# AF9(MLLT3) Function in Leukemia and Normal Hematopoiesis

> **NIH NIH R01** · UNIVERSITY OF VIRGINIA · 2020 · $617,133

## Abstract

MLLT3 (AF9) and its homolog MLLT1 (ENL) were initially identified as chromosome translocation
partners of the MLL (KMT2A) gene observed in Mixed Lineage Leukemia (MLL). The amino termini of MLLT3,
and MLLT1 proteins contain a nearly identical chromatin-binding YEATS domain which preferentially binds
crotonylated histone sites (Kcr). This distinguishes YEATS domains as crotonylation reader modules in contrast
to other acetylation reader modules, such as bromodomains. The MLLT3 YEATS domain directly links histone
Kcr readout to active gene transcription, but mechanisms underlying specific recruitment to direct target genes
are not understood. Work from different laboratories, including ours, has revealed roles of MLLT3 and MLLT1 in
at least four different complexes with critical gene regulatory functions based on direct binding to the C-terminal
ANC1 homology domain (AHD). The canonical functions of two of these complexes (AF4-containing Super
Elongation Complex; DOT1L) are to activate gene transcription whereas the other two (CBX8, BCOR) most often
function in gene repression. The factors that decide which of these four different complexes are recruited, and
whether recruitment of one complex facilitates or inhibits recruitment of another are not understood.
 Aim 1: Functional effects of CBX8 and BCOR recruitment on MLL-MLLT3/1 (MLL-AF9/-ENL) function.
We have determined 3D structures of MLLT3 AHD-CBX8 and AHD-BCOR complexes and used the structural
information to develop point mutations to selectively disrupt recruitment of CBX8 and BCOR. These will be used
to specifically delineate the role of direct recruitment of CBX8 and BCOR to MLL-MLLT3 and MLL-MLLT1 in
altering gene expression and driving leukemia, as we have done previously for the AF4 and DOT1L interactions.
 Aim 2: MLLT3 (AF9) YEATS domain is a dual reader of H3K9 (and K18, K27) crotonylation and RNA.
We have used a biochemical approach to show that the MLLT3 YEATS domain also binds to RNA, in addition
to specific binding to crotonylated H3, indicating this domain is a dual reader of both epigenetic marks and RNA.
We are proposing to fully characterize the role of the RNA binding of this domain in MLLT3 function. This includes
delineation of the RNA binding specificity, structural studies of a YEATS domain-H3K9cro-RNA ternary complex,
and development of point mutations which can selectively disrupt RNA binding and H3 peptide binding to probe
the functional role of these interactions. Similar studies will be carried out with MLLT1.
 Aim 3: MLLT3 (AF9) and MLLT1 (ENL) have non-redundant roles in hematooietic stem and progenitor
cell (HSPC) gene regulation which require their YEATS domain and C-terminal AHD functions. Using wildtype
and point mutant forms of MLLT3 and MLLT1 which can selectively disrupt either histone or RNA binding, we
will probe the functional role of the H3Kcr and RNA interactions via ChIP-seq, RNA-seq, and effects on in vitro
and in vivo HSPC functions. Wildtype and mut...

## Key facts

- **NIH application ID:** 9959369
- **Project number:** 5R01CA233749-02
- **Recipient organization:** UNIVERSITY OF VIRGINIA
- **Principal Investigator:** JOHN Hackett BUSHWELLER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $617,133
- **Award type:** 5
- **Project period:** 2019-07-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9959369

## Citation

> US National Institutes of Health, RePORTER application 9959369, AF9(MLLT3) Function in Leukemia and Normal Hematopoiesis (5R01CA233749-02). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/9959369. Licensed CC0.

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