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How do signal transduction pathways utilize transcription factors to express a specific gene?

How do signal transduction pathways utilize transcription factors to express a specific gene?


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I have an inquiry regarding the regulation of genes via extracellular signaling.

To my knowledge, in autocrine, paracrine, and endocrine cellular communication, large protein ligands that cannot directly diffuse through the plasma membrane of the target cell(s) use surface receptors to perform their desired action on the target cell(s).

I have learned that some of these ligands activate signal transduction pathways such as the MAPK/ERK and JAK/STAT pathway and drive the expression of specific genes by utilizing transcription factors (of course, in eukaryots). A simple example of this would be the action of epinephrine (adrenaline) on hepatocytes (liver cells), where the amino acid-based hormone uses the transcription factor CREB to express the gene coding for Glycogen Phosphorylase to engage in glycogenolysis.

Here I have two questions:

  1. How does the transcription factor chemically indicate which gene to express? (Is there a gene indexing system like a computer filesystem?)

  2. How does the transcription factor locate and bind to the promoter of the gene it is trying to express?

Thank you.


This is a combination of multiple regulatory systems. Most genes are not regulated by a single factor, but by many. Moreover in eukaryotic organisms there is also epigenetic, which "inactivates" permanently certain areas of the genome by compacting these zones forming heterochromatin.

Moreover, even when we are interested in a single function a TF might have, that does not mean that it only has this function in the cell. We view it as humans, and maybe for us it is logical that a response to a high metabolite in the environment simply leads to expressing the protein that metabolizes it. However, probably it is interesting for the cell to also activate other pathways and gene networks (i.e. anabolic pathways that feed off this metabolite, compensatory pathways to mantain homeostasis… ). Because that normally TFs affect more than one gene naturally.

The mechanisms of action of the TFs are multiple, but they tend to simply allow or deny the access to the adequate RNA polymerase. For more information about this check the wikipedia article on TF structure

In this image we see a typical mechanism of action, DNA bending

Different domains interact in activating a gene, enhancers which are usually long distance, promoters which tend to be in the upstream region, though they can be downstream or in the gene sequence, and epigenetic factors that change the compaction of DNA, making it inaccessible to proteins.

Just as a last thing to point out, you should acknowledge that the biological systems are far from exact and yes, a single TF might activate 200 genes, even when its main objective in that moment is to activate one, but it won't really matter as long as the those 199 activated genes express at a very low rate (for example by not having the right RNA polymerase subunit accessible, being marked as inactive by epigenetic systems or having their own inhibitors).


Transcription factor network efficiency in the regulation of Candida albicans biofilms: it is a small world

Complex biological processes are frequently regulated through networks comprised of multiple signaling pathways, transcription factors, and effector molecules. The identity of specific genes carrying out these functions is usually determined by single mutant genetic analysis. However, to understand how the individual genes/gene products function, it is necessary to determine how they interact with other components of the larger network one approach to this is to use genetic interaction analysis. The human fungal pathogen Candida albicans regulates biofilm formation through an interconnected set of transcription factor hubs and is, therefore, an example of this type of complex network. Here, we describe experiments and analyses designed to understand how the C. albicans biofilm transcription factor hubs interact and to explore the role of network structure in its overall function. To do so, we analyzed published binding and genetic interaction data to characterize the topology of the network. The hubs are best characterized as a small world network that functions with high efficiency and low robustness (high fragility). Highly efficient networks rapidly transmit perturbations at given nodes to the rest of the network. Consistent with this model, we have found that relatively modest perturbations, such as reduction in the gene dosage of hub transcription factors by one-half, lead to significant alterations in target gene expression and biofilm fitness. C. albicans biofilm formation occurs under very specific environmental conditions and we propose that the fragile, small world structure of the genetic network is part of the mechanism that imposes this stringency.

Keywords: Biofilm formation Candida albicans Epistasis Genetic networks Transcription factors.


Signal Transduction Pathway

During signal transduction, a signal may have many components. There is the primary messenger, which may be a chemical signal, electrical pulse, or even physical stimulation. Then, the receptor protein embedded in the cellular membrane must accept the signal. Upon receiving the signal, this protein goes through a conformational change. This changes its shape and thus, how it interacts with the molecules around it.

