Transcriptional program that drives human preimplantation development is largely unknown. Here, by using single-cell RNA sequencing of 348 oocytes, zygotes and single blastomeres from 2- to 3-day-old embryos, we provide a detailed analysis of the human preimplantation transcriptome. By quantifying transcript far 5′-ends (TFEs), we include in our analysis transcripts that derive from alternative promoters. We show that 32 and 129 genes are transcribed during the transition from oocyte to four-cell stage and from four- to eight-cell stage, respectively. A number of identified transcripts originates from previously unannotated genes that include the PRD-like homeobox genes ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB and LEUTX. Employing de novo promoter motif extraction on sequences surrounding TFEs, we identify significantly enriched gene regulatory motifs that often overlap with Alu elements. Our high-resolution analysis of the human transcriptome during preimplantation development may have important implications on future studies of human pluripotent stem cells and cell reprograming.
Although a mutation in the NF1 gene is the only factor required to initiate the neurocutaneous-skeletal neurofibromatosis 1 (NF1) syndrome, the pathoetiology of the multiple manifestations of this disease in different organ systems seems increasingly complex. The wide spectrum of different clinical phenotypes and their development, severity, and prognosis seem to result from the cross talk between numerous cell types, cell signaling networks, and cell-extracellular matrix interactions. The bi-allelic inactivation of the NF1 gene through a "second hit" seems to be of crucial importance to the development of certain manifestations, such as neurofibromas, café-au-lait macules, and glomus tumors. In each case, the second hit involves only one cell type, which is subsequently clonally expanded in a discrete lesion. Neurofibromas, which are emphasized in this review, and cutaneous neurofibromas in particular, are known to contain a subpopulation of NF1-diploinsufficient Schwann cells and a variety of NF1-haploinsufficient cell types. A recent study identified a multipotent precursor cell population with an NF1 ؉/؊ genotype that resides in human cutaneous neurofibromas and that has been suggested to play a role in their pathogenesis. café-au-lait macules and multiple benign cutaneous neurofibromas, which, typically, are detectable in adulthood by simple visual inspection.NF1 can affect nearly every organ system, and the complications vary between individuals, even within a single family. The clinical diagnosis is based on the presence of two or more of the following findings: six or more café-au-lait macules with diameters Ͼ5 mm in prepubertal patients and Ͼ15 mm in postpubertal patients; two or more neurofibromas of any type or one plexiform neurofibroma; axillary or inguinal freckling; optic glioma; two or more Lisch nodules of the iris; a distinctive osseous lesion, such as sphenoid wing dysplasia or pseudarthrosis; or a first-degree relative diagnosed as having NF1 according to the preceding criteria. 3 It has been suggested that a pathogenic mutation in the NF1 gene be added to the list of diagnostic criteria. 4 The most common complications of NF1 are included in the previously listed diagnostic criteria. Additional features include short stature, scoliosis, headache, speech disorders, attention deficit disorder and attention-deficit/ hyperactivity disorder, and learning disabilities. Rare complications, affecting Ͻ5% of patients, include epilepsy, hydrocephalus, cardiovascular problems, and dystrophic scoliosis. 5 The lifetime risk of malignant tumors arising from peripheral nerves is estimated to be 10% to 13%. 6 The pathoetiology of the complications in NF1 is, however, largely unknown. Genetic BackgroundNF1 is caused by mutations in the NF1 gene that encodes the tumor suppressor protein neurofibromin. 7,8 The NF1 gene is located on chromosome band 17q11.2, spanning approximately 280 kb of genomic DNA, and is composed of 57 constitutive exons and 4 alternatively spliced exons (9a, 10a2, 23a, and 48a). 9 Howeve...
CRISPR-Cas9-based gene activation (CRISPRa) is an attractive tool for cellular reprogramming applications due to its high multiplexing capacity and direct targeting of endogenous loci. Here we present the reprogramming of primary human skin fibroblasts into induced pluripotent stem cells (iPSCs) using CRISPRa, targeting endogenous OCT4, SOX2, KLF4, MYC, and LIN28A promoters. The low basal reprogramming efficiency can be improved by an order of magnitude by additionally targeting a conserved Alu-motif enriched near genes involved in embryo genome activation (EEA-motif). This effect is mediated in part by more efficient activation of NANOG and REX1. These data demonstrate that human somatic cells can be reprogrammed into iPSCs using only CRISPRa. Furthermore, the results unravel the involvement of EEA-motif-associated mechanisms in cellular reprogramming.
Leucine twenty homeobox (LEUTX) is a paired (PRD)-like homeobox gene that is expressed almost exclusively in human embryos during preimplantation development. We previously identified a novel transcription start site for the predicted human LEUTX gene based on the transcriptional analysis of human preimplantation embryos. The novel variant encodes a protein with a complete homeodomain. Here, we provide a detailed description of the molecular cloning of the complete homeodomain-containing LEUTX. Using a human embryonic stem cell overexpression model we show that the complete homeodomain isoform is functional and sufficient to activate the transcription of a large proportion of the genes that are upregulated in human embryo genome activation (EGA), whereas the previously predicted partial homeodomain isoform is largely inactive. Another PRD-like transcription factor, DPRX, is then upregulated as a powerful repressor of transcription. We propose a two-stage model of human EGA in which LEUTX acts as a transcriptional activator at the 4-cell stage, and DPRX as a balancing repressor at the 8-cell stage. We conclude that LEUTX is a candidate regulator of human EGA.
