MSL2 — the second H2B ubiquitin ligase, sitting inside the MOF complex the KMT2A fragment contacts

This is a prediction, not a measurement. No one has seen this protein in this shape. The colours say how much the software trusts itself, and that is worth reading before anything else — the orange stretches are places the model is effectively saying this part has no fixed shape.

• MSL2 provides a route to DOT1L activation that does not pass through RNF20/RNF40, so it is a pre-existing bypass of the only writer-directed strategy this beat could describe.
• Its complex partner MOF/KAT8 is among the most essential genes in DepMap (-1.452), so complex-level targeting is not available.
• MSL2 is near-dispensable itself, which means both that it is a poor target and that the assay used to say so has little power.
• one RING domain, two consequences - MSL2's H2BK34 ubiquitylation controls H3K4 and H3K79 methylation at HOXA9 and MEIS1 - the marks and loci this disease runs on - and the same RING domain drives p53 out of the nucleus without changing its abundance. An MSL2 inhibitor would therefore perturb a tumour suppressor's compartment while perturbing the oncogenic mark, and no chemical tool separates the two.

1. Does MSL2 ubiquitinate nucleosomal H2BK34 efficiently, or only after some other complex has altered the nucleosome (PMID 30930284)? If the latter, MSL2 is not an independent entry point to the mark and the bypass argument in this graph weakens.
2. Does the KMT2A fragment retained in the fusion co-purify with MSL2? A co-IP would take a week and would decide whether MSL2 is a bypass or a fusion-proximal route to the licensing mark.
3. Does H2BK34ub increase when RNF20 is degraded? The compensation question, answerable with one western blot.
4. What fraction of nucleosomal H2B ubiquitin in a leukaemia cell is at K34? Nobody in this beat's reading has measured it.
5. Does MSL2 deposit H2BK34ub at HOXA9 and MEIS1 in a KMT2A-rearranged cell, and does that ubiquitylation gate the DOT1L-dependent H3K79 methylation the fusion depends on? If it does, MSL2 sits upstream of the mark this disease is defined by and nobody has looked.

1. E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

   MSL2 in the MOF-MSL complex is a histone ubiquitin E3 ligase; with MSL1 it has robust histone ubiquitylation activity mainly targeting nucleosomal H2B on lysine 34; H2BK34ub directly regulates H3K4 and H3K79 methylation through trans-tail crosstalk in vitro and in cells; MSL1/2 activity is important for transcription activation at the HOXA9 and MEIS1 loci; and the activity is evolutionarily conserved in the Drosophila dosage compensation complex.

   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 2011
   PMID 21726816DOI
   ▸What this evidence cannot show

   Abstract read in full; full text not read. No KMT2A-rearranged cell line is named in the abstract; the HOXA9/MEIS1 result is not a leukaemia result and should not be read as one. Effect sizes are not given.

   ▸Evidence that argues against this (1)

   • The enzyme's activity on the substrate that matters is weak. Krajewski & Vassiliev assayed purified MSL1/MSL2 against free histones and against nucleosomes and found that MSL1/2 ubiquitylate histone substrates efficiently but modify NUCLEOSOMES very poorly, concluding that efficient H2BK34 ubiquitylation requires a prior structural alteration of the nucleosome. 'Robust nucleosomal H2B ubiquitylation activity' is therefore contested at the level of the in vitro assay that established it. In vitro, purified components, no cellular or leukaemia data - but it is the only independent reinvestigation of the activity this beat found. Krajewski WA et al. 2019
2. E2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

   Cryo-EM structures show H2BK34ub induces an almost identical orientation and binding pattern of Dot1L on the nucleosome as H2BK120ub and positions Dot1L for the productive conformation, but by inducing DNA and histone distortion around the modified site rather than by direct ubiquitin-Dot1L contact.

   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 2022
   PMID 35739357DOI
   ▸What this evidence cannot show

   Abstract read in full; full text not read. Reconstituted nucleosomes with chemically installed ubiquitin.

   ▸Evidence that argues against this (1)

   • Structural sufficiency is being asked to carry a claim about a mark whose deposition on chromatin is itself in doubt: the only independent reinvestigation of MSL1/MSL2 activity reports that the enzyme modifies nucleosomes very poorly and needs the nucleosome structurally altered first (PMID 30930284). A cryo-EM structure built on chemically installed K34 ubiquitin cannot speak to whether MSL2 puts it there. Krajewski WA et al. 2019
3. E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.dataset

   In the project's genome-wide DepMap KMT2A-selectivity scan, MSL2 has a mean gene effect of -0.083 across 1,208 lines with a KMT2A-rearranged minus other-leukaemia difference of -0.015 (rank 7,112 of 17,728); MSL1 is -0.365 with a difference of -0.073 (rank 2,078); KAT8/MOF in the same complex is -1.452 with a difference of +0.004.

