Project description:Histone H3 lysine 9 (H3K9) methylation is a central epigenetic modification that defines heterochromatin from unicellular to multicellular organisms. In mammalian cells, H3K9 methylation can be catalyzed by at least six distinct SET domain enzymes: Suv39h1/Suv39h2, Eset1/Eset2 and G9a/Glp. We used mouse embryonic fibroblasts (MEFs) with a conditional mutation for Eset1 and introduced progressive deletions for the other SET domain genes by CRISPR/Cas9 technology. Compound mutant MEFs for all 6 SET domain methyltransferase (KMT) genes lack all H3K9 methylation states, derepress nearly all families of repeat elements and display genomic instabilities. Strikingly, the 6KO H3K9 KMT MEFs no longer maintain heterochromatin organization and have lost electron-dense heterochromatin. This is the first analysis of H3K9 methylation deficient mammalian chromatin and reveals a crucial function for H3K9 methylation in protecting heterochromatin organization and genome integrity.
Project description:The ciliary body is required for the maintenance of intraocular pressure and immunity as well as vision accommodation. We report a comprehensive cell atlas of human ciliary body from single-cell RNA sequencing (scRNAseq)
Project description:Chromosomes of metazoan organisms are partitioned in the interphase nucleus into discrete topologically associating domains (TADs). Borders between TADs are preferentially formed in regions containing high density of active genes and clusters of architectural protein binding sites. Transcription of most genes is turned off during the heat shock response in Drosophila. Here we show that temperature stress induces relocalization of architectural proteins from TAD borders to inside TADs, and this is accompanied by a dramatic rearrangement in the 3D organization of the nucleus. TAD border strength declines, allowing for an increase in long-distance inter-TAD interactions. Similar but quantitatively weaker effects are observed upon inhibition of transcription or depletion of individual architectural proteins. New heat shock-induced inter-TAD interactions result in increased contacts among enhancers and promoters of silenced genes, which recruit Pc and form Pc bodies at the nucleolus. These results suggest that the TAD organization of metazoan genomes is plastic and can be quickly reconfigured to allow new interactions between distant sequences. Analysis of 3D chromatin organization using Hi-C in Drosophila Kc167 cells. Cells were grown at 25 C and heat shocked for 20 min at 36.5 C. Cells were also treated with flavopiridol or triptolide to inhibit transcription elongation or initiation, respectively. Cells were depeleted of Cap-H2 or Rad21 using RNAi. Finally, cells depleted of RAd21 were subjected to heat shock at 36.5 C for 20 min.
Project description:Hox genes are essential regulators of embryonic development. They are activated in a temporal sequence following their topological order within their genomic clusters. Subsequently, states of activity are fine-tuned and maintained to translate into domains of progressively overlapping gene products. While the mechanisms underlying such temporal and spatial progressions begin to be understood, many of their aspects remain unclear. We have systematically analyzed the 3D chromatin organization of Hox clusters in vivo, during their activation using high-resolution circular chromosome conformation capture (4C-seq). Initially, Hox clusters are organized as single 3D chromatin compartments decorated with bivalent chromatin marks. Their progressive transcriptional activation is associated with a dynamic bi-modal 3D organization, whereby the genes switch one after the other, from an inactive to an active 3D compartment. These local 3D dynamics occur within a larger constitutive framework of interactions within the surrounding Topological Associated Domains, which confirms previous results that regulation of this process in primarily cluster intrinsic. The local step-wise progression in time can be stopped and memorized at various body levels and hence it may accounts for the various chromatin architectures previously described at different anterior to posterior body levels for the same embryo at a later stage. Circular Chromosome Conformation Capture (4C-seq) samples from mouse ES cells and mouse embryonic samples at different stages of development. Data based on 41 biological samples.