Abstract
Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3KBTBD8) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralogue TCOF1, the mutation of which underlies the neurocristopathy Treacher Collins syndrome2,3. Ubiquitylation drives formation of a TCOF1–NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Gurdon, J. B. The egg and the nucleus: a battle for supremacy. Development 140, 2449–2456 (2013)
The Treacher Collins Syndrome Collaborative Group. Positional cloning of a gene involved in the pathogenesis of Treacher Collins syndrome. Nature Genet. 12, 130–136 (1996)
Dixon, J. et al. Tcof1/Treacle is required for neural crest cell formation and proliferation deficiencies that cause craniofacial abnormalities. Proc. Natl Acad. Sci. USA 103, 13403–13408 (2006)
Petroski, M. D. & Deshaies, R. J. Function and regulation of cullin-RING ubiquitin ligases. Nature Rev. Mol. Cell Biol. 6, 9–20 (2005)
Silverman, J. S., Skaar, J. R. & Pagano, M. SCF ubiquitin ligases in the maintenance of genome stability. Trends Biochem. Sci. 37, 66–73 (2012)
Wang, Y. et al. Deletion of the Cul1 gene in mice causes arrest in early embryogenesis and accumulation of cyclin E. Curr. Biol. 9, 1191–1194 (1999)
Arai, T. et al. Targeted disruption of p185/Cul7 gene results in abnormal vascular morphogenesis. Proc. Natl Acad. Sci. USA 100, 9855–9860 (2003)
Singer, J. D., Gurian-West, M., Clurman, B. & Roberts, J. M. Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells. Genes Dev. 13, 2375–2387 (1999)
Jiang, B. et al. Lack of Cul4b, an E3 ubiquitin ligase component, leads to embryonic lethality and abnormal placental development. PLoS ONE 7, e37070 (2012)
Voigt, J. & Papalopulu, N. A dominant-negative form of the E3 ubiquitin ligase Cullin-1 disrupts the correct allocation of cell fate in the neural crest lineage. Development 133, 559–568 (2006)
Jin, L. et al. Ubiquitin-dependent regulation of COPII coat size and function. Nature 482, 495–500 (2012)
Lander, R., Nordin, K. & LaBonne, C. The F-box protein Ppa is a common regulator of core EMT factors Twist, Snail, Slug, and Sip1. J. Cell Biol. 194, 17–25 (2011)
Skaar, J. R., Pagan, J. K. & Pagano, M. Mechanisms and function of substrate recruitment by F-box proteins. Nature Rev. Mol. Cell Biol. 14, 369–381 (2013)
Barbieri, C. E. et al. Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer. Nature Genet. 44, 685–689 (2012)
Louis-Dit-Picard, H. et al. KLHL3 mutations cause familial hyperkalemic hypertension by impairing ion transport in the distal nephron. Nature Genet. 44, 456–460 (2012)
Welcker, M. & Clurman, B. E. FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation. Nature Rev. Cancer 8, 83–93 (2008)
Yanai, I., Peshkin, L., Jorgensen, P. & Kirschner, M. W. Mapping gene expression in two Xenopus species: evolutionary constraints and developmental flexibility. Dev. Cell 20, 483–496 (2011)
Chambers, S. M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnol. 27, 275–280 (2009)
Dauwerse, J. G. et al. Mutations in genes encoding subunits of RNA polymerases I and III cause Treacher Collins syndrome. Nature Genet. 43, 20–22 (2011)
Jones, N. C. et al. Prevention of the neurocristopathy Treacher Collins syndrome through inhibition of p53 function. Nature Med. 14, 125–133 (2008)
Price, J. C., Guan, S., Burlingame, A., Prusiner, S. B. & Ghaemmaghami, S. Analysis of proteome dynamics in the mouse brain. Proc. Natl Acad. Sci. USA 107, 14508–14513 (2010)
