SUPT5H (SPT5) — the pausing factor the KMT2A field has never studied, and the counted absence that undercuts 'the fusion hijacks pause release'

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.

• SUPT5H has a mean gene effect of -1.51 across 1,208 cell lines. Any agent that inhibited DSIF would be a general transcription poison, and the KMT2A rationale would add nothing to its selectivity.
• DSIF depletion reduces the protein stability of its partner SPT4, so partial inhibition of one subunit effectively removes the complex - there is no graded regime to exploit.
• The main risk here is not a failed drug but a wasted hypothesis: an intervention designed around pause release would be aimed at a step two independent published results place off the critical path for MLL fusion-dependent transcription.
• If a future experiment does find DSIF involvement, the pan-essentiality means the only viable strategy would be a fusion-specific recruitment interface rather than the factor itself - and no such interface is known.

1. Does DSIF occupancy or SPT5 CTR phosphorylation differ at KMT2A::AFF1 target genes relative to matched non-target genes? This is a single ChIP or PRO-seq experiment and it has never been done in this disease.
2. Okuda and Yokoyama say TBP loading is rate-limiting and not elongation. Does that hold for KMT2A::AFF1 in B-lineage cells, or only for the AEP constructs they tested in myeloid transformation assays? This is the most consequential unanswered question this entry raises.
3. If DSIF is present in both the paused and the activated complex, is there any state of DSIF that is specific to a fusion target gene? If not, DSIF cannot be a fusion-selective handle at all.
4. The CRISPRi result that DSIF depletion reduces AFF4 promoter occupancy was found in an HIV model. Does DSIF loss displace SEC from fusion target genes in a KMT2A-rearranged cell?

1. E1established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper

   Cryo-EM of a paused transcription elongation complex containing Sus scrofa Pol II with human DSIF and NELF at 3.2 A shows a tilted DNA-RNA hybrid impairing NTP substrate binding; NELF binds the polymerase funnel, bridges two mobile polymerase modules, contacts the trigger loop and restrains the Pol II mobility required for pause release, and prevents binding of the anti-pausing factor TFIIS; NELF has two flexible tentacles that can contact DSIF and exiting RNA.

   Structure of paused transcription complex Pol II-DSIF-NELF · Vos SM, Farnung L, Urlaub H, Cramer P · Nature 560(7720):601-606 2018
   PMID 30135580DOI
   ▸What this evidence cannot show

   Abstract fetched and read on 2026-09-19; full text not read. Reconstituted complex with pig Pol II and human factors; no chromatin, no nucleosome, no leukaemia, and no MLL fusion protein anywhere in the experiment. A structural state is not a demonstration of what limits transcription in a cell.

   ▸Evidence that argues against this (1)

   • A beautiful structure of the paused state does not establish that pausing is rate-limiting for any particular gene programme. For MLL fusion-dependent transcription specifically, Okuda and Yokoyama report that the rate-limiting step is TBP loading through SL1, not pause release - so the structural centrality of the pause should not be read across into this disease. Okuda H et al. 2016
2. E2established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper

   Formation of an activated Pol II elongation complex in vitro requires P-TEFb kinase function plus the PAF1 complex and SPT6; the 3.1 A cryo-EM structure of Pol II-DSIF-PAF-SPT6, compared with the paused Pol II-DSIF-NELF complex, shows that PAF displaces NELF from the Pol II funnel for pause release, that P-TEFb phosphorylates the Pol II linker to the C-terminal domain, and that SPT6 binds the phosphorylated CTD linker and opens the RNA clamp formed by DSIF.

   Structure of activated transcription complex Pol II-DSIF-PAF-SPT6 · Vos SM, Farnung L, Boehning M, Wigge C, Linden A, Urlaub H, Cramer P · Nature 560(7720):607-612 2018
   PMID 30135578DOI
   ▸What this evidence cannot show

   Abstract fetched and read on 2026-09-19; full text not read. Reconstituted, chromatin-free, and with pig Pol II. The PAF1 complex used here is the general elongation factor; the paper says nothing about the KMT2A-fusion-associated PAF1 interaction the graph carries under gene-paf1.

