Kagramian 1

Simulating Various Stereoisomers of IP300w Before the

Synthesis of a DUX4 Transcription Factor Inhibitor on a

Mutated Gene for the Treatment of Facioscapulohumeral

Muscular Dystrophy

Marine Academy of Technology and Environmental Science

Research Proposal

Michelle Kagramian

michelle.kagramian@ocvts.org
SYNTHESIZING DUX4 INHIBITOR Kagramian 2

Introduction:

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive neuromuscular disease

that causes muscle atrophy in skeletal muscles in regions such as: facial, arms, around the

scapula, the pelvic girdle, and legs. Both Type 1 and Type 2 of FSHD is caused by the excess

production of the DUX4 protein (FSHD Society, 2020). FSHD2 accounts for 5% of all FSHD

patients. Type 2 occurs in numerous genes, some of which have yet to be identified, the most

common being the SMCHD1 gene. Typically, the gene is fully methylated in order to prevent its

transcription. However, in the event of FSHD2, there is a lack of methylation on the gene which

allows for the polyA tail to transcribe the DNA sequence into the DUX4 RNA leading to the

DUX4 protein (Gaillard et al., 2019; Figure 1). Type 1 of FSHD is much more common,

occurring in 95% of patients. Located on the end of chromosome 4, q35 chromatin consists of

KpnI units, or repeating chunks of DNA, that make up the D4Z4 gene (Van Der Maarel & Frants,

2005). Each KpnI unit contains a copy of the DUX4 transcription factor which can only be

translated into DUX4 RNA with a polyA tail. Normally, the polyA tail is too far to be able to

reach the units to transcribe them, however, FSHD1 patients contain an abnormally shortened

D4Z4 sequence due to the deletion of some KpnI units; this allows the polyA tail to be closer to

the KpnI units and promote DUX4 RNA to be manufactured and create protein (Figure 1). In

both types of FSHD, the abundance of the DUX4 transcription factor formed by either the

deletion of KpnI units or lack of methylation, goes on to further affect other proteins that are

synthesized (FSHD Society, 2020).

Skeletal muscles are made to withstand the stress of a muscle contraction with the help

of various proteins. All cells in the body contain the same genomic sequence, however only the

necessary genes are activated and transcribed into the needed proteins. In skeletal muscle cells,
SYNTHESIZING DUX4 INHIBITOR Kagramian 3

there are specific proteins that are manufactured for the benefit and strength of the muscle. Each

necessary gene for that cell is set up for translation and transcription, while the rest of the

unnecessary genes remain methylated, meaning gene transcription is halted (Phillips, 2014).

Each gene also requires a specific transcription factor to go along with the lack of methylation in

order to create a protein. In the case of FSHD, both of these factors play a role in unnecessary

proteins being synthesized for the detriment of the muscle cell. Genes containing the DUX4

transcription binding site also lack any methylation to block the transcription. With the excess

production of the DUX4 transcription factor, unwanted proteins get synthesized. As a result of

those proteins, muscles become more prone to oxidative stress, otherwise known as muscle

wasting (Ghiasvand & Hariri, 2016); muscle stem cells are unable to make new muscle cells to

replace the damaged ones; muscle inflammation and eventual cell death. Muscle mass becomes

replaced by fat content (FSHD Society, 2020).

A gene repressor is needed to bind to the promoter on a DNA sequence for the inhibition

of the gene transcription (Shaw, 2008; Figure 2). This research will focus on the synthesis of a

repressor protein capable of interfering with the activity of the DUX4 transcription factor.

Previous studies have shown that targeting the two proteins with histone acetyltransferase (HAT)

activity (p300 and CBP) at the DUX4 binding site regulates DUX4 cytotoxicity in the skeletal

muscle with iP300w (Le Gall et al., 2020; Bosnakovski et al., 2021). In that study, muscle cells

treated with iP300w showed a decrease in toxicity caused by DUX4. However, ZSCAN4,

another gene targeted by DUX4, was instead amplified, showing the range of genes iP300w is

able to reach. One of the promoter sequences known to provide a binding site for DUX4 is

TCAAT (Zhang et al., 2015). A similar promoter can be found in the bacteria Pediococcus

acidilactici (P. acidilactici) which uses the promoter to synthesize amino acids (Garmyn et al.,
SYNTHESIZING DUX4 INHIBITOR Kagramian 4

1995). This research will be testing different orientations of the iP300w molecule in silico on the

most common DUX4 binding site through computer software. Additionally, three concentrations

of the iP300w will be created to determine its effect on the TCAAT DNA sequence.

