DNA topological regulation by topoisomerase IIβ-DNA-PK interaction is important for controlled hypoxia-inducible gene expression

This study reveals that DNA-PK-mediated phosphorylation of topoisomerase IIβ (TOP2B) at T1403 regulates DNA topology to repress hypoxia-inducible gene expression under normoxia, while TOP2B dissociation and DNA-PK/HIF1 recruitment under hypoxia activate these genes.

The Gist

The Big Picture: A Cellular Traffic Control System

Imagine your cell's DNA as a massive, tangled library of books (genes). To read a specific book (turn on a gene), the cell needs to untangle the pages and open them up. However, if the pages are too loose or too tight, the reading machine (RNA Polymerase) can't work properly.

This paper discovers a new "traffic control" system that manages how these books are opened, specifically when the cell is in danger (low oxygen, or "hypoxia"). The two main characters in this story are:

1. TOP2B: The "Librarian" who usually keeps the books tightly closed and organized.
2. DNA-PK: The "Security Guard" who sometimes helps the Librarian, but sometimes needs to be told to step aside.

The Story: How the Cell Reacts to Low Oxygen

1. The Normal State (Normoxia): The Librarian is on Duty

When there is plenty of oxygen, the cell is calm. The Librarian (TOP2B) sits on the "Emergency Response" books (genes that help the cell survive low oxygen).

• What they do: The Librarian keeps these books tightly wound and closed. They prevent the pages from getting too loose (negative supercoiling).
• The Result: The reading machine cannot access these books. The emergency genes stay silent. This is good because you don't want to waste energy on emergency plans when everything is fine.

2. The Crisis (Hypoxia): The Alarm Goes Off

Suddenly, oxygen runs low. The cell needs to switch to emergency mode (like switching from a gas engine to a battery). It needs to open those "Emergency Response" books immediately.

• The Old Theory: Scientists thought the Librarian (TOP2B) would just leave the books alone so the reading machine could start working.
• The New Discovery: It's more complicated! The Librarian doesn't just leave; they need to be fired (released) from the book first.

3. The Twist: The Security Guard's Double Role

Here is where the Security Guard (DNA-PK) comes in.

• In a healthy cell: When oxygen drops, the Security Guard rushes to the scene. But instead of helping the reading machine directly, the Guard punches the Librarian (specifically, it adds a chemical tag called "phosphorylation" to a spot on the Librarian called T1403).
• The Effect of the Punch: This punch tells the Librarian, "You did your job keeping it closed! Now, go away and let the reading machine in!"
• The Result: The Librarian leaves, the DNA untangles just enough, and the emergency genes turn on.

4. What Happens if the Security Guard is Missing?

The researchers removed the Security Guard (DNA-PK) from the cells to see what happened.

• The Chaos: Without the Guard, the Librarian (TOP2B) refuses to leave. The Librarian stays glued to the books, thinking they are still in "normal mode."
• The Paradox: Even though the cell is in a crisis (low oxygen) and the "Emergency Manager" (HIF1a) is screaming to open the books, the Librarian won't budge.
• The Surprise: Despite the Librarian staying put, the emergency genes still turn on, and they turn on too much.
  • Why? The paper suggests that without the Security Guard, the Librarian gets confused. They stay on the DNA but stop doing their job of keeping the DNA tight. The DNA becomes a messy, tangled mess that the reading machine accidentally stumbles upon and starts reading. It's like a door that is stuck halfway open; it's not a controlled opening, it's a chaotic leak.

The Key Mechanism: The "Tightening" vs. "Loosening" Analogy

Think of the DNA like a rubber band.

• Negative Supercoiling: The rubber band is twisted too tight in the wrong direction. It's hard to pull apart.
• The Librarian's Job: The Librarian (TOP2B) usually acts like a tool that untwists the rubber band to make it smooth.
• The Security Guard's Job: The Guard (DNA-PK) tells the Librarian when to untwist.
  • Normal Time: The Guard tells the Librarian to untwist the rubber band just enough to keep the book closed but organized.
  • Crisis Time: The Guard tells the Librarian to stop untwisting and leave, allowing the rubber band to snap open so the book can be read.