   ▸Evidence that argues against this (1)

   • This structure makes PAF1 the agent of pause release, which sits awkwardly beside the KMT2A field's model in which the fusion's PAF1 contact is about recruiting the fusion to chromatin rather than about releasing paused polymerase. Both cannot be the dominant role of PAF1 at a fusion target gene, and no experiment has tested the two against each other. Okuda H et al. 2016
3. E3established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.database

   UniProtKB O00267 (SPT5H_HUMAN, Transcription elongation factor SPT5) is 1087 aa and annotates five KOW domains (aa 273-306, 420-451, 472-503, 594-627, 704-737), a SUPT4H1-interaction region at aa 176-270, an RNA polymerase II interaction region at aa 313-420, and two C-terminal repeat regions - CTR1 at aa 754-817 (nine approximate 7-residue G-S-[QR]-T-P-X-[YQ] repeats) and CTR2 at aa 844-950 (ten approximate 8-residue P-[TS]-P-S-P-[QA]-[SG]-Y repeats).

   UniProtKB O00267 (SPT5H_HUMAN), Transcription elongation factor SPT5 · UniProt Knowledgebase, reviewed (Swiss-Prot) 2026
   ▸What this evidence cannot show

   Record fetched 2026-09-19; primary gene name read back as SUPT5H and length 1087 confirmed from the same fetched record, and every coordinate above copied from that record rather than typed from memory. Repeat boundaries are annotated 'approximate' by UniProt for several of the CTR repeats.

   ▸Evidence that argues against this (1)

   • UniProt's domain boundaries and the cryo-EM models disagree in detail: the structures resolve DSIF as clamp and stalk elements around Pol II rather than as a linear series of KOW domains, and the entire CTR region is annotated disordered (aa 747-978), so it is invisible in the structures. Any figure drawn from these coordinates will show a protein the structural work cannot corroborate. Vos SM et al. 2018
4. E4moderate confidence 3 of 5: One strong study, or several smaller consistent ones.database

   As of 2026-09-19, PubMed returns 1 record for '(MLL OR KMT2A) AND (SUPT5H OR SPT5 OR SPT5H OR DSIF)', 0 for 'SPT5 AND MLL', 0 for 'SPT5 AND leukemia', 0 for 'SUPT5H AND (AML OR acute myeloid)', and 1 for '(SPT5 OR SPT5H OR SUPT5H OR hSPT5 OR Supt5 OR DSIF) AND (MLL-AF9 OR MLL-AF4 OR MLL-ENL OR MLL-rearranged OR mixed lineage leukemia)'. No published experiment tests DSIF in a KMT2A-rearranged model.

   PubMed E-utilities esearch record counts for SUPT5H/SPT5/DSIF co-occurrence with KMT2A, MLL and MLL-fusion terms, run for this beat · NCBI PubMed, queried 2026-09-19 2026
   ▸What this evidence cannot show

   Symbol set searched: SUPT5H, SPT5, SPT5H, hSPT5, Supt5, DSIF. **Not searched:** Tat-CT1, 'DSIF p160', or non-English spellings, and the single-word term SPT5 will not match a paper that writes only 'Spt4/5' or 'the DRB sensitivity-inducing factor' in full. A counted-absence claim is a floor. That SUPT5H alone returns 130 records confirms the term itself retrieves, so the zero is about co-occurrence rather than about indexing.

   ▸Evidence that argues against this (1)

   • The absence may be an artefact of how elongation papers name things. A functional DSIF result could be buried in an MLL-fusion paper that writes only 'pausing factors' or 'the pause-release machinery' without naming SPT5, in which case this query would miss it entirely. This project has already been caught once by a bounded, well-written absence claim that was wrong because of the symbol set. NCBI PubMed, queried 2026-09-19; finding originally made by this project's non-elongation-partners beat, cycle 40 2026
5. E5moderate confidence 3 of 5: One strong study, or several smaller consistent ones.database

   In the project's genome-wide KMT2A-rearranged selectivity scan of 17,728 genes, SUPT5H is pan-essential with a mean gene effect of -1.5066 across 1,208 lines; KMT2A-rearranged mean -1.5754 against other-leukaemia mean -1.4539, a difference of -0.1215, rank 767 (4.326th percentile). Its DSIF partner SUPT4H1 scores a larger difference, -0.2642 at rank 138 (0.778th percentile), with a shallower pan-essentiality of -0.6692.

   Genome-wide KMT2A-rearranged selectivity scan, 17,728 genes (DepMap Chronos via Open Targets) · Broad Institute DepMap / Open Targets Platform; analyses/depmap-kmt2a-selective-scan-full 2026
   ▸What this evidence cannot show

   Numbers read out of analyses/depmap-kmt2a-selective-scan-full/results.json on 2026-09-19. At a mean effect of -1.5 in both groups there is a severe floor effect: a difference statistic computed where every line is dying is close to uninterpretable, and the 4.3rd-percentile rank should not be read as evidence of KMT2A selectivity. Eleven KMT2A-rearranged lines, none infant-derived.

