Analysis of Metabolic Pathways In Relation to the KDM5A and Retinoblastoma 1 Gene

Aishwarya Raj

Illinois Mathematics and Science Academy

Dr. Elizaveta Benevolenskaya Lab: University of Illinois at Chicago

Background Information

Independent Variables:Rb1: The Retinoblastoma gene(Rb1) has been shown to be critical in cancer development due to its role as a tumor suppressor. Since Rb1 is responsible for positively and negatively regulating transcription it can both repress and facilitate gene expression(Kaelin, 1999). Previously, there have also been research displaying that cell differentiation correlates directly with gene expression. Therefore in certain conditions such as in cell cycle, the inactivation of Rb1 should increase differentiation as Rb1 negatively regulates transcription in cell cycle. However in all other conditions, the inactivation or knock down of Rb1 should decrease differentiation as the gene facilitates transcription

( Nicolay et al., 2013).

Independent Variable Cont.

KDM5A: (K)-specific demethylase 5A gene like the Rb1 gene has also been shown to positively and negatively regulate gene expression. However KDM5A is critical in researching the Rb1 gene as it reinforces the effects of Rb1(Beshiri et al., 2012). Since the KDM5A gene codes for enzymes that work together with protein-factors coded by the RB1 gene, the double knockout (inactivation) of Rb1 and KDM5A can be studied for restoration of gene expression. In a previous study, KDM5A was studied along with the E2F4 gene for functional restoration of permanently silenced genes with promising results however this is a novel area of study (Beshiri et al., 2012).

Dependent Variables

• Cell Regulation via Various Processes:• Cell Metabolism: Metabolism via the Alanine, Aspartate, and Glutamate(AAG) pathway as well as the

Glutathione pathway are effective in studying gene expression as metabolism is directly impacted by Rb1 regulation. In addition, the AAG pathway is more closely related to the Glutathione pathway than other metabolic pathways and therefore excellent pathways for comparable research on Rb1 and KDM5A effects.

• Cell Cycle: One of the main roles of the protein-factor coded by the Rb1 gene is to negatively regulate(repress) gene expression in the cell cycle process. Therefore when the Rb1 gene is inactivated, the result should be higher expression of cell cycle related genes. Through research and experimental data, this notion can be verified to prove previous findings and to add to the accuracy of hypothesis.

• Muscle System: Muscle differentiation is another process impacted by the Rb1 gene for gene expression regulation. Therefore the inactivation of Rb1 should impact muscle differentiation similar to cell metabolism.

These three specific cell regulation processes were chosen for the focus of this study due to the Rb1 gene’s direct impact on these processes as well as because of the critical role cell regulation plays in cancer development. This is because the loss of tumor suppressors such as in this case with Rb1, lead to a high rate of glucose fermentation and consequently, high rate of cancer cell growth (Dang, 2012). Thus, survival of cancer cell growth can directly be attributed to changes in cell metabolism and indirectly to loss of tumor suppressors.

Thus , the study of different cell regulation processes allows for study of KDM5A and Rb1 effects on a micro level in order to make conclusions about triggers for cancer development and greater insight into cancer at a macro level.

Purpose & Inquiry

• The purpose of this research inquiry was to determine how the inactivation of the Rb 1 gene, the KDM5A gene, and both Rb1 and KDM5A would affect cell metabolism.

• Inquiry Objectives:• Analyze effects on cell metabolism via Rb1 gene

inactivation• Analyze effects on cell metabolism via KDM5A gene

inactivation• Analyze effects on cell metabolism via Rb1 and KDM5A

inactivation

Hypothesis

• The inactivation of the Rb1 gene would negatively affect cell regulation and therefore decrease gene expression in metabolic pathways.

• The inactivation of both the Rb1 gene and the KDM5A gene would restore gene expression in cell regulation to wild type(normal gene expression).

• The inactivation of the KDM5A gene would also decrease gene expression in cell regulation due to its negative effect on cell metabolism.

Methods

• This research inquiry was conducted using data and computer software including GI tools, Microsoft Excel, Photoshop elements(diagram creation), as well as experimental data.

