Does the fusion carry an H2B ubiquitin ligase? A co-IP and an H2BK34ub CUT&RUN in KMT2A::AFF1 cells
also called MSL2 co-IP in SEM · H2BK34ub profiling at fusion targets
Pull the fusion protein out of a leukaemia cell and look for MSL2, then map where the K34 ubiquitin tag sits across the genome. Two standard experiments that together decide whether the ubiquitin layer is something the fusion controls or something it merely borrows.
The same thing, for a scientist
Two-part, low-risk test of `hyp-msl-route-to-h2bub-at-fusion-targets`. Part A: endogenous tag on the KMT2A::AFF1 fusion in SEM (KMT2A::AFF1 B-ALL) and MV4;11, immunoprecipitate, and probe for MSL2, MSL1 and MOF/KAT8 by western and by mass spectrometry, with the wild-type KMT2A allele as the internal comparator. Part B: CUT&RUN for H2BK34ub and H2BK120ub in the same lines, with and without acute fusion degradation (dTAG) and with and without a menin inhibitor, read out at HOXA9, MEIS1 and genome-wide against H3K79me2 and nascent transcription. Part A is a fortnight; Part B depends entirely on whether a CUT&RUN-grade H2BK34ub antibody exists, which this beat did not verify and which is the experiment's single point of failure.
Order of operations, and the cheap kill
Step 0, before anything else. Determine whether the KMT2A–MOF contact maps to a region retained at the KMT2A::AFF1 breakpoint. This graph's subtraction-kmt2a-mof-contact is the place to look. If the contact is on a discarded region the hypothesis is dead and no bench work is needed. This step costs an afternoon of reading and should not be skipped.
Step 1, the co-IP. Endogenous knock-in tag rather than overexpression, because overexpressed KMT2A fragments pull down chromatin complexes indiscriminately and this project has previously had to discount such results. Include an isotype control, a wild-type-KMT2A-only line, and a non-KMT2A leukaemia line.
Step 2, the mark. CUT&RUN for H2BK34ub. Validate the antibody on nucleosomes carrying chemically installed K34 ubiquitin versus K120 ubiquitin versus unmodified before spending a sequencing run on it; antibodies against ubiquitinated histones cross-react and a bad one would produce a confident, wrong genome-wide map.
Step 3, causality. Degrade the fusion (dTAG) and re-measure. If H2BK34ub at HOXA9/MEIS1 falls with the fusion, the fusion is delivering it. If it does not move, the mark is there for transcription-general reasons and the hypothesis is refuted.
Step 2, revised after the 2026-09-19 coverage repair: do the mass spectrometry first. Two findings make the CUT&RUN the wrong first move. Purified MSL1/MSL2 modify nucleosomes very poorly in vitro and appear to need the nucleosome structurally altered first (PMID 30930284), so the mark may be near the detection floor in ordinary chromatin and a negative map would be uninterpretable. And in a survey of more than 200 histone-modification antibodies, over 25% failed specificity outright and over 20% of the survivors failed in ChIP (PMID 21131980) - the quantified version of this entry's own worry. Add to that a second modification at the same residue: H2BK34 is enriched for methylation during meiosis in mouse testes (PMID 32933557), so the validation panel needs a K34-methylated nucleosome alongside K34ub, K120ub and unmodified. Antibody-free MS on acid-extracted histones answers 'is K34ub present at all in these cells' for a fraction of the cost and should gate the sequencing run.
| design | Part A: endogenous-tag co-immunoprecipitation of KMT2A::AFF1 from SEM and MV4;11, probing for MSL2, MSL1 and KAT8 by western and mass spectrometry, with wild-type-KMT2A and non-KMT2A comparator lines. Part B: CUT&RUN for H2BK34ub and H2BK120ub with and without acute fusion degradation and with and without menin inhibition. |
|---|---|
| readout | Presence of MSL2/MSL1 in the fusion pulldown; H2BK34ub occupancy at HOXA9 and MEIS1 and genome-wide; co-variation with H3K79me2 and nascent transcription; change on fusion degradation. |
| controls | Isotype IP; wild-type KMT2A allele; non-KMT2A leukaemia line; antibody validation on chemically ubiquitinated K34 vs K120 vs unmodified nucleosomes before sequencing. Added 2026-09-19: the antibody validation panel must include a K34-METHYLATED nucleosome (PMID 32933557), not only K34ub vs K120ub vs unmodified. |
| cost tier | low |
| timeline | Part A 2-4 weeks. Part B 2-3 months, contingent on a validated H2BK34ub antibody. |
| prerequisite | Confirm from subtraction-kmt2a-mof-contact that the KMT2A-MOF contact is retained at the KMT2A::AFF1 breakpoint; if it is not, do not run the experiment. |
| revised order | MS on acid-extracted histones first, to establish K34ub is detectable, before any CUT&RUN - MSL1/2 modify nucleosomes poorly in vitro (PMID 30930284) and >20% of specific histone antibodies fail in ChIP (PMID 21131980) |
tests hypotheses
[ "hyp-msl-route-to-h2bub-at-fusion-targets" ]
- A negative co-IP does not exclude a transient or substoichiometric interaction, which is the usual fate of chromatin co-IPs and the usual way an interesting hypothesis dies without being settled.
