The AU-rich elements (AREs) encoded within many mRNA 3= untranslated regions (3=UTRs) are targets for factors that control transcript longevity and translational efficiency. Hsp70, best known as a protein chaperone with well-defined peptide-refolding properties, is known to interact with ARE-like RNA substrates in vitro. Here, we show that cofactor-free preparations of Hsp70 form direct, high-affinity complexes with ARE substrates based on specific recognition of U-rich sequences by both the ATP-and peptide-binding domains. Suppressing Hsp70 in HeLa cells destabilized an ARE reporter mRNA, indicating a novel ARE-directed mRNA-stabilizing role for this protein. Hsp70 also bound and stabilized endogenous ARE-containing mRNAs encoding vascular endothelial growth factor (VEGF) and Cox-2, which involved a mechanism that was unaffected by an inhibitor of its protein chaperone function. Hsp70 recognition and stabilization of VEGF mRNA was mediated by an ARE-like sequence in the proximal 3=UTR. Finally, stabilization of VEGF mRNA coincided with the accumulation of Hsp70 protein in HL60 promyelocytic leukemia cells recovering from acute thermal stress. We propose that the binding and stabilization of selected ARE-containing mRNAs may contribute to the cytoprotective effects of Hsp70 following cellular stress but may also provide a novel mechanism linking constitutively elevated Hsp70 expression to the development of aggressive neoplastic phenotypes.

Melanocyte stem cells (McSCs) are the undifferentiated melanocytic cells of the mammalian hair follicle (HF) responsible for recurrent generation of a large number of differentiated melanocytes during each HF cycle. HF McSCs reside in both the CD34+ bulge/lower permanent portion (LPP) and the CD34- secondary hair germ (SHG) regions of the HF during telogen. Using Dct- H2BGFP mice, we separate bulge/LPP and SHG McSCs using FACS with GFP and anti-CD34 to show that these two subsets of McSCs are functionally distinct. Genome-wide expression profiling results support the distinct nature of these populations, with CD34- McSCs exhibiting higher expression of melanocyte differentiation genes and with CD34+ McSCs demonstrating a profile more consistent with a neural crest stem cell. In culture and in vivo , CD34- McSCs regenerate pigmentation more efficiently whereas CD34+ McSCs selectively exhibit the ability to myelinate neurons. CD34+ McSCs, and their counterparts in human skin, may be useful for myelinating neurons in vivo , leading to new therapeutic opportunities for demyelinating diseases and traumatic nerve injury.

Hsp70 is a protein chaperone that prevents protein aggregation and aids protein folding by binding to hydrophobic peptide domains through a reversible mechanism directed by an ATPase cycle. However, Hsp70 also binds U-rich RNA including some AU-rich elements (AREs) that regulate the decay kinetics of select mRNAs and has recently been shown to bind and stabilize some ARE-containing transcripts in cells. Previous studies indicated that both the ATP- and peptide-binding domains of Hsp70 contributed to the stability of Hsp70-RNA complexes and that ATP might inhibit RNA recruitment. This suggested the possibility that RNA binding by Hsp70 might mimic features of its peptide-directed chaperone activities. Here, using purified, cofactor-free preparations of recombinant human Hsp70 and quantitative biochemical approaches, we found that high-affinity RNA binding requires at least 30 nucleotides of RNA sequence but is independent of Hsp70's nucleotide-bound status, ATPase activity, or peptide-binding roles. Furthermore, although both the ATP- and peptide-binding domains of Hsp70 could form complexes with an ARE sequence from mRNA, only the peptide-binding domain could recover cellular mRNA in ribonucleoprotein immunoprecipitations. Finally, Hsp70-directed stabilization of mRNA in cells was mediated exclusively by the protein's peptide-binding domain. Together, these findings indicate that the RNA-binding and mRNA-stabilizing functions of Hsp70 are independent of its protein chaperone cycle but also provide potential mechanical explanations for several well-established and recently discovered cytoprotective and RNA-based Hsp70 functions.

Melanocytes are neural crest-derived cells that are responsible for mammalian hair follicle (HF) pigmentation. The Dct-LacZ transgenic mouse is extensively used to study melanocyte biology but lacks conditionally-inducible labelling and fluorescent labelling, enabling specific, viable isolation of melanocytes using fluorescence-activated cell sorting (FACS). Here, we have generated a Tet-off bitransgenic mouse model, Dct-H2BGFP, containing Dct-tTA and TRE-H2BGFP transgenes. Characterization of Dct-H2BGFP mice confirmed a pattern of Dct-H2BGFP expression in melanoblasts, melanocyte stem cells (McSCs), and terminally differentiated melanocytes similar to the expression pattern of previously published mouse models Dct-LacZ and iDct-GFP. GFP expression is regulated by doxycycline. GFP is shown to co-localize with melanocyte label-retaining cells (LRCs) identified through BrdU retention. The GFP-expressing cells identified in vivo in the bulge and the secondary hair germ of telogen HFs of Dct-H2BGFP mice express the melanocyte and melanocyte stem cell markers Dct and Kit. Using Dct-H2BGFP mice, we separated GFP-expressing cells from the telogen HF based on FACS and showed that GFP-expressing cells express high levels of Kit and Dct, and lower levels of HF epithelial keratin genes. We also show that GFP-expressing cells express high levels of the melanocyte differentiation genes Tyr, Tyrp1, and Pmel17, further substantiating their identity within the melanocyte lineage. Thus, Dct-H2BGFP mice are not only useful for the in vivo identification of melanocytic cells, but also for isolating them viably and studying their molecular and biological properties.

