CTCF

A Glucose-Starvation Model Explained Why CTCF Levels Didn't Rise Consistently After DNA Damage

McCord Genome Lab, with Heng LiMay 2022 – May 2023Presented — 1st-authored poster, UT Knoxville undergraduate research exhibitionCell culture (GM12878, BJ-5ta), qPCR, Western blot, glucose-starvation modeling

2 cell lines (GM12878, BJ-5ta)3 glucose conditions per line (0 / 12.5 / 25 mM)6 replicate rounds (R1–R3, R11–R13)1st-authored poster — UT undergraduate research exhibition, May 2023

Context

What the system looked like before.

My next project was still in McCord Lab. I worked with Heng Li, a grad student working towards his PhD, under an AURA grant. This time we'd moved deeper into the cells: I was working with fibroblast cells and blood cells, and instead of cancer damaging cells, I was watching Heng damage cells himself with X-ray exposure. One of the ways he verified cellular DNA damage was by measuring CTCF — a protein that binds to chromatin at sites of DNA damage to coordinate repair and maintain genome structural integrity.

Question

We expected a straightforward increase in CTCF 24 hours after X-ray-induced DNA damage — so why is there an inconsistent change in CTCF levels instead?

After checking the literature and finding a recent study stating that cellular stress can cause CTCF to phosphorylate and disassociate from its genome binding site, we hypothesized that glucose starvation — not the damage itself — was behind the inconsistency.

Approach & rationale

To test that, we designed a glucose-starvation model, using it as a proxy for cellular starvation more broadly. We cultured two cell lines — GM12878 (lymphoblastoid) and BJ-5ta (fibroblasts) — in 0 mM, 12.5 mM, and 25 mM glucose media for 24 hours. We used qPCR to check GLUT-1 expression, a glucose transporter gene that rises under starvation, giving us three sample types: definitely starved, definitely unstarved ("Collect" — collected directly, no media change), and an unknown condition ("DTCG" — medium not changed for 24 hours, mimicking how the original X-ray samples were actually collected). Once GLUT-1 confirmed starvation status, we measured CTCF protein levels by Western blot on the same samples.

Methodology & iteration

Including the paths that did not hold.

Approach A — trust the CTCF signal directly

Assumed24 hours after X-ray damage, CTCF should show a consistent, straightforward increase as it accumulates at repair sites.

Fell shortThe actual data showed CTCF changing inconsistently across replicates — sometimes even decreasing — and it persisted across multiple rounds of data collection.

Approach B — rule out a confound before trusting the readout

TriedRather than assuming the DNA-damage signal itself was noisy, I checked the literature for alternative explanations and found the LATS-kinase pathway linking cellular stress to CTCF phosphorylation and dissociation from chromatin.

Held upThis reframed "inconsistent CTCF" as a possible artifact of starvation state at collection time, testable independently of the X-ray experiment.

Final approach

TriedBuilt a standalone glucose-starvation model, confirmed starvation status with GLUT-1 qPCR before trusting any CTCF reading, normalized CTCF Western blots to a loading control (H3), and only then compared CTCF between starved and unstarved conditions.

Held upBefore trusting any CTCF measurement, I confirmed the cells were actually starved. Only then did I read CTCF — the inconsistency resolved: some samples had been starved all along.

Western blots and quantified bar charts, CTCF and H3 loading control — Western blots (CTCF + H3 loading control) and quantified bar charts, both cell lines.

In short

In the GM12878 cell line, the DTCG sample (medium not changed for 24h) showed CTCF protein levels consistent with a starvation response compared to the Collect sample.

Results

Numbers first, not buried in prose.

In the GM12878 cell line, the DTCG sample (medium not changed for 24h) showed CTCF protein levels consistent with a starvation response compared to the Collect sample. In the BJ-5ta cell line, the DTCG sample also showed a starvation-consistent response, with the difference reaching statistical significance (BJ-5ta: p<0.001 for 12.5 mM vs. Collect, p<0.01 for DTCG vs. Collect; GM12878 comparisons were not statistically significant). Starved cells showed lower CTCF than non-starved cells in both lines — consistent with the hypothesis that some samples in the original X-ray experiment had been inadvertently starved.

GM12878 didn't reach significance while BJ-5ta did — a cell-type-specific difference in starvation sensitivity that's still an open question.

Significance

Where this went.

The work became a first-authored poster, funded by an NIH grant (NIGMS R35GM133557), and won at the university's undergraduate research exhibition in May 2023.

The finished CTCF glucose-starvation poster — The complete poster — background, experiments, results, and mechanism diagram.

Reflection

I ended up presenting this at our undergrad research symposium, and I won. But to be completely honest, this was one of the hardest and most soul-sucking things I had done throughout undergrad — soul-sucking because I was struggling to understand the research and maintain my course load. While this had a happy ending, my grades and courses suffered, and I wasn't sure what to do with the outcomes of each. Being in the lab and watching research unfold made me question everything about research, and I left the experience thinking I would never pursue a career or degree in research.

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