Nuclear import of every KMT2A fusion is a property of retained KMT2A sequence, and the determinant was mapped in 1997 to a region containing the AT-hook motifs
also called MLL nuclear localisation determinant · why MLL-AF6 is nuclear · KMT2A NLS region
Some of the proteins that MLL fuses to normally live outside the nucleus, yet the fusion always ends up inside it. A 1997 experiment found that the part of MLL responsible for pulling the fusion into the nucleus is the stretch that carries its DNA-gripping hooks, which every leukemia fusion keeps.
The same thing, for a scientist
The statement that KMT2A fusions are nuclear because the KMT2A half carries the import information is repeated throughout this graph, usually without a source. It has one. Joh and colleagues asked the question in the hardest available case - MLL-AF6, whose partner AF6/AFDN is a cytoplasmic Ras-binding protein - and showed by immunofluorescence **and cell fractionation** that the chimera is nuclear, then mapped the responsible region of the MLL N-terminus and found it to be **a region containing the AT-hook motifs** [E1]. The same group had already shown the nuclear localisation of MLL, MLL-AF9, MLL-ENL and the truncated N-terminal MLL product by the same two methods [E2]. The consequence is a systematic one: **a nuclear result carries almost no information about which fusion is present**, because the determinant is in the part every fusion retains. It also means 'the fusion is nuclear' is not a fusion phenotype at all - it is a wild-type KMT2A phenotype inherited by the chimera, and the graph should not spend confidence on it.
The mapping and what it does not say
The abstract's wording is precise and worth keeping precise: the region responsible 'was found to be a region containing AT-hook motifs' 1. That is a region assignment, not a demonstration that the hooks themselves are the signal. A classical import signal is a short basic stretch recognised by importin-alpha, and the KMT2A N-terminus is basic across a span far wider than the hooks; a deletion that removes the hooks removes a good deal else.
This matters because this graph's AT-hook entry already carries a correction - that the one experiment on MLL's own hooks found they read DNA structure (four-way cruciform) rather than AT-richness. A DNA-structure reader and a nuclear import signal are different jobs, and the 1997 mapping does not decide which job the deleted region was doing. Nuclear retention by chromatin binding looks identical to nuclear import in a fixed-cell image: a protein that enters passively and is held by DNA scores as nuclear, and so does a protein that is actively imported.
That ambiguity is resolvable and has not been resolved. It needs a cytoplasmic-retention or import-block assay, not a picture.
Why this collapses a whole class of published claims
Three separate 1996-97 papers report that a KMT2A chimera is nuclear: MLL-AF9 and MLL-ENL 2, MLL-AF6 1, and MLL-ELL in transfected COS7 3. A fourth adds MLL-SEPT6 as nuclear speckles while SEPT6 alone stays cytoplasmic, and a fifth reports the truncated N-terminal MLL product as nuclear too 2.
Given the mapping, all of these were predictable before they were done, and none of them discriminates between partners. They are consistent with, and are usually cited as supporting, the claim that the partner is irrelevant to localisation - but the experiment that would show a partner does matter has to look inside the nucleus, and inside the nucleus nobody has an attributable readout.
What is lost at the junction, and whether it could matter
The canonical KMT2A::AFF1 junction joins KMT2A residue 1406 to AFF1 residue 347 4. Everything the 1997 mapping implicates is well upstream of that and is retained. But the fusion also loses the clustered PHD region, which in KMT2A begins at approximately residue 1416 - ten residues downstream of the junction - and which has independently been shown to target active promoters and enhancers 5. So the fusion keeps the module that gets it into the nucleus and loses the module that would fine-tune where it goes once inside. That asymmetry is the cleanest available structural statement about fusion localisation and it has, as far as this beat could find, never been stated.
| determinant region | N-terminal region of KMT2A containing the AT-hook motifs |
|---|---|
| mapped in | 9349501 |
| method | immunofluorescence + cell fractionation of deletion constructs |
| retained in all fusions | true |
| ambiguity not resolved | active nuclear import versus nuclear retention by chromatin binding |
phd cluster lost
{
"construct": "2A13",
"start": 1416,
"end": 1640,
"uniprot": "Q03164"
}- Because the import determinant is retained KMT2A sequence shared with wild-type KMT2A, any agent that blocks it would block wild-type KMT2A import as well, and KMT2A is required in normal haematopoiesis.
- A mislocalisation strategy - forcing the fusion out of the nucleus - would have to overcome a determinant that is also present on the abundant wild-type protein, so selectivity would have to come from somewhere else entirely.
- Is the KMT2A N-terminal determinant an import signal or a chromatin-retention element? An importin-binding assay or a cytoplasmic-anchor competition would separate them.
- Does the triple AT-hook alanine mutant this graph already proposes change nuclear entry, or only chromatin residence?
- Do any KMT2A fusion breakpoints retain part of the clustered PHD region, and do those patients differ?
- E1strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
Immunofluorescence staining and cell fractionation showed that MLL-AF6 chimeric products localise in the nuclei despite AF6 itself being cytoplasmic, and the region in the N-terminal portion of MLL responsible for this nuclear localisation was examined and found to be a region containing AT-hook motifs.