   ▸Evidence that argues against this (1)

   • The project's own essentiality-confound analysis found essentiality explains only a small fraction of variance across the whole scan, but that average does not license reading the extreme tail, and SUPT5H sits in it. The honest reading is that DepMap cannot resolve whether DSIF has any KMT2A-specific role, not that it has one. Krasnopolsky S et al. 2021
6. E6moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

   Okuda and Yokoyama state the standard model - 'it is hypothesized that relieving the promoter proximal pausing of RNA polymerase II is the rate-limiting step of MLL fusion-dependent transcription' - and report the contrary: the key activity responsible for the oncogenic property of MLL-AEP fusion proteins is TBP loading activity through the SL1 complex, and not mediator recruitment or transcriptional elongation activities.

   TBP loading by AF4 through SL1 is the major rate-limiting step in MLL fusion-dependent transcription · Okuda H, Takahashi S, Takaori-Kondo A, Yokoyama A · Cell Cycle 15(20):2712-22 2016
   PMID 27564129DOI
   ▸What this evidence cannot show

   Abstract fetched and read on 2026-09-19; the quoted phrases are verbatim from that abstract. Full text not read. This is a structure-function dissection of MLL-AEP fusion constructs in murine myeloid transformation assays, so 'rate-limiting' means rate-limiting for transformation by engineered constructs, not necessarily for transcription at a native fusion target in a patient blast. It is also a single laboratory's conclusion and the AEP/AF4 framework is that laboratory's own.

   ▸Evidence that argues against this (1)

   • The pause-release model is not without support: ENL, an MLL fusion partner, initiates multivalent phase separation of SEC that concentrates P-TEFb and controls transcriptional pause release, and MLL-ENL fusion is reported to BOOST SEC phase separation. If the fusion increases the capacity of the pause-release machinery, pause release cannot be irrelevant even if TBP loading is rate-limiting. Guo C et al. 2020
7. E7moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

   Native chromatin proteomics identified ECAS, a bipartite assembly linking an AFF4-ELL2-EAF1 elongation module to a U2 snRNP-associated intron binding complex, occupying promoter-proximal chromatin and molecularly distinct from canonical P-TEFb-containing SEC; acute perturbation of AQR/XAB2-dependent ECAS functions impairs productive gene-body elongation WITHOUT altering pause release, increases intron retention and preferentially affects long genes, and in mouse haematopoietic cells Aqr perturbation profoundly impairs leukaemia initiation, maintenance and leukaemia stem cell function in MLL-rearranged leukaemia.

   A dual-module ECAS complex couples transcription elongation and RNA processing · Xiang Y, Wang H, Yin Z, Xiao R, Song Z, Lu R, Dai R, Ye J, Li C, Cui Y, Wang M, Lv J, Li Q, Wang F, Li Z, Zhang K, Zhang Y, Wu D, Fang P, Liang K · Molecular Cell, online ahead of print 16 September 2026 2026
   PMID 42748909DOI
   ▸What this evidence cannot show

   Abstract fetched and read on 2026-09-19; full text not read. Very recent and not independently replicated. The leukaemia phenotype is reported for Aqr, not for SPT5 or DSIF; the 'without altering pause release' statement is about AQR perturbation and is used here as evidence about where the ECAS mechanism acts, not as a measurement of SPT5 function.

   ▸Evidence that argues against this (1)

   • This project's own genome-wide scan does not support AQR as a KMT2A-rearranged-selective dependency and points the other way (AQR rank 17,654, difference +0.244). The ECAS leukaemia result is an in-vivo stem-cell phenotype that DepMap cannot see, so the two are not strictly in conflict - but the ECAS claim is the weaker leg of the argument against pause-release centrality, and the Okuda result is the stronger one. Broad Institute DepMap / Open Targets Platform; analyses/depmap-kmt2a-selective-scan-full 2026
8. E8moderate confidence 3 of 5: One strong study, or several smaller consistent ones.paper

   CRISPR interference knockdown of Spt4, Spt5 or the whole DSIF complex inhibited transcription primarily at the step of transactivation, decreased promoter occupancy of Pol II and Cdk9 and, to a lesser extent, AFF4; targeting either DSIF subunit reduced the protein stability of its partner; and depletion impaired cell growth without causing cell death.

   CRISPRi-mediated depletion of Spt4 and Spt5 reveals a role for DSIF in the control of HIV latency · Krasnopolsky S, Novikov A, Kuzmina A, Taube R · Biochimica et Biophysica Acta - Gene Regulatory Mechanisms 1864(1):194656 2021
   PMID 33333262DOI
   ▸What this evidence cannot show

   Abstract fetched and read on 2026-09-19; full text not read. The system is HIV-1 Tat transactivation in Jurkat cells, not leukaemia, and the AFF4 occupancy change is reported as a lesser effect without quantification in the abstract. This is the only citation in this entry that connects DSIF to a SEC subunit at all, and it does so in an entirely unrelated disease.