Cambridge, S. B. et al. Systems-wide proteomic analysis in mammalian cells reveals conserved, functional protein turnover. J. Proteome Res. 10, 5275–5284 (2011)
Tuck, A. C. & Tollervey, D. A transcriptome-wide atlas of RNP composition reveals diverse classes of mRNAs and lncRNAs. Cell 154, 996–1009 (2013)
Jack, K. et al. rRNA pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells. Mol. Cell 44, 660–666 (2011)
Yoon, A. et al. Impaired control of IRES-mediated translation in X-linked dyskeratosis congenita. Science 312, 902–906 (2006)
Kondrashov, N. et al. Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning. Cell 145, 383–397 (2011)
Xue, S. & Barna, M. Specialized ribosomes: a new frontier in gene regulation and organismal biology. Nature Rev. Mol. Cell Biol. 13, 355–369 (2012)
McCann, K. L. & Baserga, S. J. Genetics. Mysterious ribosomopathies. Science 341, 849–850 (2013)
Freed, E. F., Prieto, J. L., McCann, K. L., McStay, B. & Baserga, S. J. NOL11, implicated in the pathogenesis of North American Indian childhood cirrhosis, is required for pre-rRNA transcription and processing. PLoS Genet. 8, e1002892 (2012)
Sondalle, S. B. & Baserga, S. J. Human diseases of the SSU processome. Biochim. Biophys. Acta 1842, 758–764 (2014)
Rape, M., Reddy, S. K. & Kirschner, M. W. The processivity of multiubiquitination by the APC determines the order of substrate degradation. Cell 124, 89–103 (2006)
Jin, L., Williamson, A., Banerjee, S., Philipp, I. & Rape, M. Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Cell 133, 653–665 (2008)
Khokha, M. K. et al. Techniques and probes for the study of Xenopus tropicalis development. Develop. Dyn. 225, 499–510 (2002)
Sowa, M. E., Bennett, E. J., Gygi, S. P. & Harper, J. W. Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009)
Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. & Weissman, J. S. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nature Protocols 7, 1534–1550 (2012)
Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010)
Acknowledgements
We thank R. Harland, J. Schaletzky, R. Zoncu, J. Corn, A. Manford and E. Oh for advice and critical reading of the manuscript, and all members of the Rape and Ingolia laboratories for helpful discussions. A.W. was funded in part by a postdoctoral fellowship the California Institute of Regenerative Medicine; S.I. is supported by a Human Frontier Science Fellowship; C.A.M. was supported by an NSF graduate student fellowship; and S.M.-R. was supported by a UC Mexus-Conacyt fellowship and NIH GM42341. This work used the Vincent J. Coates Genomics Sequencing Laboratory and Proteomics/Mass Spectrometry Laboratory at UC Berkeley (NIH grants S10RR029668, S10RR027303 and S10RR025622). This work was funded by a Basic Biology Award from the California Institute of Regenerative Medicine to M.R. (RB-02222). M.R. is an investigator of the Howard Hughes Medical Institute.
Author information
Authors and Affiliations
Contributions
A.W. designed and performed most experiments and helped to write the manuscript; S.I. performed ribosomal profiling and RNA processing experiments; C.A.M. and S.M.-R. performed Xenopus experiments; N.T. performed binding studies; I.F. wrote code for analysis of CompPASS mass spectrometry; N.T.I. supervised ribosomal profiling studies; M.R. helped design experiments and wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
M.R. is a co-founder and consultant to Nurix, a biotech company in the ubiquitin space.
