Apoptotic dysregulation mediates stem cell competition and tissue regeneration.

Since adult stem cells are responsible for replenishing tissues throughout life, it is vital to understand how failure to undergo apoptosis can dictate stem cell behavior both intrinsically and non-autonomously. Here, we report that depletion of pro-apoptotic Bax protein bestows hair follicle stem cells with the capacity to eliminate viable neighboring cells by sequestration of TNFα in their membrane. This in turn induces apoptosis in "loser" cells in a contact-dependent manner. Examining the underlying mechanism, we find that Bax loss-of-function competitive phenotype is mediated by the intrinsic activation of NFκB. Notably, winner stem cells differentially respond to TNFα, owing to their elevated expression of TNFR2. Finally, we report that in vivo depletion of Bax results in an increased stem cell pool, accelerating wound-repair and de novo hair follicle regeneration. Collectively, we establish a mechanism of mammalian cell competition, which can have broad therapeutic implications for tissue regeneration and tumorigenesis.

Thy1 marks a distinct population of slow-cycling stem cells in the mouse epidermis.

The presence of distinct stem cells that maintain the interfollicular epidermis is highly debated. Here, we report a population of keratinocytes, marked by Thy1, in the basal layer of the interfollicular epidermis. We find that epidermal cells expressing differential levels of Thy1 display distinct transcriptional signatures. Thy1+ keratinocytes do not express T cell markers, express a unique transcriptional profile, cycle significantly slower than basal epidermal progenitors and display significant expansion potential in vitro. Multicolor lineage tracing analyses and mathematical modeling reveal that Thy1+ basal keratinocytes do not compete neutrally alike interfollicular progenitors and contribute long-term to both epidermal replenishment and wound repair. Importantly, ablation of Thy1+ cells strongly impairs these processes, thus indicating the non-redundant function of Thy1+ stem cells in the epidermis. Collectively, these results reveal a distinct stem cell population that plays a critical role in epidermal homeostasis and repair.

THY1-mediated mechanisms converge to drive YAP activation in skin homeostasis and repair.

Anchored cells of the basal epidermis constantly undergo proliferation in an overcrowded environment. An important regulator of epidermal proliferation is YAP, which can be controlled by both cell-matrix and cell-cell interactions. Here, we report that THY1, a GPI-anchored protein, inhibits epidermal YAP activity through converging molecular mechanisms. THY1 deficiency leads to increased adhesion by activating the integrin-ß1-SRC module. Notably, regardless of high cellular densities, the absence of THY1 leads to the dissociation of an adherens junction complex that enables the release and translocation of YAP. Due to increased YAP-dependent proliferation, Thy1-/- mice display enhanced wound repair and hair follicle regeneration. Taken together, our work reveals THY1 as a crucial regulator of cell-matrix and cell-cell interactions that controls YAP activity in skin homeostasis and regeneration.

Apoptotic cells represent a dynamic stem cell niche governing proliferation and tissue regeneration.

Stem cells (SCs) play a key role in homeostasis and repair. While many studies have focused on SC self-renewal and differentiation, little is known regarding the molecular mechanism regulating SC elimination and compensation upon loss. Here, we report that Caspase-9 deletion in hair follicle SCs (HFSCs) attenuates the apoptotic cascade, resulting in significant temporal delays. Surprisingly, Casp9-deficient HFSCs accumulate high levels of cleaved caspase-3 and are improperly cleared due to an essential caspase-3/caspase-9 feedforward loop. These SCs are retained in an apoptotic-engaged state, serving as mitogenic signaling centers by continuously releasing Wnt3 and instructing proliferation. Investigating the underlying mechanism, we reveal a caspase-3/Dusp8/p38 module responsible for Wnt3 induction, which operates in both normal and Casp9-deleted HFSCs. Notably, Casp9-deleted mice display accelerated wound repair and de novo hair follicle regeneration. Taken together, we demonstrate that apoptotic cells represent a dynamic SC niche, from which emanating signals drive SC proliferation and tissue regeneration.

Blimp1+ cells generate functional mouse sebaceous gland organoids in vitro.

Most studies on the skin focus primarily on the hair follicle and interfollicular epidermis, whereas little is known regarding the homeostasis of the sebaceous gland (SG). The SG has been proposed to be replenished by different pools of hair follicle stem cells and cells that resides in the SG base, marked by Blimp1. Here, we demonstrate that single Blimp1+ cells isolated from mice have the potential to generate SG organoids in vitro. Mimicking SG homeostasis, the outer layer of these organoids is composed of proliferating cells that migrate inward, undergo terminal differentiation and generating lipid-filled sebocytes. Performing confocal microscopy and mass-spectrometry, we report that these organoids exhibit known markers and a lipidomic profile similar to SGs in vivo. Furthermore, we identify a role for c-Myc in sebocyte proliferation and differentiation, and determine that SG organoids can serve as a platform for studying initial stages of acne vulgaris, making this a useful platform to identify potential therapeutic targets.

ARTS mediates apoptosis and regeneration of the intestinal stem cell niche.

