Single-stranded DNA-binding proteins are essential components of the architectural LDB1 protein complex

Transcriptional enhancers are brought into proximity with promoters via chromatin looping. The architectural transcription cofactor LDB1 facilitates spatial connectivity among enhancers and promoters, but whether this occurs through simple dimerization or requires heterotypic protein assemblies is unknown. Here, we investigated the role of single-stranded DNA-binding proteins (SSBPs) in regulating LDB1-mediated chromatin looping and transcription. SSBP2, SSBP3, and SSBP4 colocalize with LDB1 genome wide. Among these, only SSBP3 is essential for murine erythroid cell viability, LDB1 function, and transcription. LDB1, but not single-stranded DNA, is the predominant genome-wide tether of SSBP3 to chromatin. SSBP3 depletion in SSBP2/4 knockout cells globally weakened LDB1-dependent chromatin loops and lowered nascent transcription without impacting LDB1’s chromatin binding. Chromatin tethering experiments revealed that SSBP3 and LDB1 mutually depend on each other to form looped contacts. SSBP3 stabilizes LDB1 dimerization in vitro, providing a possible mechanism. SSBPs emerge as key functional components of the architectural LDB1 complex.