Enhancer-Promoter Looping Deciphers Dosage of the Haploinsufficient Transcription Factor, CUX1

One third of tumor suppressor genes encode haploinsufficient transcriptional regulators, including transcription factors and chromatin remodelers. This presents a major barrier in oncology, as tumor suppressor genes and transcription factors are inherently difficult to target therapeutically. It remains unknown how a 50% reduction of a transcriptional regulator translates at the cis-regulatory level into a malignant transcriptional program. It is imperative to address this question, in order to predict and target aberrant downstream pathways. CUX1 encodes a quintessential haploinsufficient transcription factor that is recurrently mutated or deleted, monoallelically, in high-risk myeloid neoplasms and across solid tumor types. We hypothesized that the transcriptional response to changes in CUX1 level is encoded within the cis-regulatory architecture of dosage-sensitive CUX1 target genes. In this study, we determined that CUX1 primarily binds distal enhancers, in a tissue-specific manner. CUX1 binding sites are significantly enriched for co-binding with transcriptional activators and cohesin components. Haploinsufficiency of CUX1 in K562 myeloid leukemia cells led to altered expression of mitotic and apoptotic genes with concomitant increased cellular proliferation. Surprisingly, ChIP-seq of CUX1 in the haploinsufficient state revealed a unimodal decrease in CUX1 occupancy genome-wide, with no relevance to differential gene expression. Thus we used a machine-learning algorithm to identify characteristics of CUX1 binding sites at dosage-sensitive target genes and revealed a relationship with distance to the transcription start site, chromatin accessibility, and enhancer activity. Finally, we demonstrate that CUX1 binding sites at dosage-sensitive genes loop to the promoter, and those genes with an intermediate number of Hi-C loops are most responsive to changes in CUX1 abundance. Overall, these data point to a novel mechanism of transcription factor dose-responsiveness mediated by enhancer-promoter looping.