The many different receptor proteins act in different ways. Above is a simple representation of the many different signal transduction pathways in mammals. Do not be overwhelmed by the complexity of the drawing. The important thing to realize is that all of these signal transduction pathways contain the same elements. A signal is received by a receptor protein, and the protein transfers the signal through the cell membrane and into the cell. The kinds of receptors and the second messengers they create can be very different. This is based on the action which the signal must stimulate. There are some examples in the next section which will help shed light on the many differences and similarities between pathways.


REGULATION OF GENE EXPRESSION

One of the mechanisms by which eukaryotes regulate gene expression is through modifications to chromatin structure. When chromatin is condensed, DNA is not accessible for transcription. Acetylation of histone tails reduces the attraction between neighboring nucleosomes, causing chromatin to assume a looser structure and allowing access to the DNA for transcription. If the histone tails undergo deacetylation, chromatin can recondense, once again making DNA inaccessible for transcription.

Recent evidence suggests that methylation of histone tails can promote either the condensation or the decondensation of chromatin, depending on where the methyl groups are located on the histones. Thus, methylation can either inactivate or activate transcription, and demethylation can reverse the effect of methylation.

Which statements about the regulation of transcription initiation in these genes are true?
Select all that apply.


DIMORPHISM, AN IMPORTANT VIRULENCE FACTOR

The terms 𠇍imorphism” and 𠇍imorphic fungus” (i.e., existing in two morphological forms) are commonly accepted in reference to C. albicans. Strictly speaking, however, this fungus has the ability to adopt a spectrum of morphologies thus, C. albicans can be considered a “polymorphic” or “pleomorphic” organism (71, 289). The production of germ tubes results in conversion to a filamentous growth phase or hypha, also called the mycelial form. The formation of pseudohyphae occurs by polarized cell division when yeast cells growing by budding have elongated without detaching from adjacent cells. Under certain nonoptimal growth conditions, C. albicans can undergo the formation of chlamydospores, which are round, retractile spores with a thick cell wall. These morphological transitions often represent a response of the fungus to changing environmental conditions and may permit adaptation to a different biological niche. The transition from a commensal to pathogenic lifestyle may also involve changes in environmental conditions and dispersion within the human host. Although progress has been achieved in recent years, the molecular mechanisms governing these morphogenetic conversions are still not fully understood, partly due to the difficulty of genetic manipulations with C. albicans (164), an issue we address briefly below.


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Conclusions

Computational simulation is an important systems biology approach to the analysis of signaling pathways and gene regulatory networks. In this work, we present a software tool called Sig2GRN which is able to link the cellular signaling pathways with the downstream gene expression regulation. A generalized logical model is used in modeling the upstream signaling pathways, while a Boolean Network and a thermodynamic model are employed in modeling the downstream gene expression based on the simulated activities of transcription factors. We have shown two case studies on simulating the cell responses to the extracellular perturbations and validated the simulations with wet-lab experimental data. As a Cytoscape plugin, Sig2GRN is designed to be extensible so that more computational models of gene regulation (e.g., epigenetic modifications) can be integrated to facilitates studies in systems biology. Compared with existing methods to link signaling pathways with gene regulation, such as in [24], Sig2GRN is a parameter-free software which requires no kinetic parameters of the pathways, and thus it is still applicable when only insufficient prior knowledge of the underlying mechanisms is available. Moreover, Sig2GRN is able to predict the gene expression time-course data given the perturbations to the signaling pathways, whereas in [24] the gene expression data are required as the input of their model, which is therefore unable to predict new gene expression patterns.


9.3 Response to the Signal

Inside the cell, ligands bind to their internal receptors, allowing them to directly affect the cell’s DNA and protein-producing machinery. Using signal transduction pathways, receptors in the plasma membrane produce a variety of effects on the cell. The results of signaling pathways are extremely varied and depend on the type of cell involved as well as the external and internal conditions. A small sampling of responses is described below.

Gene Expression

Some signal transduction pathways regulate the transcription of RNA. Others regulate the translation of proteins from mRNA. An example of a protein that regulates translation in the nucleus is the MAP kinase ERK. ERK is activated in a phosphorylation cascade when epidermal growth factor (EGF) binds the EGF receptor (see Figure 9.10). Upon phosphorylation, ERK enters the nucleus and activates a protein kinase that, in turn, regulates protein translation (Figure 9.14).

