Reiss, I. H.; Cooper, J. D.; Musiek, E. S.; Mitra, R. D.

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. Moreover, astrocyte circadian gene expression patterns are profoundly reprogrammed in disease, yet the underlying mechanisms remain unclear, primarily due to a lack of methods for identifying DNA binding sites of BMAL1 in specific cell types in the brain. Here, we have 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. We applied this strategy to the Cln3{Delta}ex7/8 mouse model of CLN3 disease, a fatal neurodegenerative disorder, which exhibits early astrocyte dysfunction prior to widespread neuronal loss as well as circadian dysregulation. This allowed us to test how the progressive pathological cascade caused by CLN3 deficiency reprograms BMAL1 binding in astrocytes. 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 Cln3{Delta}ex7/8 mice. To define the mechanisms underlying BMAL1 retargeting in Cln3{Delta}ex7/8 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 transcription factor occupancy mapping in astrocytes and reveals the mechanisms behind early rewiring of circadian regulatory programs within a model of progressive pathology leading to neurodegeneration. Our data also shed new light on astrocyte dysfunction in CLN3 disease.