Gene bivalency at Polycomb domains regulates cranial neural crest positional identity

INTRODUCTION Craniofacialmorphogenesis involves the cranial neural crest( NC) cells, a vertebrate-specific multipotent cell population that provides most of the head skeletogenic mesenchyme. Cranial NCcells delaminate from different points along the developing neural tube and migrate into distinct facial and pharyngeal archprocesses, where they give rise to distinctly shaped cartilage and bone elements, which in turn assemble into a harmonious face. How do distinct cranial NCcell subpopulations acquire their regional identity, allowing them to generate the specific subsets of craniofacialelements appropriate to their position? Premigratory NCcells that contribute to frontonasal, maxillary, or mandibular processesshare similar patterning potential, because they can replace each other in building a whole craniofacialskeleton. Such plasticity is maintained during and after migration until subpopulation-specific transcriptional identity and positional patterning programs are established as a result of interactions with their local surrounding environment. We asked how chromatinregulation may allow cranial NCcells to maintain broad patterning competence through migration while being poisedto respond to local cues and induce position-specific transcriptional subprograms. RATIONALE We used genome-wide RNA sequencing (RNA-seq), chromatinimmunoprecipitation followed by sequencing (ChIP-seq), and assay for transposase-accessible chromatinwith high-throughput sequencing ( ATAC-seq) and integrated the information to propose a model to explain how cranial NCsubpopulations maintain broad patterning competence through chromatinepigenetic regulation and how transcription factor–dependent responses to local cues can modify the chromatinpattern to establish unique subpopulation-specific transcriptional subprograms. To this aim, we microdissected the Hox-free frontonasal, maxillary, mandibular, and the Hox -expressing second pharyngeal archprocesses of E10.5 mouse embryos. We isolated the NCcell subpopulations from each of these prominences by cell sorting and analyzed their transcriptional state, as well as the H3K27me3, H3K4me2, and H3K27Ac histone modification and chromatinaccessibility profiles at promoters and enhancers. We then compared these data sets with the transcriptional, histone mark, and chromatinaccessibility profiles of the Hox-free NCpremigratory progenitors and of E10.5 frontonasal, maxillary, mandibular, and second pharyngeal arch NCcell subpopulations in which we conditionally inactivated the Polycomb H3K27 methyltransferase gene enhancer of zestehomolog 2 ( Ezh2cKO mutants). RESULTS Early postmigratory NCsubpopulations contributing to distinct mouse craniofacialstructures displayed similar chromatinaccessibility patterns yet differed transcriptionally. The differentially expressed genes (positional genes) displayed accessible and H3K27me3 + /H3K4me2 + bivalent enhancers and promoters, and were embedded in large Ezh2-dependent Polycomb domains, in the NCcell subpopulations in which they were silenced, indicating transcriptional poising. These postmigratory chromatindomains of poisedgene regulation were inherited from NCpremigratory progenitors. At Polycomb domains, H3K27me3 antagonized H3K4me2 deposition, which was restricted to accessible promoter and enhancer elements, preventing ectopic activation at inappropriate positions. DISCUSSION Our findings explain how cranial NCcell plasticity is maintained through migration until postmigratory stages. We propose that an Ezh2-dependent poised chromatinorganization underlies the positional plasticity of cranial premigratory NCcell progenitors. This chromatinprepattern is maintained through migration. In response to position-specific environmental signals encountered by the NCcells during or after their migration, the regulatory elements and promoters of positional genes switch from a poisedto an active chromatinstate, contributing to establish NCsubpopulation–specific transcriptional identities. This work contributes novel insights into the epigenetic regulation of face morphogenesis.