Testing OMAC-seq, A New Way to Study Chromatin Accessibility

Project Description

My project was testing the validity of OMAC-seq, or Optical Mapping of Accessible Chromatin

using sequencing, my lab’s approach to analyzing chromatin accessibility in living cells in their

native setting. OMAC-seq fragments a cell’s accessible DNA into small pieces, differentiating it

from the rest of the DNA so it can be extracted and sequenced. Through genetic engineering, the

necessary protein complexes for this process are naturally formed in target cells, and the proteins

are activated on command through optogenetics. Thus, membrane permeabilization and DNA

modification, required steps in current techniques for studying chromatin accessibility which kill

the cell and prevent observation of the chromatin’s native conformation, are avoided altogether.

I tested the efficacy of OMAC-seq by seeing whether its results reflect differing chromatin

condensation levels through varying fragmentation levels. OMAC-seq should fragment more

DNA if chromatin is less condensed, meaning more accessible, and less DNA if chromatin is

more condensed, meaning less accessible. The first step was to show that I can truly change the

chromatin condensation levels in the genetically engineered model cell, mouse embryonic stem

cells (mESCs). To relax the DNA, I used a histone deacetylase (HDAC) inhibitor called TSA.

First, I tested what length and concentration of TSA treatment I could use on the cells while still

maintaining cell health. Then, I did Western Blots with antibodies for histone acetylation to

prove that the TSA treatment I chose did cause higher acetylation of DNA as intended. To

further condense the DNA, I used an ATP depletion treatment of a glucose-free medium, sodium

azide, and 2-deoxy-D-glucose. I just found the maximum length of this treatment that still

maintained cell health as I did with the TSA treatment. I did not perform Western Blots on these

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cells because this combination has been proven to deplete ATP, the energy source in cells, which

leads to decreased accessibility of DNA. Once I chose treatments and showed the TSA to be

effective, I performed OMAC-seq on treated and control cells, extracted and purified the DNA,

then performed gel electrophoresis. This process visualizes the fragmentation levels of DNA, as

smaller fragments of DNA run faster through the gel along with the current and end up at the

bottom of the gel. Darker bands of short DNA show higher fragmentation, and lighter bands

show lower fragmentation. Thus, I expected to see darker bands from the TSA-treated cells

compared to the control, and lighter bands from the ATP-depleted cells compared to the control.

Project Results

As I treated the mESCs with higher concentration TSA for longer, I saw different signs of

decreasing cell health, including cells disassociating from colonies, deflating and spreading out,

differentiating, detaching from the plate, or dying. I observed samples treated with 100ng TSA

over time until the cells seemed too unhealthy to assume that the native state was unchanged.

Once the maximum treatment was reached, I also checked that the red and green fluorescent

proteins attached to the OMAC-seq protein complexes were still equally fluorescing as an

additional sign of sufficient cell health. The maximum length I observed was 7 hours (Fig 1).

Additionally, I concluded cell health and fluorescence was maintained enough in samples treated

with 1ng, 2.5ng, and 5ng for 18 hours (Fig 2). When I depleted the cell’s ATP, I saw similar signs

of decreasing cell health. The maximum amount of time I could treat the cells for ATP depletion

before the cells seemed too unhealthy was 3 hours (Fig 3).

Next, I performed Western Blot on both the 100ng TSA samples at different treatment lengths

(control, 1 hr, 3hr, and 5hr) and the 18hr TSA samples at different concentrations (control, 1ng,

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2.5ng, and 5ng). I used a primary antibody targeted for acetyl K5 on Histone 4; this meant that

the antibody should appear brighter if there is more acetylation on Lysine 5, and there should be

more acetylation in an effective TSA treatment. I also used a general Histone 4 antibody as a

control to make sure the TSA treatment didn’t affect the DNA as a whole; all conditions should

have the same amount of brightness from this control antibody because they should all have

equal amounts of H4. For the H4 K5 antibody, results showed a strong steady increase as the

time of 100ng treatment increased, and a less prominent gradual increase as the concentration

increased on the 18hr treatments. The longest time of 100ng treatments (5hr) was darker than the

highest concentration of the 18hr treatments (5ng). The brightness of the control antibody was

even in each set of cells (Fig 4). Following this, I probed both types of TSA treatments with H4

K8 and K12 antibodies, which search other lysines for acetylation and would show more proof of

de-acetylation inhibition. Both of these antibodies’ results appeared similar to H4 K5’s results —

strong increase in 100ng over time, gradual increase in 18hr over increasing concentration, and

the 5hr 100ng treatment coming out as significantly darker than the 18hr 5ng treatment (Fig 5).

Finally, I performed OMAC-seq on the treated and controlled cells, extracted and purified the

DNA, and did gel electrophoresis to compare fragmentation levels. The 100ng 7hr treatment

showed extremely promising results, as the treated cells showed significantly darker bottom

bands and more blur at the top band showing full DNA length (another sign of fragmentation)

when compared to the control (Fig 6). The 18hr 5ng treatment also showed similar results to this

(Fig 7). Additionally, the ATP depletion cells’ fragmentation also appeared as expected because

the treated cells’ DNA had lighter bottom bands and less blur on the top band when compared to

the control cells (Fig 8). All of these results showed that the cells experienced much less

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fragmentation when there was no light causing the OMAC-seq proteins to bind. This ‘dark’

condition is an additional control showing OMAC-seq works as intended.

Since the Western Blot results most strongly support the efficiency of the 100ng 7hr treatment,

this treatment plan is the best to use in the future of this project (this length was not specifically

tested, but since the 5hr treatment was tested and was quite brightly tagged by the antibodies, we

can assume that the 7hr treatment is equally promising). OMAC-seq fragmentation levels

changed as expected in cells proven to have varied condensation levels. The next step is to

sequence the fragments OMAC-seq make in these cells and compare the range of DNA that was

cut in treated and control cells. This will show whether or not OMAC-seq cuts the expanded or

decreased ranges as it is intended to. This fall, I plan to use the TSA and ATP depletion

treatments I concluded are most effective and isolate the fragmented DNA for sequencing.

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Figure 1: Pictures depicting the stages of decreasing cell health over time for the 100ng TSA
treatment cells until the maximum treatment length, and the fluorescence of the control cells
versus the fluorescence of the maximum treatment length cells.

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Figure 2: Pictures depicting the decreasing cell health of 18hr TSA treatment cells as the
concentration increases until the maximum concentration treatment, and the fluorescence of the
control cells versus the fluorescence of the maximum concentration treatment cells.

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Figure 3: Pictures comparing cell health and fluorescence between control cells and cells that
received ATP depletion treatment for 3hrs, the maximum length before substantial cell death.

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Figure 4: General Histone H4 and Histone H4 K5 Western Blots on cells treated with both types
of TSA treatment.

Figure 5: Histone H4 K5, K8, and K12 Western Blots on cells treated with both types of TSA
treatment.

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Figure 6: Two sets of gel electrophoresis results (auto exposure and high exposure) depicting, in
each picture from left to right: Ladder, Control Cells Dark, Control Cells Light, TSA 100ng 7hr
Dark, and TSA 100ng 7hr Light.

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Figure 7: Gel electrophoresis results (auto exposure and high exposure) depicting, in each
picture from left to right: Ladder, Control Cells Dark, Control Cells Light, TSA 18hr 5ng Dark,
and TSA 18hr 5ng Light.

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Figure 8: Gel electrophoresis results (auto exposure and high exposure) depicting, in each
picture from left to right: Ladder, Control Cells Dark, Control Cells Light, ATP Depletion 3hr
Dark, and ATP Depletion 3hr Light.

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