Trichothiodystrophy-associated MPLKIP maintains DBR1 levels for proper lariat debranching and ectodermal differentiation.

The brittle hair syndrome Trichothiodystrophy (TTD) is characterized by variable clinical features, including photosensitivity, ichthyosis, growth retardation, microcephaly, intellectual disability, hypogonadism, and anaemia. TTD-associated mutations typically cause unstable mutant proteins involved in various steps of gene expression, severely reducing steady-state mutant protein levels. However, to date, no such link to instability of gene-expression factors for TTD-associated mutations in MPLKIP/TTDN1 has been established. Here, we present seven additional TTD individuals with MPLKIP mutations from five consanguineous families, with a newly identified MPLKIP variant in one family. By mass spectrometry-based interaction proteomics, we demonstrate that MPLKIP interacts with core splicing factors and the lariat debranching protein DBR1. MPLKIP-deficient primary fibroblasts have reduced steady-state DBR1 protein levels. Using Human Skin Equivalents (HSEs), we observed impaired keratinocyte differentiation associated with compromised splicing and eventually, an imbalanced proteome affecting skin development and, interestingly, also the immune system. Our data show that MPLKIP, through its DBR1 stabilizing role, is implicated in mRNA splicing, which is of particular importance in highly differentiated tissue.

Single-cell intracellular epitope and transcript detection reveals signal transduction dynamics.

To further our understanding of how biochemical information flows through cells upon external stimulation, we require single-cell multi-omics methods that concurrently map changes in (phospho)protein levels across signaling networks and the associated gene expression profiles. Here, we present quantification of RNA and intracellular epitopes by sequencing (QuRIE-seq), a droplet-based platform for single-cell RNA and intra- and extracellular (phospho)protein quantification through sequencing. We applied QuRIE-seq to quantify cell-state changes at both the signaling and the transcriptome level after 2-, 4-, 6-, 60-, and 180-min stimulation of the B cell receptor pathway in Burkitt lymphoma cells. Using the multi-omics factor analysis (MOFA+) framework, we delineated changes in single-cell (phospho)protein and gene expression patterns over multiple timescales and revealed the effect of an inhibitory drug (ibrutinib) on signaling and gene expression landscapes.

The corepressor NCOR1 and OCT4 facilitate early reprogramming by suppressing fibroblast gene expression.

Reprogramming somatic cells to induced pluripotent stem cells (iPSC) succeeds only in a small fraction of cells within the population. Reprogramming occurs in distinctive stages, each facing its own bottlenecks. It initiates with overexpression of transcription factors OCT4, SOX2, KLF4 and c-MYC (OSKM) in somatic cells such as mouse embryonic fibroblasts (MEFs). OSKM bind chromatin, silencing the somatic identity and starting the stepwise reactivation of the pluripotency programme. However, inefficient suppression of the somatic lineage leads to unwanted epigenetic memory from the tissue of origin, even in successfully generated iPSCs. Thus, it is essential to shed more light on chromatin regulators and processes involved in dissolving the somatic identity. Recent work characterised the role of transcriptional corepressors NCOR1 and NCOR2 (also known as NCoR and SMRT), showing that they cooperate with c-MYC to silence pluripotency genes during late reprogramming stages. NCOR1/NCOR2 were also proposed to be involved in silencing fibroblast identity, however it is unclear how this happens. Here, we shed light on the role of NCOR1 in early reprogramming. We show that siRNA-mediated ablation of NCOR1 and OCT4 results in very similar phenotypes, including transcriptomic changes and highly correlated high-content colony phenotypes. Both NCOR1 and OCT4 bind to promoters co-occupied by c-MYC in MEFs. During early reprogramming, downregulation of one group of somatic MEF-expressed genes requires both NCOR1 and OCT4, whereas another group of MEF-expressed genes is downregulated by NCOR1 but not OCT4. Our data suggest that NCOR1, assisted by OCT4 and c-MYC, facilitates transcriptional repression of genes with high expression in MEFs, which is necessary to bypass an early reprogramming block; this way, NCOR1 facilitates early reprogramming progression.

The interplay of chromatin and transcription factors during cell fate transitions in development and reprogramming.

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.

WDR5, BRCA1, and BARD1 Co-regulate the DNA Damage Response and Modulate the Mesenchymal-to-Epithelial Transition during Early Reprogramming.

