Distinct structural bases for sequence-specific DNA binding by mammalian BEN domain proteins

Area of Science:

• Molecular Biology
• Structural Biology
• Genomics

Background:

• The BEN domain is a DNA-binding module found in metazoans and viruses, often acting as transcriptional repressors.
• Limited understanding exists regarding how human BEN domain factors identify their DNA targets, especially compared to Drosophila factors.
• Existing X-ray structures are primarily from Drosophila BEN domain proteins, which lack direct vertebrate orthologs.

Purpose of the Study:

• To characterize mammalian BEN domain (BD) factors, including NACC family BTB-BEN proteins and BEND3.
• To elucidate the mechanisms of sequence-specific DNA binding and target identification by mammalian BEN domain proteins.
• To provide high-resolution structural insights into BEND3-DNA interactions.

Main Methods:

• In vitro DNA selection assays to identify sequence-specific binding activities of isolated BEN domains.
• Functional, genomic, and structural studies focused on the BEND3 protein.
• High-resolution X-ray crystallography of the BEND3-BD4 domain bound to DNA.
• Comparative structural analysis with invertebrate BEN domains and computational predictions (AlphaFold2, RoseTTAFold).

Main Results:

• Isolated BEN domains from mammalian factors, including BEND3, exhibit sequence-specific DNA binding.
• The BD4 domain of BEND3 is crucial for its in vivo association with and repression of endogenous targets.
• A high-resolution structure of BEND3-BD4 reveals its DNA target recognition mechanism, distinct from its non-DNA-binding BD1 domain.
• Distinct DNA recognition strategies exist among different types of BEN domain proteins across species.

Conclusions:

• Mammalian BEN domain factors possess sequence-specific DNA binding capabilities.
• BEND3 utilizes its BD4 domain for specific DNA target recognition and transcriptional repression.
• These findings expand the known DNA-binding activities of BEN factors and offer structural insights into mammalian BEN protein function.