Melanocyte stem cells (McSCs) are key components of the hair follicle (HF) stem cell system that regenerate differentiated melanocytes during successive HF cycles.To facilitate continued research on melanocyte development and differentiation and McSCs, we backcrossed inducible Dct-H2BGFP mice into the C57BL/6J background (B6-Dct-H2BGFP). We compared the expression pattern of B6-Dct-H2BGFP to that of Dct-H2BGFP mice on a mixed genetic background reported previously. To characterize B6-Dct-H2BGFP mice, we confirmed not only the expression of GFP in all melanocyte lineage cells, but also doxycycline regulation of GFP expression. Furthermore, ex vivo culture of the McSC subsets isolated by fluorescence-activated cell sorting (FACS) showed the propensity of bulge/CD34+ McSCs to differentiate with expression of non-melanocytic, neural crest lineage markers including glia (Gfap and CNPase, 73 ± 1% and 77 ± 2%, respectively), neurons (Tuj1 26 ± 5%), and smooth muscle (α-Sma, 31 ± 9%). In contrast, CD34−/secondary hair germ (SHG)McSCs differentiated into pigmented melanocytes, with higher expression of melanogenic markers Tyr (71 ± 1%), Tyrp1 (68 ± 4%), and Mitf (75 ± 7%). These results establish the utility of B6-Dct-H2BGFP bitransgenic mice for future in vivo studies of melanocytes requiring a defined genetic background.

Tools to visualize genetic alterations within tissues remain underdeveloped despite the growth of spatial transcriptomic technologies, which measure gene expression in different regions of tissues. Since genetic alterations can be detected in RNA-sequencing data, we explored the feasibility of observing somatic alterations in spatial transcriptomics data. Extracting genetic information from spatial transcriptomic data would illuminate the spatial distribution of clones and allow for correlations with regional changes in gene expression to support genotype-phenotype studies. Recent work demonstrates that copy number alterations can be inferred from spatial transcriptomics data1. Here, we describe new software to further enhance the inference of copy number from spatial transcriptomics data. Moreover, we demonstrate that single nucleotide variants are also detectable in spatial transcriptomic data. We applied these approaches to map the location of point mutations, copy number alterations, and allelic imbalances in spatial transcriptomic data of two cutaneous squamous cell carcinomas. We show that both tumors are dominated by a single clone of cells, suggesting that their regional variations in gene expression2 are likely driven by non-genetic factors. Furthermore, we observe mutant cells in histologically normal tissue surrounding one tumor, which were not discernible upon histopathologic evaluation. Finally, we detected mono-allelic expression of immunoglobulin heavy chains in B-cells, revealing clonal populations of plasma cells surrounding one tumor. In summary, we put forward solutions to add the genetic dimension to spatial transcriptomic datasets, augmenting the potential of this new technology.

Melanocyte stem cells (McSCs) are key components of the hair follicle (HF) stem cell system that are derived from neural crest during embryogenesis and are responsible for regeneration of differentiated melanocytes during successive HF cycles. Our previous research has shown presence of two subsets of phenotypically and functionally distinct McSCs exist in murine telogen HFs, CD34+ McSCs in the bulge/lower permanent portion (LPP) and CD34− McSCs in the secondary hair germ (SHG). Whether these subsets are maintained independently or exist in a developmental hierarchy is not yet known. Using Dct-H2BGFP mice, we analyzed the quiescent and proliferative properties of McSCs and melanocytes in anagen and telogen. We found unexpectedly that Kit+Nestin− quiescent melanocytes are maintained outside of the bulge/LPP region throughout anagen in addition to the Kit+Nestin+ quiescent melanocytes of the bulge/LPP. Both subpopulations express lower levels of melanocyte differentiation markers Mitf, Pax3, Dct, Tyrp1 and Tyr compared to differentiated melanocytes of the HF bulb/matrix. These results suggest that quiescent melanocytes localized in the outer root sheath, both in and below the bulge/LPP) retain the stem cell phenotype observed in quiescent McSCs during telogen. This finding has implications for maintenance of distinct subsets of McSCs throughout successive HF cycles.