The Breakthrough: The paper found that the Security Guard (DNA-PK) actually helps the Librarian do its job of keeping things closed during normal times. If you remove the Guard, the Librarian gets confused, stays on the DNA, but fails to keep the DNA organized. This leads to a chaotic, uncontrolled opening of the emergency genes.

Why Does This Matter?

1. Cancer Connection: Cancer cells often live in low-oxygen environments (hypoxia). They rely heavily on these emergency genes to survive and spread. If we understand how the Librarian and Security Guard interact, we might be able to design drugs that jam this system, forcing cancer cells to either die or stop growing.
2. Gene Regulation: It changes how we think about gene regulation. We used to think proteins just "turn things on." This paper shows that proteins can also "turn things off" by managing the physical shape (topology) of the DNA, and that this process is a delicate dance between two proteins.

Summary in One Sentence

This paper reveals that a protein called TOP2B acts as a gatekeeper that keeps emergency genes closed, but it needs a "kick" from a protein called DNA-PK to know when to leave; without that kick, the gatekeeper gets confused, stays on the DNA, and accidentally causes the emergency genes to go haywire.

Technical Summary

1. Problem Statement

Hypoxia (low oxygen) triggers a critical cellular stress response mediated by Hypoxia-Inducible Factor 1α (HIF1α), leading to the activation of Hypoxia-Inducible Genes (HIGs). While the role of HIF1α in recruiting transcriptional machinery is well-established, the mechanisms governing the DNA topology (supercoiling states) required for HIG activation remain unclear.

• The Gap: Previous studies identified Topoisomerase IIβ (TOP2B) as a transcriptional activator for immediate early genes (IEGs) and hormone-responsive genes, often involving programmed DNA breaks. However, its role in HIGs was unknown. Furthermore, the interplay between TOP2B and DNA-dependent protein kinase (DNA-PK)—a known regulator of transcription elongation and DNA repair—during hypoxic stress had not been characterized.
• The Core Question: How do TOP2B and DNA-PK interact to regulate the DNA topology and transcriptional output of HIGs under normoxic versus hypoxic conditions?

2. Methodology

The authors employed a multi-faceted approach combining cellular biology, biochemistry, genomics, and structural biology:

• Cell Models: Human neuroblastoma (SH-SY5Y) and colorectal carcinoma (HCT116) cells, including wild-type (WT), DNA-PK knockout (KO), DNA-PK kinase-dead (KD), and TOP2B KO lines.
• Hypoxia Induction: Chemical hypoxia mimetics were used: Cobalt Chloride (CoCl₂) and Deferoxamine (DFO).
• Genomic & Topological Assays:
  • ChIP-qPCR/ChIP-seq: To map the occupancy of TOP2A, TOP2B, DNA-PK, pDNA-PK (activated form), and HIF1α at gene loci.
  • TMP-seq (Trimethylpsoralen sequencing): To map negatively supercoiled (underwound) DNA regions genome-wide.
  • MNase-seq: To assess chromatin accessibility.
  • ICE Assay (In vivo Complex of Enzyme): To measure the amount of TOP2 trapped in covalent cleavage complexes (indicating catalytic activity).
• Biochemical Assays:
  • Co-Immunoprecipitation (Co-IP): To verify physical interactions between TOP2B and DNA-PK.
  • In vitro Kinase Assays: Using purified human TOP2B and DNA-PK, followed by MALDI-TOF-TOF mass spectrometry to identify phosphorylation sites.
• Mutagenesis & Rescue: Generation of TOP2B phospho-null mutants (e.g., T1403A) expressed in TOP2B KO cells to determine the functional necessity of specific phosphorylation sites.
• Transcriptomics: RNA-seq to analyze global gene expression changes under various genetic and environmental conditions.

3. Key Contributions & Findings

A. TOP2B Acts as a Transcriptional Repressor for HIGs

Contrary to its known role as an activator for other gene classes, the study reveals that TOP2B represses HIG transcription under normoxic conditions.

• Under normoxia, TOP2B binds to HIG promoters, suppressing transcription.
• Upon hypoxic stress, TOP2B dissociates from HIG loci, allowing transcriptional activation.
• Inhibiting TOP2B catalytic activity (using ICRF193) or trapping it (using etoposide) paradoxically increased HIG expression, suggesting that TOP2B's catalytic activity is required to maintain repression.

