Developmental Biology BY1101 P. Murphy Lecture 7 Cellular differentiation and the regulation of gene expression. In this lecture we looked at two main questions: How is gene expression regulated? (revision of some material in chapters 11, 17 and 18) and How does this relate to cellular differentiation? As you heard in lecture 1, Cellular differentiation is one of the 3 main processes needed to form a complex organism from a single fertilized egg cell (Cell division, cell differentiation and morphogenesis). A complex organism requires many hundreds of different cell types to form structures and carry out specific functions. For example, red blood cells are required to carry oxygen, muscle cells are required for movement, neurons are required to receive and transmit nerve signals. If all the cells arise from a single fertilised egg cell and all contain the same DNA in their nuclei, how do they become different to each other or differentiate? This is what we call cellular differentiation. Cellular differentiation is brought about by differential gene expression: the cells become different because they express different genes. e.g. Muscle cells must express the myosin gene so that they have one of the structural proteins needed (myosin) to enable a muscle fiber to contract and red blood cells must express globin genes in order to produce haemoglobin to transport oxygen. Red blood cells do not express the myosin gene and muscle cells do not express globin genes.- these different cell types follow different differentiation programmes. So in order to understand how this can be brought about, we recapped on what it means to express a gene (turn it on) and how the decision to be expressed (or not) is controlled in a cell. How are genes turned on and off? = How are genes regulated? Some terms revised: DNA (deoxyribonucleic acid): The substance that constitutes the hereditary material of an organism. It resides in the nucleus of all eukaryotic cells, organised into linear units called chromosomes. It is a double stranded polymer of deoxyribonucleotides of which there are 4 types (see genetic code below)
A gene: A unit of hereditary information consisting of a particular nucleotide sequence of DNA (generally). Many genes are organised along a chromosome. (There are many definitions of a gene depending on the perspective you take) The genetic code: DNA is made up of 4 types of nucleotide: A (adenine), C (cytosine), G (guanine) and T (thymine). The sequence in which these nucleotides occur determines the protein that a gene will encode. Transcription: When a gene is turned on its DNA sequence is used as a template for the synthesis of a complimentary RNA (RiboNucleic Acid) molecule (messenger RNA; mrna) by a process called transcription. mrna: A single stranded polymer of ribonucleotides produced by transcription of a gene. It directs the production of a protein during translation. The sequence of ribonucleotides is complimentary to the DNA sequence being transcribed. (U instead of T) Translation: The production of protein from RNA, the sequence of amino acids that make up the protein depending on the genetic code carried by the RNA. See Campbell and Reece Fig 17.4 for an illustration of transcription and translation To view what happens when a gene is on (being expressed) in a eukaryotic cell, see Campbell and Reece figure 17.3. A gene includes more than coding sequences (sequences that are transcribed and translated). It also includes regulatory sequences that determine which cells express that gene and when they turn it on. Remember this when you need to define or describe a gene; a gene is not just coding sequence but also the regulatory sequences that determine when and where it will be expressed (turned on). Regulatory sequences at the start of the coding sequence (Promoters) are needed for the transcriptional machinery to assemble and begin to transcribe the DNA sequence into an RNA message or transcript (mrna). These are similar in all eukaryotic genes. See Campbell and Reece figure 17.8 Other regulatory sequences are gene specific and these determine when and where a gene will be turned on. They can be situated close to the coding sequence (proximal control elements) or at large distances (distal control elements). These are often called enhancers and can be positioned upstream (before) or downstream (after) or within the coding sequence (in an intron) See Campbell and Reece figure 18.8 Transcription initiation is controlled by proteins that interact with DNA (regulatory
