No abstract

Chromatin looping is key to gene regulation, yet no broadly applicable methods to selectively modify chromatin loops have been described. We have engineered a method for chromatin loop reorganization using CRISPR-dCas9 (CLOuD9) to selectively and reversibly establish chromatin loops. We demonstrate the power of this technology to selectively modulate gene expression at targeted loci.

Hedgehog pathway-dependent cancers can escape smoothened (SMO) inhibition through canonical pathway mutations, however, 50% of resistant BCCs lack additional variants in hedgehog genes. Here we use multi-dimensional genomics in human and mouse resistant BCCs to identify a non-canonical hedgehog activation pathway driven by the transcription factor, serum response factor (SRF). Active SRF along with its co-activator megakaryoblastic leukemia 1 (MKL1) form a novel protein complex and share chromosomal occupancy with the hedgehog transcription factor GLI1, causing amplification of GLI1 transcriptional activity. We show cytoskeletal activation by Rho and the formin family member Diaphanous (mDia) are required for SRF/MKL-driven GLI1 activation and tumor cell viability. Remarkably, we use nuclear MKL1 staining in mouse and human patient tumors to define drug responsiveness to MKL inhibitors highlighting the therapeutic potential of targeting this pathway. Thus, our studies illuminate for the first time cytoskeletal-driven transcription as a personalized therapeutic target to combat drug resistant malignancies.

Graphical Abstract Highlights d Epigenome landscape maps reveal key transitions during epidermal lineage commitment d Network modeling identifies master regulators of lineage initiation and maturation d TFAP2C drives chromatin dynamics during initiation and primes p63-dependent maturation d Crosstalk between TFAP2C and p63 drives epigenetic transitions during differentiation

Tumor heterogeneity and lack of knowledge about resistant cell states remain a barrier to targeted cancer therapies. Basal cell carcinomas (BCCs) depend on Hedgehog (Hh)/Gli signaling, but can develop mechanisms of Smoothened (SMO) inhibitor resistance. We previously identified a nuclear myocardin-related transcription factor (nMRTF) resistance pathway that amplifies noncanonical Gli1 activity, but characteristics and drivers of the nMRTF cell state remain unknown. Here, we use single cell RNA-sequencing of patient tumors to identify three prognostic surface markers (LYPD3, TACSTD2, and LY6D) which correlate with nMRTF and resistance to SMO inhibitors. The nMRTF cell state resembles transit-amplifying cells of the hair follicle matrix, with AP-1 and TGFß cooperativity driving nMRTF activation. JNK/AP-1 signaling commissions chromatin accessibility and Smad3 DNA binding leading to a transcriptional program of RhoGEFs that facilitate nMRTF activity. Importantly, small molecule AP-1 inhibitors selectively target LYPD3+/TACSTD2+/LY6D+ nMRTF human BCCs ex vivo, opening an avenue for improving combinatorial therapies.