Differentiated cells are epigenetically stable, but can be reprogrammed to pluripotency by expression of the OSKM transcription factors. Despite significant effort, relatively little is known about the cellular requirements for reprogramming and how they affect the properties of induced pluripotent stem cells. We have performed high-content screening with small interfering RNAs targeting 300 chromatin-associated factors and extracted colony-level quantitative features. This revealed five morphological phenotypes in early reprogramming, including one displaying large round colonies exhibiting an early block of reprogramming. Using RNA sequencing, we identified transcriptional changes associated with these phenotypes. Furthermore, double knockdown epistasis experiments revealed that BRCA1, BARD1, and WDR5 functionally interact and are required for the DNA damage response. In addition, the mesenchymal-to-epithelial transition is affected in Brca1, Bard1, and Wdr5 knockdowns. Our data provide a resource of chromatin-associated factors in early reprogramming and underline colony morphology as an important high-dimensional readout for reprogramming quality.

Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells.

Environmental stimuli often lead to heterogeneous cellular responses and transcriptional output. We developed single-cell RNA and Immunodetection (RAID) to allow combined analysis of the transcriptome and intracellular (phospho-)proteins from fixed single cells. RAID successfully recapitulated differentiation-state changes at the protein and mRNA level in human keratinocytes. Furthermore, we show that differentiated keratinocytes that retain high phosphorylated FAK levels, a feature associated with stem cells, also express a selection of stem cell associated transcripts. Our data demonstrates that RAID allows investigation of heterogeneous cellular responses to environmental signals at the mRNA and phospho-proteome level.

BLNCR is a long non-coding RNA adjacent to integrin beta-1 that is rapidly lost during epidermal progenitor cell differentiation.

As our understanding of transcriptional regulation improves so does our appreciation of its complexity. Both coding and (long) non-coding RNAs provide cells with multiple levels of control and thereby flexibility to adapt gene expression to the environment. However, few long non-coding RNAs (lncRNAs) have been studied in human epidermal stem cells. Here, we characterized the expression of 26 lncRNAs in human epidermal keratinocytes, 7 of which we found to be dynamically expressed during differentiation. We performed in depth analysis of a lncRNA located proximal to the epidermal stem cell marker integrin beta-1 (ITGB1) and transcribed in the opposite direction. We dubbed this gene Beta1-adjacent long non-coding RNA, or BLNCR, and found that its expression is regulated by p63 and AP1 transcription factors. Furthermore, BLNCR expression is regulated downstream the integrin and EGF signaling pathways that are key to epidermal stem cell maintenance. Finally, we found that BLNCR expression is rapidly reduced upon induction of differentiation, preceding the down regulation of integrin beta-1 expression. These dynamics closely mirror the loss of proliferative and adhesion capacity of epidermal stem cells in colony formation assays. Together, these results suggest that loss of BLNCR expression marks the switch from a proliferative state towards terminal differentiation in human epidermal stem cells.

Mutant p63 Affects Epidermal Cell Identity through Rewiring the Enhancer Landscape.

Transcription factor p63 is a key regulator of epidermal keratinocyte proliferation and differentiation. Mutations in the p63 DNA-binding domain are associated with ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome. However, the underlying molecular mechanism of these mutations remains unclear. Here, we characterized the transcriptome and epigenome of p63 mutant keratinocytes derived from EEC patients. The transcriptome of p63 mutant keratinocytes deviated from the normal epidermal cell identity. Epigenomic analyses showed an altered enhancer landscape in p63 mutant keratinocytes contributed by loss of p63-bound active enhancers and unexpected gain of enhancers. The gained enhancers were frequently bound by deregulated transcription factors such as RUNX1. Reversing RUNX1 overexpression partially rescued deregulated gene expression and the altered enhancer landscape. Our findings identify a disease mechanism whereby mutant p63 rewires the enhancer landscape and affects epidermal cell identity, consolidating the pivotal role of p63 in controlling the enhancer landscape of epidermal keratinocytes.

Single-Cell ID-seq Reveals Dynamic BMP Pathway Activation Upstream of the MAF/MAFB-Program in Epidermal Differentiation.

