Single-Cell RNA Sequencing of Donor-Reactive T Cells Reveals Role of Apoptosis in Donor-Specific Hyporesponsiveness of Kidney Transplant Recipients

After kidney transplantation (KT), donor-specific hyporesponsiveness (DSH) of recipient T cells develops over time. Recently, apoptosis was identified as a possible underlying mechanism. In this study, both transcriptomic profiles and complete V(D)J variable regions of TR transcripts from individual alloreactive T cells of kidney transplant recipients were determined with single-cell RNA sequencing. Alloreactive T cells were identified by CD137 expression after stimulation of peripheral blood mononuclear cells obtained from KT recipients (N = 7) prior to and 3–5 years after transplantation with cells of their donor or a third party control. The alloreactive T cells were sorted, sequenced and the transcriptome and T cell receptor profiles were analyzed using unsupervised clustering. Alloreactive T cells retain a highly polyclonal T Cell Receptor Alpha/Beta repertoire over time. Post transplantation, donor-reactive CD4+ T cells had a specific downregulation of genes involved in T cell cytokine-mediated pathways and apoptosis. The CD8+ donor-reactive T cell profile did not change significantly over time. Single-cell expression profiling shows that activated and pro-apoptotic donor-reactive CD4+ T cell clones are preferentially lost after transplantation in stable kidney transplant recipients.</p

Identification of candidate enhancers controlling the transcriptome during the formation of interphalangeal joints

The formation of the synovial joint begins with the visible emergence of a stripe of densely packed mesenchymal cells located between distal ends of the developing skeletal anlagen called the interzone. Recently the transcriptome of the early synovial joint was reported. Knowledge about enhancers would complement these data and lead to a better understanding of the control of gene transcription at the onset of joint development. Using ChIP-sequencing we have mapped the H3-signatures H3K27ac and H3K4me1 to locate regulatory elements specific for the interzone and adjacent phalange, respectively. This one-stage atlas of candidate enhancers (CEs) was used to map the association between these respective joint tissue specific CEs and biological processes. Subsequently, integrative analysis of transcriptomic data and CEs identified new putative regulatory elements of genes expressed in interzone (e.g., GDF5, BMP2 and DACT2) and phalange (e.g., MATN1, HAPLN1 and SNAI1). We also linked such CEs to genes known as crucial in synovial joint hypermobility and osteoarthritis, as well as phalange malformations. These analyses show that the CE atlas can serve as resource for identifying, and as starting point for experimentally validating, putative disease-causing genomic regulatory regions in patients with synovial joint dysfunctions and/or phalange disorders, and enhancer-controlled synovial joint and phalange formation

Comparison of Single Cell Transcriptome Sequencing Methods:Of Mice and Men

Single cell RNAseq has been a big leap in many areas of biology. Rather than investigating gene expression on a whole organism level, this technology enables scientists to get a detailed look at rare single cells or within their cell population of interest. The field is growing, and many new methods appear each year. We compared methods utilized in our core facility: Smart-seq3, PlexWell, FLASH-seq, VASA-seq, SORT-seq, 10X, Evercode, and HIVE. We characterized the equipment requirements for each method. We evaluated the performances of these methods based on detected features, transcriptome diversity, mitochondrial RNA abundance and multiplets, among others and benchmarked them against bulk RNA sequencing. Here, we show that bulk transcriptome detects more unique transcripts than any single cell method. While most methods are comparable in many regards, FLASH-seq and VASA-seq yielded the best metrics, e.g., in number of features. If no equipment for automation is available or many cells are desired, then HIVE or 10X yield good results. In general, more recently developed methods perform better. This also leads to the conclusion that older methods should be phased out, and that the development of single cell RNAseq methods is still progressing considerably.</p

Zeb2 DNA-Binding Sites in Neuroprogenitor Cells Reveal Autoregulation and Affirm Neurodevelopmental Defects, Including in Mowat-Wilson Syndrome

Functional perturbation and action mechanism studies have shown that the transcription factor Zeb2 controls cell fate decisions, differentiation, and/or maturation in multiple cell lineages in embryos and after birth. In cultured embryonic stem cells (ESCs), Zeb2’s mRNA/protein upregulation is necessary for the exit from primed pluripotency and for entering general and neural differentiation. We edited mouse ESCs to produce Flag-V5 epitope-tagged Zeb2 protein from one endogenous allele. Using chromatin immunoprecipitation coupled with sequencing (ChIP-seq), we mapped 2432 DNA-binding sites for this tagged Zeb2 in ESC-derived neuroprogenitor cells (NPCs). A new, major binding site maps promoter-proximal to Zeb2 itself. The homozygous deletion of this site demonstrates that autoregulation of Zeb2 is necessary to elicit the appropriate Zeb2-dependent effects in ESC-to-NPC differentiation. We have also cross-referenced all the mapped Zeb2 binding sites with previously obtained transcriptome data from Zeb2 perturbations in ESC-derived NPCs, GABAergic interneurons from the ventral forebrain of mouse embryos, and stem/progenitor cells from the post-natal ventricular-subventricular zone (V-SVZ) in mouse forebrain, respectively. Despite the different characteristics of each of these neurogenic systems, we found interesting target gene overlaps. In addition, our study also contributes to explaining developmental disorders, including Mowat-Wilson syndrome caused by ZEB2 deficiency, and also other monogenic syndromes.</p