Extended data figures and tables
Extended Data Figure 1 KBTBD8 is a developmentally regulated CUL3 adaptor.
a, Gene expression analysis by microarray of hESCs differentiated into embryoid bodies (EB) for 6 days (n > 30,000 transcripts, mean of 3 biological replicates, analysis of variance (ANOVA) P value <0.05; blue, downregulated genes; red, upregulated genes). b, Expression analysis of all CRL substrate adaptors, including KBTBD8, with data derived from the experiment described above. c, Expression analysis of CUL3 adaptors during hESC differentiation into hEBs (blue, downregulation; yellow, upregulation). d, mRNA levels of pluripotency markers and KBTBD8 during hESC differentiation into EBs, as determined by qRT–PCR (mean of 3 technical replicates ± s.e.m.). e, Protein levels of KBTBD8 during hESC differentiation into EBs, as seen by western blot (OCT4, NANOG: pluripotency; PAX6: CNS precursors; TFAP2: neural crest marker). f, KBTBD8 is expressed in hESCs, but not in somatic cell lines, as determined by qRT–PCR (mean of 3 technical replicates ± s.e.m.). g, Abundance of KBTBD8 in H9 hESCs, D3 mESCs, or somatic cell lines was determined by western blot analysis. h, KBTBD8 expression is downregulated during mouse embryonic stem cell (mESC) differentiation into mouse embryoid bodies, as determined by qRT–PCR (mean of 3 technical replicates ± s.e.m.). i, KBTBD8 protein levels are reduced during mESC differentiation, as shown by western blot analysis.
Extended Data Figure 2 KBTBD8 controls neural crest formation.
a, Stable depletion of KBTBD8 from H1 hESCs, as determined by western blot analysis. b, KBTBD8 depletion does not significantly change the cell cycle profile of hESCs, as determined by propidium iodide staining and FACS. c, Control or KBTBD8-depleted hESCs were counted at indicated times after seeding (mean of 3 biological replicates, ±s.d.). d, KBTBD8 depletion does not induce apoptosis in hESCs, as shown by immunostaining against cleaved caspase 3 (red) or DNA (blue) (200 cells per condition; scale bar, 10 μm). e, KBTBD8 depletion does not affect the gene expression profile of hESCs, as determined by microarray analysis (genes > 2.5-fold change, n > 30,000; mean of 3 biological replicates, ANOVA P-value <0.05). f, Loss of KBTBD8 causes a decrease in the expression of neural crest cell markers during EB formation, as shown by comparative microarray analysis (genes > 2.5-fold change, n > 30,000; mean of 3 biological replicates, ANOVA P-value <0.05). g, mRNA levels of pluripotency and differentiation markers in EBs stably expressing control or KBTBD8 shRNAs were measured by qRT–PCR (3 technical replicates ± s.e.m.).
Extended Data Figure 3 KBTBD8 controls neural crest specification.
a, Depletion of KBTBD8 from hESCs subjected to neural conversion results in loss of neural crest cells, as determined by immunofluorescence against HNK1, TFAP2 and p75 (n > 200 cells, mean of 3 biological replicates ± s.d.). b, H1 hESCs transduced with control (green) or KBTBD8 shRNAs (red) were subjected to neural conversion, and expression of neural crest markers SOX10 (circles) and SNAIL2 (boxes) was monitored by qRT–PCR (mean of 3 technical replicates ± s.e.m.). c, H1 hESCs described above were subjected to neural conversion, and abundance of CNS precursor markers SOX2 (circles) and PAX6 (boxes) was measured by qRT–PCR. d, H1 hESCs described above were subjected to neural conversion, and abundance of telencephalon markers SIX3 (circles) and FOXG1 (boxes) was measured by qRT–PCR. e, Expression of OCT4 was monitored by qRT–PCR during neural conversion in the presence or absence of KBTBD8. f, hESCs stably expressing control or KBTBD8 shRNAs were subjected to neural conversion and analysed for expression of pluripotency (OCT4, CDH1), neural crest (SOX10, SNAIL2, AP2), or CNS precursor markers (PAX6) by western blotting. To provide consistency, samples were taken from the same experiment as shown in Fig. 5d (asterisks mark blots that are also shown in Fig. 5d). g, Loss of neural crest occurs in response to KBTBD8 depletion by two independent shRNAs, as shown by western blot analysis. h, hESCs were subjected to neural conversion and analysed by immunofluorescence microscopy against SOX10 (neural crest), PAX6 (CNS precursor), and OCT4 (pluripotency) (confocal, original magnification 20×).
