Human Satellite 3 DNA encodes megabase-scale transcription factor binding platforms.

Eukaryotic genomes are frequently littered with large arrays of tandem repeats, called satellite DNA, which underlie the constitutive heterochromatin often found around centromeric regions. While some satellite DNA types have well-established roles in centromere biology, other abundant satellite DNAs have poorly characterized functions. For example, Human Satellite 3 (HSat3), which makes up roughly 2% of the human genome, forms enormous arrays up to tens of megabases, but these arrays play no known roles in centromere function and were almost fully excluded from genome assemblies until recently. As a result, these massive genomic regions have remained relatively understudied, and the potential functional roles of HSat3 have remained largely unknown. To address this, we performed a systematic screen for novel HSat3 binding factors. Our work revealed HSat3 arrays contain high densities of transcription factor (TF) motifs that are bound by factors related to multiple, highly conserved signaling pathways. Unexpectedly, the most enriched TFs in HSat3 belong to the Hippo pathway transcription effector family TEAD. We found that TEAD recruits the co-activator YAP to HSat3 regions in a cell-state specific manner. Using RNA polymerase-I reporter assays, targeted repression of HSat3, inducible degradation of YAP, and super-resolution microscopy, we show that HSat3 arrays can localize YAP/TEAD inside the nucleolus, where YAP regulates RNA Polymerase-I activity. Beyond revealing a direct relationship between the Hippo pathway and ribosomal DNA regulation, this work demonstrates that satellite DNA can encode multiple transcription factor binding motifs, defining a new role for these enormous genomic elements.