   DepMap CRISPR (Chronos) genome-wide KMT2A-selectivity scan of 17,728 protein-coding genes, 11 Cellosaurus-annotated KMT2A-rearranged leukaemia lines vs 43 other leukaemia lines · Broad Institute DepMap; Cellosaurus; Open Targets Platform; analysis run by this project (analyses/depmap-kmt2a-selective-scan-full) · Database and derived analysis 2026
   ▸What this evidence cannot show

   A gene with near-zero pan-essentiality has little dynamic range in this assay, so the absence of a selectivity difference for MSL2 is weak evidence. Eleven KMT2A-rearranged lines, AML-weighted, none infant-derived.

   ▸Evidence that argues against this (1)

   • The same residue carries a different modification in a physiological setting: H2BK34 is enriched for METHYLATION during meiosis in mouse testes, identified alongside its yeast counterpart H2BK37me. An entry that treats K34 as the MSL2 ubiquitination site should record that the site is shared, because any future K34 read-out in leukaemia has to exclude the methyl mark. Crespo M et al. 2020
4. E_q65_loc1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

   MSL2, together with MSL1 in the MOF-MSL complex, is a histone ubiquitin E3 ligase that targets nucleosomal H2B at lysine 34; H2BK34 ubiquitylation directly regulates H3K4 and H3K79 methylation through trans-tail crosstalk both in vitro and in cells, and the activity is important for transcriptional activation at the HOXA9 and MEIS1 loci.

   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, et al. · Mol Cell 2011;43:132-44 2011
   PMID 21726816DOI
   ▸What this evidence cannot show

   A genomic address (HOXA9, MEIS1) and a mark, not a subnuclear compartment. Human cells but not leukaemia and not a KMT2A-rearranged system; the H3K79 link is the trans-tail crosstalk this graph's DOT1L axis depends on, but it is established here without a fusion present (brief rule 73). Abstract only.

   ▸Evidence that argues against this (1)

   • MSL2's best-characterised single-substrate activity is not on chromatin at all: it ubiquitylates p53 and drives p53 into the cytoplasm without changing p53 levels, so an MSL2 perturbation in a leukaemia cell would move a tumour suppressor's compartment as well as a histone mark, and the two effects have never been separated. Kruse JP et al. 2009
5. E_q65_loc2strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper

   MSL2 is an E3 ubiquitin ligase for p53 that promotes ubiquitin-dependent CYTOPLASMIC localisation of p53; unlike MDM2 and most other p53 E3 ligases, MSL2-mediated p53 ubiquitination does not affect p53 stability, and the effect is MDM2-independent.

   MSL2 promotes Mdm2-independent cytoplasmic localization of p53. · Kruse JP, Gu W · J Biol Chem 2009;284:3250-3263 2009
   PMID 19033443DOI
   ▸What this evidence cannot show

   Non-leukaemic human cells. A relocation without a stability change is easy to miss in a western blot and easy to over-read in an image; the paper's claim is about p53's compartment, not MSL2's. Abstract only.

   ▸Evidence that argues against this (1)

   • MSL2 also ubiquitylates p53 at K351 in the context of histone H2BK34 work by the same field, and the chromatin and p53 activities use the same RING domain, so a selective MSL2 perturbation that separates the nuclear chromatin function from the p53-export function does not exist. Wu L et al. 2011
6. E_q65_loc3moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

   MSL2 is a component of the vertebrate DNA damage response: Msl2-null chicken DT40 cells are viable with minor growth defects but show aberrant levels of several histone modifications involved in DNA damage response pathways, and both Msl2-null chicken cells and hMSL2-depleted human cells are defective in non-homologous end joining repair.

   Msl2 is a novel component of the vertebrate DNA damage response. · Lai Z, et al. · PLoS One 2013;8:e68549 2013
   PMID 23874665DOI
   ▸What this evidence cannot show

   Chicken DT40 plus human knockdown; DT40 repair phenotypes do not always transfer to human cells. Cited for the damage-response role, which bears on this graph's break-repair beat, not for a localisation. Abstract only.

   ▸Evidence that argues against this (1)

   • An NHEJ defect in an MSL2-null cell could be a downstream consequence of the H2BK34ub/H3K4/H3K79 crosstalk rather than a direct repair function, and the paper's own chromatin-modification survey is consistent with that reading. Wu L et al. 2011
7. E_q69_ct1moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

   Proteomic analysis of the immunoglobulin heavy chain Emu enhancer identifies the male-specific lethal (MSL)/MOF complex as a component of the YY1 protein network; MSL2 recruitment depends on YY1, gene knockout of Msl2 in PRIMARY PRE-B CELLS reduces mu gene expression and reduces chromatin looping of Emu to the 3' regulatory region and to the VH promoter, and Mof heterozygosity in mice impairs mu expression and early B-cell differentiation, so the MSL/MOF complex regulates Igh gene expression by augmenting YY1-mediated enhancer-promoter communication.

   YY1-mediated enhancer-promoter communication in the immunoglobulin μ locus is regulated by MSL/MOF recruitment. · Phongbunchoo Y, Braikia FZ, Pessoa-Rodrigues C, Ramamoorthy S, Ramachandran H, Grosschedl A, Ma F, Cauchy P, Akhtar A, Sen R, Mittler G, Grosschedl R · Cell Rep 2024
   PMID 38990722DOI
   ▸What this evidence cannot show

   Abstract read in full; full text not read. Murine primary pre-B cells and Mof-heterozygous mice - normal B lymphopoiesis, not leukaemia and not human (brief rule 73). The differentiation phenotype is a MOF allele-dosage result, not an MSL2 result; the MSL2 arm shows transcription and looping changes only. One study, 2024.

