MLL protects CpG clusters from methylation within the Hoxa9 gene, maintaining transcript expression.

Area of Science:

• Epigenetics
• Molecular Biology
• Cancer Research

Background:

• Homeobox (HOX) genes are crucial for cell fate determination in embryogenesis and hematopoiesis.
• MLL-related leukemias exhibit elevated expression of HOX genes, such as HOXA9.
• The mechanism by which MLL maintains target gene expression remains unclear.

Purpose of the Study:

• To elucidate the mechanism of MLL-mediated transcriptional maintenance.
• To investigate the role of MLL in regulating DNA methylation at the HOXA9 locus.

Main Methods:

• Chromatin immunoprecipitation to identify MLL binding sites.
• DNA methylation analysis (e.g., bisulfite sequencing) at CpG clusters within the Hoxa9 locus.
• Short hairpin RNA (shRNA) knock-down of MLL.
• Reconstitution of MLL expression in MLL-null cells.
• Analysis of MLL-AF4 fusion protein activity.

Main Results:

• MLL binds to specific CpG clusters within the Hoxa9 locus, regulating multiple transcripts.
• MLL binding protects these CpG clusters from DNA methylation.
• MLL depletion or absence leads to increased DNA methylation at these sites.
• MLL re-expression reverses DNA methylation, demonstrating a dominant protective effect.
• The oncogenic MLL-AF4 fusion partially reverses DNA methylation at a subset of CpGs.

Conclusions:

• MLL maintains target gene expression, including HOXA9, by protecting specific CpG-rich regions from DNA methylation.
• This mechanism of epigenetic regulation by MLL contributes to transcriptional maintenance and may extend to other DNA-binding proteins.
• Protection from DNA methylation is a key epigenetic inheritance mechanism influencing DNA methyltransferases.