Epidermal homeostasis requires balanced and coordinated adult stem cell renewal and differentiation. These processes are controlled by both extracellular signaling and by cell intrinsic transcription regulatory networks, yet how these control mechanisms are integrated to achieve this is unclear. Here, we developed single-cell Immuno-Detection by sequencing (scID-seq) and simultaneously measured 69 proteins (including 34 phosphorylated epitopes) at single-cell resolution to study the activation state of signaling pathways during human epidermal differentiation. Computational pseudo-timing inference revealed dynamic activation of the JAK-STAT, WNT, and BMP pathways along the epidermal differentiation trajectory. We found that during differentiation, cells start producing BMP2-ligands and activate the canonical intracellular effectors SMAD1/5/9. Mechanistically, the BMP pathway is responsible for activating the MAF/MAFB/ZNF750 transcription factor network to drive late-stage epidermal differentiation. Our work indicates that incorporating signaling pathway activation into this transcription regulatory network enables coordination of transcription programs during epidermal differentiation.

Splicing and Chromatin Factors Jointly Regulate Epidermal Differentiation.

Epidermal homeostasis requires balanced progenitor cell proliferation and loss of differentiated cells from the epidermal surface. During this process, cells undergo major changes in their transcriptional programs to accommodate new cellular functions. We found that transcriptional and post-transcriptional mechanisms underlying these changes jointly control genes involved in cell adhesion, a key process in epidermal maintenance. Using siRNA-based perturbation screens, we identified DNA and/or RNA binding regulators of epidermal differentiation. Computational modeling and experimental validation identified functional interactions between the matrin-type 2 zinc-finger protein ZMAT2 and the epigenetic modifiers ING5, SMARCA5, BRD1, UHRF1, BPTF, and SMARCC2. ZMAT2 is an interactor of the pre-spliceosome that is required to keep cells in an undifferentiated, proliferative state. RNA immunoprecipitation and transcriptome-wide RNA splicing analysis showed that ZMAT2 associates with and regulates transcripts involved in cell adhesion in conjunction with ING5. Thus, joint control by splicing regulation, histone, and DNA modification is important to maintain epidermal cells in an undifferentiated state.

Immuno-detection by sequencing enables large-scale high-dimensional phenotyping in cells.

Cell-based small molecule screening is an effective strategy leading to new medicines. Scientists in the pharmaceutical industry as well as in academia have made tremendous progress in developing both large-scale and smaller-scale screening assays. However, an accessible and universal technology for measuring large numbers of molecular and cellular phenotypes in many samples in parallel is not available. Here we present the immuno-detection by sequencing (ID-seq) technology that combines antibody-based protein detection and DNA-sequencing via DNA-tagged antibodies. We use ID-seq to simultaneously measure 70 (phospho-)proteins in primary human epidermal stem cells to screen the effects of ~300 kinase inhibitor probes to characterise the role of 225 kinases. The results show an association between decreased mTOR signalling and increased differentiation and uncover 13 kinases potentially regulating epidermal renewal through distinct mechanisms. Taken together, our work establishes ID-seq as a flexible solution for large-scale high-dimensional phenotyping in fixed cell populations.

Adaptation trajectories during adhesion and spreading affect future cell states.

Cells are complex systems in which dynamic gene expression and protein-interaction networks adapt to changes in the environment. Seeding and subsequent spreading of cells on substrates represents an example of adaptation to a major perturbation. The formation of adhesive interactions and self-organisation of the cytoskeleton during initial spreading might prime future cell behaviour. To elucidate the role of these events on later cellular behaviour, we mapped the trajectories by which cells respond to seeding on substrates with different physical properties. Our experiments on cell spreading dynamics on collagen- or fibrin-coated polyacrylamide gels and collagen or fibrin hydrogels show that on each substrate, cells follow distinct trajectories of morphological changes, culminating in fundamentally different cell states as quantified by RNA-expression levels, YAP/TAZ localisation, proliferation and differentiation propensities. The continuous adaptation of the cell to environmental cues leaves traces due to differential cellular organisation and gene expression profiles, blurring correlations between a particular physical property and cellular phenotype.

Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity.

In this study, we originally aimed to characterize the potential role of Argonaute 2 (AGO2) in the nucleus, a key protein of the miRNA machinery. We combined Chromatin Immunoprecipitation (ChIP) with high throughput sequencing (ChIP-seq) and quantitative mass spectrometry (ChIP-MS) using the broadly used AGO2 11A9 antibody to determine interactions with chromatin and nuclear proteins. We found a previously described interaction between AGO2 and SWI/SNF on chromatin with ChIP-MS and observed enrichment at enhancers and transcription start sites using ChIP-seq. However, antibody specificity issues can produce misleading results for ChIP, RNA-seq and Mass spectrometry. Therefore, we developed a CRISPR/Cas9 engineered AGO2-/- HEK293T cell line to validate our findings. ChIP-qPCR and immunoprecipitation combined with MS (IP-MS) showed that the 11A9 antibody associates with chromatin and SWI/SNF in the absence of AGO2. Furthermore, stoichiometry, IP-MS and co-IP analysis suggests a direct interaction of this antibody with SMARCC1, a component of the SWI/SNF complex. For this reason, particular care should be taken in performing and interpreting experiments in which the 11A9 antibody is used to study a nuclear role of AGO2.