3D chromatin reprogramming primes human memory T_H2 cells for rapid recall and pathogenic dysfunction

Memory T cells provide long-lasting defense responses through their ability to rapidly reactivate, but how they efficiently "recall" an inflammatory transcriptional program remains unclear. Here, we show that human CD4+ memory T helper 2 (TH2) cells carry a chromatin landscape synergistically reprogrammed at both one-dimensional (1D) and 3D levels to accommodate recall responses, which is absent in naive T cells. In memory TH2 cells, recall genes were epigenetically primed through the maintenance of transcription-permissive chromatin at distal (super)enhancers organized in long-range 3D chromatin hubs. Precise transcriptional control of key recall genes occurred inside dedicated topologically associating domains ("memory TADs"), in which activation-associated promoter-enhancer interactions were preformed and exploited by AP-1 transcription factors to promote rapid transcriptional induction. Resting memory TH2 cells from patients with asthma showed premature activation of primed recall circuits, linking aberrant transcriptional control of recall responses to chronic inflammation. Together, our results implicate stable multiscale reprogramming of chromatin organization as a key mechanism underlying immunological memory and dysfunction in T cells.</p

BMP4 and Temozolomide Synergize in the Majority of Patient-Derived Glioblastoma Cultures

One of the main causes of poor prognoses in patient with glioblastoma (GBM) is drug resistance to current standard treatment, which includes chemoradiation and adjuvant temozolomide (TMZ). In addition, the concept of cancer stem cells provides new insights into therapy resistance and management also in GBM and glioblastoma stem cell-like cells (GSCs), which might contribute to therapy resistance. Bone morphogenetic protein-4 (BMP4) stimulates astroglial differentiation of GSCs and thereby reduces their self-renewal capacity. Exposure of GSCs to BMP4 may also sensitize these cells to TMZ. A recent phase I trial has shown that local delivery of BMP4 is safe, but a large variation in survival is seen in these treated patients and in features of their cultured tumors. We wanted to combine TMZ and BMP4 (TMZ + BMP4) therapy and assess the inter-tumoral variability in response to TMZ + BMP4 in patient-derived GBM cultures. A phase II trial could then benefit a larger group of patients than those treated with BMP4 only. We first show that simultaneous treatment with TMZ + BMP4 is more effective than sequential treatment. Second, when applying our optimized treatment protocol, 70% of a total of 20 GBM cultures displayed TMZ + BMP4 synergy. This combination induces cellular apoptosis and does not inhibit cell proliferation. Comparative bulk RNA-sequencing indicates that treatment with TMZ + BMP4 eventually results in decreased MAPK signaling, in line with previous evidence that increased MAPK signaling is associated with resistance to TMZ. Based on these results, we advocate further clinical trial research to test patient benefit and validate pathophysiological hypothesis.</p

MAU2 and NIPBL variants impair the heterodimerization of the cohesin loader subunits and cause Cornelia de Lange syndrome

The NIPBL/MAU2 heterodimer loads cohesin onto chromatin. Mutations inNIPBLaccount for most cases ofthe rare developmental disorder Cornelia de Lange syndrome (CdLS). Here we report aMAU2 variant causing CdLS, a deletion of seven amino acids that impairs the interaction between MAU2 and the NIPBL N terminus.Investigating this interaction, we discovered that MAU2 and the NIPBL N terminus are largely dispensable fornormal cohesin and NIPBL function in cells with a NIPBL early truncating mutation. Despite a predicted fataloutcome of an out-of-frame single nucleotide duplication inNIPBL, engineered in two different cell lines,alternative translation initiation yields a form of NIPBL missing N-terminal residues. This form cannot interactwith MAU2, but binds DNA and mediates cohesin loading. Altogether, our work reveals that cohesin loading can occur independently of functional NIPBL/MAU2 complexes and highlights a novel mechanism protectiveagainst out-of-frame mutations that is potentially relevant for other genetic conditions

Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Background: Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human. Results: We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS. Conclusions: Our data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases

Origin and clinical relevance of chromosomal aberrations other than the common trisomies detected by genome-wide NIPS: Results of the TRIDENT study

PurposeNoninvasive prenatal screening (NIPS) using cell-free DNA in maternal blood is highly sensitive for detecting fetal trisomies 21, 18, and 13. Using a genome-wide approach, other chromosome anomalies can also be detected. We report on the origin

The relative value of operon predictions

For most organisms, computational operon predictions are the only source of genome-wide operon information. Operon prediction methods described in literature are based on (a combination of) the following five criteria: (i) intergenic distance, (ii) conserved gene clusters, (iii) functional relation, (iv) sequence elements and (v) experimental evidence. The performance estimates of operon predictions reported in literature cannot directly be compared due to differences in methods and data used in these studies. Here, we survey the current status of operon prediction methods. Based on a comparison of the performance of operon predictions on Escherichia coli and Bacillus subtilis we conclude that there is still room for improvement.We expect that existing and newly generated genomics and transcriptomics data will further improve accuracy of operon prediction methods.