Data availabilityData associated with this study have been deposited in the NCBI Gene Expression Omnibus under accession numbers GSE126231, GSE126734 and GSE146637 respectively for the microarray, ATAC-seq and single-cell RNA-seq. Data supporting the findings of this study are available within the article (and its Supplementary Information files). Source data behind Figures 1-4 and Extended Data Figures 1-12 are available within the manuscript files.
Code availabilityCustom computer code and algorithm used to generate results that are reported in the paper are available within the article (and its Supplementary Information files) and from the corresponding authors on reasonable request. The code used for the modeling of the clonal data has been deposited in GitHub (available at https://github.com/BenSimonsLab/Aragona_Nature_2020). In relation with the single-cells analysis, sequencing reads were preprocessed using cutadapt (version 1.13, https://pypi.org/project/ cutadapt/), alignments were generated using STAR (version 2.5.2b
Highlights d Linear growth of the epidermis during postnatal development d Constant imbalance of self-renewal and decreasing proliferation leads to IFE growth d Higher molecular homogeneity in developmental progenitors d Orientation of clonal growth follows orientation of collagen fibers
Author contributions A.C., S.L. and C.B. designed the experiments and performed data analysis. A.C. and S.L. performed most of the biological experiments. E.T. performed the experiments and data analysis on prostate glands. A.Sifrim., M.M., Y.S., J.V.H. and T.V. performed the bioinformatic analysis. A.D. and G.B. provided technical help. C.D. performed FACS experiments. N.D. provided technical help with single-cell RNA sequencing. A.C., S.L., M.F., A.W. and A.V.K. performed immunostainings, blocking antibodies and small-molecule treatments and experiments with follow-up mice. A.Sahay contributed genetic tools. V.d.M. performed statistical analysis. C.W.S. provided the Notch antibodies. A.C., A.V.K. and C.B. wrote the manuscript. All authors read and approved the final manuscript.
paaR2–paaA2–parE2 is a three-component toxin–antitoxin module found in prophage CP-993P of Escherichia coli O157:H7. Transcription regulation of this module occurs via the 123-amino-acid regulator PaaR2, which forms a large oligomeric structure. Despite appearing to be well folded, PaaR2 withstands crystallization, as does its N-terminal DNA-binding domain. Native mass spectrometry was used to screen for nanobodies that form a unique complex and stabilize the octameric structure of PaaR2. One such nanobody, Nb33, allowed crystallization of the protein. The resulting crystals belong to space group F432, with unit-cell parameter a = 317 Å, diffract to 4.0 Å resolution and are likely to contain four PaaR2 monomers and four nanobody monomers in the asymmetric unit. Crystals of two truncates containing the N-terminal helix–turn–helix domain also interact with Nb33, and the corresponding co-crystals diffracted to 1.6 and 1.75 Å resolution.
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