

Fig. 10-3. Preparation of radioactively labeled hnRNA. Following the incorporation of the label and cell lysis, RNA polymerase molecules continue the elongation of the same RNA molecules in vitro, yielding a highly active preparation of the sequences currently being transcribed in The Cell (primary RNA transcripts).

Fig. 10-4. Schematic diagram of a standard method for detecting radioactively labeled RNA molecules with a specific nucleotide sequence. If the RNA molecules can form a hybrid RNA/DNA duplex with a single-stranded DNA fragment used as a specific probe, these molecules remain intact during nuclease Treatment and are easily detected by their radioactive label. In the experiments described in the text, purified DNA segments corresponding to various genes were obtained by cloning cDNA molecules, which in turn had been synthesized by reverse transcription of total liver mRNA. Each of these DNA fragments was used as a probe in a separate experiment (see Fig. 10-5).

10-4

10.1.4. Regulatory proteins can either activate or suppress gene transcription

Eukaryotic cells contain A large number of site-specific DNA-binding proteins, whose primary function is to turn genes on or off. Each of these regulatory proteins, present in a few copies per cell (~1 molecule per 3,000 nucleosomes or 10⁴ copies per mammalian cell), recognizes specific DNA sequences 8–15 NUCLEOTIDES in length. The binding of such proteins to DNA can either stimulate the transcription of a nearby gene (positive regulation) or inhibit it (negative regulation) (Fig. 10-6). We will discuss some of the mechanisms involved in these processes later (see Section 10.2.7). Different cell types of a multicellular organism possess different sets of regulatory proteins, with the result that each cell type transcribes its own unique set of genes.

10.1.5. Combinations of several regulatory proteins controlling gene activity can determine the development of numerous cell types [4]

A schematic diagram illustrating the combinatorial Regulation of Gene activity is shown in Fig. 10-7, where each numbered element represents a distinct regulatory protein. In this purely hypothetical diagram, a single mother cell gives rise to two cell types, A and B, which differ solely in that one synthesizes regulatory protein 1 while the other does not. As these cells develop further, some of them acquire regulatory proteins 2 and 3, and subsequently 4 and 5. Consequently, 8 distinct cell types are formed (designated by letters G through N), characterized by different combinations of the five regulatory proteins. Adding two more regulatory proteins (6 and 7) to this scheme would lead to the generation of 16 cell types at the next stage. Another 10 similar stages would yield a total of 10,000 different cell types—all achieved through the interaction of just 25 regulatory proteins.

Thus, combinatorial gene regulation is remarkably efficient: a small number of regulatory elements can specify the patterning of complex biological objects. Chapter 16 will demonstrate how this system Functions during Drosophila embryonic development. A set of regulatory genes directs the sequential subdivision of the embryo into distinct compartments (see Section 16.5.5).

Fig. 10-5. Experimental design demonstrating that gene expression in mammalian cells is controlled primarily at the transcriptional level (see Fig. 10-4).

Fig. 10-6. Comparison of positive and negative regulation. Although this example illustrates control at the transcriptional level, these Two Types of control can operate at any of the regulatory steps depicted in Fig. 10-2.

Fig. 10-7. Simplified scheme of embryonic cell differentiation, illustrating how various combinations of a small number of regulatory proteins can generate numerous cell types. This highly simplified model assumes that following each Cell Division, a choice is made to synthesize one of two alternative regulatory proteins (indicated by the numbered circles). According to this convention, daughter cells positioned to the left within the overall embryonic plan (and relative to the book page) always induce the synthesis of even-numbered proteins, whereas daughter cells positioned to the right induce the synthesis of odd-numbered proteins. The synthesis of each regulatory protein is assumed to be a self-sustaining process (see Figs. 10-33 and 10-35). Consequently, cells within the expanding clone will contain a progressively increasing repertoire of distinct regulatory proteins, each controlling an entire battery of genes.

10.1.6. Gene activity typically depends on the action of multiple regulatory proteins [5]

At first glance, the scheme of combinatorial gene regulation presented in Fig. 10-7 might suggest that differences between cell generations accumulate in a stepwise fashion. For example, one might assume that adding regulatory protein 2 to cells C and E would result in the same set of additional proteins in both cell types (namely, those encoded by genes activated by regulatory protein 2). This view is incorrect for a very simple reason. Combinatorial gene regulation is far more complex because various regulatory proteins interact with one another. Even in Bacteria, turning on a single gene sometimes requires the cooperative interaction of two different regulatory proteins (see Section 10.2.2). In higher eukaryotes, the transcription of a given gene generally demands the combined action of an entire cluster of activator proteins (see Section 10.2.9). For instance, when protein 2 interacts with activator protein 1, it may activate a different set of genes in cell E than it does in cell C. This presumably explains why a single steroid hormone receptor protein (an example of a regulatory protein) dictates the synthesis of different sets of proteins in various mammalian cell types (see Section 12.2.2). Overall, the specific changes in gene expression triggered by the synthesis of a regulatory protein depend on the cell's history, since these prior events determine which regulatory proteins are already present in the cell (Fig. 10-8).