Extended Data Figure 4 KBTBD8 is required for differentiation into functional neural crest cells.
a, H1 hESCs stably expressing control or KBTBD8 shRNAs were subjected to neural conversion for 43 days and analysed by immunofluorescence microscopy against GFAP (glia), smooth muscle actin (SMA; mesenchymal cells), and neurofilament L (neurons). b, Control H1 hESCs or hESCs depleted of KBTBD8 were subjected to neural conversion for 43 days and expression of markers for glia (GFAP), mesenchyme (smooth muscle actin, SMA), melanocytes (TYRP1, DCT), chondrocytes (COL2A1), or CNS derivatives (PAX6, NESTIN, neurofilament L) was analysed by qRT–PCR (mean of 3 technical replicates ± s.e.m.). c, Xenopus tropicalis embryos were injected at the two-cell stage with splice-blocking morpholinos (sMO) against CUL3 or KBTBD8, or with a dominant-negative construct of CUL3 that allows KBTBD8 to bind, but not ubiquitylate, substrates. Neural crest formation was monitored by SOX10 in situ hybridization. Quantification included experiment shown in Fig. 1d (mean of 3 biological replicates ± s.d.; ∼20 embryos per condition and replicate).
Extended Data Figure 5 Biochemical characterization of the substrate adaptor role of KBTBD8.
a, Domain structure of KBTBD8, including the residues mutated to generate ubiquitylation- (Y74A) and substrate-binding-deficient KBTBD8 (F550A, W579A). b, Effects of point mutations in predicted KELCH domain loops on binding of KBTBD8 to candidate substrates were determined by affinity purification and western blot analysis. c, Effects of point mutations in BTB domain on binding of KBTBD8 to CUL3 were determined by affinity purification and western blotting. Dimerization of Flag–KBTBD8 with KBTBD8–HA was analysed in the same experiment to provide a folding control. d, Binding of recombinant CUL3 to immobilized recombinant MBP–KBTBD8 variants was analysed by Coomassie. e, Binding of in vitro-transcribed/translated 35S-NOLC1 to immobilized recombinant KBTBD8 variants was analysed by autoradiography. f, Binding of in vitro-transcribed/translated 35S-TCOF1 to immobilized recombinant KBTBD8 variants was analysed by autoradiography. g, Endogenous β-arrestin proteins in reticulocyte lysates binds immobilized, recombinant KBTBD8, as detected by western blot analysis. h, 293T cells were transfected with control- or β-arrestin 1/2-siRNAs and reconstituted with Flag–KBTBD8. Binding of KBTBD8 to endogenous TCOF1 and NOLC1 was analysed by anti-Flag affinity purification and western blot analysis. i, Ubiquitylation of HA–TCOF1 in 293T cells depleted of β-arrestin 1/2 and reconstituted with KBTBD8 was determined after denaturing Ni-NTA purification by western blotting as described above. j, Ubiquitylation of HA–NOLC1 was detected in 293T cells depleted of β-arrestins and reconstituted with KBTBD8, as described above.
Extended Data Figure 6 KBTBD8 specifies neural crest fate through TCOF1 and NOLC1.
