Defining the Role of Nuclear Factor (NF)-κB p105 Subunit in Human Macrophage by Transcriptomic Analysis of NFKB1 Knockout THP1 Cells.

Since its discovery over 30 years ago the NF-ĸB family of transcription factors has gained the status of master regulator of the immune response. Much of what we understand of the role of NF-ĸB in immune development, homeostasis and inflammation comes from studies of mice null for specific NF-ĸB subunit encoding genes. The role of inflammation in diseases that affect a majority of individuals with health problems globally further establishes NF-ĸB as an important pathogenic factor. More recently, genomic sequencing has revealed loss of function mutations in the NFKB1 gene as the most common monogenic cause of common variable immunodeficiencies in Europeans. NFKB1 encodes the p105 subunit of NF-ĸB which is processed to generate the NF-ĸB p50 subunit. NFKB1 is the most highly expressed transcription factor in macrophages, key cellular drivers of inflammation and immunity. Although a key role for NFKB1 in the control of the immune system is apparent from Nfkb1-/- mouse studies, we know relatively little of the role of NFKB1 in regulating human macrophage responses. In this study we use the THP1 monocyte cell line and CRISPR/Cas9 gene editing to generate a model of NFKB1-/- human macrophages. Transcriptomic analysis reveals that activated NFKB1-/- macrophages are more pro-inflammatory than wild type controls and express elevated levels of TNF, IL6, and IL1B, but also have reduced expression of co-stimulatory factors important for the activation of T cells and adaptive immune responses such as CD70, CD83 and CD209. NFKB1-/- THP1 macrophages recapitulate key observations in individuals with NFKB1 haploinsufficiency including decreased IL10 expression. These data supporting their utility as an in vitro model for understanding the role of NFKB1 in human monocytes and macrophages and indicate that of loss of function NFKB1 mutations in these cells is an important component in the associated pathology.

Defining the structure of the NF-ĸB pathway in human immune cells using quantitative proteomic data.

The NF-ĸB transcription factor is a critical regulator of immune homeostasis and inflammatory responses and is a critical factor in the pathogenesis of inflammatory disease. The pathways to NF-ĸB activation are paradigms for signal-induced ubiquitination and proteasomal degradation, control of transcription factor function by subcellular localisation, and the control of gene transcription and physiological processes by signal transduction mechanisms. Despite the importance of NF-ĸB in disease, the NF-ĸB pathway remains unexploited for the treatment of inflammatory disease. Our understanding of NF-ĸB comes mostly from studies of transgenic mice and cell lines where components of the pathway have been deleted or over expressed. Recent advances in quantitative proteomics offer new opportunities to understand the NF-ĸB pathway using the absolute abundance of individual pathway components. We have analysed available quantitative proteomic datasets to establish the structure of the NF-ĸB pathway in human immune cells under both steady state and activated conditions. This reveals a conserved NF-κB pathway structure across different immune cell lineages and identifies important differences to the current model of the NF-ĸB pathway. These include the findings that the IKK complex in most cells is likely to consist predominantly of IKKß homodimers, that the relative abundancies of IκB proteins show strong cell type variation, and that the components of the non-canonical NF-ĸB pathway are significantly increased in activated immune cells. These findings challenge aspects of our current view of the NF-κB pathway and identify outstanding questions important for defining the role of key components in regulating inflammation and immunity.

Transcriptional dynamics of pluripotent stem cell-derived endothelial cell differentiation revealed by single-cell RNA sequencing.