The second kind of protein with which PKC can interact is a protein that acts as an inhibitor. An inhibitor is a molecule that binds to a protein and prevents it from functioning or reduces its function. In this case, the inhibitor is a protein called Iκ-B, which binds to the regulatory protein NF-κB. (The symbol κ represents the Greek letter kappa.) When Iκ-B is bound to NF-κB, the complex cannot enter the nucleus of the cell, but when Iκ-B is phosphorylated by PKC, it can no longer bind NF-κB, and NF-κB (a transcription factor) can enter the nucleus and initiate RNA transcription. In this case, the effect of phosphorylation is to inactivate an inhibitor and thereby activate the process of transcription.

Increase in Cellular Metabolism

The result of another signaling pathway affects muscle cells. The activation of β-adrenergic receptors in muscle cells by adrenaline leads to an increase in cyclic AMP (cAMP) inside the cell. Also known as epinephrine, adrenaline is a hormone (produced by the adrenal gland attached to the kidney) that readies the body for short-term emergencies. Cyclic AMP activates PKA (protein kinase A), which in turn phosphorylates two enzymes. The first enzyme promotes the degradation of glycogen by activating intermediate glycogen phosphorylase kinase (GPK) that in turn activates glycogen phosphorylase (GP) that catabolizes glycogen into glucose. (Recall that your body converts excess glucose to glycogen for short-term storage. When energy is needed, glycogen is quickly reconverted to glucose.) Phosphorylation of the second enzyme, glycogen synthase (GS), inhibits its ability to form glycogen from glucose. In this manner, a muscle cell obtains a ready pool of glucose by activating its formation via glycogen degradation and by inhibiting the use of glucose to form glycogen, thus preventing a futile cycle of glycogen degradation and synthesis. The glucose is then available for use by the muscle cell in response to a sudden surge of adrenaline—the “fight or flight” reflex.

Cell Growth

Cell signaling pathways also play a major role in cell division. Cells do not normally divide unless they are stimulated by signals from other cells. The ligands that promote cell growth are called growth factors . Most growth factors bind to cell-surface receptors that are linked to tyrosine kinases. These cell-surface receptors are called receptor tyrosine kinases (RTKs). Activation of RTKs initiates a signaling pathway that includes a G-protein called RAS, which activates the MAP kinase pathway described earlier. The enzyme MAP kinase then stimulates the expression of proteins that interact with other cellular components to initiate cell division.

Career Connection

Cancer Biologist

Cancer biologists study the molecular origins of cancer with the goal of developing new prevention methods and treatment strategies that will inhibit the growth of tumors without harming the normal cells of the body. As mentioned earlier, signaling pathways control cell growth. These signaling pathways are controlled by signaling proteins, which are, in turn, expressed by genes. Mutations in these genes can result in malfunctioning signaling proteins. This prevents the cell from regulating its cell cycle, triggering unrestricted cell division and cancer. The genes that regulate the signaling proteins are one type of oncogene which is a gene that has the potential to cause cancer. The gene encoding RAS is an oncogene that was originally discovered when mutations in the RAS protein were linked to cancer. Further studies have indicated that 30 percent of cancer cells have a mutation in the RAS gene that leads to uncontrolled growth. If left unchecked, uncontrolled cell division can lead tumor formation and metastasis, the growth of cancer cells in new locations in the body.

Cancer biologists have been able to identify many other oncogenes that contribute to the development of cancer. For example, HER2 is a cell-surface receptor that is present in excessive amounts in 20 percent of human breast cancers. Cancer biologists realized that gene duplication led to HER2 overexpression in 25 percent of breast cancer patients and developed a drug called Herceptin (trastuzumab). Herceptin is a monoclonal antibody that targets HER2 for removal by the immune system. Herceptin therapy helps to control signaling through HER2. The use of Herceptin in combination with chemotherapy has helped to increase the overall survival rate of patients with metastatic breast cancer.

More information on cancer biology research can be found at the National Cancer Institute website (http://www.cancer.gov/cancertopics/understandingcancer/targetedtherapies).

Cell Death

When a cell is damaged, superfluous, or potentially dangerous to an organism, a cell can initiate a mechanism to trigger programmed cell death, or apoptosis . Apoptosis allows a cell to die in a controlled manner that prevents the release of potentially damaging molecules from inside the cell. There are many internal checkpoints that monitor a cell’s health if abnormalities are observed, a cell can spontaneously initiate the process of apoptosis. However, in some cases, such as a viral infection or uncontrolled cell division due to cancer, the cell’s normal checks and balances fail. External signaling can also initiate apoptosis. For example, most normal animal cells have receptors that interact with the extracellular matrix, a network of glycoproteins that provides structural support for cells in an organism. The binding of cellular receptors to the extracellular matrix initiates a signaling cascade within the cell. However, if the cell moves away from the extracellular matrix, the signaling ceases, and the cell undergoes apoptosis. This system keeps cells from traveling through the body and proliferating out of control, as happens with tumor cells that metastasize.

