Developmental abnormalities, cancer, and premature aging each have been linked to defects in the DNA damage response (DDR). Mutations in the ATR checkpoint regulator cause developmental defects in mice (pregastrulation lethality) and humans (Seckel syndrome). Here we show that eliminating ATR in adult mice leads to defects in tissue homeostasis and the rapid appearance of age-related phenotypes, such as hair graying, alopecia, kyphosis, osteoporosis, thymic involution, fibrosis, and other abnormalities. Histological and genetic analyses indicate that ATR deletion causes acute cellular loss in tissues in which continuous cell proliferation is required for maintenance. Importantly, thymic involution, alopecia, and hair graying in ATR knockout mice were associated with dramatic reductions in tissue-specific stem and progenitor cells and exhaustion of tissue renewal and homeostatic capacity. In aggregate, these studies suggest that reduced regenerative capacity in adults via deletion of a developmentally essential DDR gene is sufficient to cause the premature appearance of age-related phenotypes.

Adult stem cells (SCs) reside in niches which balance self-renewal with lineage selection and progression during tissue homeostasis. Following injury, culture or transplantation, SCs outside their niche often display fate flexibility1-4. Here we show that super-enhancers5 underlie the identity, lineage commitment and plasticity of adult SCs in vivo. Using hair follicle (HF) as model, we map the global chromatin domains of HFSCs and their committed progenitors in their native microenvironments. We show that super-enhancers and their dense clusters (‘epicenters’) of transcription factor (TF) binding sites change upon lineage progression. New fate is acquired by decommissioning old and establishing new super-enhancers and/or epicenters, an auto-regulatory process that abates one master regulator subset while enhancing another. We further show that when outside their niche, either in vitro or in wound-repair, HFSCs dynamically remodel super-enhancers in response to changes in their microenvironment. Intriguingly, some key super-enhancers shift epicenters, enabling them to remain active and maintain a transitional state in an ever-changing transcriptional landscape. Finally, we identify SOX9 as a crucial chromatin rheostat of HFSC super-enhancers, and provide functional evidence that super-enhancers are dynamic, dense TF-binding platforms which are acutely sensitive to pioneer master regulators whose levels define not only spatial and temporal features of lineage-status, but also stemness, plasticity in transitional states and differentiation.

SUMMARY Aged skin heals wounds poorly, increasing susceptibility to infections. Restoring homeostasis after wounding requires the coordinated actions of epidermal and immune cells. Here we find that both intrinsic defects and communication with immune cells are impaired in aged keratinocytes, diminishing their efficiency in restoring the skin barrier after wounding. At the wound-edge, aged keratinocytes display reduced proliferation and migration. They also exhibit a dampened ability to transcriptionally activate epithelial-immune crosstalk regulators, including a failure to properly activate/maintain dendritic epithelial T-cells (DETCs), which promote re-epithelialization following injury. Probing mechanism, we find that aged keratinocytes near the wound edge don’t efficiently up-regulate Skints or activate STAT3. Notably, when epidermal Stat3, Skints or DETCs are silenced in young skin, re-epithelialization following wounding is perturbed. These findings underscore epithelial-immune crosstalk perturbations in general, and Skints in particular, as critical mediators in the age-related decline in wound-repair.

Chromosomal abnormalities are frequently caused by problems encountered during DNA replication. Although the ATR-Chk1 pathway has previously been implicated in preventing the collapse of stalled replication forks into doublestrand breaks (DSB), the importance of the response to fork collapse in ATR-deficient cells has not been well characterized. Herein, we demonstrate that, upon stalled replication,

Trp53 (p53) loss of function has previously been shown to rescue tissue maintenance and developmental defects resulting from DNA damage or DNA repair gene mutations1–12. Herein, we report that p53 deficiency significantly exacerbates tissue degeneration caused by mosaic deletion of the essential genome maintenance regulator ATR. Combined loss of ATR and p53 (p53−/−ATRmKO) led to severe defects in hair follicle regeneration, localized inflammation (Mac1+Gr1+ infiltrates), accelerated deterioration of the intestinal epithelium, and synthetic lethality in adult mice. Tissue degeneration in p53−/−ATRmKO mice was characterized by the accumulation of cells maintaining high levels of DNA damage. Moreover, the elevated presence of these damaged cells in both progenitor and downstream compartments in the skin coincided with delayed compensatory tissue renewal from residual ATR-expressing cells. Together, our results indicate that combined loss of ATR and p53 in adult mice leads to the accumulation of highly damaged cells, which consequently impose a barrier to regeneration from undamaged progenitors.