• Figure 1 was the result of data collected on 4 different cell regulation processes: Mitochondrion, Cell Cycle, Muscle System, and the AAG pathway. Then within each process, 6 standardized conditions were tested which are:

baseMean WT: normal differentiation meaning that the genes within the process were not altered so that differentiation patterns in this condition could be used as a control.

baseMean KDM5A: KDM5A was inactivated in this condition so that defects of KDM5A could be observed through patterns of differentiation.

baseMean Rb: Rb1 was inactivated in this condition so that defects of Rb1 could be observed through patterns of differentiation.

baseMean DKO: Both KDM5A and Rb1 were inactivated so that gene expression restoration could be observed by comparing differentiation patterns to wild type.

baseMean WT-myo: This condition is when a purified population of wild type differentiation is taken in order to observe gene expression accurately. This is because some genes will lack expression regardless of conditions and therefore those genes must be removed in order to observe changes in gene expression accurately.

baseMean DKO-myo: This condition is when a purified population of double knockout differentiation is take in order to observe and compare gene expression accurately for the same reason above. By eliminating genes from both wild type and double knock out that never express themselves, accurate comparisons can be made in analyzing differentiation patterns and accordingly, gene expression.

Methods Cont.

• Figures 2 & 3 was the result of data taken from several databases including: GeneCards, Kyoto Encyclopedia of Genes and Genomes(Kegg database), and experimental data from lab.

• The databases provided information on the pathway diagrams and I had experimental data on gene expression for genes within the AAG pathway and the Glutathione pathway. Then I grouped the experimental data by pathway and gene using Microsoft Excel so that I could label the information on each pathway. Finally, photo shop was used to label each gene according to name, expression, and restoration on the respective pathway diagrams.

• The different labels on the figures include:

• Rescue ES Targets (*): Genes that are supposed to be rescued through double knockout(inactivation) of Rb1 and KDM5A.

• Rescue (+): Genes that were actually rescued through double knockout of Rb1 and KDM5A.• Increased Expression: Genes that increased expression through inactivation of Rb1.• Decreased Expression: Genes that decreased expression through inactivation of Rb1• Increased DKO Expression (WT): Genes that increased expression through inactivation of Rb1

and KDM5A.• Decreased DKO Expression (WT): Genes that decreased expression through inactivation of Rb1

and KDM5A.

*Note: Gene expression when KDM5A was inactivated was not labeled due to insignificant changes after inactivation.

Data & Results

Figure 1.

Low differentiation

High differentiation

Explanation of Figure 1

• Figure 1 is a heat map displaying different conditions of the KDM5A gene and Rb1 gene that were tested for each cell regulation process(cell cycle, metabolism: AAG, mitochondrion, muscle system). The different conditions included:

baseMean WT: wild type or normal differentiation

baseMean KDM5A: KDM5A defected differentiation

baseMean Rb: Rb1 defected differentiation

baseMean DKO: double knockout, or inactivation of both KDM5A and Rb1, differentiation

baseMean WT-myo: purified population of normal differentiation

baseMean DKO-myo: purified population of double knockout differentiation

Explanation of Figure 1 Cont.

• Cell regulation processes: Mitochondrion; Muscle System; Alanine, Aspartate, Glutamate; were examined with six different standardized conditions. The conditions of the wild type were similar to KDM5A as the green color indicated comparable levels of repressed differentiation. The wild type- myo and the double knockout-myo, also expressed similar levels of differentiation. This signified that the double knockout was effective in restoring repressed genes caused by Rb1 inactivation. However, comparison of only double knockout to wild type displayed disparity in differentiation levels as the double knockout has higher levels of differentiation than wild type. As expected, KDM5A has lower levels of differentiation repression than Rb1 because KDM5a only aids the inhibition caused by Rb1 but does not directly cause gene expression inhibition.

• Cell cycle: had an inverse relationship with all other cell regulation processes as Rb1 and KDM5A are negative regulators in cell cycle. Thus, the results exhibited that differentiation repression is higher in wild type differentiation than in Rb1 defected differentiation, KDM5A defected differentiation and double knockout differentiation. This comparison is also clearly seen in comparison of the wild type- myo and double knockout-myo, as the double knockout-myo has significantly higher levels of cell differentiation than wild type.

Figure 2.

Figure 3.

Explanation of Figures 2 & 3

• Figure 2 & 3 display the metabolic pathways: Alanine, Aspartate and Glutamate pathway as well as Glutathione pathway in diagram form in order to visualize expression of specific genes within the pathways. Rescue (+) stands for genes that were rescued through the double knock out of Rb1 and KDM5A. Rescue ES targets (*) stands for genes that were supposed to return to normal through inactivation of KDM5A and Rb1.

