1. 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

2. 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).
Background Information

3. 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).
Independent Variable
Cont.

4. •
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.
Dependent Variables

5. • 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
Purpose & Inquiry

6. • 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.
Hypothesis

7. •
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

8. • 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.
Methods Cont.

9. Data & Results
Figure 1.
Low
differentiation
High
differentiation

10. • 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

11. • 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.
Explanation of Figure 1 Cont.

12. Figure
2.

13. Figure 3.

14. • 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.
Explanation of Figures 2 & 3

15. • 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.
Conclusions

16. 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.
Conclusion Cont.

17. • 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.
Real World Application

18. • 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
Future Studies

19. • 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.
References

20. 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.
Acknowledgments