Environmental Genomics and Systems Biology Division, Lawrence Berkeley National, Berkeley, United States; Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland
Svetalana Markman
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
Dena Leshkowitz
Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot, Israel
Sharon Krief
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
Ronnie Blecher-Gonen
Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
Shani Ben-Moshe
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel
Department of Human Evolutionary Biology, Harvard University, Department of Human Evolutionary Biology, United States; Broad Institute of Harvard and MIT, Cambridge, United States
Environmental Genomics and Systems Biology Division, Lawrence Berkeley National, Berkeley, United States; U.S. Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, United States; School of Natural Sciences, University of California, Merced, Merced, United States
The mechanical challenge of attaching elastic tendons to stiff bones is solved by the formation of a unique transitional tissue. Here, we show that murine tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, under regulation of shared regulatory elements and Krüppel-like factors (KLFs) transcription factors. High-throughput bulk and single-cell RNA sequencing of humeral attachment cells revealed expression of hundreds of chondrogenic and tenogenic genes, which was validated by in situ hybridization and single-molecule ISH. ATAC sequencing showed that attachment cells share accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis revealed enhancer signatures for most of these regions. Transgenic mouse enhancer reporter assays verified the shared activity of some of these enhancers. Finally, integrative chromatin and motif analyses and transcriptomic data implicated KLFs as regulators of attachment cells. Indeed, blocking expression of both Klf2 and Klf4 in developing limb mesenchyme impaired their differentiation.