AIMS: Pluripotent stem cell-derived endothelial cell products possess therapeutic potential in ischaemic vascular disease. However, the factors that drive endothelial differentiation from pluripotency and cellular specification are largely unknown. The aims of this study were to use single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape and cellular dynamics of directed differentiation of human embryonic stem cell-derived endothelial cells (hESC-EC) and to compare these cells to mature endothelial cells from diverse vascular beds. METHODS AND RESULTS: A highly efficient directed 8-day differentiation protocol was used to generate a hESC-derived endothelial cell product (hESC-ECP), in which 66% of cells co-expressed CD31 and CD144. We observed largely homogeneous hESC and mesodermal populations at Days 0 and 4, respectively, followed by a rapid emergence of distinct endothelial and mesenchymal populations. Pseudotime trajectory identified transcriptional signatures of endothelial commitment and maturation during the differentiation process. Concordance in transcriptional signatures was verified by scRNA-seq analysis using both a second hESC line RC11, and an alternative hESC-EC differentiation protocol. In total, 105 727 cells were subjected to scRNA-seq analysis. Global transcriptional comparison revealed a transcriptional architecture of hESC-EC that differs from freshly isolated and cultured human endothelial cells and from organ-specific endothelial cells. CONCLUSION: A transcriptional bifurcation into endothelial and mesenchymal lineages was identified, as well as novel transcriptional signatures underpinning commitment and maturation. The transcriptional architecture of hESC-ECP was distinct from mature and foetal human EC.

CRISPR/Cas9 editing reveals novel mechanisms of clustered microRNA regulation and function.

MicroRNAs (miRNAs) are important regulators of diverse physiological and pathophysiological processes. MiRNA families and clusters are two key features in miRNA biology. Here we explore the use of CRISPR/Cas9 as a powerful tool to delineate the function and regulation of miRNA families and clusters. We focused on four miRNA clusters composed of miRNA members of the same family, homo-clusters or different families, hetero-clusters. Our results highlight different regulatory mechanisms in miRNA cluster expression. In the case of the miR-497~195 cluster, editing of miR-195 led to a significant decrease in the expression of the other miRNA in the cluster, miR-497a. Although no gene editing was detected in the miR-497a genomic locus, computational simulation revealed alteration in the three dimensional structure of the pri-miR-497~195 that may affect its processing. In cluster miR-143~145 our results imply a feed-forward regulation, although structural changes cannot be ruled out. Furthermore, in the miR-17~92 and miR-106~25 clusters no interdependency in miRNA expression was observed. Our findings suggest that CRISPR/Cas9 is a powerful gene editing tool that can uncover novel mechanisms of clustered miRNA regulation and function.

Vegfc acts through ERK to induce sprouting and differentiation of trunk lymphatic progenitors.

Vascular endothelial growth factor C (Vegfc) activates its receptor, Flt4, to induce lymphatic development. However, the signals that act downstream of Flt4 in this context in vivo remain unclear. To understand Flt4 signaling better, we generated zebrafish bearing a deletion in the Flt4 cytoplasmic domain that eliminates tyrosines Y1226 and 1227. Embryos bearing this deletion failed to initiate sprouting or differentiation of trunk lymphatic vessels and did not form a thoracic duct. Deletion of Y1226/7 prevented ERK phosphorylation in lymphatic progenitors, and ERK inhibition blocked trunk lymphatic sprouting and differentiation. Conversely, endothelial autonomous ERK activation rescued lymphatic sprouting and differentiation in flt4 mutants. Interestingly, embryos bearing the Y1226/7 deletion formed a functional facial lymphatic network enabling them to develop normally to adulthood. By contrast, flt4 null larvae displayed hypoplastic facial lymphatics and severe lymphedema. Thus, facial lymphatic vessels appear to be the first functional lymphatic network in the zebrafish, whereas the thoracic duct is initially dispensable for lymphatic function. Moreover, distinct signaling pathways downstream of Flt4 govern lymphatic morphogenesis and differentiation in different anatomical locations.

Construction and application of site-specific artificial nucleases for targeted gene editing.

Artificial nucleases have developed into powerful tools for introducing precise genome modifications in a wide variety of species. In this chapter the authors provide detailed protocols for rapidly constructing zinc finger nucleases (ZFNs) and TALE nucleases (TALENs) and evaluating their activity for the targeted generation of InDels within the zebrafish genome.

Targeted chromosomal deletions and inversions in zebrafish.

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%-15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.

Zebrafish rest regulates developmental gene expression but not neurogenesis.

