MLL3/4 methyltransferases regulate the differentiation of pluripotent stem cells via cellular respiration

This study reveals that the epigenetic regulators MLL3/4 govern the differentiation of pluripotent stem cells by activating cellular respiration through the upregulation of the glycolytic enzyme HK2 and the mitochondrial OGDH complex.

The Gist

Imagine your body is a bustling city, and the stem cells are the master architects and construction crews waiting to build everything from your heart to your brain. These cells start out in a "naive" state—they are flexible, ready to become anything, but they need a specific set of instructions to know when to stop being flexible and start building.

In this scientific paper, researchers discovered that two very important "foremen" in the cell, called MLL3 and MLL4, are actually the ones handing out the blueprints for the city's power grid.

Here is the story of what they found, broken down into simple concepts:

1. The Problem: The Construction Crew is Stuck

When the researchers removed MLL3 and MLL4 from the stem cells, the construction crew got confused. They couldn't stop being "flexible" and start building. The cells failed to differentiate (turn into specific body parts).

But why? The researchers suspected it wasn't just a lack of blueprints; it was a lack of fuel.

2. The Power Plant Analogy: Glycolysis vs. The Engine

Cells get energy in two main ways, kind of like a car:

• Glycolysis (The Quick Start): Like a hybrid car running on a battery. It's fast but not very efficient. Stem cells usually use this a lot.
• Mitochondrial Respiration (The Engine): Like a gas-powered engine. It's slower to start but provides deep, sustained power needed for complex tasks like building organs.

Healthy stem cells are "hybrids"—they can switch between the battery and the engine. This is called metabolic bivalency.

What went wrong?
When MLL3 and MLL4 were missing, the cells lost their ability to run both systems.

• The "battery" (glycolysis) died because the foremen forgot to order the Hexokinase 2 (HK2) part, which is the key switch that turns on the battery.
• The "engine" (mitochondria) sputtered because a crucial component called OGDH (part of the engine's transmission) wasn't getting its oil change (a process called lipoylation).

Without both power sources, the construction crew (the stem cell) couldn't finish the job. They were stuck in the "waiting room" and couldn't grow into a real tissue.

3. The "Shadow" Connection

The most fascinating part of the discovery is how MLL3 and MLL4 do this.

Usually, we think of these proteins as "epigenetic regulators"—they sit on DNA and tell genes to turn on or off, like a dimmer switch on a light.

• The Discovery: The researchers found that MLL3/4 act as the dimmer switch for the HK2 gene (turning it up high).
• The Twist: They also found that MLL3/4 act like a mechanic for the OGDH engine part. They don't just tell the cell to make more of it; they ensure the part is "oiled" (lipoylated) so it actually works. If the oil is missing, the engine part sits there uselessly.

4. The Rescue Mission: The "Double-Boost"

To prove this was the cause of the problem, the researchers played a game of "fix-it."

• They took the broken cells (missing MLL3/4).
• They manually forced the cells to install more HK2 (to fix the battery).
• They also forced the cells to install more OGDH (to fix the engine).

The Result: The cells woke up! They regained their power, started building again, and successfully turned into different body tissues. It was as if the researchers bypassed the missing foremen by manually turning on the power switches and oiling the engine themselves.

Why Does This Matter?

This study connects two worlds that scientists used to think were separate:

1. Epigenetics: The software that tells cells what to be.
2. Metabolism: The hardware (fuel and energy) that powers the cell.

The paper shows that you can't have a healthy body without the software (MLL3/4) correctly managing the hardware (energy).

The Big Picture:
Mutations in MLL3 and MLL4 cause serious human diseases like Kabuki syndrome and Kleefstra syndrome, which involve developmental delays and facial differences. This research suggests that these diseases happen because the cells literally run out of the right kind of energy to build the body correctly.

The Takeaway:
Think of MLL3 and MLL4 as the Chief Energy Officers of the cell. If they are missing, the cell's power plant goes dark, and the construction of your body grinds to a halt. By understanding this, scientists might one day find ways to "jump-start" these power plants to treat developmental diseases.

Technical Summary

1. Problem Statement

The epigenetic modifiers MLL3 (KMT2C) and MLL4 (KMT2D) are critical enhancer regulators required for mammalian development and stem cell differentiation. Loss-of-function mutations in these genes are linked to severe congenital disorders (Kabuki and Kleefstra syndromes) and various cancers. While recent studies suggest a link between MLL3/4 and cellular metabolism, the specific mechanisms by which they regulate metabolic pathways to govern cell fate transitions (specifically the exit from pluripotency) remain elusive. The central question addressed is: How do MLL3/4 regulate the metabolic balance between glycolysis and mitochondrial respiration to facilitate the differentiation of naive pluripotent stem cells (PSCs)?

