Does WDR5 move between complexes? An acute KANSL1 degron with WDR5 co-IP, WDR5 CUT&RUN and H4K5/K8/K16ac readout in a KMT2A-rearranged line
also called KANSL1 degron WDR5 exchange experiment
Tag the NSL scaffold so it can be destroyed within an hour, then ask whether the shared hub protein WDR5 moves over to the KMT2A-type machinery - in the test tube, on the DNA, and in the chemical marks left on histones.
The same thing, for a scientist
A three-readout acute-degradation experiment in a KMT2A-rearranged line, designed to decide `hyp-wdr5-partition-nsl-versus-compass` in one pass. Endogenous **KANSL1 is tagged with a degron** (auxin-inducible or dTAG) in SEM (KMT2A::AFF1, B-lineage) and MV4;11 or MOLM-13 (myeloid), so that both halves of this project's lineage split are represented - the standing rule that a myeloid-only result is not a result for this disease applies to bench work as much as to DepMap. After 2, 4 and 6 hours of degradation: **(1)** WDR5 immunoprecipitation followed by quantitative western or mass spectrometry for RBBP5, ASH2L, DPY30, SETD1A, SETD1B and KMT2A - does WDR5's partner distribution shift towards the WRAD core? **(2)** WDR5 CUT&RUN at HOXA9 and MEIS1 - does the released WDR5 arrive at the loci that matter? **(3)** Quantitative histone mass spectrometry for **H4K5ac, H4K8ac and H4K16ac** - does MOF output shift from the NSL pattern towards the MSL pattern [E2]? The critical design feature is the **short timescale**: the competing explanation for any steady-state observation is transcriptional adaptation, and 2-6 hours is too short for it. Absolute WDR5 and partner abundances should be measured in the same lysates, because if WDR5 is in molar excess the hypothesis is dead regardless of the other readouts [E1].
Controls that decide whether the result means anything
- Reciprocal arm. Degrade a SET1/MLL scaffold and ask whether WDR5 moves into NSL immunoprecipitates. A one-directional result is much weaker: it is compatible with KANSL1 loss destabilising WDR5 or changing its modification state rather than reallocating it.
- WDR5 abundance control. Measure total WDR5 at every timepoint. If WDR5 protein level falls with KANSL1 loss, partner-ratio changes are confounded.
- A KANSL1 mutant that cannot bind WDR5. The published structure-based mutants exist 1; expressing one in place of wild-type KANSL1 tests the partition without degrading the protein at all, and separates 'WDR5 binding' from 'NSL integrity'.
- Both lineages. SEM and a myeloid line. This graph has repeatedly found myeloid-only effects that do not hold in B-lineage KMT2A-rearranged cells, and the index disease is infant B-ALL.
- Mark-readout validity control. The H4K5/K8 versus K16 discrimination was established in vitro on nucleosomal substrates 2; before relying on it, confirm in the same cells that KAT8 depletion moves all three marks and that KANSL1 depletion moves K5/K8 more than K16.
What each outcome means
| Result | Interpretation |
|---|---|
| WDR5-WRAD association rises and WDR5 appears at HOXA9/MEIS1 and H4K5/K8ac falls more than H4K16ac | The partition is real and functionally coupled. NSL-directed agents are contraindicated in this disease; the WIN-site dossier needs revisiting with NSL in the model. |
| Association rises but no change at HOXA9/MEIS1 | Partition real, locally irrelevant. Record and close. |
| No association change within 6 h | Hypothesis falsified, or exchange is slower than assembly turnover - distinguish with a 24 h arm, accepting the adaptation confound. |
| WDR5 in molar excess of partners | Hypothesis dead on stoichiometry alone, and the cheapest of the readouts says so. |
The last row is why the abundance measurement is not optional: it can kill the hypothesis from the same lysates that would otherwise be used for the expensive readouts.
