The CLOCK/BMAL1 protein interaction landscape at the chromatin reveals homeodomain transcription factors as modulators of tissue-specific circadian transcription

Although the molecular mechanism of the circadian clock is conserved across cells, its transcriptional outputs are highly tissue specific. To experimentally explore how the core CLOCK/BMAL1 heterodimer achieves this specificity, we investigated its protein interaction landscape at the chromatin by employing a method that combines chromatin immunoprecipitation with mass spectrometry-based quantitative proteomics (ChIP–MS). This approach yielded the first two-dimensional (temporal and tissue-specific) interaction map of CLOCK/BMAL1 at the chromatin and revealed a complex organ-dependent landscape of clock-associated protein interactions. Among these we identified three homeodomain containing transcription factors -PROX1, HFN1B and HOXA5- in liver, kidney and lung, respectively. Functional analyses demonstrated that these factors colocalize with CLOCK/BMAL1 at the chromatin physically interacting via their homeodomain and the BMAL1 C-terminal transactivation domain and act as transcriptional repressors. Deletion of these homeodomain factors in organ-derived cell lines led to upregulation of core clock genes and disrupted rhythmic transcription of tissue-specific targets. Our findings uncover a new class of tissue-specific circadian transcriptional repressors and suggest that the molecular clock does not solely impose a universal temporal program but is instead modulated by chromatin-bound cofactors—such as PROX1, HNF1B, and HOXA5—that shape tissue-specific transcriptional outputs