Chimeric MLL products with a Ras binding cytoplasmic protein AF6 involved in t(6;11) (q27;q23) leukemia localize in the nucleus · Joh T, et al. · Oncogene 1997What this evidence cannot show
Abstract only; the Europe PMC core record returns no pmcid so routes 2-4 were not reachable. The abstract does not name the cell system or the deletion boundaries, and a region assignment is not a demonstration that the hooks are the signal. Nuclear retention by chromatin binding is not distinguished from active import by this design.
Evidence that argues against this (1)
- The one experiment performed on MLL's own AT-hooks found they recognise DNA structure - the four-way cruciform - rather than AT-richness, so the region's characterised function is DNA binding, which would produce nuclear retention rather than import and would look the same in a fixed-cell image. Yano T et al. 1997
- E2moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper
MLL and the chimeric MLL-LTG9 (AF9) and MLL-LTG19 (ENL) products, and the truncated N-terminal MLL product common to various 11q23 translocations, were all shown to be synthesised and localised in the nuclei by immunofluorescence and cell fractionation.
Identification of MLL and chimeric MLL gene products involved in 11q23 translocation and possible mechanisms of leukemogenesis by MLL truncation · Joh T, et al. · Oncogene 1996What this evidence cannot show
Abstract only, no PMCID, no DOI in the PubMed record. The cell system is not stated and the fractionation resolves nucleus versus cytoplasm only.
Evidence that argues against this (1)
- The truncated N-terminal MLL product being nuclear by the same criterion shows the readout cannot separate a fusion from a truncation, so nuclear localisation is uninformative about which product is present. Kanda Y et al. 1997
- E3moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper
MEN (ELL), MLL/MEN chimeric and truncated MLL proteins expressed from cDNA in COS7 cells were nuclear by immunostaining and subcellular fractionation.
Subcellular localization of the MEN, MLL/MEN and truncated MLL proteins expressed in leukemic cells carrying the t(11;19)(q23;p13.1) translocation · Kanda Y, et al. · International journal of hematology 1997What this evidence cannot show
Transfected COS7 monkey kidney fibroblasts, not leukaemic cells, although the paper's closing sentence asserts localisation 'in the nucleus of leukemic cells'. Abstract only; no PMCID.
Evidence that argues against this (1)
- Construct-level localisation can be perturbed by the construct itself: an N-terminal tag on MLL::AF9 failed to reproduce the untagged construct's leukaemogenesis, attributed to interference with chromatin localisation. Olsen SN et al. 2022
- E4established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper
The t(4;11)(q21;q23) translocation generates an MLL-AF4 fusion product, the chimera whose retained KMT2A N-terminus carries the import determinant.
Acute mixed-lineage leukemia t(4;11)(q21;q23) generates an MLL-AF4 fusion product · Domer PH, et al. · Proceedings of the National Academy of Sciences of the United States of America 1993What this evidence cannot show
The founding report of the fusion product. Exact junction residues vary with breakpoint and should be read from this graph's breakpoint entries rather than assumed.
Evidence that argues against this (1)
- Breakpoint heterogeneity within KMT2A intron 9-11 changes how much KMT2A sequence is retained, so the amount of the implicated region present is not identical across patients. Butler LH et al. 1997
- E5strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
The clustered PHD domains of KMT2A (construct 2A13, aa 1416-1640 of UniProt Q03164) localise to H3K4me3 and H3 acetylation-rich active promoters and enhancers, and fusing the KMT2A CXXC domain to those PHDs drastically enhances their preference for promoters over enhancers.
Clustered PHD domains in KMT2/MLL proteins are attracted by H3K4me3 and H3 acetylation-rich active promoters and enhancers · Stroynowska-Czerwinska AM, et al. · Cellular and molecular life sciences : CMLS 2023What this evidence cannot show
Read from the Europe PMC full-text XML (PMC9813062). The constructs are NLS-EYFP-tagged isolated domains expressed from lentivirus in HeLa S3 cells and read out by greenCUT&RUN, not full-length KMT2A and not a leukaemia cell; the NLS is supplied by the tag, so the construct cannot report on import. The authors note the nuclear EYFP signal was diffuse rather than punctate.
Evidence that argues against this (1)
- An isolated domain cluster carries no constraint from the rest of the protein, so its genome-wide distribution need not match the distribution of the same domains inside full-length KMT2A; the authors' own Jaccard comparison shows only partial overlap with full-length KMT2A peaks. Stroynowska-Czerwinska AM et al. 2023
- ← depends onKMT2A / MLL1 — the gene that breaks
nuclear import maps to the AT-hook region; UniProt annotates no NLS motif on KMT2A
the KMT2A import determinant is retained in all seven baits; which importin-alpha reads it has never been asked
adds the 1997 nuclear-localisation mapping to the AT-hook region, and flags that import and retention are not separated
supplies the primary citation for that entry's otherwise unsourced claim that the KMT2A half carries the NLS
- related to →KMT2A::AFDN (MLL-AF6), t(6;11)(q27;q23)
MLL-AF6 was the test case: a cytoplasmic partner whose chimera is nuclear
- part of →KMT2A / MLL1 — the gene that breaks
the import determinant is retained KMT2A sequence, shared with the wild-type protein
the fusion keeps the module that gets it into the nucleus and loses the PHD module that would tune where it goes inside
the nuclear outcome is a property of retained KMT2A sequence, not of the partner
two independent groups place the targeting information in the retained amino terminus
rests on the 1997 mapping of the localisation determinant to retained KMT2A sequence
KMT2A's own NLS has never been mapped as an NLS; the 1997 region assignment is all there is