   ▸Evidence that argues against this (1)

   • Using an HIV latency model to make inferences about SEC in leukaemia is exactly the kind of transfer this project has been careful to refuse elsewhere. HIV Tat recruits P-TEFb through a mechanism (TAR RNA) that has no counterpart at a KMT2A fusion target gene, so reduced AFF4 occupancy on DSIF depletion in Jurkat cells may say nothing at all about the fusion. Guo C et al. 2020
9. E9established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper

   RCSB PDB entry 7OKX, 'Structure of active transcription elongation complex Pol II-DSIF (SPT5-KOW5)-ELL2-EAF1 (composite structure)' at 3.3 A, models SPT5 (UniProt O00267, residues 1-1087) together with ELL2 (O00472, 1-640) and EAF1 (Q96JC9, 1-268) on RNA polymerase II; the primary citation is Chen et al., Molecular Cell 2021, which reports allosteric transcription stimulation by the RNA polymerase II super elongation complex.

   Allosteric transcription stimulation by RNA polymerase II super elongation complex · Chen Y, Vos SM, Dienemann C, Ninov M, Urlaub H, Cramer P · Molecular Cell 81(16):3386-3399.e10 2021
   PMID 34265249DOI
   ▸What this evidence cannot show

   PDB entity descriptions, UniProt accessions and modelled ranges fetched from data.rcsb.org on 2026-09-19; the PMID, DOI, title, authors, journal, volume and pages fetched from PubMed in the same session and copied from those records. This is a **composite** deposition, so the stated ranges are more generous than the density in any single reconstruction; 'SPT5 1-1087' does not mean all 1,087 residues are ordered, and UniProt annotates aa 747-978 of SPT5 as disordered. The Pol II subunits are Sus scrofa. **This is a reconstituted complex on naked DNA and says nothing about a KMT2A fusion.**

   ▸Evidence that argues against this (1)

   • A reconstituted structure containing DSIF and an ELL2-EAF1 module does not establish that DSIF and SEC co-occupy a gene in a cell, still less at a fusion target. The structure was assembled to study how the ELL2-EAF1 pair stimulates elongation allosterically, and DSIF is present because it is part of the elongating polymerase in any such reconstitution - its presence is a property of the experimental design as much as of biology. Xiang Y et al. 2026

• ← part ofSUPT6H (SPT6) - the histone chaperone that rides with RNA polymerase II, which puts it closer to the fusion's proposed mechanism than anything else in this beat

  both subunits of the activated Pol II-DSIF-PAF-SPT6 elongation complex

• binds →SUPT4H1 (SPT4) — zero papers linking it to KMT2A, and the 138th most KMT2A-selective gene out of 17,728

  DSIF heterodimer: SPT5 aa 176-270 binds SPT4; each subunit stabilises the other's protein level

• related to →P-TEFb (CDK9 + cyclin T1) — the deepest dependency and the clearest therapeutic-index wall in the interactome

  SPT5 CTR1 (aa 754-817) and CTR2 (aa 844-950) are P-TEFb substrates; the graph's CDK9 axis had no substrate entry before this one

• related to →PAF1 — pan-essential subunit of a complex the fusion needs, where the window is in the interface and not in the protein

  PAF1 displaces NELF from the Pol II funnel for pause release while DSIF is retained (cryo-EM)

• related to →KMT2A::AFF1 (MLL-AF4), t(4;11)(q21;q23)

  NOT an interactor: 1 PubMed record across all aliases co-occurring with MLL/KMT2A; no functional test in any KMT2A-rearranged model

• ← bindsNELF — the brake that has to come off for elongation, absent from the KMT2A literature, and less required in KMT2A-rearranged lines than in any other leukaemia

  NELF tentacles contact DSIF in the paused complex Pol II-DSIF-NELF

• ← co-occurs withEAF1 — the interactor with an actual subnuclear phenotype: an MLL fusion evicts it from Cajal bodies, and the bodies themselves fall apart

  EAF1, ELL2 and SPT5 are modelled together on Pol II in PDB 7OKX - the one structure placing DSIF and a SEC-family activation module in the same complex

• ← supportsInteractome round 4: what was counted, what was written, what was rejected, and the two alias traps this beat fell into and caught

  documents the DSIF counted absence and the two published results placing the rate-limiting step away from pause release