Astrocyte circadian regulation in neurodegeneration: in vivo BMAL1 occupancy mapping using MACS-Calling Cards

Astrocytes maintain neuronal homeostasis through cellular programs that are frequently disrupted early in neurodegenerative disease, many of which are regulated by the circadian clock. The core clock transcription factor BMAL1 is required for normal astrocyte function, yet whether its regulatory activity is altered during neurodegeneration remains unclear. BMAL1 expression changes in disease are often inconsistent, suggesting that transcription factor binding, rather than expression levels, may be a more informative readout of BMAL1 function. We used a CLN3Δex7/8 model of neurodegeneration, which exhibits early astrocyte dysfunction prior to widespread neuronal loss, to test whether degenerative pathology reprograms BMAL1 binding in astrocytes. We developed an in vivo strategy to record transcription factor genomic occupancy specifically in astrocytes (MACS-Calling Cards, MACS-CC) and used it to map BMAL1 binding in wild-type and CLN3Δex7/8 mice. BMAL1 binding was extensively redistributed, with comparable numbers of wild-type–specific and disease-specific sites. Wild-type-specific BMAL1 binding was enriched near genes involved in circadian regulation and astrocyte differentiation, whereas disease-specific sites lacked coherent functional enrichment. Consistent with these changes, RNA-seq of sorted astrocytes revealed reduced expression of mature astrocyte markers in CLN3Δex7/8 mice. To define the mechanisms underlying BMAL1 retargeting in CLN3Δex7/8 astrocytes, we tested whether changes in chromatin accessibility could explain disease-associated gains and losses of BMAL1 binding. Although chromatin accessibility was broadly remodeled, differential accessibility did not predict BMAL1 binding gains or losses. Instead, motif analyses supported a model in which loss of normal cooperative transcription factor partnerships contributes to BMAL1 retargeting. Together, these findings demonstrate that MACS-CC enables in vivo TF occupancy mapping in astrocytes and reveals the mechanisms behind early rewiring of circadian regulatory programs within a model of neurodegeneration.