a, mRNA levels of KBTBD8, NOLC1 and TCOF1 were determined in hESCs or differentiating cells transduced with lentiviruses expressing the indicated shRNAs by qRT–PCR (mean of 3 technical replicates ± s.e.m.). b, hESCs stably depleted of KBTBD8 and reconstituted with either wild-type KBTBD8, KBTBD8(W579A), or KBTBD8(Y74A) were subjected to neural conversion (9 days) and analysed for the expression of marker proteins by qRT–PCR (mean of 3 technical replicates ± s.e.m.). c, hESCs stably depleted of KBTBD8, TCOF1, or NOLC1 were subjected to neural conversion (9 days) and analysed for marker expression by qRT–PCR (mean of 3 technical replicates ± s.e.m.). d, Depletion of TCOF1 or NOCL1 from hESCs results in loss of neural crest cells, as determined by triple staining immunofluorescence against the neural crest markers HNK1, TFAP2 and p75 (n > 200 cells, mean of 3 biological replicates ± s.d.). Scale bar, 10 μm. e, hESCs were transduced with lentiviruses expressing control or BRD2 shRNAs, subjected to puromycin selection for 7 days, and analysed by western blot analysis. f, Depletion efficiency for shRNAs against various KBTBD8 binding partners, as determined by qRT–PCR (mean of 3 technical replicates ± s.e.m.).
Extended Data Figure 7 Characterization of TCOF1 regulation by ubiquitylation.
a, hESCs depleted of either KBTBD8 or TCOF1 were subjected to neural conversion and analysed for expression of indicated proteins by western blot. b, Control or KBTBD8-depleted hESCs were fixed and subjected to indirect immunofluorescence analysis against endogenous TCOF1 or NOLC1. Scale bar, 10 μm. c, Total spectral counts of proteins associated with TCOF1–NOLC1 complexes purified by sequential immunoprecipitation in the presence of KBTBD8 compared to single TCOF1 affinity purification in the absence of KBTBD8, as determined by mass spectrometry (sum of 3 biological replicates). d, 293T cells were reconstituted with CUL3KBTBD8 and depleted of TCOF1 and NOLC1 by siRNAs. Endogenous RNA polymerase I was immunoprecipitated and binding to the SSU processome (NOP58, CSK2A) was analysed by western blot.
Extended Data Figure 8 KBTBD8 is not required for general ribosome biogenesis.
a, hESCs stably depleted of KBTBD8 were subjected to neural conversion and levels of 5S rRNA, 18S rRNA and mRNAs encoding RPS6, RPS28, RPL10A and RPL28 were measured by qRT–PCR (mean of 3 technical replicates ± s.e.m.). b, hESCs stably depleted of KBTBD8 were subjected to neural conversion, and total RNA was subjected to a bioanalyser assay to monitor processing of ribosomal RNAs. c, hESCs stably depleted of KBTBD8 were subjected to neural conversion (3 days), and nucleoli were analysed by anti-fibrillarin (original magnification: 60 ×, confocal) immunofluorescence microscopy. d, Quantification of nucleolar analysis described above (mean of 3 technical replicates ± s.e.m.). e, hESCs stably depleted of KBTBD8 were analysed for localization of 5.8S rRNA by anti-5.8S rRNA immunofluorescence microscopy (original magnification: 60×, confocal). f, hESCs depleted of KBTBD8 were subjected to neural conversion and analysed by anti-5.8S rRNA immunofluorescence microscopy (original magnification: 60×, confocal). g, Polysomes were purified from control or KBTBD8-depleted hESCs and differentiated counterparts subjected to neural conversion via sucrose gradient centrifugation followed by fractionation and UV detection. h, KBTBD8-depleted hESCs were subjected to neural conversion for 9 days and analysed for apoptosis by immunofluorescence analysis against cleaved caspase 3 (red) and DNA (Hoechst, blue). Cells with active caspase 3 staining were quantified (∼200 cells per condition; scale bar, 10 μm) (original magnification: 40×, confocal).