Another example of external signaling that leads to apoptosis occurs in T-cell development. T-cells are immune cells that bind to foreign macromolecules and particles, and target them for destruction by the immune system. Normally, T-cells do not target “self” proteins (those of their own organism), a process that can lead to autoimmune diseases. In order to develop the ability to discriminate between self and non-self, immature T-cells undergo screening to determine whether they bind to so-called self proteins. If the T-cell receptor binds to self proteins, the cell initiates apoptosis to remove the potentially dangerous cell.

Apoptosis is also essential for normal embryological development. In vertebrates, for example, early stages of development include the formation of web-like tissue between individual fingers and toes (Figure 9.15). During the course of normal development, these unneeded cells must be eliminated, enabling fully separated fingers and toes to form. A cell signaling mechanism triggers apoptosis, which destroys the cells between the developing digits.

Termination of the Signal Cascade

The aberrant signaling often seen in tumor cells is proof that the termination of a signal at the appropriate time can be just as important as the initiation of a signal. One method of stopping a specific signal is to degrade the ligand or remove it so that it can no longer access its receptor. One reason that hydrophobic hormones like estrogen and testosterone trigger long-lasting events is because they bind carrier proteins. These proteins allow the insoluble molecules to be soluble in blood, but they also protect the hormones from degradation by circulating enzymes.

Inside the cell, many different enzymes reverse the cellular modifications that result from signaling cascades. For example, phosphatases are enzymes that remove the phosphate group attached to proteins by kinases in a process called dephosphorylation. Cyclic AMP (cAMP) is degraded into AMP by phosphodiesterase , and the release of calcium stores is reversed by the Ca 2+ pumps that are located in the external and internal membranes of the cell.


TEACHING DISCUSSION

The in-class exercises have been used in a Developmental Biology class (L. Emtage, Fall 2015). The exercises in Supporting Files S2 and S4 were given in class in the week after two lectures on regulation of gene expression and a lecture on signaling mechanisms. The instructor fielded many more questions on the actual material of the lesson (signaling pathways) after the case study exercise than after more traditional lectures.

OUTCOMES

The effect of these exercises on student understanding were measured in two ways. First, we compared students in a traditionally-taught Cell Biology course (Emtage, Spring 2015) with students in the Developmental Biology course described above (Emtage, Fall 2015). The Cell Biology course covered both gene expression and signaling pathways in a traditional lecture format. In the session following the lecture on signaling mechanisms and signaling pathway components, the students were given a quiz question similar to the question included here (Supporting File S8), but on G protein-coupled receptors. Only one out of 35 was able to correctly answer the question (Figure 1). In Developmental Biology, the students participated in the active learning exercises given in Supporting Files S2 and S4. In the class session after the active-learning exercises S2 and S4, they were given a quiz on the Wnt signaling pathway. Three out of fourteen students received full credit for the first quiz question given in S8 (Figure 1), while a further six students received partial credit (not shown).


Figure 1. Student improvement with case study. The fraction of students receiving full credit on a quiz question requiring analysis of a signaling pathway after a traditional lecture (35 students), and after the active-learning activity described here (14 students).

In the following year's Developmental Biology class (Fall 2016), the students again participated in the in-class exercises S2 and S4, were assigned the homework exercise S6, and were given the quiz in S8. We did not discuss the homework assignment prior to the quiz the 2016 cohort performed similarly to their peers from the previous year on the quiz. However, we did review the homework and quiz during the next lecture. Three weeks later, the students were given an exam including two short-answer questions on the β-catenin signaling pathway. One was a simple question on the mechanics of the pathway. Eleven out of 18 students were able to answer this question correctly (Figure 2, Simple exam question). The second question was analytical it asked the students to predict the probable outcome of a manipulation of the pathway. Six out of 18 students were able to answer the question correctly and give a reasonable explanation for their answer (Figure 2, Analytical exam question).


Figure 2. Student performance with case study and homework. Effect of an analytical, challenging homework exercise on student performance in active learning setting.

The fraction of students able to answer analytical questions does gradually increase with practice (Figures 1 and 2). These results indicate that the combination of active-learning exercises and the creation of opportunities to discuss challenging questions can improve both the students' basic understanding of signaling pathways and their ability to analyze and manipulate the information that they have learned.

