Brice E. Keyes

RNAi screens in mice identify physiological regulators of oncogenic growth

Tissue growth is the multifaceted outcome of a cell’s intrinsic capabilities and its interactions with the surrounding environment. Decoding these complexities is essential for understanding human development and tumorigenesis. Here we tackle this problem by carrying out the first genome-wide RNA-interference-mediated screens in mice. Focusing on skin development and oncogenic (HrasG12V-induced) hyperplasia, our screens uncover previously unknown as well as anticipated regulators of embryonic epidermal growth. Among the top oncogenic screen hits are Mllt6 and the Wnt effector β-catenin, which maintain HrasG12V-dependent hyperproliferation. We also expose β-catenin as an unanticipated antagonist of normal epidermal growth, functioning through Wnt-independent intercellular adhesion. Finally, we validate functional significance in mouse and human cancers, thereby establishing the feasibility of in vivo mammalian genome-wide investigations to dissect tissue development and tumorigenesis. By documenting some oncogenic growth regulators, we pave the way for future investigations of other hits and raise promise for unearthing new targets for cancer therapies.

SOX9: a stem cell transcriptional regulator of secreted niche signaling factors

Hair follicles (HFs) undergo cyclical periods of growth, which are fueled by stem cells (SCs) at the base of the resting follicle. HF-SC formation occurs during HF development and requires transcription factor SOX9. Whether and how SOX9 functions in HF-SC maintenance remain unknown. By conditionally targeting Sox9 in adult HF-SCs, we show that SOX9 is essential for maintaining them. SOX9-deficient HF-SCs still transition from quiescence to proliferation and launch the subsequent hair cycle. However, once activated, bulge HF-SCs begin to differentiate into epidermal cells, which naturally lack SOX9. In addition, as HF-SC numbers dwindle, outer root sheath production is not sustained, and HF downgrowth arrests prematurely. Probing the mechanism, we used RNA sequencing (RNA-seq) to identify SOX9-dependent transcriptional changes and chromatin immunoprecipitation (ChIP) and deep sequencing (ChIP-seq) to identify SOX9-bound genes in HF-SCs. Intriguingly, a large cohort of SOX9-sensitive targets encode extracellular factors, most notably enhancers of Activin/pSMAD2 signaling. Moreover, compromising Activin signaling recapitulates SOX9-dependent defects, and Activin partially rescues them. Overall, our findings reveal roles for SOX9 in regulating adult HF-SC maintenance and suppressing epidermal differentiation in the niche. In addition, our studies expose a role for SCs in coordinating their own behavior in part through non-cell-autonomous signaling within the niche.

Nfatc1 orchestrates aging in hair follicle stem cells

Significance Signs of aging often first appear in our skin and hair. As animals age, hair follicles spend more time resting instead of generating hair. Here we show that this decline is rooted in age-related changes in systemic, local, and intrinsic factors, which collaborate to reduce hair follicle stem cell (HFSC) activity. We uncover a unique and hitherto-undescribed age-related role for bone morphogenic protein signaling and a downstream effector, nuclear factor of activated T-cell c1 (NFATc1). In young stem cells, NFATc1 is on when they are quiescent and wanes when they make hair. In aging follicles, NFATc1 and its target genes remain high too long. Importantly, NFATc1 inhibitors restore youthful behavior to aging HFSCs, providing unique insights into age-related changes in skin physiology. Hair production is fueled by stem cells (SCs), which transition between cyclical bouts of rest and activity. Here, we explore why hair growth wanes with age. We show that aged hair follicle SCs (HFSCs) in mice exhibit enhanced resting and abbreviated growth phases and are delayed in response to tissue-regenerating cues. Aged HFSCs are poor at initiating proliferation and show diminished self-renewing capacity upon extensive use. Only modestly restored by parabiosis, these features are rooted in elevated cell-intrinsic sensitivity and local elevation in bone morphogenic protein (BMP) signaling. Transcriptional profiling presents differences consistent with defects in aged HFSC activation. Notably, BMP-/calcium-regulated, nuclear factor of activated T-cell c1 (NFATc1) in HFSCs becomes recalcitrant to its normal down-regulating cues, and NFATc1 ChIP-sequencing analyses reveal a marked enrichment of NFATc1 target genes within the age-related signature. Moreover, aged HFSCs display more youthful levels of hair regeneration when BMP and/or NFATc1 are inhibited. These results provide unique insights into how skin SCs age.