BackgroundEvolutionarily conserved RFX transcription factors (TFs) regulate their target genes through a DNA sequence motif called the X-box. Thereby they regulate cellular specialization and terminal differentiation. Here, we provide a comprehensive analysis of all the eight human RFX genes (RFX1–8), their spatial and temporal expression profiles, potential upstream regulators and target genes.ResultsWe extracted all known human RFX1–8 gene expression profiles from the FANTOM5 database derived from transcription start site (TSS) activity as captured by Cap Analysis of Gene Expression (CAGE) technology. RFX genes are broadly (RFX1–3, RFX5, RFX7) and specifically (RFX4, RFX6) expressed in different cell types, with high expression in four organ systems: immune system, gastrointestinal tract, reproductive system and nervous system. Tissue type specific expression profiles link defined RFX family members with the target gene batteries they regulate. We experimentally confirmed novel TSS locations and characterized the previously undescribed RFX8 to be lowly expressed. RFX tissue and cell type specificity arises mainly from differences in TSS architecture. RFX transcript isoforms lacking a DNA binding domain (DBD) open up new possibilities for combinatorial target gene regulation. Our results favor a new grouping of the RFX family based on protein domain composition. We uncovered and experimentally confirmed the TFs SP2 and ESR1 as upstream regulators of specific RFX genes. Using TF binding profiles from the JASPAR database, we determined relevant patterns of X-box motif positioning with respect to gene TSS locations of human RFX target genes.ConclusionsThe wealth of data we provide will serve as the basis for precisely determining the roles RFX TFs play in human development and disease.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4564-6) contains supplementary material, which is available to authorized users.
Cutaneous neurofibromas are the hallmarks of neurofibromatosis type 1 (NF1). They are composed of multiple cell types, and traditionally they are believed to arise from small nerve tributaries of the skin. A key finding in the context of this view has been that subpopulations of tumor Schwann cells harbor biallelic inactivation of the NF1 gene (NF1 ؊/؊ ). In the present study, our aim was to clarify further the pathogenesis of cutaneous neurofibromas. First, we detected cells expressing multipotency-associated biomarkers in cutaneous neurofibromas. Second, we developed a method for isolating and expanding multipotent neurofibroma-derived precursor cells ( Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene that encodes the tumor suppressor protein neurofibromin, an inactivator of Ras. Clinical diagnosis of the disease is based on the presence of café-au-lait macules, axillary freckling, hamartomas of the iris (Lisch nodules), optic pathway gliomas, distinctive osseous lesions such as sphenoid wing dysplasia or pseudarthrosis, and neurofibromas. 1 Neurofibromas can be classified according to their anatomical location: cutaneous, subcutaneous, intraneural, and plexiform. 2 Plexiform neurofibromas are congenital tumor masses involving nerve trunks and often extend to the skin, whereas cutaneous neurofibromas are not detectable at birth and usually appear during adolescence.Histologically, neurofibromas are mixed tumors consisting of cells with divergent differentiation characteristics. The use of traditional histological stains, as well as immunohistochemistry with a variety of biomarkers and electron microscopy, has been taken as proof for the involvement of Schwann cells, perineurial cells, and fibroblasts. 3-5 Neurofibromas also contain numerous mast cells and axonal processes, all of which are embedded in an abundant collagenous extracellular matrix. 4,6 Unlike plexiform neurofibromas, which carry a risk for malignant transformation and may form tumor masses of several kilograms, cutaneous neurofibromas invariably retain their benign phenotype. Their neoplastic cells never undergo malignant transformation and the tumor diameter usually varies from millimeters to 2 cm, rarely exceeding 3 cm.In light of previous reports and the current study, a feasible explanation for neurofibroma development involves a biallelic inactivation of the NF1 gene. 7,8 This inactivation has been detected in cultured cells displaying characteristics typical of Schwann cells, including a bipolar morphology and the expression of S100 protein.Previous studies have observed diploinsufficiency in 29 of 29 cutaneous neurofibromas in which two separate mutations were found in 26 of 29 tumors and loss of heterozygosity was found in 3 of 29 7 It should be noted that each of the second mutations was unique.
S U M M A R Y The findings of this study show that Class III b-tubulin is a component of the mitotic spindle in multiple cell types. Class III b-tubulin has been widely used as a neuronspecific marker, but it has been detected also in association with breast and pancreatic cancers. In this study, we describe a novel finding of Class III b-tubulin in a subpopulation of cells in malignant peripheral nerve sheath tumor. The findings of this study also show that Class III b-tubulin is expressed by normal mesenchymal and epithelial cells (fibroblasts and keratinocytes), two transitional cell carcinoma cell lines, and neurofibroma Schwann cells, as shown by immunolabeling and Western transfer analysis using two different Tuj-1 antibodies that are specific for Class III b-tubulin. The corresponding mRNA was detected using RT-PCR and whole human genome microarrays. Both antibodies localized Class III b-tubulin to the mitotic spindle and showed a colocalization with a-tubulin. The immunoreaction became visible in early prophase, and the most intense immunoreaction was detected during metaphase and anaphase when microtubules were connected to the kinetochores on chromosomes. Class III b-tubulin-specific immunoreaction lasted to the point when the midbody of cytokinesis became detectable.
PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes, ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB, NOBOX, TPRX1 and TPRX2, were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1, CPHX2, ARGFX) or repressors (DPRX, DUXA, TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development.
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