MODIFICATIONS

The out-of-class exercise was developed to give the students an opportunity to apply the principles that they used in analyzing the MAP kinase pathway to the Wnt signaling pathway, which is more complicated than the MAP kinase pathway. The exercise includes guiding questions and a diagram of the pathway, but no explanatory text. However, the students are free to find additional materials to help them understand the diagram. Our goal is to challenge students to continue to develop build on their problem-solving skills and to encourage them to interpret information that is presented diagrammatically.

The lesson can be pared down to only the Noonan Case Study, to suit an introductory course covering signaling, or expanded upon in a variety of directions for a more in-depth analysis suitable to upper level courses in cellular, molecular genetics or developmental biology. One possible expansion would be to connect mutations that affect signaling and congenital disorders of development. If an instructor prefers, the out-of-class assignment could be altered or expanded to cover a different signaling pathway, such as G protein-coupled receptors Essential Cell Biology, for example, covers GPCRs but not Wnt signaling( )(18). The small group exercise could be expanded with a discussion of oncogenes and tumor suppressors, and chemotherapeutics that target particular oncogenes (personalized medicine). Finally, the instructor could reverse the order of the case study and interleaving exercise if the unit on regulation of gene expression comes after the unit on signaling.


Conclusion

The recent evidence to date has strongly cemented the fact that Wnt signaling plays a critical role in pattern formation during embryogenesis. Many studies over the last two decades have identified numerous signaling components that have helped to build a molecular framework for the many branches of the Wnt signal transduction pathway. However, the diverse function, integration and specificity of the Wnt signaling are still unclear. Furthermore, we lack a clear understanding of many of the biochemical aspects within this signaling framework. Recent studies have demonstrated a strong correlation and at times causative relationships between deregulated Wnt signaling and human diseases. Thus the investigation of Wnt signaling remains an important goal for dually understanding both the basic mechanism of embryonic development and human diseases. Undoubtedly, the future holds many important breakthrough studies in Wnt signaling that will further our understanding of this important pathway and we all await these discoveries with eager enthusiasm.


6. CONCLUSIONS

The regulation of gene expression by E2 is a multi-factorial process, involving both genomic and non-genomic actions that converge at certain response elements located in the promoters of target genes. The final gene responses, however, could depend on a number of conditions such as the combination of transcription factors bound to a specific gene promoter, the cellular localization of ERs, the levels of various co-regulator proteins and signal transduction components, as well as the nature of extra-cellular stimuli. These variables are highly specific for cell types. Thus, E2 could use different signaling pathways depending both on the cellular type and on the physiological status of the cell. In this way E2 evokes distinct gene responses in different types of target cells [15, 16, 97, 162].

The possibility that E2 could act on ER pools localized in different cell compartments (i.e., membrane versus cytoso-lic) gives rise to questioning the ability of these different ER pools to send parallel or synergic signals to the nucleus. For example, it has been observed that a naturally occurring variant of the metastatic tumor antigen 1 sequesters ER in the cytoplasm of breast cancer cells. The result of this cyto-solic retention is the reduction of E2-mediated transcription and the enhancement of E2-initiated ERK activation [136]. These data suggest that the same ER molecule is involved in genomic and in rapid signal transduction cascade. More data are needed to confirm this hypothesis and the use of dynamic imaging in the near future will help to clarify this issue.

Based upon findings highlighted in this review, one may envisage a dynamic integrated model of action for ERs inside the cell. In this model, ERs would shuttle from cell membrane to the cytoplasm and to the nucleus, in a dynamic equilibrium between different cell compartments. Each could play a different role in a multi step process of target gene activation by ER and co-activators from their upstream non-genomic to their downstream genomic responses would lead to activation of transcription (Fig. ( ​ (2 2 )).

The cell context specific environment (e.g., differentiation, ER level, and ER co-expression) has an impact on the integration of rapid signaling by E2 from the membrane and on subsequent nuclear transcription. This leads to different signal cascades, different gene expression in response to the same hormone, and different cell biological outcome.

The field is moving quickly. The challenges in the near future are to continue identifying the discrete actions of each ER intracellular pool, in order to clarify the role of ERβ, and to identify the potential cross-talk between ERs and other nuclear receptors. As we gain a deeper understanding of the complex controls exerted by ER and start identifying the critical players, it is likely that some of these putative molecules might emerge target candidates for therapeutic development in the treatment of hormone-responsive diseases, such as for different types of cancer.



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