Although DUX4 is necessary in embryonic development (Quintero et al., 2022), it is

necessary for this gene to eventually be silenced. The results of excess expression of DUX4 leads

to the progressive weakening and eventual apoptosis of skeletal muscle cells. As of right now,

there are no cures or treatments available for FSHD. The repression of DUX4 as a transcription

factor may prevent harmful proteins from being produced and further destroying the muscle.

Figure 1: Each segment is a KpnI unit containing a copy of DUX4. The strand
with FSHD1 is shown to be shorter than the normal stand above it, bringing the
polyA tail closer to the units. Meanwhile, FSHD2 is the same length but contains
less methylation.
SYNTHESIZING DUX4 INHIBITOR Kagramian 5

Figure 2: A representation of how a repressor protein acts to prevent the RNA

polymerase from binding to the promoter and transcribing the gene.

Hypothesis:

The iP300w with the concentration of 5mM will repel the DUX4 transcription factor and

prevent it from transcribing the gene the best because the concentration is high enough to where

it will spread out enough to DNA sequence.

Methodology:

1. Study site

a. iP300w will be synthesized under a fume hood in the chemistry laboratory at

MATES High School and stored below -20℃.

2. Visualize various molecular orientations using UCSF Chimera

a. Determine the possible stereoisomers of iP300w

b. Construct and dock three versions of iP300w with various spatial arrangements
SYNTHESIZING DUX4 INHIBITOR Kagramian 6

c. Calculate the in silico binding affinity of the ligand to the binding spot for each

compound using AutoDock Vina

3. Synthesize the compound

a. Alternatively, 6 mg of the compound will be purchased.

4. 20 plates of P. acidilactici will be cultured using Coliscan Easygel (control)

a. Use an inoculation loop to gather P. acidilactici from the vial

b. Dip the inoculation loop into the Luria Broth to culture the bacteria and swirl

c. Pipette 0.5mL of the mixed culture into the Coliscan Easygel medium bottle.

Close the bottle and swirl around for 15 seconds.

i. Pour onto petri dish

d. Incubate petri dishes for 24 hours at 37°C.

5. Create solutions with iP300w at different concentrations

a. Following the calculations in Table 1, the solvent (Dimethyl sulfoxide) and

solute (iP300w) will be mixed.

i. For obtaining better solubility, the tube will be warmed to 37 ℃ and

well-shaken before mixing

Table 1: The different concentrations of the solution with the desired compound will be mixed
according to these calculations.
Concentration (mM) Mass of iP300w Volume of solvent Dimethyl
sulfoxide (mL)

1 371.13 μg 0.6

5 1.8556 mg 0.6

10 3.7113 mg 0.6
SYNTHESIZING DUX4 INHIBITOR Kagramian 7

6. 20 plates of P. acidilactici for each concentration of iP300w will be cultured using

Coliscan Easygel

a. Use an inoculation loop to gather P. acidilactici from the vial

b. Dip the inoculation loop into the Luria Broth to culture the bacteria

c. Pipette 0.5mL of the mixed culture into the Coliscan Easygel medium bottle.

i. Pipette 0.5mL of the iP300w solution into the medium bottle with the

bacteria.

ii. Close the bottle and swirl around for 15 seconds.

d. Pour onto petri dishes and incubate for 24 hours at 37°C.

7. Results

a. Binding affinity will be calculated using the aforementioned software

b. A chromatography test will show whether the synthesized compound is active

c. An NMR test will be performed to characterize the purity of the compound

d. Use the program ImageJ to quantify the bacterial colonies in both the control and

altered petri dishes.

