Question

1. Compare and contrast the different mechanisms for regulating differential gene expression.
2. Predict different mechanisms that could be responsible for control of gene expression in development.
3. Explain extrinsic stem cell regulatory mechanisms and the role of niches

Answer 1

Answer :

Differential Gene Expression

If the genome is the same in all somatic cells within an organism (with the exception of the above-mentioned lymphocytes), how do the cells become different from one another? If every cell in the body contains the genes for hemoglobin and insulin proteins, how are the hemoglobin proteins made only in the red blood cells, the insulin proteins made only in certain pancreas cells, and neither made in the kidney or nervous system? Based on the embryological evidence for genomic equivalence (and on bacterial models of gene regulation), a consensus emerged in the 1960s that cells differentiate through differential gene expression. The three postulates of differential gene expression are as follows:

1.

Every cell nucleus contains the complete genome established in the fertilized egg. In molecular terms, the DNAs of all differentiated cells are identical.

2.

The unused genes in differentiated cells are not destroyed or mutated, and they retain the potential for being expressed.

3.

Only a small percentage of the genome is expressed in each cell, and a portion of the RNA synthesized in the cell is specific for that cell type.

The first two postulates have already been discussed. The third postulate—that only a small portion of the genome is active in making tissue-specific products—was first tested in insect larvae. Fruit fly larvae have certain cells whose chromosomes become polytene. These chromosomes, beloved by Drosophila geneticists, undergo DNA replication in the absence of mitosis and therefore contain 512 (29), 1024 (210), or even more parallel DNA double helices instead of just one (Figure 4.13A,Figure 4.13B). These cells do not undergo mitosis, and they grow by expanding to about 150 times their original volume. Beermann (1952) showed that the banding patterns of polytene chromosomes were identical throughout the larva, and that no loss or addition of any chromosomal region was seen when different cell types were compared. However, he and others showed that in different tissues, different regions of these chromosomes were making organ-specific RNA. In certain cell types, particular regions of the chromosomes would loosen up, “puff” out, and transcribe mRNA. In other cell types, these regions would be “silent,” but other regions would puff out and synthesize mRNA.

The idea that the genes of chromosomes were differentially expressed in different cell types was confirmed using DNA-RNA hybridization (Figure 4.13C). This technique involves annealing single-stranded pieces of RNA and DNA to allow complementary strands to form double-stranded hybrids. While some mRNAs from one cell type were also found in other cell types (as expected for mRNAs encoding enzymes concerned with cell metabolism), many mRNAs were found to be specific for a particular type of cell and were not expressed in other cell types, even though the genes encoding them were present (Wetmur and Davidson 1968). Thus, differential gene expression was shown to be the way a single genome derived from the fertilized egg could generate the hundreds of different cell types in the body. The question then became, How does this differential gene expression occur? The answers to that question will be the topic of the next chapter. To understand the results that will be presented there, however, one must become familiar with some of the techniques of molecular biology that are being applied to the study of development. These include techniques to determine the spatial and temporal location of specific mRNAs, as well as techniques to determine the functions of these messages.

Differential gene expression analysis

Differential expression analysis means taking the normalised read count data and performing statistical analysis to discover quantitative changes in expression levels between experimental groups. For example, we use statistical testing to decide whether, for a given gene, an observed difference in read counts is significant, that is, whether it is greater than what would be expected just due to natural random variation.

Methods for differential expression analysis

There are different methods for differential expression analysis such as edgeR and DESeq based on negative binomial (NB) distributions or baySeq and EBSeq which are Bayesian approaches based on a negative binomial model. It is important to consider the experimental design when choosing an analysis method. While some of the differential expression tools can only perform pair-wise comparison, others such as edgeR, limma-voom, DESeq and maSigPro can perform multiple comparisons.

Regulation of gene expression, or gene regulation, includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA). Sophisticated programs of gene expression are widely observed in biology, for example to trigger developmental pathways, respond to environmental stimuli, or adapt to new food sources. Virtually any step of gene expression can be modulated, from transcriptional initiation, to RNA processing, and to the post-translational modification of a protein. Often, one gene regulator controls another, and so on, in a gene regulatory network.

Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed. Although as early as 1951, Barbara McClintock showed interaction between two genetic loci, Activator (Ac) and Dissociator (Ds), in the color formation of maize seeds, the first discovery of a gene regulation system is widely considered to be the identification in 1961 of the lac operon, discovered by François Jacob and Jacques Monod, in which some enzymes involved in lactose metabolism are expressed by E. coli only in the presence of lactose and absence of glucose.

In multicellular organisms, gene regulation drives cellular differentiation and morphogenesis in the embryo, leading to the creation of different cell types that possess different gene expression profiles from the same genome sequence. Although this does not explain how gene regulation originated, evolutionary biologists include it as a partial explanation of how evolution works at a molecular level, and it is central to the science of evolutionary developmental biology ("evo-devo").

Extrinsic stem cell regulatory mechanism

Extrinsic and intrinsic mechanisms that determine stem cell fate. HSC quiescence, self-renewal and differentiation are regulated by both intrinsic and extrinsic mechanisms. Extrinsic mechanisms include changes in stem cell fate that are dictated by the environment, i.e. niche. Once physical association between the osteoblast and the HSC has occurred, release of different growth factors will trigger diverse signal transduction pathways that will initiate expression of downstream target genes. Intrinsic mechanisms are niche-independent and, for example, they can affect the epigenetic state of HSC, as it is controlled by chromatin remodelers. (TF-transcription factors).