Diverse Functions of Spindle Assembly Checkpoint Genes in Saccharomyces cerevisiae

The spindle assembly checkpoint regulates the metaphase-to-anaphase transition from yeast to humans. We examined the genetic interactions with four spindle assembly checkpoint genes to identify nonessential genes involved in chromosome segregation, to identify the individual roles of the spindle assembly checkpoint genes within the checkpoint, and to reveal potential complexity that may exist. We used synthetic genetic array (SGA) analysis using spindle assembly checkpoint mutants mad1, mad2, mad3, and bub3. We found 228 synthetic interactions with the four spindle assembly checkpoint mutants with substantial overlap in the spectrum of interactions between mad1, mad2, and bub3. In contrast, there were many synthetic interactions that were common to mad1, mad2, and bub3 that were not shared by mad3. We found shared interactions between pairs of spindle assembly checkpoint mutants, suggesting additional complexity within the checkpoint and unique interactions for all of the spindle assembly checkpoint genes. We show that most genes in the interaction network, including ones with unique interactions, affect chromosome transmission or microtubule function, suggesting that the complexity of interactions reflects diverse roles for the checkpoint genes within the checkpoint. Our analysis expands our understanding of the spindle assembly checkpoint and identifies new candidate genes with possible roles in chromosome transmission and mitotic spindle function.

BMP signaling and its pSMAD1/5 target genes differentially regulate hair follicle stem cell lineages.

Hair follicle stem cells (HFSCs) and their transit amplifying cell (TAC) progeny sense BMPs at defined stages of the hair cycle to control their proliferation and differentiation. Here, we exploit the distinct spatial and temporal localizations of these cells to selectively ablate BMP signaling in each compartment and examine its functional role. We find that BMP signaling is required for HFSC quiescence and to promote TAC differentiation along different lineages as the hair cycle progresses. We also combine in vivo genome-wide chromatin immunoprecipitation and deep-sequencing, transcriptional profiling, and loss-of-function genetics to define BMP-regulated genes. We show that some pSMAD1/5 targets, like Gata3, function specifically in TAC lineage-progression. Others, like Id1 and Id3, function in both HFSCs and TACs, but in distinct ways. Our study therefore illustrates the complex differential roles that a key signaling pathway can play in regulation of closely related stem/progenitor cells within the context of their overall niche.

ETS family transcriptional regulators drive chromatin dynamics and malignancy in squamous cell carcinomas.

Tumor-initiating stem cells (SCs) exhibit distinct patterns of transcription factors and gene expression compared to healthy counterparts. Here, we show that dramatic shifts in large open-chromatin domain (super-enhancer) landscapes underlie these differences and reflect tumor microenvironment. By in vivo super-enhancer and transcriptional profiling, we uncover a dynamic cancer-specific epigenetic network selectively enriched for binding motifs of a transcription factor cohort expressed in squamous cell carcinoma SCs (SCC-SCs). Many of their genes, including Ets2 and Elk3, are themselves regulated by SCC-SC super-enhancers suggesting a cooperative feed-forward loop. Malignant progression requires these genes, whose knockdown severely impairs tumor growth and prohibits progression from benign papillomas to SCCs. ETS2-deficiency disrupts the SCC-SC super-enhancer landscape and downstream cancer genes while ETS2-overactivation in epidermal-SCs induces hyperproliferation and SCC super-enhancer-associated genes Fos, Junb and Klf5. Together, our findings unearth an essential regulatory network required for the SCC-SC chromatin landscape and unveil its importance in malignant progression. DOI: http://dx.doi.org/10.7554/eLife.10870.001

Spatiotemporal antagonism in mesenchymal-epithelial signaling in sweat versus hair fate decision.