B. The TOP2B-DNA-PK Regulatory Axis

The study identifies a novel, antagonistic yet correlated relationship between TOP2B and DNA-PK:

• Physical Interaction: TOP2B physically interacts with DNA-PK.
• Regulation of Release: DNA-PK is required for the hypoxia-induced dissociation of TOP2B. In DNA-PK KO cells, TOP2B fails to dissociate from HIGs even under hypoxia, yet paradoxically, HIG expression is elevated. This suggests that without DNA-PK, TOP2B becomes "stuck" but loses its repressive function, or that DNA-PK is needed to activate TOP2B's repressive mechanism.
• Recruitment Dynamics: In the absence of TOP2B, DNA-PK and HIF1α are hyper-recruited to HIGs, and DNA-PK phosphorylation (at T2609) is elevated, leading to uncontrolled transcription.

C. Mechanism: Phosphorylation at T1403 Controls Topology

• Phosphorylation Site: Mass spectrometry and kinase assays identified Threonine 1403 (T1403) in the C-terminal domain (CTD) of TOP2B as a direct substrate of DNA-PK.
• Functional Consequence:
  • Phosphorylated TOP2B (WT): Stimulates TOP2B catalytic activity to relax negative DNA supercoiling (remove underwinding) at promoters. This maintains a "closed" chromatin state, preventing premature HIG activation.
  • Non-phosphorylatable Mutant (T1403A): Fails to relax negative supercoiling. Consequently, DNA remains underwound (open), leading to constitutive HIG activation even under normoxia.
• The Paradox Resolved: DNA-PK promotes the repressive function of TOP2B by phosphorylating it. When DNA-PK is absent, TOP2B cannot be phosphorylated at T1403, loses its ability to relax negative supercoils, and fails to repress HIGs, leading to their overexpression.

D. Genome-Wide Topological Changes

• TMP-seq Results: Under hypoxia in WT cells, HIG promoters exhibit a dramatic increase in negative supercoiling (DNA underwinding), which correlates with transcriptional activation.
• TOP2B KO Phenotype: In TOP2B KO cells, the genome is globally more open (underwound) under normoxia. However, the specific, regulated "spike" in underwinding at HIGs during hypoxia is lost.
• Conclusion: TOP2B normally restricts DNA openness to prevent HIG activation. DNA-PK-mediated phosphorylation of TOP2B is the switch that allows TOP2B to actively remove negative supercoils, thereby acting as a "brake" on transcription.

4. Significance

• Novel Regulatory Mechanism: This study redefines TOP2B's role, showing it acts as a context-dependent repressor for HIGs, distinct from its activator role in other genes.
• DNA Topology as a Control Point: It establishes that the regulation of DNA supercoiling (specifically the removal of negative supercoils) is a critical, active step in controlling gene expression, mediated by the TOP2B-DNA-PK axis.
• Dual Role of DNA-PK: The paper clarifies that DNA-PK has biphasic roles: it acts as a transcriptional activator for IEGs (via TRIM28 phosphorylation) but as a repressor for HIGs (by phosphorylating TOP2B to maintain repression).
• Therapeutic Implications: Understanding how DNA-PK and TOP2B regulate the hypoxic response (crucial in cancer progression, angiogenesis, and metastasis) offers new potential targets. Inhibiting the DNA-PK-TOP2B interaction or the T1403 phosphorylation site could modulate the hypoxic response in tumors.
• Model of Transcription Control: The authors propose a model where DNA-PK phosphorylates TOP2B to relax negative supercoils under normoxia (repression). Upon hypoxia, TOP2B is released (mechanism unknown), DNA-PK is recruited, and the resulting DNA underwinding facilitates Pol II elongation.

Summary Model (Figure 8)

1. Normoxia: DNA-PK phosphorylates TOP2B at T1403. This stimulates TOP2B to relax negative supercoils at HIG promoters, keeping DNA "tight" and repressing transcription.
2. Hypoxia: TOP2B dissociates from HIGs. DNA-PK and HIF1α are recruited. The removal of TOP2B allows negative supercoiling (DNA underwinding) to accumulate, opening the chromatin and enabling productive RNA Polymerase II transcription.
3. Loss of DNA-PK: TOP2B cannot be phosphorylated at T1403. It remains bound but fails to relax supercoils effectively, leading to a loss of repression and elevated HIG expression despite reduced HIF1α stability.