Wounding induces dedifferentiation of epidermal Gata6+ cells and acquisition of stem cell properties.

The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6+ cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair.

A covalent and cleavable antibody-DNA conjugation strategy for sensitive protein detection via immuno-PCR.

Immuno-PCR combines specific antibody-based protein detection with the sensitivity of PCR-based quantification through the use of antibody-DNA conjugates. The production of such conjugates depends on the availability of quick and efficient conjugation strategies for the two biomolecules. Here, we present an approach to produce cleavable antibody-DNA conjugates, employing the fast kinetics of the inverse electron-demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO). Our strategy consists of three steps. First, antibodies are functionalized with chemically cleavable NHS-s-s-tetrazine. Subsequently, double-stranded DNA is functionalized with TCO by enzymatic addition of N3-dATP and coupling to trans-Cyclooctene-PEG12-Dibenzocyclooctyne (TCO-PEG12-DBCO). Finally, conjugates are quickly and efficiently obtained by mixing the functionalized antibodies and dsDNA at low molar ratios of 1:2. In addition, introduction of a chemically cleavable disulphide linker facilitates release and sensitive detection of the dsDNA after immuno-staining. We show specific and sensitive protein detection in immuno-PCR for human epidermal stem cell markers, ITGA6 and ITGB1, and the differentiation marker Transglutaminase 1 (TGM1). We anticipate that the production of chemically cleavable antibody-DNA conjugates will provide a solid basis for the development of multiplexed immuno-PCR experiments and immuno-sequencing methodologies.

Insulin/IGF-1-mediated longevity is marked by reduced protein metabolism.

Mutations in the daf-2 gene of the conserved Insulin/Insulin-like Growth Factor (IGF-1) pathway double the lifespan of the nematode Caenorhabditis elegans. This phenotype is completely suppressed by deletion of Forkhead transcription factor daf-16. To uncover regulatory mechanisms coordinating this extension of life, we employed a quantitative proteomics strategy with daf-2 mutants in comparison with N2 and daf-16; daf-2 double mutants. This revealed a remarkable longevity-specific decrease in proteins involved in mRNA processing and transport, the translational machinery, and protein metabolism. Correspondingly, the daf-2 mutants display lower amounts of mRNA and 20S proteasome activity, despite maintaining total protein levels equal to that observed in wild types. Polyribosome profiling in the daf-2 and daf-16;daf-2 double mutants confirmed a daf-16-dependent reduction in overall translation, a phenotype reminiscent of Dietary Restriction-mediated longevity, which was independent of germline activity. RNA interference (RNAi)-mediated knockdown of proteins identified by our approach resulted in modified C. elegans lifespan confirming the importance of these processes in Insulin/IGF-1-mediated longevity. Together, the results demonstrate a role for the metabolism of proteins in the Insulin/IGF-1-mediated extension of life.

Posterior association networks and functional modules inferred from rich phenotypes of gene perturbations.

Combinatorial gene perturbations provide rich information for a systematic exploration of genetic interactions. Despite successful applications to bacteria and yeast, the scalability of this approach remains a major challenge for higher organisms such as humans. Here, we report a novel experimental and computational framework to efficiently address this challenge by limiting the 'search space' for important genetic interactions. We propose to integrate rich phenotypes of multiple single gene perturbations to robustly predict functional modules, which can subsequently be subjected to further experimental investigations such as combinatorial gene silencing. We present posterior association networks (PANs) to predict functional interactions between genes estimated using a Bayesian mixture modelling approach. The major advantage of this approach over conventional hypothesis tests is that prior knowledge can be incorporated to enhance predictive power. We demonstrate in a simulation study and on biological data, that integrating complementary information greatly improves prediction accuracy. To search for significant modules, we perform hierarchical clustering with multiscale bootstrap resampling. We demonstrate the power of the proposed methodologies in applications to Ewing's sarcoma and human adult stem cells using publicly available and custom generated data, respectively. In the former application, we identify a gene module including many confirmed and highly promising therapeutic targets. Genes in the module are also significantly overrepresented in signalling pathways that are known to be critical for proliferation of Ewing's sarcoma cells. In the latter application, we predict a functional network of chromatin factors controlling epidermal stem cell fate. Further examinations using ChIP-seq, ChIP-qPCR and RT-qPCR reveal that the basis of their genetic interactions may arise from transcriptional cross regulation. A Bioconductor package implementing PAN is freely available online at http://bioconductor.org/packages/release/bioc/html/PANR.html.