sequences) and with each other- see figure 18.11 These proteins are called transcription factors and operate by binding to the specific regulatory sequence elements (control elements- enhancers) described above. Cell specific transcription factors influence the efficiency with which the general transcription factors (transcription initiation complex) assemble on the promoter sequence and initiates expression of the gene. Distant control elements, enhancers, may be thousands of nucleotides away from the promoter or even downstream of the gene or within an intron. Factors that turn a gene on in this way are called activators. Eukaryotic genes can also be influenced by repressor proteins that bind to DNA regulatory sequences and tend to destabilize transcription and turn the gene off (can also be called silencers). So to recap: Activator If the balance is favourable Transcription Three important points to note about cellular differentiation: Point 1 Cellular differentiation is usually a result of transcriptional regulation: turning genes on and off. Point 2: During embryonic development, cells become obviously different in
structure and function as they differentiate. But differentiation does not happen suddenly. Differentiation happens progressively as the embryo develops. When differentiated cells appear they already produce the proteins that allow them to carry out their specialised roles in the organism e.g eye lens cells, 80% of their capacity for protein synthesis makes crystallins. However changes will be taking place inside a cell long before it visibly differentiates. These include a gradual reprogramming of the genes that are expressed. This would show up only at the molecular level. We looked at the example of progressive myoblast differentiation under the control of the cell specific transcription factor MyoD to illustrate progressive differentiation. This is well covered in the text book: See Campbell and Reece Fig 18.18 and from bottom of page 414 Point 3: The genes that encode transcription factors that control cellular differentiation (e.g. MyoD) are called Master regulatory genes. These control the expression of sets of target genes (downstream genes), the products of which are needed for the cell to differentiate. Many of the downstream genes may also be regulatory genes controlling the expression of more target genes. This is how a cascade event along a differentiation pathway may be controlled and explains why differentiation is progressive. These master regulatory genes, or developmental regulators, are the genes of most interest to developmental biologists. ------------------------------------------------------ This leaves much unexplained How is the pathway initiated? How are the master regulators (e.g. MyoD) spurred into action? How do cells receive instructions about which master regulators to turn on? We will begin to address these questions in lecture 8 Key concepts in lecture 7 1. Cellular differentiation, one of the three major processes that must take place during
development, is brought about by different cells expressing different sets of genes. 2. The genes that are expressed in a cell give it its special characteristics and allow it to carry out its particular functions, e.g. muscle must contract, neurons must receive and transmit signals, lens cells must transmit and focus light, and blood cells must transport oxygen. 3. Consideration of what a gene is and how its expression is regulated is therefore fundamental to working out how development is controlled. The basic facts about gene expression were therefore revised. 4. The primary level at which gene expression is controlled is transcription: the decision about whether or not to make an mrna copy of the coding sequence of the gene. 5. Regulatory sequences (control regions) outside the coding sequence of the gene determine when and where a gene is transcribed. They do this by acting as binding sites for regulatory proteins called transcription factors. Most transcription factors are activators of transcription but some can act as repressors. 6. The genes that are expressed in a cell therefore depend on the transcription factors that are present. Muscle structural genes are therefore active in muscle cells because the cells possess the right transcription factors (e.g. MyoD) to turn them on. Lecture 7: Learning outcomes: you should be able to A) Define cellular differentiation and describe its importance during embryonic development giving examples of cell types that must be established, mentioning how differential gene expression is the basis of cellular differentiation. B) Describe how cell specific transcription factors binding to enhancers in the control regions of genes, regulate the turn on of different genes in different cells. (N.B. you can use the lac operon in bacteria as an example of gene regulatory mechanisms but make sure you know that the lac operon operates in bacteria and is not involved in the differentiation of cells in a complex multicellular organism.) C) Use the example of muscle differentiation and the experiment used to find the regulator MyoD to illustrate the importance of gene regulation during differentiation. Key terms to be familiar with: differential gene expression, gene regulation, DNA, gene, mrna, transcription, translation, regulatory sequences, promoter, enhancer, transcriptional machinery, transcription factors and cell specific transcription factors, activators, repressors, myod, determination, cascade of events, master regulatory genes,