Conclusions

• Overall the results proved that 2 of the hypothesized outcomes and disproved 1 of the hypothesized outcomes. The proven hypothesized outcomes were: The inactivation of the Rb1 gene would negatively affect cell regulation

and therefore decrease gene expression in metabolic pathways. The inactivation of both the Rb1 gene and the KDM5A gene would

restore gene expression in cell regulation to wild type(normal gene expression).

• However the disproven hypothesized outcome was:X The inactivation of the KDM5A gene would also decrease gene

expression in cell regulation due to its negative effect on cell metabolism.

Conclusion Cont.

Due to previous research on the Rb1 gene, it was expected that Rb1 would show lack of differentiation in the heat maps and in proportion, negatively effect gene expression. However restoration of Rb1 gene defects through double knockout of Rb1 and KDM5A was hypothesized based on evidence from previous research on Rb1 and KDM5A and evidence proved this outcome to be true. These conclusions were based on differentiation patterns depicted in Figure 1 as all cell regulation processes except cell cycle displayed similar trends. These were that cell differentiation between double knockout of KDM5A and Rb1 and wild type were at or near the same level. In addition, Rb1 defected differentiation depicted significant decrease in gene expression in comparison to wild type and therefore proved that that Rb1 negatively affected cell metabolism and regulation.

Although two of the hypothesis were proven true, the hypothesis concerning KDM5A was disproven as there was insignificant data to suggest that KDM5A negatively affected cell regulation if it affected cell regulation at all. This is because differentiation patterns in Figure 1 depicted that there was little change between wild type differentiation and KDM5A defected differentiation. In addition, there was insignificant data for KDM5A defected gene expression in cell metabolism and therefore could not be labeled on the pathway diagrams. As a result this hypothesis was disproved but can still be researched further in the future.

Real World Application

• This research conducted on the two genes(KDM5A and Rb1) exhibit viability for pharmaceutical development as KDM5A is an ideal drug target in comparison to Rb1. Where Rb1 codes for a protein factor, KDM5A codes for an enzyme which is much easier to target and therefore feasible. In addition, the hypothesized outcome of restoring Rb1 defects through inactivation of KDM5A and Rb1 was proven true. As previously mentioned, the Rb1 gene defects is a primary cause of cancer development and metabolism. Therefore, cancer related drug development targeted on KDM5A are prospective applications of my project for beneficial causes.

Future Studies

• In depth analysis of KDM5A and Rb1 effects on cell cycle(due to disparity in results)

• Focus on homologs of Rb1• Continue research through data analysis of Generation

Precursor pathway in relation to AAG pathway and Glutathione

References

• Beshiri, M. L., Holmes, K. B., Richter, W. F., Hess, S., Islam, A. B. M. M. K., Yan, Q., … Benevolenskaya, E. V. (2012). Coordinated repression of cell cycle genes by KDM5A and E2F4 during differentiation. Proceedings of the National Academy of Sciences of the United States of America, 109(45), 18499–504. doi:10.1073/pnas.1216724109

• Ciavarra, G., & Zacksenhaus, E. (2010). Rescue of myogenic defects in Rb-deficient cells by inhibition of autophagy or by hypoxia-induced glycolytic shift. The Journal of cell biology, 191(2), 291–301. doi:10.1083/jcb.201005067

• Dang, C. (2012). Links between metabolism and cancer. Genes & development, 877–890. doi:10.1101/gad.189365.112.GENES

• Henley, S. a, & Dick, F. a. (2012). The retinoblastoma family of proteins and their regulatory functions in the mammalian cell division cycle. Cell division, 7(1), 10. doi:10.1186/1747-1028-7-10

• Iacobuzio-Donahue, C. a. (2009). Epigenetic changes in cancer. Annual review of pathology, 4, 229–49. doi:10.1146/annurev.pathol.3.121806.151442

• Kaelin, W. G. (1999). Functions of the retinoblastoma protein. BioEssays : news and reviews in molecular, cellular and developmental biology, 21(11), 950–8. doi:10.1002/(SICI)1521-1878(199911)21:11<950::AID-BIES7>3.0.CO;2-D

• Nicolay, B., & Gameiro, P. (2013). Loss of RBF1 changes glutamine catabolism. Genes & Development, 182–196. doi:10.1101/gad.206227.112.permissive

• Robinsion(editor), R. (2003). Cancer. In Genetics. Macmillan Reference USA.

Acknowledgments

I would like to thank my advisors Dr. Elizaveta, Mr. Mike Beshiri (Graduate Student) and Ms. Katie Holmes (Post Doctorial Research Associate) as well as my parents for

their support and guidance.