Data Analysis:

An ANOVA test will be done to compare the means of bacterial colonies in the control

versus the colonies with added iP300w at different concentrations (P < 0.05).
SYNTHESIZING DUX4 INHIBITOR Kagramian 8

Estimated Timeline:

September:

● Development and Research of topic

● Create an improved plan to follow when carrying out the experiment

October - November:

● Purchase/prepare materials needed for experiment

● Start journal

● Use UCSF Chimera to find stereoisomers and record results of each ligand’s orientation

December-January:

● Create the control bacteria cultures

● Synthesize the selected compound and test on the bacteria

● Record all results

February:

● Gather, organize, and explain the data collected

● Create and analyze graphs

● Create a poster ready for the science fair

SYNTHESIZING DUX4 INHIBITOR Kagramian 9

References:

Bosnakovski, D., Ener, E. T., Cooper, M. S., Gearhart, M. D., Knights, K. A., Xu, N. C.,

Palumbo, C. A., Toso, E. A., Marsh, G. P., Maple, H. J., & Kyba, M. (2021). Inactivation

of the CIC-DUX4 oncogene through P300/CBP inhibition, a therapeutic approach for

CIC-DUX4 sarcoma. Oncogenesis, 10(10), 1–11.

https://doi.org/10.1038/s41389-021-00357-4

FSHD Society. (2020, July 2). FSHD 101 - What causes FSHD? With Matt Harms, MD.

Www.youtube.com. https://www.youtube.com/watch?v=K19T3lr3xrE

Gaillard, M.-C., Broucqsault, N., Morere, J., Laberthonnière, C., Dion, C., Badja, C., Roche, S.,

Nguyen, K., Magdinier, F., & Robin, J. D. (2019). Analysis of the 4q35 chromatin

organization reveals distinct long-range interactions in patients affected with

Facio-Scapulo-Humeral Dystrophy. Scientific Reports, 9(1).

https://doi.org/10.1038/s41598-019-46861-x

Garmyn, D., Ferain, T., Bernard, N., Hols, P., & Delcour, J. (1995). Cloning, nucleotide

sequence, and transcriptional analysis of the Pediococcus acidilactici L-(+)-lactate

dehydrogenase gene. Applied and Environmental Microbiology, 61(1), 266–272.

https://doi.org/10.1128/aem.61.1.266-272.1995

Ghiasvand, R., & Hariri, M. (2016). Muscle and oxidative stress. Oxidative Stress and

Antioxidant Protection, 205–220. https://doi.org/10.1002/9781118832431.ch14

Le Gall, L., Sidlauskaite, E., Mariot, V., & Dumonceaux, J. (2020). Therapeutic Strategies

Targeting DUX4 in FSHD. Journal of Clinical Medicine, 9(9), 2886.

https://doi.org/10.3390/jcm9092886
SYNTHESIZING DUX4 INHIBITOR Kagramian 10

Phillips, T. (2014). The Role of Methylation in Gene Expression | Learn Science at Scitable.

Nature.com; Nature Education.

https://www.nature.com/scitable/topicpage/the-role-of-methylation-in-gene-expression-1

070/

Quintero, J., Saad, N. Y., Pagnoni, S. M., Jacquelin, D. K., Gatica, L. V., Harper, S. Q., & Rosa,

A. L. (2022). The DUX4 protein is a co-repressor of the progesterone and glucocorticoid

nuclear receptors. FEBS Letters, 596(20), 2644–2658.

https://doi.org/10.1002/1873-3468.14416

Shaw, K. (2008). Negative Transcription Regulation in Prokaryotes | Learn Science at Scitable.

Www.nature.com; Nature Education.

https://www.nature.com/scitable/topicpage/negative-transcription-regulation-in-prokaryot

es-1013/

Van Der Maarel, S. M., & Frants, R. R. (2005). The D4Z4 Repeat–Mediated Pathogenesis of

Facioscapulohumeral Muscular Dystrophy. The American Journal of Human Genetics,

76(3), 375–386. https://doi.org/10.1086/428361

Zhang, Y., Lee, J. K., Toso, E. A., Lee, J. S., Choi, S. H., Slattery, M., Aihara, H., & Kyba, M.

(2015). DNA-binding sequence specificity of DUX4. Skeletal Muscle, 6(1).

https://doi.org/10.1186/s13395-016-0080-z