10.1.7. Master regulatory proteins activate many genes simultaneously [6]

As noted earlier, cells contain numerous regulatory proteins, each of which, acting in combination with other proteins of this class, controls multiple genes. There appears to be an intricate network of Protein Interactions in which individual regulatory proteins control genes encoding other regulatory proteins, and so on.

Fig. 10-8. The Effect of newly synthesized regulatory proteins on a cell. This effect depends on the regulatory proteins already present within the cell and, consequently, on its developmental history. The diagram depicts the same gene in cells A and B. Initially, this gene is inactive in both cells. However, cell A produces a protein (shown on the far left) that is absent in cell B. For simplicity, each regulatory protein is assumed to exert either a positive or negative effect on transcription, with the overall outcome determined by their combined influence. In reality, the net effect is not necessarily additive. For example, in some cases, two regulatory proteins interact upon binding to DNA, altering each other's activity.

Fig. 10-9. Diagram illustrating how a "decision" to synthesize a single master regulatory protein can influence The production of a wide variety of proteins within a cell.

However, not all regulatory proteins are equal. Some of them (master regulatory proteins) exert a decisive, coordinating influence on The activity of numerous genes (Fig. 10-9). For example, Chapter 16 will show how a series of Mutations in a single Drosophila gene can transform one body part of the fly into another. Mutations causing such transformations are known as homeotic mutations. One such mutation, called Antennapedia, directs the synthesis of an aberrant master regulatory protein. As a result, an entire group of cells that normally form an antenna radically alters its behavior and develops into a leg, yielding a fly with a leg growing out of its HEAD. Evidence for the existence of master regulatory proteins in vertebrates (including humans) comes from findings such as the following: the absence of a single protein (the testosterone steroid hormone receptor) causes a genetic male embryo (XY) to develop into a phenotypically near-normal female (see Section 12.2.3). Direct proof of the involvement of master regulatory proteins in vertebrate development has recently been obtained from studies of Skeletal Muscle cells.

10-5

10.1.8. A single master regulatory protein can convert a fibroblast into a myoblast [7]

A mammalian skeletal muscle cell is typically very large and multinucleated. It forms through the fusion of numerous precursor cells called myoblasts (see Section 17.6.1). A mature muscle cell differs from other cells by its high Abundance of cell-specific proteins, including specialized isoforms of Actin, Myosin, Tropomyosin, and troponin (Components of the contractile apparatus), creatine kinase (which Supports specialized muscle metabolism), and acetylcholine receptors (required for membrane sensitivity to neural stimulation). Proliferating myoblasts lack these muscle-specific proteins and their corresponding mRNAs, or contain them only in trace amounts. As myoblasts fuse, the concentrations of muscle-specific proteins and their mRNAs increase coordinately in the resulting multinucleated cells. Consequently, the expression of these genes is controlled at the transcriptional level. The synthesis rate of many muscle-specific proteins rises at least 500-fold. Two-dimensional polyacrylamide gel electrophoresis reveals that the concentrations of many other proteins change simultaneously: some cease to be synthesized, others peak in production and then decline, and still others shift from one synthesis rate to another, and so on.

When the gene for a myoblast regulatory protein (myoD1) is introduced into cultured Skin fibroblasts (cells that normally never express muscle-specific genes), these cells acquire The ability to fuse and exhibit other characteristics of muscle cells (Fig. 10-10). Clearly, myoD1 acts as a master regulatory protein that normally specifies the "myoblast" fate. The myoD1 protein is localized in the Cell Nucleus. Its Amino Acid Sequence was deduced from The nucleotide sequence of its DNA. Studies of the myoD1 protein have demonstrated that it binds to regulatory regions of muscle-specific genes. The properties of this remarkable protein strongly support the hypothesis that master regulatory proteins can dictate cell fate conversion.

Fig. 10-10. Immunofluorescence micrograph of embryonic chicken skin fibroblasts converted into muscle cells following the artificially Induced Expression of the myoD1 gene. These fibroblasts were grown in culture and transformed with recombinant plasmid DNA three days prior to photography. The plasmid contained sequences encoding the myoD protein linked to a viral promoter/enhancer active in chick embryos. A small percentage of the fibroblasts took up the DNA and produced the myoD protein. These cells fused to form elongated myofibrils, which are highlighted in this photograph using Antibodies directed against a muscle-specific protein. A control culture transfected with a different plasmid lacks muscle cells. (Kindly provided by Stephen Tapscott, Andrew Lassar, Robert Davis, and Harold Weintraub.)

Summary

In multicellular organisms, cell differentiation occurs as a result of the expression of different genes from the same genome, although cell types differ surprisingly little from one another in their protein content. The expression of most genes is controlled at the transcriptional level, which does not rule out a significant role for Post-transcriptional Control. Transcriptional control depends on regulatory proteins that bind to specific DNA sequences. As a result of the binding of such proteins, the corresponding genes are either turned on (positive control) or turned off (negative control). Genes in higher eukaryotes are typically regulated through the combinatorial action of multiple regulatory proteins exerting both positive and negative control. Master regulatory proteins play a special role in the gene activity regulation system by influencing the activity of many genes simultaneously: for example, the expression of the MyoD1 gene can convert a fibroblast into a myoblast.