Nature - USA (2020-01-02)

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nature research | reporting summary

October 2018

For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design

All studies must disclose on these points even when the disclosure is negative.

Sample size For most high-throughput sequencing experiments, two independent biological replicates were used (e.g. RNA-seq, and most of ChIP-seq).

Additional independent experiments were repeated and ChIP-qPCR and RT-qPCR were used to validate key results obtained from high-

throughput sequencing.

Sample sizes for other assays were not predetermined and were chosen based on our prior experience and common standards in the field for

detecting statistically significant differences between conditions.

Data exclusions No data was excluded from the analysis.

Replication Each experiment was repeated (See Figure Legends) and all findings were reproducible. In most assays, the conclusions about functional

difference between wild-type and mutant ENL were drawn from comparing wild-type and multiple distinct mutants (T1, T2, T3), which further

strengthens the conclusions.

Randomization Samples were allocated to groups according to genotype or treatment. No randomization was required as the starting materials (e.g. parental

cell lines before generation of sub-lines with different genotypes) are identical.

Blinding The investigators were not blinded to allocation during experiments and outcome assessment except for pathological analysis in Figure. 1g.

Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material,

system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Materials & experimental systems

n/a Involved in the study

Antibodies

Eukaryotic cell lines

Palaeontology

Animals and other organisms

Human research participants

Clinical data

Methods

n/a Involved in the study

ChIP-seq

Flow cytometry

MRI-based neuroimaging

Antibodies

Antibodies used All antibodies used have been provided in Supplementary Tables with details (e.g. supplier name, catalog number, application

and dilution)

Validation For Immunoblot, the correct size of the detected bands was assessed based on the protein marker.

1. Flag: Manufacturer indicates reactivity by immunoblot to detect Flag epitope-tagged proteins. We have demonstrated in prior
   work (Wan et al. 2017) that this antibody can be used for ChIP-seq and confirmed its specificity using cells without Flagged
   transgenes.


2. ENL: Manufacturer indicates human reactivity by immunoblot. We have demonstrated its specificity in prior work (Wan et al.

2017. using ENL KD cells.

3. b-actin: Manufacturer indicates human and mouse reactivity by immunoblot. It is widely used in western blot as a loading
   control.


4. GAPDH: Manufacturer indicates human and mouse reactivity by immunoblot. It is widely used in western blot as a loading
   control.


5. b-tubulin: Manufacturer indicates human and mouse reactivity by immunoblot. It is widely used in western blot as a loading
   control.


6. AFF1: Manufacturer indicates human reactivity by immunoblot. We have demonstrated its specificity in prior work (Wan et al.

2017. using AFF1 KD cells.

7. PAF1: Manufacturer indicates human reactivity by immunoblot. We have demonstrated its specificity using PAF1 KD cells.


8. DOT1L: Manufacturer indicates human reactivity by immunoblot. We showed it can be used for western blot in current study.


9. Myc: Manufacturer indicates reactivity by immunoblot to detect Myc epitope-tagged proteins. We have demonstrated its
   specificity in prior work (Wan et al. 2017) in co-immunoprecipitation assays.


10. CDK9: Manufacturer indicates human and mouse reactivity by immunoblot. We showed that it can be used for ChIP-seq in
    current study.


11. Pol II (ser-2p): Manufacturer indicates human and mouse reactivity by immunoplot. We have demonstrated in prior work
    (Wan et al. 2017) that this antibody can be used for ChIP-seq.