2. Methodology

The authors employed a multi-faceted approach using murine embryonic stem cells (ESCs) in a ground-state (naive) condition:

• Genetic Models: Utilization of wild-type (WT) and MLL3/4 double knockout (DKO) ESCs.
• Metabolic Flux Analysis:
  • Seahorse XF Analyzer: Measured Extracellular Acidification Rate (ECAR) for glycolysis and Oxygen Consumption Rate (OCR) for mitochondrial respiration.
  • Stable Isotope Tracing: Used $[^{13}\text{C}_6]$-glucose and $[^{13}\text{C}_5]$-glutamine labeling followed by LC-MS/MS to trace metabolic flux through glycolysis and the TCA cycle.
  • Glucose Uptake Assays: Measured to distinguish between import defects and enzymatic defects.
• Molecular Biology:
  • Rescue Experiments: Created doxycycline (DOX)-inducible overexpression systems for Hexokinase 2 (HK2) and $\alpha$-ketoglutarate dehydrogenase (OGDH) in DKO cells.
  • Protein Analysis: Western blotting to assess protein levels of metabolic enzymes (HK1, HK2, OGDH, DLST, DLD) and post-translational modifications (lipoylation).
  • ChIP-seq & RNA-seq: Analyzed enhancer binding at the Hk2 locus and performed transcriptomic profiling during embryoid body (EB) differentiation.
• Differentiation Assays: Generated embryoid bodies (EBs) to assess morphological changes and differentiation capacity.

3. Key Contributions

• Discovery of Metabolic Regulation: Established that MLL3/4 are essential for maintaining the "metabolic bivalency" (co-utilization of glycolysis and oxidative phosphorylation) characteristic of naive PSCs.
• Mechanistic Insight into Glycolysis: Identified that MLL3/4 regulate glycolysis primarily by activating the expression of Hexokinase 2 (HK2) via specific enhancers.
• Mechanistic Insight into TCA Cycle: Revealed that MLL3/4 are required for the proper lipoylation of the E2 subunit (DLST) of the $\alpha$-ketoglutarate dehydrogenase complex (OGDHc), rather than simply regulating the transcription of the enzyme itself.
• Functional Rescue: Demonstrated that the simultaneous overexpression of HK2 and OGDH is sufficient to partially rescue both the metabolic defects and the differentiation failure caused by MLL3/4 loss.

4. Key Results

A. MLL3/4 Loss Impairs Both Glycolysis and Mitochondrial Respiration

• Glycolysis: MLL3/4 DKO ESCs showed significantly reduced ECAR (glycolytic rate and capacity). Stable isotope tracing revealed reduced incorporation of $^{13}\text{C}$ into glycolytic intermediates (F6P/G6P) and lactate.
  • Mechanism: Glucose uptake was normal, but HK2 protein and mRNA levels were significantly downregulated. ChIP-seq confirmed MLL4 binding to putative enhancers near the Hk2 locus, which lost H3K27ac marks upon MLL3/4 loss.
• Mitochondrial Respiration: MLL3/4 DKO ESCs exhibited reduced basal and maximal OCR.
  • Metabolic Flux: While citrate and $\alpha$-KG levels remained stable, downstream TCA metabolites (succinate, fumarate, malate) were significantly reduced in both glucose and glutamine tracing experiments.
  • Mechanism: This blockage occurred downstream of $\alpha$-KG. Protein levels of OGDH and other TCA enzymes were unchanged, but lipoylated DLST (a critical cofactor for OGDHc activity) was significantly reduced. This indicates a defect in the post-translational modification (lipoylation) required for OGDHc function.

B. Simultaneous Overexpression Rescues Metabolic Defects

• Overexpressing HK2 alone in DKO cells increased glycolysis but further reduced mitochondrial respiration (OCR), suggesting a shift toward glycolytic dependence.
• Simultaneous overexpression of HK2 and OGDH in DKO cells:
  • Restored glycolytic capacity (ECAR).
  • Restored basal and maximal mitochondrial respiration (OCR).
  • Partially restored levels of reduced metabolites (succinate, fumarate, lactate).

C. Rescue of Differentiation Defects

• Phenotype: MLL3/4 DKO ESCs failed to differentiate properly, forming smaller embryoid bodies (EBs) and retaining high levels of pluripotency markers while failing to upregulate lineage-specific markers (mesoderm/endoderm).
• Rescue: The HK2/OGDH double-overexpressing DKO cells showed:
  • Significant increase in EB size (morphological rescue).
  • Downregulation of pluripotency genes and upregulation of differentiation markers.
  • Reactivation of ~34% of the "MLL3/4-dependent differentiation genes" identified in RNA-seq, including genes involved in embryogenesis and cytoskeleton organization.

5. Significance

• Epigenetic-Metabolic Crosstalk: The study provides a concrete mechanistic link showing how enhancer-regulating epigenetic modifiers (MLL3/4) directly control cell fate by governing specific metabolic nodes (HK2 expression and OGDHc lipoylation).
• Developmental Biology: It explains the severe gastrulation defects and neonatal lethality observed in MLL3/4-null embryos, attributing them to a failure in the metabolic reprogramming required for differentiation.
• Therapeutic Implications: The findings suggest that diseases driven by MLL3/4 loss-of-function (e.g., Kabuki syndrome, Kleefstra syndrome 2, and certain cancers) might be amenable to therapeutic strategies that target cellular respiration or bypass the specific metabolic block (e.g., via metabolic supplementation or enzyme activation).
• Novel Regulation of Lipoylation: The discovery that MLL3/4 influence the lipoylation status of DLST (potentially via interaction with ABHD11 or other factors) opens a new avenue for understanding how epigenetic machinery regulates mitochondrial enzyme activity post-translationally.