LEDGF does NOT hold the fusion on chromatin: knocking it down INCREASES fusion occupancy, and an IBD-binding-dead fusion still binds its targets
also called LEDGF is not a fusion tether · Zhu 2016 correction
The standard picture is that LEDGF is a clamp holding the cancer protein onto DNA. When researchers removed LEDGF from leukaemia cells, the cancer protein stuck to DNA more, not less. Whatever LEDGF does for the fusion, holding it in place is not it.
The same thing, for a scientist
The tethering model - menin bridges the KMT2A fusion to LEDGF, whose PWWP reads H3K36me2/3 and anchors the complex - is the framing this graph uses in `mechanism-menin-ledgf-chromatin-tethering` and in several dependent entries. It is contradicted in the direction that matters. In MLL-AF9-transformed HSPCs, LEDGF shRNA **decreased** wild-type MLL occupancy at Hoxa9 and Meis1 while **increasing** MLL-AF9 occupancy at the same loci, by ChIP with both anti-AF9 and anti-Flag antibodies; the same reciprocal pattern held for MLL-AF10 and MLL-ENL. A missense MLL-AF9 carrying **F129A**, which cannot interact with LEDGF, retained the ability to associate with MLL target genes. The authors' own sentence is unambiguous: *'LEDGF is required for retention of WT MLL, but not MLL fusion proteins, at target gene loci in MLL-transformed HSPCs'*, and in the discussion, *'depletion of either ASH1L or LEDGF resulted in increased occupancy of MLL fusion proteins suggesting a likely competition for chromatin sites vacated by WT MLL.'* An independent group reproduced the direction in a mouse ESC TetR reporter, though at only 30% knockdown. The reconciliation is not that LEDGF is irrelevant - IBM mutations abolish transformation - but that its role is downstream of recruitment; see `mechanism-ledgf-recruits-elongation-machinery-not-the-fusion`.
What was measured
In MLL-AF9-transformed murine HSPCs with LEDGF shRNA 1:
| Factor | Occupancy at Hoxa9/Meis1 after LEDGF knockdown |
|---|---|
| wild-type MLL | down |
| MLL-AF9 (anti-AF9 and anti-Flag) | up |
| MLL-AF10, MLL-ENL | up |
| H3K4me3 | unchanged |
| H4K16ac | down |
| BRD4, P-TEFb, Ser2P Pol II | substantially down |
| AF4, CDK9 (AEP/SEC components) | significantly down |
And the reciprocal: MLL shRNA reduced LEDGF binding at proximal promoters, so MLL promotes or stabilises LEDGF chromatin binding rather than only the other way round. The authors call the association mutually dependent.
The corroborating experiment, and its limits
The Milne group tested the same thing in a mouse ESC TetR/TetO reporter and reported 'we noticed a slight increase in MLL(N) ChIP at TetO in the presence of Ledgf siRNA', adding immediately that 'we were only able to achieve a 30% Ledgf knockdown at the RNA level' 2. In their discussion they describe this as partly supporting Zhu et al.
So the direction has been seen twice, in two systems, by two groups; the magnitude has been established once. That is why this entry is graded strong rather than established for the general claim and established only for the measurement in the originating paper - the confidence-grading rule this project adopted in cycle 67.
What this changes in the graph
mechanism-menin-ledgf-chromatin-tethering is titled 'The clamp: menin plus LEDGF/PSIP1 hold the fusion on chromatin' and its summary states that the combination 'gives stable, high-avidity occupancy' to the fusion. For wild-type MLL that is supported. For the fusion it is contradicted by the only direct occupancy measurement. The entry is not owned by this beat and is not edited here; the correction is reported with this citation.
The practical consequence is for modality choice. If LEDGF anchored the fusion, a PWWP ligand that peeled the complex off chromatin would be the obvious drug. If LEDGF instead licenses elongation from a complex that stays bound regardless, a PWWP occupancy inhibitor addresses the wrong step, and the IBD arm - which is what the IBM2 transformation mutants actually interrogate - is the arm with genetic support.
Why the fusion can do without it
The published explanation is that wild-type MLL has the more complex chromatin requirement - CXXC/RD1 plus PHD3 reading H3K4me3 plus PAF1c - while for the fusion, 'the CXXC/RD1 domain alone, which binds CpG-rich DNA as well as PAF1, is sufficient for the recruitment of MLL fusion proteins' 1. This sits directly beside the finding that MLL-AF4 binding is confined to unmethylated-CpG regions and that MLL-AF4 and menin co-stabilise one another 2. In other words the fusion's address is written in DNA sequence and CpG methylation state, and the histone-mark arm is the wild-type protein's.
| correction target | mechanism-menin-ledgf-chromatin-tethering |
|---|---|
| what survives | menin bridges KMT2A to LEDGF; LEDGF is required for wild-type MLL retention; IBM mutations abolish transformation |
| what does not | that LEDGF holds the KMT2A FUSION on chromatin |
| direct measurements | 2 |
| independent groups | 2 |
- A PWWP-directed chromatin-anchoring inhibitor is designed against a mechanism the direct measurement says the fusion does not use.
