PROJECT SUMMARY Growth plates are highly specialized cartilage structures that ensure skeletal growth and endochondral ossification during fetal and postnatal development. They are formed and maintained by chondrocytes, as these cells follow a spatially and temporally tightly controlled multi-step differentiation program. The present project focuses on transcription factors that have pivotal roles in effecting this program, but whose modes of actions remain incompletely deciphered. It will test the paradigm-shifting hypothesis that SOX9, its cofactors SOX5 and SOX6, and RUNX2 and RUNX3 fulfill many of their main functions in a cooperative manner. This hypothesis is based on a solid scientific premise that includes co-expression of the factors in growth plate chondrocytes, the presence of RUNT-domain motifs in many chondrocyte-specific enhancers bound by SOX9, and preliminary evidence that the SOX and RUNX proteins have synergistic activities in enhancer activation. Specific Aim 1 is to determine whether SOX5/6, SOX9 and RUNX2/3 genetically interact during growth plate formation in mouse fetuses and in the maintenance of active growth plates in juvenile mice. Specific Aim 2 is to profile the whole genetic targetomes of SOX5/6, SOX9 and RUNX2/3 in growth plate chondrocytes, and to assess and validate their overlap. Specific Aim 3 is to identify mechanisms underlying SOX5/6, SOX9 and RUNX2/3 cooperativity. The proteins will be tested for roles in inducing chromatin accessibility and three- dimensional connectivity, and for cooperativity in DNA binding and recruitment of functional partners. New findings should have a significant impact on current understanding of fundamental mechanisms governing the formation and maintenance of growth plates and other cartilage types. They should thereby help uncover the molecular basis of many types of pathologies, including chondrodysplasias, tumors and joint degenerative diseases, and also suggest novel, innovative and efficient treatments for these still unsatisfactorily treatable diseases.