JMJD6 and YBX1 physically interact and regulate HOTAIR proximal promoter

Area of Science:

• Molecular Biology
• Cancer Research
• Epigenetics

Background:

• Previous work identified jumonji domain containing protein 6 (JMJD6) binding to the HOTAIR promoter.
• Further investigation revealed an additional regulatory region upstream of the JMJD6 binding site.
• In silico and ENCODE data suggested Y-box interacting protein 1 (YBX1) binds to this upstream region.

Purpose of the Study:

• To validate the interaction between JMJD6 and YBX1 in breast cancer cell lines.
• To elucidate the functional consequences of the JMJD6-YBX1 interaction on HOTAIR expression.
• To determine the mechanism by which YBX1 influences JMJD6 recruitment to the HOTAIR promoter.

Main Methods:

• Co-immunoprecipitation assays to confirm protein-protein interactions.
• Domain mapping using deletion constructs.
• Luciferase reporter assays to assess promoter activity.
• siRNA-mediated depletion and gene knockout (YKO) to study functional effects.
• Chromatin immunoprecipitation (ChIP and ChIP-re-ChIP) and electrophoretic mobility shift assays (EMSA).

Main Results:

• JMJD6 and YBX1 interact directly, with the A/P domain of YBX1 binding the JMJC domain of JMJD6.
• YBX1 enhances HOTAIR promoter activity, and its depletion or mutation of the YBX1 interaction region (YIR) reduces activity.
• Both JMJD6 and YBX1 co-occupy the HOTAIR promoter, and YBX1 is crucial for JMJD6 recruitment.
• A positive feed-forward loop exists where JMJD6 and YBX1 mutually regulate each other's expression, leading to increased HOTAIR induction.

Conclusions:

• YBX1 is essential for recruiting JMJD6 to the HOTAIR promoter in breast cancer cells.
• The JMJD6-YBX1 interaction and their positive feedback loop drive HOTAIR expression.
• This mechanism contributes to tumor progression, highlighting JMJD6 and YBX1 as potential therapeutic targets.