How to grow hair or sweat glands Unlike other mammals that must pant or seek shade or water when overheated, humans are able to self-cool to tolerate extreme heat. Sweat glands, which enable humans to run in marathons, are instrumental for this remarkable feat. Lu et al. investigated skin appendage diversity during development of the furry backs and sweaty paws of mice (see the Perspective by Lai and Chuong). They also examined human skin, which is capable of making both hairs and sweat glands in the same area of the body. Epithelialmesenchymal interactions, with varied signaling pathways that act at specific times in development, are key to producing different skin appendages for adaptation to the environment. Science, this issue p. 10.1126/science.aah6102; see also p. 1533 Mice and humans exploit the epithelial-mesenchymal circuitry in different ways to direct sweat gland or hair follicle generation. INTRODUCTION Across the vertebrate kingdom, epithelial appendages—including mammary, sweat, and salivary glands; hair follicles (HFs); teeth; scales; and feathers—begin to form during embryogenesis when WNT signaling instructs progenitors within the epithelial sheet to organize into morphologically similar placodes. Most animals restrict these epidermal appendages to distinct body regions. This paradigm shifted late in mammalian evolution; the dual presence of HFs and eccrine sweat glands (SwGs) in the skin is a recent acquisition of primates. Classical tissue recombination experiments from the 1960s revealed that mesenchyme directs the divergent downstream events that determine appendage specification and patterning. Relatively little is known about the specific spatiotemporal cross-talk and molecular mechanisms that underlie epithelial fate specification in response to mesenchymal signals. Elucidating the epithelial-mesenchymal cross-talk involved in regional skin appendage specification is integral to understanding how humans have adjusted these mechanisms to endow them with a greater capacity than that of their hairy cousins to live in diverse environments. RATIONALE The acquisition of SwGs and their importance in thermoregulation and water balance are underscored by human patients who suffer from the life-threatening condition of lacking SwGs, either from loss in severe burns or from genetic disorders. Conversely, a gain-of-function variant that elevates SwG numbers has been expanding among the Southeast Asian population, where excessive SwGs are desirable. By elucidating the underlying mechanisms that distinguish humans from other mammals in their ability to make both glands and follicles over their body skin, our findings could pave the way for future therapeutic advances in skin regeneration with dual appendages. RESULTS Using mouse as a model, we explored the differences between the back skin mesenchyme, which is only competent to specify HFs, and the foot skin mesenchyme, which can only make SwGs. Using genome-wide analyses and functional studies, we discovered that just after the formation of morphologically similar epidermal buds, appendage choice is determined through regional skin differences in mesenchymal expression of bone morphogenetic proteins (BMPs). Probing into mechanisms, we showed that when BMPs are elevated in foot skin mesenchyme, BMP signaling is activated in both dermis and epidermis. This triggers a cascade of downstream signaling events. WNT signaling is elevated in the dermis and reduced in the epidermis. Mesenchymal fibroblast growth factors (FGFs) appear and affect the overlying epithelium. These converging pathways lead to suppression of sonic hedgehog (SHH) in the epithelium. This BMP:SHH antagonism within the epithelial bud specifies SwG fate and prevents HF fate. Moreover, by manipulating gene expression in vivo at specific developmental stages, we demonstrated that this signaling circuitry acts only within a narrow window of time during mouse embryogenesis. Thus, when SHH is ectopically expressed in foot skin epithelium during the permissive phase, HF-specific gene expression is up-regulated in the epithelial bud, whereas SHH signaling in the mesenchyme stimulates expression of BMP antagonists, further suppressing local BMP signaling and blocking SwG fate. In human skins, this antagonistic interplay of BMP:SHH signaling occurs temporally, in addition to spatially. The first bud waves are specified as HFs, and then a tipping in the balance of BMP:SHH signaling results in the last waves of buds becoming SwGs. CONCLUSION Our findings revealed a differential impact of BMP signaling on appendage fate specification that has ancient roots and occurs repeatedly throughout vertebrate evolution. In the evolutionary developmental biology view of BMP signaling and fate specification of integument, chicken scales and mammalian SwGs require BMP signaling to specify their fate, whereas feathers and HFs must suppress it. Our discovery of BMP-SHH antagonism in bud fate choice uncovered additional evolutionary parallels, this time between two even more distantly related epidermal appendages, the mammalian SwG and the fly wing. Our studies provide new insights into how elevated mesenchymal BMP signaling triggers a self-perpetuating molecular cascade that culminates in silencing of SHH signaling to suppress one appendage fate and specify another. In most mammals, the BMP:SHH antagonism is regulated spatially. Humans, however, have evolved to regulate it temporally, endowing them with greater ability to run marathons and survive in extreme climates. BMP-SHH antagonism specifies SwG versus HF fate. To specify SwGs, mesenchymal-derived BMPs and FGFs signal to epithelial buds and suppress epithelially derived SHH production. Conversely, hair follicles are specified when mesenchymal BMP signaling is inhibited, permitting SHH production. This antagonism is spatially restricted in most mammals but temporally regulated in humans, permitting the presence of HFs (pink), SwGs (purple), or both HFs and SwGs (pink with purple droplet) throughout our body skin. The gain of eccrine sweat glands in hairy body skin has empowered humans to run marathons and tolerate temperature extremes. Epithelial-mesenchymal cross-talk is integral to the diverse patterning of skin appendages, but the molecular events underlying their specification remain largely unknown. Using genome-wide analyses and functional studies, we show that sweat glands are specified by mesenchymal-derived bone morphogenetic proteins (BMPs) and fibroblast growth factors that signal to epithelial buds and suppress epithelial-derived sonic hedgehog (SHH) production. Conversely, hair follicles are specified when mesenchymal BMP signaling is blocked, permitting SHH production. Fate determination is confined to a critical developmental window and is regionally specified in mice. In contrast, a shift from hair to gland fates is achieved in humans when a spike in BMP silences SHH during the final embryonic wave(s) of bud morphogenesis.

A Redundant Function for the N-terminal Tail of Ndc80 in Kinetochore-microtubule Interaction in Saccharomyces cerevisiae

The N-terminal tail of Ndc80 is essential for kinetochore–microtubule binding in human cells but is not required for viability in yeast. We show that the yeast Ndc80 tail is required for timely mitotic progression and accurate chromosome segregation. The tail is essential when cells are limited for Dam1, demonstrating a redundant function for the Ndc80 and Dam1 complexes in vivo.

Sister Chromatids Segregate at Mitosis Without Mother–Daughter Bias in Saccharomyces cerevisiae

There is evidence accumulating for nonrandom segregation of one or more chromosomes during mitosis in different cell types. We use cell synchrony and two methods to show that all chromatids of budding yeast segregate randomly and that there is no mother–daughter bias with respect to Watson and Crick-containing strands of DNA.

Stem cells: Aging and transcriptional fingerprints

Keyes and Fuchs discuss the decline in stem cell renewal and function with aging and the ensuing consequences on tissue homeostasis and regeneration.