| design | Degron-tagged endogenous KANSL1 (AID or dTAG) in SEM (KMT2A::AFF1, B-lineage) and MV4;11 or MOLM-13 (myeloid). Degrade for 2, 4, 6 h with a 24 h fallback arm. Reciprocal arm degrades a SET1/MLL scaffold. Separate arm expresses a structure-based KANSL1 WDR5-binding mutant in place of wild type. |
|---|---|
| readout | (1) WDR5 IP followed by quantitative western or MS for RBBP5, ASH2L, DPY30, SETD1A, SETD1B, KMT2A; (2) WDR5 CUT&RUN at HOXA9 and MEIS1 with an expression-matched control gene set; (3) quantitative histone MS for H4K5ac, H4K8ac, H4K16ac; (4) absolute abundance of WDR5 and of its partners in the same lysates. |
| controls | total WDR5 abundance at every timepoint; reciprocal degradation arm; KANSL1 WDR5-binding point mutant; both lineage halves; KAT8-depletion control to validate the H4 mark readout in cells; expression-matched control loci for CUT&RUN. |
| cost tier | med |
| timeline | 4-6 months including degron line generation; the abundance measurement alone could be done in 2 weeks in existing lines and can kill the hypothesis on its own. |
| decides | hyp-wdr5-partition-nsl-versus-compass |
| kill criterion | WDR5 in molar excess of the summed abundance of its NSL and SET1/MLL partners, or no change in WDR5-WRAD association within 6 h of KANSL1 degradation. |
- A positive result would argue AGAINST an intervention rather than for one, since releasing WDR5 towards SET1/MLL complexes is the wrong direction in this disease.
- Degron tagging can partially deplete the tagged protein at baseline, blunting the very exchange being measured and biasing towards a false negative.
- The histone mass spectrometry readout depends on an in vitro mark discrimination that has not been shown to hold in cells, so one of the three readouts may be uninterpretable before the experiment starts.
- Neither outcome identifies a therapeutic window. This experiment resolves a mechanism and a sign; it does not produce a target.
- Does WDR5 exchange between assembled complexes at all, or is allocation fixed at assembly? If the latter, no acute experiment can see the partition and only dilution through division would reveal it.
- Is a 2-6 hour window genuinely free of transcriptional adaptation for WDR5 partner composition, or does the WRAD core turn over fast enough to confound it?
- Could the same design be run with a WIN-site inhibitor instead of a degron, which would be cheaper and would directly model the pharmacology the graph cares about?
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
The interactions of WDR5 with the MOF-containing NSL complex and with MLL/COMPASS histone methyltransferase complexes are mutually exclusive; WDR5 is recruited into NSL via conserved linear motifs of KANSL1 and KANSL2, and structure-based KANSL1 mutants in transgenic flies show the KANSL1-WDR5 interaction is required for NSL assembly and efficient recruitment to target promoters.
Structural analysis of the KANSL1/WDR5/KANSL2 complex reveals that WDR5 is required for efficient assembly and chromatin targeting of the NSL complex · Dias J, Van Nguyen N, Georgiev P, Gaub A, Brettschneider J, Cusack S, Kadlec J, Akhtar A · Genes & Development 28(9):929-42 2014What this evidence cannot show
Abstract fetched from PubMed and read on 2026-09-19; full text not read, identifiers copied from that fetched record. Crystallography plus Drosophila genetics; no mammalian cellular stoichiometry is established, which is exactly the gap this experiment addresses. The structure-based mutants proposed as a control here are described in the abstract but their residue identities were not read.
Evidence that argues against this (1)
- The experiment assumes WDR5 exchange is fast enough to observe within 6 hours. If complexes assemble co-translationally or exchange only on dilution through division, a negative result would be uninformative rather than falsifying - which is why a 24 hour arm is specified as a fallback despite its adaptation confound. Cai Y et al. 2010
- E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
Assembly of MOF into the MSL versus the NSL complex controls its substrate specificity: MSL-associated MOF acetylates nucleosomal H4 almost exclusively on K16, whereas NSL-associated MOF has relaxed specificity and also acetylates H4 on K5 and K8.
Subunit composition and substrate specificity of a MOF-containing histone acetyltransferase distinct from the male-specific lethal (MSL) complex · Cai Y, Jin J, Swanson SK, Cole MD, Choi SH, Florens L, Washburn MP, Conaway JW, Conaway RC · Journal of Biological Chemistry 285(7):4268-72 2010What this evidence cannot show
Abstract fetched from PubMed and read on 2026-09-19; full text not read, identifiers copied from that fetched record. **Measured in vitro on nucleosomal substrates**; the discrimination has not, in anything read here, been demonstrated in cells, which is why this experiment includes a validity control for the readout before relying on it.
Evidence that argues against this (1)
- If the in vitro mark discrimination does not hold in cells, readout (3) is uninterpretable and the experiment rests on readouts (1) and (2) alone. That is survivable but reduces the design from three independent measurements to two. Dias J et al. 2014
The experiment designed to decide this hypothesis in one pass.
KANSL1 is the degron-tagged protein.
KAT8 depletion is the control that validates the H4K5/K8 versus K16 mark readout in cells.
- related to →KMT2A::AFF1 (MLL-AF4), t(4;11)(q21;q23)
Run in SEM, a KMT2A::AFF1 B-lineage line, alongside a myeloid line so both halves of the lineage split are represented.