# Deciphering the histone interactions and reader functions of ASH1L in biology and leukemia

> **NIH NIH F32** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2022 · $49,038

## Abstract

Project Summary
 Leukemia is a rare, but often fatal, form of cancer stemming from progenitor cells in the blood that are
defective in cellular differentiation. A particularly aggressive and hard to treat form called mixed-lineage leukemia
(MLL) arises from aberrant chromosomal translocations of the MLL gene with various genes encoding elongation
factors. These MLL-fusions improperly drive transcription of homeotic genes like those in the Hox family, thereby
maintaining a highly proliferative, stem cell-like population. These “leukemic stem cells” mature into leukemic
blasts that can form tumors and metastasize throughout the body via the blood stream. However, the specific
mechanisms of MLL-fusion driven leukemogenesis are still not well understood and the high frequency of
relapses upon treatment call for further characterization of the main drivers of this cancer. One such factor that
has recently been shown to be critical for regulating MLL-fusion driven leukemogenesis is ASH1L, a histone
H3K36 dimethyltransferase with a putative BRD-PHD-BAH histone reader domain module. A scaffold protein
called LEDGF was shown to bind to H3K36me2 and recruit MLL to homeotic genes in an ASH1L-dependent
manner, but it is unclear whether H3K36me2 and/or ASH1L are directly recruiting LEDGF-MLL complexes to
these genes. Additionally, it is completely unknown how ASH1L specifically localizes to homeotic genes rather
than much of the rest of the genome. To address how ASH1L functions in non-leukemic and leukemic cells, I
will employ a suite of innovative histone-binding assays, structural methodologies, and cellular-based analyses.
For Aim 1, I will determine the histone binding specificity and mode of the putative ASH1L reader domain module
using modernized histone peptide and nucleosome binding assays in parallel with Cryo-EM of purified ASH1L
bound to modified nucleosomes. For Aim 2, I will interrogate how ASH1L reads the chromatin landscape in the
well-studied and easy to genetically manipulate HEK293T cell line using a knockout/complementation system. I
will assess whether ASH1L colocalizes with histone modifications that we identify in Aim 1 as well as proteins
thought to associate with ASH1L like LEDGF using CUT&RUN. Additionally, I will examine how
loss/complementation of ASH1L in these cells affects known gene target expression. For Aim 3, I will employ a
knockdown/complementation system in MLL-fusion leukemia models to 1) assess whether ASH1L is required
for viability, 2) determine where ASH1L genomically localizes in MLL, and 3) characterize how ASH1L loss and
complementation affects recruitment of co-localizing gene regulators to impact transcription. This work aims to
further decipher the “Histone Code” and provide insights into how ASH1L functions as a driver of MLL-fusion
leukemogenesis. I will also lay out a foundation for testing ASH1L as a drug target for treating these aggressive
diseases. Finally, this work serves as a strong training platform ...

## Key facts

- **NIH application ID:** 10389050
- **Project number:** 1F32CA261015-01A1
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Nathaniel T Burkholder
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $49,038
- **Award type:** 1
- **Project period:** 2022-03-01 → 2022-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10389050

## Citation

> US National Institutes of Health, RePORTER application 10389050, Deciphering the histone interactions and reader functions of ASH1L in biology and leukemia (1F32CA261015-01A1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10389050. Licensed CC0.

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