The transcriptional repressor Rest (Nrsf) recruits chromatin-modifying complexes to RE1 'silencer elements', which are associated with hundreds of neural genes. However, the requirement for Rest-mediated transcriptional regulation of embryonic development and cell fate is poorly understood. Conflicting views of the role of Rest in controlling cell fate have emerged from recent studies. To address these controversies, we examined the developmental requirement for Rest in zebrafish using zinc-finger nuclease-mediated gene targeting. We discovered that germ layer specification progresses normally in rest mutants despite derepression of target genes during embryogenesis. This analysis provides the first evidence that maternal rest is essential for repression of target genes during blastula stages. Surprisingly, neurogenesis proceeds largely normally in rest mutants, although abnormalities are observed within the nervous system, including defects in oligodendrocyte precursor cell development and a partial loss of facial branchiomotor neuron migration. Mutants progress normally through embryogenesis but many die as larvae (after 12 days). However, some homozygotes reach adulthood and are viable. We utilized an RE1/NRSE transgenic reporter system to dynamically monitor Rest activity. This analysis revealed that Rest is required to repress gene expression in mesodermal derivatives including muscle and notochord, as well as within the nervous system. Finally, we demonstrated that Rest is required for long-term repression of target genes in non-neural tissues in adult zebrafish. Our results point to a broad role for Rest in fine-tuning neural gene expression, rather than as a widespread regulator of neurogenesis or cell fate.

Churchill and Sip1a repress fibroblast growth factor signaling during zebrafish somitogenesis.

Cell-type specific regulation of a small number of growth factor signal transduction pathways generates diverse developmental outcomes. The zinc finger protein Churchill (ChCh) is a key effector of fibroblast growth factor (FGF) signaling during gastrulation. ChCh is largely thought to act by inducing expression of the multifunctional Sip1 (Smad Interacting Protein 1). We investigated the function of ChCh and Sip1a during zebrafish somitogenesis. Knockdown of ChCh or Sip1a results in misshapen somites that are short and narrow. As in wild-type embryos, cycling gene expression occurs in the developing somites in ChCh and Sip1a compromised embryos, but expression of her1 and her7 is maintained in formed somites. In addition, tail bud fgf8 expression is expanded anteriorly in these embryos. Finally, we found that blocking FGF8 restores somite morphology in ChCh and Sip1a compromised embryos. These results demonstrate a novel role for ChCh and Sip1a in repression of FGF activity.

The role of the SPT6 chromatin remodeling factor in zebrafish embryogenesis.

Somitogenesis is a highly controlled process that results in segmentation of the paraxial mesoderm. Notch pathway activity in the presomitic mesoderm is fundamental for management of synchronized gene expression which is necessary for regulation of somitogenesis. We have isolated an embryonic lethal mutation, SBU2, that causes somite formation defects very similar to Notch pathway mutants. SBU2 mutants generate only 6-7 asymmetrically arranged somites. However, in contrast to Notch pathway mutants, these mutants do not maintain previously formed somite boundaries and by 24 hpf, almost no somite boundaries remain. Other developmental processes disrupted in SBU2 mutants include tail morphogenesis, muscle fiber elongation, pigmentation, circulatory system development and neural differentiation. We demonstrated that these defects are the result of a nonsense mutation within the spt6 gene. spt6 encodes a transcription elongation factor that genetically interacts with the Paf-1 chromatin remodeling complex. SBU2 mutant phenotypes could be rescued by microinjection of spt6 mRNA and microinjection of spt6 morpholinos phenocopied the mutation. Our real-time PCR analysis revealed that Spt6 is essential for the transcriptional response to activation of the Notch pathway. Analysis of sbu2;mib double mutants indicates that Spt6 deficiency suppresses the neurogenic effects of the mib. Altogether, these results demonstrate that Spt6 is critical for somite formation in zebrafish and suggest that some defects observed in spt6 mutants result from alterations in Notch signaling. However, additional Spt6 mutant phenotypes are likely caused by vital functions of Spt6 in other pathways.