INTRODUCTION Adult tissues must balance growth and differentiation to develop and maintain homeostasis. Excessive differentiation can lead to aging and poor wound healing. Too much growth is observed in hyperproliferative disorders and cancers. How tissue imbalances arise in disease states is poorly understood. Skin is an excellent system for understanding the importance of this balance. Essential for keeping harmful microbes out and retaining body fluids, the skin barrier is maintained by an inner layer of proliferative basal progenitors, which generate a constant outward flux of terminally differentiating cells. It is known that when epidermal progenitors accumulate mutations that will give rise to malignancy, they change their program of gene expression. However, the extent to which cancer progression involves a gain of proliferation versus a loss of differentiation is unclear. A detailed molecular knowledge of how normal basal epidermal progenitors transition from a proliferative, undifferentiated state to a terminally differentiated state allows us to investigate how this process goes awry in a tumorigenic state. We use a genetic screen to identify which of the gene changes that occur in both early cell commitment and cancer are integral to maintaining the balance between growth and differentiation. RATIONALE Epithelial cancers are among the most prevalent and life-threatening cancers worldwide. Despite intensive research, the mechanisms by which these cancers evade regulatory systems working to balance differentiation and proliferation remain poorly understood. To provide new insights into how malignancies arise and how this might be exploited in advancing cancer therapeutics, we tackled this problem in the developing skin where these regulatory systems are established. RESULTS To understand how the balance between growth and differentiation is controlled, we first devised a strategy to transcriptionally profile epidermal stem cells and their terminally differentiating progeny. Using this method, we defined the earliest molecular events associated with the commitment of epidermal progenitors to their differentiation program. Of the many changes that occur, we focused on the cohort of genes that are also mutated in human epithelial cancers. To sift through which of these genes are functional drivers in cancers and how they perturb homeostasis, we conducted an in vivo epidermal RNA interference (RNAi) screen to identify candidates that are selectively enriched or depleted in proliferative progenitors relative to their differentiating progeny. We focused on PEX11b, a protein associated with peroxisomes, organelles involved in fatty acid and energy metabolism. PEX11b deficiency compromised epidermal terminal differentiation and barrier formation. Without PEX11b, peroxisomes functioned but failed to localize and therefore segregate properly during mitosis. Probing deeper, we discovered that in normal cells, peroxisomes take on stereotyped positions during mitosis. However, after depletion of PEX11b, peroxi...

DNA synthesis is a remarkably vulnerable phase in the cell cycle. In addition to introduction of errors during semi-conservative replication, the inherently labile structure of the replication fork, as well as numerous pitfalls encountered in the course of fork progression, make the normally stable double stranded molecule susceptible to collapse and recombination. As described in this issue, maintenance of genome integrity in the face of such events is essential to prevent the premature onset of agerelated diseases. At the organismal level, the roles for such maintenance are numerous; however, the preservation of stem and progenitor cell pools may be particularly important as indicated by several genetically engineered mouse models. Stresses on stem and progenitor cell pools, in the form of telomere shortening (Terc −/− ) or other genome maintenance failures (ATR mKO , Ku86 −/− , LIG4 Y288C , XPD R722W/R722W , etc.), have been shown to degrade tissue renewal capacity and accelerate the appearance of age-related phenotypes. In the case of telomere shortening, exhaustion of replicative potential appears to be at least partially dependent on the cell cycle regulatory component of the DNA damage response. Therefore, both the genome maintenance mechanisms that counter DNA damage and the cell cycle checkpoint responses to damage strongly influence the onset of age-related diseases and do so, at least in part, by affecting long-term stem and progenitor cell potential.

To spatially co-exist and differentially specify fates within developing tissues, morphogenetic cues must be correctly positioned and interpreted. Here, we investigate mouse hair follicle development to understand how morphogens operate within closely spaced, fate-diverging progenitors. Coupling transcriptomics with genetics, we show that emerging hair progenitors produce both WNTs and WNT inhibitors. Surprisingly, however, instead of generating a negative feedback loop, the signals oppositely polarize, establishing sharp boundaries and consequently a short-range morphogen gradient that we show is essential for three-dimensional pattern formation. By establishing a morphogen gradient at the cellular level, signals become constrained. The progenitor preserves its WNT signaling identity and maintains WNT signaling with underlying mesenchymal neighbors, while its overlying epithelial cells become WNT-restricted. The outcome guarantees emergence of adjacent distinct cell types to pattern the tissue.