   ▸Evidence that argues against this (1)

   • MSL1/MSL2's ubiquitin ligase activity is poor on nucleosomes in vitro, requiring a prior structural alteration of the nucleosome for efficient H2BK34 modification. An Igh looping phenotype may therefore reflect the MSL complex acting as a scaffold rather than MSL2 acting as an enzyme, and the study does not separate those. Krajewski WA et al. 2019

• related to →H2BK34ub — a second ubiquitination site on H2B that also stimulates DOT1L, written by MSL1/MSL2, and important at HOXA9 and MEIS1

  Counter-evidence (Krajewski and Vassiliev 2019, PMID 30930284): purified MSL1/MSL2 ubiquitylate free histone substrates efficiently but modify nucleosomes very poorly, implying efficient H2BK34 ubiquitylation needs the nucleosome structurally altered first - the founding claim of robust nucleosomal activity is contested at the level of the assay that established it

• ← related toKAT8 (MOF) — a genuine KMT2A partner that binds the half of KMT2A the fusion discards

  Counter-evidence (Krajewski and Vassiliev 2019, PMID 30930284): the MOF-MSL complex's second activity is weak on the substrate that matters - MSL1/MSL2 modify nucleosomes very poorly in vitro - so the word 'robust' has been withdrawn from this entry's own voice

• ← related toH2BK34ub — a second ubiquitination site on H2B that also stimulates DOT1L, written by MSL1/MSL2, and important at HOXA9 and MEIS1

  Counter-evidence (Crespo 2020, PMID 32933557): H2BK34 is not a residue dedicated to ubiquitin - the mouse paralogue of yeast H2BK37 is enriched for METHYLATION during meiosis in the testes - so any K34 read-out must identify the modification rather than assume it

• related to →The fusion may reach the H2B ubiquitin mark through MOF-MSL rather than through RNF20/RNF40 — the one configuration in which this layer could be selective

  Counter-evidence (Krajewski and Vassiliev 2019, PMID 30930284): this is the published reason the prior is 0.15, and it was previously asserted without a source - MSL1/MSL2 modify nucleosomes very poorly, so recruitment is not sufficient if the enzyme cannot act on ordinary chromatin once it arrives

• related to →Does the fusion carry an H2B ubiquitin ligase? A co-IP and an H2BK34ub CUT&RUN in KMT2A::AFF1 cells

  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

• causes →H2BK34ub — a second ubiquitination site on H2B that also stimulates DOT1L, written by MSL1/MSL2, and important at HOXA9 and MEIS1

  RING E3 that ubiquitinates nucleosomal H2B at K34 with MSL1

• part of →KAT8 (MOF) — a genuine KMT2A partner that binds the half of KMT2A the fusion discards

  same MOF-MSL complex; two distinct activities, H4K16ac and H2BK34ub

• related to →Lost from the KMT2A half: the MOF/KAT8 contact - required in the cell, but not shown to matter to the fusion

  if the retained KMT2A fragment contacts MOF it is in contact with an H2B ubiquitin ligase

• ← bindsMSL1 - the MSL scaffold that carries MOF and MSL2 to HOXA9 and MEIS1, with zero records against KMT2A and a mapped, drawable domain architecture

  MSL1 binds MSL2 through residues 223-237 and is required with it for H2BK34 ubiquitylation.

• ← related toH4K16ac - the one mark rule 80 actually rescued in this beat: elevated at MLL-AF9 targets in a supplementary figure, and the reason MLL1's own enzyme turns out to be dispensable

  KAT8 writes this mark inside the MSL complex

• part of →The soluble-nucleoplasm versus chromatin-bound axis - the one localisation readout that keeps a size dimension, and nobody has run it on a KMT2A fusion

  MSL2 acts on nucleosomal H2BK34 at the HOXA9 and MEIS1 loci, controlling H3K4 and H3K79 methylation in trans - a genomic address at the canonical KMT2A-fusion targets, never measured in a fusion-bearing cell

• related to →Two pools of one protein: the chromatin-bound XPO1 that matters here has never been separated biochemically from the soluble export machine

  MSL2 is a nuclear chromatin ligase that drives p53 OUT of the nucleus by ubiquitylation without changing its stability - a second, non-XPO1 route onto this graph's compartment-control axis

• depends on →Pre-B cell — the stage KMT2A::AFF1 leukaemia never reaches, and why that boundary is informative

  Msl2 knockout in primary pre-B cells reduces Igmu expression and Emu-to-3'RR/VH promoter looping; Mof heterozygosity impairs early B-cell differentiation (murine; PMID 38990722)

• part of →KAT8 (MOF) — a genuine KMT2A partner that binds the half of KMT2A the fusion discards

  MSL2 and MOF/KAT8 act as one complex at the Igh Emu enhancer; the differentiation phenotype in that study belongs to the MOF allele, not to MSL2 (PMID 38990722)