Extended Data Figure 9 Characterization of KBTBD8- and TCOF1-depleted hESCs during neural conversion.
a, hESCs were treated with increasing concentrations of rapamycin, subjected to neural conversion for 9 days, and analysed for expression of neural crest or CNS precursor markers by qRT–PCR. For comparison, effects of KBTBD8, TCOF1, or NOLC1 depletion (extracted from Fig. 3a) are shown. b, hESCs were treated with rapamycin, subjected to neural conversion, and analysed for marker expression by western blotting. c, hESCs were depleted of KBTBD8 or TCOF1, subjected to neural conversion for 3 days, and analysed for expression of 5S and 18S rRNA by qRT–PCR (mean of 3 technical replicates ± s.e.m.). d, hESCs depleted of KBTBD8 or TCOF1 were subjected to neural conversion for 3 days and analysed for p53 activation by RNA-seq against p53 targets. e, hESCs were depleted of KBTBD8 or TCOF1, subjected to neural conversion for 3 days, and analysed for apoptosis by immunofluorescence microscopy against cleaved caspase 3. Quantification shown below (∼200 cells per condition). f, hESCs depleted of KBTBD8 were subjected to neural conversion for 9 days and analysed for expression levels of 5S and 18S rRNA by qRT–PCR (mean of 3 technical replicates ± s.e.m.). g, hESCs stably depleted of NOLC1 or TCOF1 were subjected to neural conversion for 9 days and analysed by immunofluorescence microscopy against cleaved caspase 3 (red) or DNA (Hoechst, blue). Quantification is shown below (∼200 cells per condition; scale bar, 10 μm).
Extended Data Figure 10 KBTBD8 controls translation.
a, hESCs stably depleted of KBTBD8 were subjected to neural conversion for 3 days, and hESCs and differentiating cells were analysed by RNA deep sequencing and ribosomal profiling to determine translation efficiency. Distribution of translation efficiency changes for 7,725 mRNAs brought about by KBTBD8 depletion is shown. b, hESCs stably depleted of either TCOF1 or KBTBD8 were subjected to neural conversion for 3 days, and translation efficiency was determined by RNA-seq and ribosome profiling. c, Translation efficiency blot of differentiating hESCs transduced with control or KBTBD8 shRNAs was labelled for significantly affected transcripts in general (blue), with links to CNS precursor formation (gold), or with links to neural crest formation (green). d, hESCs stably depleted of KBTBD8 or TCOF1 were subjected to neural conversion for 3 days, and expression levels of indicated proteins were analysed by western blotting. e, hESCs stably depleted of KBTBD8 were subjected to neural conversion for 3 days, and levels of ATRX1 and PCM1 mRNA were determined by qRT–PCR (mean of 3 technical replicates ± s.e.m.). f, hESCs stably depleted of KBTBD8 were subjected to neural conversion for 3 days, and protein stability of ATRX1 and PCM1 was determined by cycloheximide chase and western blotting (mean of 3 biological replicates ± s.d., ATRX1 and PCM1 levels were normalized relative to actin levels and 0 h time point set to 100%).
Supplementary information
Supplementary Figures
This file contains Extended Data Figure 11. (PDF 927 kb)
Supplementary Table 1
This table contains the GO enrichment analysis of genes significantly down-regulated more than 2.5 fold in EB6 upon KBTBD8 depletion. (XLSX 218 kb)
Supplementary Table 2
This table contains the qRT-PCR primer sequences. (XLSX 45 kb)
Rights and permissions
About this article
Cite this article
Werner, A., Iwasaki, S., McGourty, C. et al. Cell-fate determination by ubiquitin-dependent regulation of translation. Nature 525, 523–527 (2015). https://doi.org/10.1038/nature14978
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature14978
This article is cited by
-
Insights into the diverse mechanisms and effects of variant CUL3-induced familial hyperkalemic hypertension
Cell Communication and Signaling (2023)
-
Detecting recurrent passenger mutations in melanoma by targeted UV damage sequencing
Nature Communications (2023)
-
Roles of NOLC1 in cancers and viral infection
Journal of Cancer Research and Clinical Oncology (2023)
-
FGF12 is a novel component of the nucleolar NOLC1/TCOF1 ribosome biogenesis complex
Cell Communication and Signaling (2022)
-
TCOF1 upregulation in triple-negative breast cancer promotes stemness and tumour growth and correlates with poor prognosis
British Journal of Cancer (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.