RESUMEN
Mesendodermal specification is one of the earliest events in embryogenesis, where cells first acquire distinct identities. Cell differentiation is a highly regulated process that involves the function of numerous transcription factors (TFs) and signaling molecules, which can be described with gene regulatory networks (GRNs). Cell differentiation GRNs are difficult to build because existing mechanistic methods are low throughput, and high-throughput methods tend to be non-mechanistic. Additionally, integrating highly dimensional data composed of more than two data types is challenging. Here, we use linked self-organizing maps to combine chromatin immunoprecipitation sequencing (ChIP-seq)/ATAC-seq with temporal, spatial, and perturbation RNA sequencing (RNA-seq) data from Xenopus tropicalis mesendoderm development to build a high-resolution genome scale mechanistic GRN. We recover both known and previously unsuspected TF-DNA/TF-TF interactions validated through reporter assays. Our analysis provides insights into transcriptional regulation of early cell fate decisions and provides a general approach to building GRNs using highly dimensional multi-omic datasets.
Asunto(s)
Endodermo/embriología , Redes Reguladoras de Genes , Genómica , Mesodermo/embriología , Xenopus/embriología , Xenopus/genética , Animales , Cromatina/metabolismo , Secuencia de Consenso/genética , ADN/metabolismo , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Unión Proteica , ARN/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
During vertebrate gastrulation, mesoderm is induced in pluripotent cells, concomitant with dorsal-ventral patterning and establishing of the dorsal axis. We applied single-cell chromatin accessibility and transcriptome analyses to explore the emergence of cellular heterogeneity during gastrulation in Xenopus tropicalis. Transcriptionally inactive lineage-restricted genes exhibit relatively open chromatin in animal caps, whereas chromatin accessibility in dorsal marginal zone cells more closely reflects transcriptional activity. We characterized single-cell trajectories and identified head and trunk organizer cell clusters in early gastrulae. By integrating chromatin accessibility and transcriptome data, we inferred the activity of transcription factors in single-cell clusters and tested the activity of organizer-expressed transcription factors in animal caps, alone or in combination. The expression profile induced by a combination of Foxb1 and Eomes most closely resembles that observed in the head organizer. Genes induced by Eomes, Otx2, or the Irx3-Otx2 combination are enriched for maternally regulated H3K4me3 modifications, whereas Lhx8-induced genes are marked more frequently by zygotically controlled H3K4me3. Taken together, our results show that transcription factors cooperate in a combinatorial fashion in generally open chromatin to orchestrate zygotic gene expression.
Asunto(s)
Cromatina/genética , Análisis de la Célula Individual/métodos , Factores de Transcripción/metabolismo , Proteínas de Xenopus/genética , Xenopus/embriología , Animales , Tipificación del Cuerpo , Cromatina/metabolismo , Gastrulación , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Análisis de Secuencia de ARN , Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
Reprogramming to induced pluripotency through expression of OCT4, SOX2, KLF4, MYC (OSKM) factors is often considered the dedifferentiation of somatic cells. This would suggest that reprogramming represents the reversal of embryonic differentiation. Indeed, molecular events involving the activity of the pluripotency network occur in opposite directions. However, reprogramming and development substantially differ as OSKM bind to accessible regulatory elements in the genome of somatic cells due to their overexpression, including regulatory elements never bound by these factors during normal differentiation. In addition, rewiring the transcriptional network back to pluripotency involves overcoming molecular barriers that protect or stabilize the somatic identity, whereas extrinsic and intrinsic cues will drive differentiation in an energetically favorable landscape in the embryo. This review focuses on how cell fate transitions in reprogramming and development are differentially governed by interactions between transcription factors and chromatin. We also discuss how these interactions shape chromatin architecture and the transcriptional output. Major technological advances have resulted in a better understanding of both differentiation and reprogramming, which is essential to exploit reprogramming regimes for regenerative medicine.
Asunto(s)
Linaje de la Célula/genética , Reprogramación Celular/genética , Cromatina/genética , Factores de Transcripción/genética , Animales , Regulación del Desarrollo de la Expresión Génica/genética , Genoma/genética , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción SOXB1/genéticaRESUMEN
The DNA of eukaryotic genomes is packaged into chromatin by nucleosomes. This not only compacts the DNA but also plays a central role in gene regulation and establishment of cellular identity during development. Because of this packaging, the DNA is relatively inaccessible to nucleoplasmic factors; however, regulatory elements such as promoters, enhancers, and insulators are largely kept nucleosome-free. The assay for transposase-accessible chromatin (ATAC-seq) can be used to identify genomic locations of "open" chromatin, footprints of DNA-binding proteins, and positioned nucleosomes. It therefore is a powerful tool for unraveling the dynamic regulatory landscape of chromatin. The method exploits the action of hyperactive prokaryotic Tn5-transposase, which preferentially cuts DNA in accessible chromatin and tags the sites with sequencing adaptors. Here we describe an ATAC-seq protocol for use with Xenopus tropicalis embryos.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina/métodos , ADN/metabolismo , Transposasas/metabolismo , Animales , Unión Proteica , Xenopus/embriologíaRESUMEN
Crustins are cationic antimicrobial peptides of ca. 7-14kDa with a characteristic four-disulphide core containing WAP domain, present in the hemocytes of crustaceans. The present study reports the first crustin sequences from two portunid crabs, viz. the mud crab Scylla tranquebarica (St-Crustin, JQ965930) and the blue swimmer crab Portunus pelagicus (Pp-Crustin, JQ753312). St-Crustin and Pp-Crustin represented the complete cDNA sequence of Type I crustin, with an ORF of 336bp encoding 111aa with a predicted molecular weight of 10kDa and a pI of 8. The signal sequence contained 21aa residues, which was followed by a mature peptide with a WAP domain at the C-terminus. Peptide model of St-Crustin and Pp-Crustin indicated a randomly coiled structure enclosing two ß-sheets but no helices. St-Crustin and Pp-Crustin shared significant similarities with crustins of portunid crabs (68-95%) and other crabs (60-73%). Phylogenetic analysis showed that St-Crustin and Pp-Crustin possess the same ancestral origin and have a similar evolutionary status like other crustins, which has subsequently diverged at different phases of evolution. St-Crustin and Pp-Crustin were closely related to crab crustins rather than to the crustins of other crustacean groups. The wide distribution of crustins in crustaceans indicates the importance of these AMPs in the innate immunity. Discovery of novel crustins might pave way to the discovery of promising therapeutic/prophylactic agents in health management and disease control in crustacean aquaculture.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Braquiuros/metabolismo , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/genética , Secuencia de Bases , Braquiuros/genética , Clonación Molecular , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Isoformas de Proteínas , ARN/química , ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
A marine yeast Candida aquaetextoris S527 as a source of immunostimulant in Penaeus monodon was studied. Yeast diet was prepared by incorporating 10% C. aquaetextoris S527 biomass into a standard shrimp diet and administered in P. monodon at different frequencies (daily, once in three days, once in seven days and once in ten days) followed by challenge with white spot syndrome virus (WSSV). Immune parameters such as total protein, total hemocyte count, pro-phenoloxidase, nitroblue tetrazolium reduction, alkaline phosphatase activity and acid phosphatase activity were tested. Expression profile of antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5; immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, peroxinectin, prophenol oxidase (proPO) and transglutaminase, and WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, ribonucleotide reductase, thymidine kinase and VP28 were analyzed. The study demonstrated that marine yeast diet administered once every seven days conferred better protection to P. monodon against WSSV infection, supported by the hematological and immune gene expression profiles analyzed.