- More than 25% of histone-modification antibodies fail specificity and more than 20% of the specific ones fail in ChIP (PMID 21131980), so the most likely single outcome of Part B is a confident genome-wide map of the wrong thing.
- Does a CUT&RUN-grade H2BK34ub antibody exist? This beat did not check, and the whole of Part B depends on it.
- Would mass spectrometry on acid-extracted histones be a better first measurement than CUT&RUN, given it needs no antibody and would immediately say whether K34ub is present at all in these cells?
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
MSL2 with MSL1 ubiquitylates nucleosomal H2B at lysine 34; the mark regulates H3K4 and H3K79 methylation in trans; and MSL1/2 activity is important for transcription activation at the HOXA9 and MEIS1 loci - the premise that makes this experiment worth running.
The RING finger protein MSL2 in the MOF complex is an E3 ubiquitin ligase for H2B K34 and is involved in crosstalk with H3 K4 and K79 methylation. · Wu L, Zee BM, Wang Y, Garcia BA, Dou Y · Mol Cell 2011What this evidence cannot show
Abstract read in full; full text not read. Not a leukaemia system.
Evidence that argues against this (1)
- The mark this experiment proposes to map may be near the detection floor in ordinary chromatin. Krajewski & Vassiliev found purified MSL1/MSL2 ubiquitylate free histone substrates efficiently but modify NUCLEOSOMES very poorly, inferring that efficient H2BK34 ubiquitylation requires the nucleosome to be structurally altered first. That makes the experiment's most likely outcome a negative CUT&RUN that cannot distinguish 'the fusion does not deliver MSL2' from 'MSL1/2 cannot ubiquitinate this chromatin'. An orthogonal, antibody-free measurement of total K34ub by mass spectrometry on acid-extracted histones should come first - the entry's own second open question, promoted here to a design objection. Krajewski WA et al. 2019
- E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
H2BK34ub positions Dot1L for the productive conformation on the nucleosome in essentially the same orientation as H2BK120ub, so a K34 mark at fusion target genes would be functionally sufficient to license DOT1L there.
H2B Lys34 Ubiquitination Induces Nucleosome Distortion to Stimulate Dot1L Activity. · Ai H, Sun M, Liu A, Sun Z, Liu T, Cao L, Liang L, Qu Q, Li Z, Deng Z, Tong Z, Chu G, Tian X, Deng H, Zhao S, et al. · Nat Chem Biol 2022What this evidence cannot show
Abstract read in full; full text not read. Structural sufficiency in a reconstituted nucleosome is not cellular sufficiency.
Evidence that argues against this (2)
- The failure mode that looks like a result is the antibody, and it is quantified. Egelhofer et al. tested more than 200 antibodies against 57 histone modifications: more than 25% failed specificity by dot blot or western, and of those that passed, more than 20% failed in chromatin immunoprecipitation. A cross-reactive H2BK34ub antibody would deliver a confident genome-wide map of something else, and the prior probability of that is not small. This entry's step-2 validation plan is necessary and, on these numbers, may well fail. Egelhofer TA et al. 2011
- The same residue carries a second modification, so specificity has to be demonstrated against it and not only against K120ub. Crespo et al. identified methylation of yeast H2BK37 as a meiotic mark and showed the mouse paralogue H2BK34 is enriched for methylation during meiosis in the testes. Validation on chemically ubiquitinated K34 versus K120 versus unmodified nucleosomes, as this entry specifies, does not include a K34-methylated control and would not detect that cross-reactivity. Crespo M et al. 2020
- ← related toMSL2 — the second H2B ubiquitin ligase, sitting inside the MOF complex the KMT2A fragment contacts
Design objection (Krajewski and Vassiliev 2019, PMID 30930284): MSL1/MSL2 modify nucleosomes very poorly in vitro, so the mark may sit near the detection floor and a negative CUT&RUN could not distinguish 'the fusion does not deliver MSL2' from 'MSL1/2 cannot ubiquitinate this chromatin' - antibody-free mass spectrometry should gate the sequencing run
Design objection (Egelhofer 2011, PMID 21131980; Crespo 2020, PMID 32933557): more than 25% of 200+ histone-modification antibodies failed specificity and more than 20% of the survivors failed in ChIP, and H2BK34 also carries a conserved meiotic methyl mark - so the validation panel needs a K34-methylated control, and the most likely single outcome of Part B is a confident map of the wrong thing
co-IP plus H2BK34ub CUT&RUN in KMT2A::AFF1 cells