Noncoding RNAs (ncRNA) participate in epigenetic regulation but are poorly understood. Here we characterize the transcriptional landscape of the four human HOX loci at five base pair resolution in 11 anatomic sites and identify 231 HOX ncRNAs that extend known transcribed regions by more than 30 kilobases. HOX ncRNAs are spatially expressed along developmental axes and possess unique sequence motifs, and their expression demarcates broad chromosomal domains of differential histone methylation and RNA polymerase accessibility. We identified a 2.2 kilobase ncRNA residing in the HOXC locus, termed HOTAIR, which represses transcription in trans across 40 kilobases of the HOXD locus. HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of ncRNA may demarcate chromosomal domains of gene silencing at a distance; these results have broad implications for gene regulation in development and disease states.
Cell-fate transitions involve the integration of genomic information encoded by regulatory elements, such as enhancers, with the cellular environment1,2. However, identification of genomic sequences that control human embryonic development represents a formidable challenge3. Here we show that in human embryonic stem cells (hESCs), unique chromatin signatures identify two distinct classes of genomic elements, both of which are marked by the presence of chromatin regulators p300 and BRG1, monomethylation of histone H3 at lysine 4 (H3K4me1), and low nucleosomal density. In addition, elements of the first class are distinguished by the acetylation of histone H3 at lysine 27 (H3K27ac), overlap with previously characterized hESC enhancers, and are located proximally to genes expressed in hESCs and the epiblast. In contrast, elements of the second class, which we term ‘poised enhancers’, are distinguished by the absence of H3K27ac, enrichment of histone H3 lysine 27 trimethylation (H3K27me3), and are linked to genes inactive in hESCs and instead are involved in orchestrating early steps in embryogenesis, such as gastrulation, mesoderm formation and neurulation. Consistent with the poised identity, during differentiation of hESCs to neuroepithelium, a neuroectoderm-specific subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos, poised enhancers are able to direct cell-type and stage-specific expression characteristic of their proximal developmental gene, even in the absence of sequence conservation in the fish genome. Our data demonstrate that early developmental enhancers are epigenetically pre-marked in hESCs and indicate an unappreciated role of H3K27me3 at distal regulatory elements. Moreover, the wealth of new regulatory sequences identified here provides an invaluable resource for studies and isolation of transient, rare cell populations representing early stages of human embryogenesis.
The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.
type six1 with activating or repressing co-factors (eya1 and groucho, respectively), we demonstrate that Six1 inhibits neural crest and epidermal genes via transcriptional repression and enhances PPE genes via transcriptional activation. Ectopic expression of neural plate, neural crest and epidermal genes in the PPE demonstrates that these factors mutually influence each other to establish the appropriate boundaries between these ectodermal domains.Key words: Pre-placodal ectoderm, Neural crest, foxD3, zic2, sox2, sox3, keratin, dlx5, dlx6 Research article 5872 Woda et al., 2003). Zic genes are initially expressed throughout the neural plate in response to anti-BMP factors, and as they become restricted to its lateral border they initiate neural crest fates (Nakata et al., 1997;Nakata et al., 1998; Brewster et al., 1998;Kuo et al., 1998;Mizuseki et al., 1998). The roles that border genes play to specify the fates of the different ectodermal subdomains remain to be elucidated. Although placodes have long been recognized as important embryonic structures, their transient nature and the lack of specific molecular markers have made it difficult to study the mechanisms by which they form. Recently, however, markers of the PPE during the initial induction of the placodes have been identified in Xenopus. six1 is homologous to Drosophila sine oculis; it is characterized by a homeobox DNA-binding domain and a protein-protein interaction domain called the Six domain. It is initially expressed in a band surrounding the anterior neural plate and later in all neurogenic placodes (Pandur and Moody, 2000). eya1 is homologous to Drosophila eyes absent (eya); it functions as a co-factor for Six genes of the Six1/2 and Six4/5 subfamilies (Pignoni et al., 1997;Ohto et al., 1999;Ikeda et al., 2002) and is expressed in a pattern very similar to that of six1 (David et al., 2001). We have used these markers to demonstrate that gradients of both neural inducer and anteroposterior signals are required for proper PPE formation. Moreover, we show that six1 expression is required for the establishment of the PPE, and it promotes the PPE at the expense of the neural crest and epidermis by both activating and repressing target gene expression. Finally, we demonstrate that several genes expressed in the embryonic ectoderm mutually influence each other to define its distinct subdomains. Materials and methods Expression constructsThe full open-reading frames of Xenopus six1 and Drosophila groucho (Dgroucho; LD33829, Berkeley Drosophila Genome Project) were cloned into expression vectors (pDH105, pCS2+). To generate a chimeric transactivating six1 construct, the Six domain plus the homeodomain (SDHD; amino acids 9-183) was amplified by PCR and ligated upstream of the VP16 activation domain in pCS2VP16 (from M. Whitman). To generate a chimeric repressive six1 construct, the SDHD region was ligated downstream of the Engrailed repressor (EnR) domain in pCS2EnR (from D. Kessler). RNA microinjectionTranscripts of six1 (400-600 pg), six1VP16 (100 ...
Summary Neural Crest Cells (NCC) are a transient, embryonic cell population characterized by unusual migratory ability and developmental plasticity. To annotate and characterize cis-regulatory elements utilized by the human NCC we coupled a hESC differentiation model with genome-wide profiling of histone modifications, coactivator and transcription factor (TF) occupancy. Sequence analysis predicted major TFs binding at epigenomically annotated hNCC enhancers, including master NC regulator, TFAP2A, and nuclear receptors NR2F1 and NR2F2. Although many TF binding events occur outside enhancers, sites coinciding with enhancer chromatin signatures show significantly higher sequence constraint, nucleosomal depletion, correlation with gene expression and functional conservation in NCC isolated from chicken embryos. Simultaneous co-occupancy of TFAP2A and NR2F1/F2 is associated with permissive enhancer chromatin states, characterized by high levels of p300 and H3K27ac. Our results provide first global insights into human NC chromatin landscapes and a rich resource for studies of craniofacial development and disease.
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