- If LEDGF loss raises fusion occupancy, a partially effective LEDGF-directed drug could transiently increase fusion binding at target genes before any downstream effect appears - a pharmacodynamic readout that would look like the drug making things worse.
- The wild-type MLL allele is the one that loses LEDGF, and wild-type MLL is itself required for MLL-fusion leukaemogenesis, so a LEDGF drug attacks the fusion through a collaborator rather than through the oncoprotein.
- Has anyone measured fusion occupancy after LEDGF loss in a HUMAN KMT2A::AFF1 line? Both existing measurements are murine or mouse-ESC, and the fusion with the strongest CXXC-independent partner biology is the one nobody tested.
- If the fusion's occupancy rises on LEDGF loss because it is competing with wild-type MLL for vacated sites, what happens in ML-2, which expresses no wild-type MLL? That is a single ChIP experiment in an existing line and it would settle the competition interpretation.
- Does the same dissociation hold for menin? Menin inhibitors evict the fusion, so the two arms of the clamp may not be symmetrical at all, and nobody has compared them in one system.
- E1established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper
LEDGF knockdown in MLL-AF9-transformed HSPCs decreased wild-type MLL occupancy but consistently increased MLL fusion protein occupancy at target loci; the same held for MLL-AF10 and MLL-ENL; an MLL-AF9 F129A mutant unable to bind LEDGF still associated with MLL target genes; and the authors conclude LEDGF is required for retention of wild-type MLL but not of MLL fusion proteins.
ASH1L Links Histone H3 Lysine 36 Dimethylation to MLL Leukemia. · Zhu L, Li Q, et al. · Cancer Discov 2016What this evidence cannot show
Murine MLL-AF9/AF10/ENL-transformed HSPCs, ChIP-qPCR at Hoxa9 and Meis1 rather than genome-wide, knockdown rather than knockout. Read in full via efetch db=pmc (36,284 characters, body element present) after Europe PMC fullTextXML returned HTTP 500. The increase in fusion occupancy is attributed by the authors to competition for sites vacated by wild-type MLL, which is an interpretation rather than a measurement.
Evidence that argues against this (1)
- The conditional Psip1 knockout study reaches the opposite conclusion about the fusion, writing that its data 'corroborate the function of LEDGF/p75 in the tethering the MLL-FP'. Its evidence is that an F148A/F151A MLL-ENL cannot transform - a functional assay, not an occupancy measurement - so the two papers are not measuring the same thing and the disagreement is partly terminological. El Ashkar S et al. 2018
- E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
An independent group reproduced the direction of the effect in a mouse ESC TetR/TetO recruitment system, observing a slight increase in MLL(N) ChIP at the TetO array with Ledgf siRNA, while achieving only 30% Ledgf knockdown at the RNA level; the same paper shows MLL-AF4 binding in SEM cells is confined to unmethylated CpG regions and that MLL-AF4 and menin co-stabilise each other.
MLL-AF4 Spreading Identifies Binding Sites that Are Distinct from Super-Enhancers and that Govern Sensitivity to DOT1L Inhibition in Leukemia. · Kerry J, Godfrey L, et al. · Cell Rep 2017What this evidence cannot show
30% knockdown is weak and the authors describe their result as only partly supporting Zhu et al. The TetO array is an artificial recruitment platform in mouse ESCs, not a leukaemic locus.
Evidence that argues against this (1)
- At 30% knockdown a null or small effect is expected regardless of the underlying biology, so this experiment cannot distinguish 'LEDGF is not required' from 'not enough LEDGF was removed'. Kerry J et al. 2017
- E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
The graph's existing framing, that menin plus LEDGF form a clamp that holds the fusion on chromatin with high avidity, traces to the demonstration that menin is the adaptor linking KMT2A to LEDGF and that LEDGF is required for KMT2A-dependent transcription and transformation.
Menin critically links MLL proteins with LEDGF on cancer-associated target genes. · Yokoyama A, Cleary ML · Cancer Cell 2008What this evidence cannot show
This founding paper establishes the physical bridge and the functional requirement. It does not establish that the fusion's chromatin retention depends on LEDGF, which is the specific proposition the later occupancy measurement contradicts.
Evidence that argues against this (1)
- Later occupancy measurements from the same laboratory report increased fusion occupancy on LEDGF loss, which is the opposite of what a tether predicts. Zhu L et al. 2016
direct occupancy measurement contradicts the fusion-tether claim
what removing this protein does to fusion occupancy
- related to →KMT2A / MLL1 — the gene that breaks
LEDGF is required for wild-type KMT2A retention, not the fusion's
constrains what the PWWP arm can be doing for the fusion
supplies the positive role that replaces the tether model
the in-cis versus in-trans reconciliation
UNRESOLVED TENSION, FLAGGED NOT SUPPRESSED: if LEDGF is dispensable for retaining the FUSION on chromatin, that does not settle whether it retains MENIN once the fusion is gone - but the two entries pull in opposite directions and should be adjudicated before either is relied on.