Diverse epigenetic strategies interact to control epidermal differentiation.

It is becoming clear that interconnected functional gene networks, rather than individual genes, govern stem cell self-renewal and differentiation. To identify epigenetic factors that impact on human epidermal stem cells we performed siRNA-based genetic screens for 332 chromatin modifiers. We developed a Bayesian mixture model to predict putative functional interactions between epigenetic modifiers that regulate differentiation. We discovered a network of genetic interactions involving EZH2, UHRF1 (both known to regulate epidermal self-renewal), ING5 (a MORF complex component), BPTF and SMARCA5 (NURF complex components). Genome-wide localization and global mRNA expression analysis revealed that these factors impact two distinct but functionally related gene sets, including integrin extracellular matrix receptors that mediate anchorage of epidermal stem cells to their niche. Using a competitive epidermal reconstitution assay we confirmed that ING5, BPTF, SMARCA5, EZH2 and UHRF1 control differentiation under physiological conditions. Thus, regulation of distinct gene expression programs through the interplay between diverse epigenetic strategies protects epidermal stem cells from differentiation.

FRMD4A upregulation in human squamous cell carcinoma promotes tumor growth and metastasis and is associated with poor prognosis.

New therapeutic strategies are needed to improve treatment of head and neck squamous cell carcinoma (HNSCC), an aggressive tumor with poor survival rates. FRMD4A is a human epidermal stem cell marker implicated previously in epithelial polarity that is upregulated in SCC cells. Here, we report that FRMD4A upregulation occurs in primary human HNSCCs where high expression levels correlate with increased risks of relapse. FRMD4A silencing decreased growth and metastasis of human SCC xenografts in skin and tongue, reduced SCC proliferation and intercellular adhesion, and stimulated caspase-3 activity and expression of terminal differentiation markers. Notably, FRMD4A attenuation caused nuclear accumulation of YAP, suggesting a potential role for FRMD4A in Hippo signaling. Treatment with the HSP90 inhibitor 17-DMAG or ligation of CD44 with hyaluronan caused nuclear depletion of FRMD4A, nuclear accumulation of YAP and reduced SCC growth and metastasis. Together, our findings suggest FRMD4A as a novel candidate therapeutic target in HNSCC based on the key role in metastatic growth we have identified.

Mapping dynamic histone acetylation patterns to gene expression in nanog-depleted murine embryonic stem cells.

Embryonic stem cells (ESC) have the potential to self-renew indefinitely and to differentiate into any of the three germ layers. The molecular mechanisms for self-renewal, maintenance of pluripotency and lineage specification are poorly understood, but recent results point to a key role for epigenetic mechanisms. In this study, we focus on quantifying the impact of histone 3 acetylation (H3K9,14ac) on gene expression in murine embryonic stem cells. We analyze genome-wide histone acetylation patterns and gene expression profiles measured over the first five days of cell differentiation triggered by silencing Nanog, a key transcription factor in ESC regulation. We explore the temporal and spatial dynamics of histone acetylation data and its correlation with gene expression using supervised and unsupervised statistical models. On a genome-wide scale, changes in acetylation are significantly correlated to changes in mRNA expression and, surprisingly, this coherence increases over time. We quantify the predictive power of histone acetylation for gene expression changes in a balanced cross-validation procedure. In an in-depth study we focus on genes central to the regulatory network of Mouse ESC, including those identified in a recent genome-wide RNAi screen and in the PluriNet, a computationally derived stem cell signature. We find that compared to the rest of the genome, ESC-specific genes show significantly more acetylation signal and a much stronger decrease in acetylation over time, which is often not reflected in a concordant expression change. These results shed light on the complexity of the relationship between histone acetylation and gene expression and are a step forward to dissect the multilayer regulatory mechanisms that determine stem cell fate.