ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/pharmacology , Argonaute Proteins/antagonists & inhibitors , Chromosomal Proteins, Non-Histone/metabolism , Transcription Factors/metabolism , CRISPR-Cas Systems , Chromatin Assembly and Disassembly , Chromatin Immunoprecipitation , Chromosomal Proteins, Non-Histone/antagonists & inhibitors , Chromosomal Proteins, Non-Histone/genetics , Gene Knockdown Techniques , Genetic Engineering , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , Mass Spectrometry , Protein Binding , Transcription Factors/antagonists & inhibitors , Transcription Factors/geneticsABSTRACT
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.
