DOT1L complex regulates transcriptional initiation in human erythroleukemic cells

Area of Science:

• Molecular Biology
• Epigenetics
• Cancer Biology

Background:

• DOT1L is the sole H3K79 methyltransferase in humans, forming complexes with AF10, AF17, and ENL/AF9.
• Dysregulation of DOT1L is implicated in mixed-lineage leukemias (MLLr).
• DOT1L was previously thought to regulate transcriptional elongation based on its association with RNA polymerase II (Pol II) and gene methylation patterns.

Purpose of the Study:

• To investigate the precise role of DOT1L in transcriptional regulation.
• To determine whether DOT1L primarily affects elongation or initiation.
• To elucidate the mechanism by which DOT1L influences gene expression.

Main Methods:

• Depletion of DOT1L in erythroleukemic cells.
• 4sUDRB-seq to assess Pol II traveling ratio and elongation rate.
• Analysis of TATA binding protein (TBP) and Pol II occupancy.
• Proteomic and biochemical studies.
• Assessing H2Bub1 levels and Spt-Ada-Gcn5-acetyltransferase (SAGA) complex recruitment.

Main Results:

• DOT1L depletion reduced its global occupancy but did not affect Pol II elongation rate.
• Depletion of DOT1L or ENL led to decreased TBP occupancy at thousands of genes.
• DOT1L and ENL depletion reduced TBP and Pol II occupancy at direct DOT1L target genes, indicating a role in transcription initiation.
• DOT1L complex appears to facilitate transcription factor IID recruitment/stabilization, possibly via H2Bub1.
• DOT1L enhances H2Bub1 levels by limiting SAGA complex recruitment.

Conclusions:

• DOT1L plays a critical role in transcription initiation, not elongation, in erythroleukemic cells.
• The DOT1L complex's function in initiation involves facilitating transcription factor recruitment, potentially modulated by H2Bub1.
• These findings offer new insights into DOT1L's function and have implications for MLLr leukemias.