Stem cells (SCs) play a pivotal role in fueling homeostasis and regeneration. While much focus has been given to self-renewal and differentiation pathways regulating SC fate, little is known regarding the specific mechanisms utilized for their elimination. Here, we report that the pro-apoptotic protein ARTS (a Septin4 isoform) is highly expressed in cells comprising the intestinal SC niche and that its deletion protects Lgr5+ and Paneth cells from undergoing apoptotic cell death. As a result, the Sept4/ARTS-/- crypt displays augmented proliferation and, in culture, generates massive cystic-like organoids due to enhanced Wnt/ß-catenin signaling. Importantly, Sept4/ARTS-/- mice exhibit resistance against intestinal damage in a manner dependent upon Lgr5+ SCs. Finally, we show that ARTS interacts with XIAP in intestinal crypt cells and that deletion of XIAP can abrogate Sept4/ARTS-/--dependent phenotypes. Our results indicate that intestinal SCs utilize specific apoptotic proteins for their elimination, representing a unique target for regenerative medicine.

Exiting the dark side: A vital role for Caspase-3 in Yap signaling.

Caspase-3 is known to play a critical function in the process of apoptosis. Recently, we have discovered a non-apoptotic role of Caspase-3 as a key regulator of cell proliferation and organ size. Caspase-3 cleaves α-Catenin, which sequesters Yes-associated protein 1 (Yap1) in the cytoplasm, thus facilitating the activation and nuclear translocation of Yap1. These findings reveal that the apoptotic machinery can be refocused to regulate cell proliferation and organ size.

Caspase-3 Regulates YAP-Dependent Cell Proliferation and Organ Size.

Apoptosis culminates in the activation of caspase-3, which plays an important role in implementing the cell death program. Here, we reveal a non-apoptotic role of caspase-3 as a key regulator of cell proliferation and organ size. Caspase-3 is specifically activated in the proliferating cells of the sebaceous gland, but does not instruct cell elimination. Deletion or chemical inhibition of caspase-3 diminishes cell proliferation, decreases cell number and reduces sebaceous gland size in vivo. Exploring the underlying mechanism, we demonstrate that α-catenin is cleaved by caspase-3, thus facilitating the activation and nuclear translocation of yes-associated protein (YAP), a vital regulator of organ size. Accordingly, activation of caspase-3 leads to YAP-dependent organ size augmentation. Finally, we show that X-linked inhibitor of apoptosis protein (XIAP) serves as an endogenous feedback antagonist for the caspase-3/YAP signaling module. Taken together, we report here a molecular mechanism wherein the apoptotic machinery is refocused to regulate cell proliferation and orchestrate organ size.

Tet Enzymes, Variants, and Differential Effects on Function.

Discovery of the ten-eleven translocation 1 (TET) methylcytosine dioxygenase family of enzymes, nearly 10 years ago, heralded a major breakthrough in understanding the epigenetic modifications of DNA. Initially described as catalyzing the oxidation of methyl cytosine (5mC) to hydroxymethyl cytosine (5hmC), it is now clear that these enzymes can also catalyze additional reactions leading to active DNA demethylation. The association of TET enzymes, as well as the 5hmC, with active regulatory regions of the genome has been studied extensively in embryonic stem cells, although these enzymes are expressed widely also in differentiated tissues. However, TET1 and TET3 are found as various isoforms, as a result of utilizing alternative regulatory regions in distinct tissues. Some of these isoforms, like TET2, lack the CXXC domain which probably has major implications on their recruitment to specific loci in the genome, while in certain contexts TET1 is seen paradoxically to repress transcription. In this review we bring together these novel aspects of the differential regulation of these Tet isoforms and the likely consequences on their activity.

Multifaceted Targeting of the Chromatin Mediates Gonadotropin-Releasing Hormone Effects on Gene Expression in the Gonadotrope.

Gonadotropin-releasing hormone (GnRH) stimulates the expression of multiple genes in the pituitary gonadotropes, most notably to induce synthesis of the gonadotropins, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), but also to ensure the appropriate functioning of these cells at the center of the mammalian reproductive endocrine axis. Aside from the activation of gene-specific transcription factors, GnRH stimulates through its membrane-bound receptor, alterations in the chromatin that facilitate transcription of its target genes. These include changes in the histone and DNA modifications, nucleosome positioning, and chromatin packaging at the regulatory regions of each gene. The requirements for each of these events vary according to the DNA sequence which determines the basal chromatin packaging at the regulatory regions. Despite considerable progress in this field in recent years, we are only beginning to understand some of the complexities involved in the role and regulation of this chromatin structure, including new modifications, extensive cross talk, histone variants, and the actions of distal enhancers and non-coding RNAs. This short review aims to integrate the latest findings on GnRH-induced alterations in the chromatin of its target genes, which indicate multiple and diverse actions. Understanding these processes is illuminating not only in the context of the activation of these hormones during the reproductive life span but may also reveal how aberrant epigenetic regulation of these genes leads to sub-fertility.

An epigenetic switch repressing Tet1 in gonadotropes activates the reproductive axis.

The TET enzymes catalyze conversion of 5-methyl cytosine (5mC) to 5-hydroxymethyl cytosine (5hmC) and play important roles during development. TET1 has been particularly well-studied in pluripotent stem cells, but Tet1-KO mice are viable, and the most marked defect is abnormal ovarian follicle development, resulting in impaired fertility. We hypothesized that TET1 might play a role in the central control of reproduction by regulating expression of the gonadotropin hormones, which are responsible for follicle development and maturation and ovarian function. We find that all three TET enzymes are expressed in gonadotrope-precursor cells, but Tet1 mRNA levels decrease markedly with completion of cell differentiation, corresponding with an increase in expression of the luteinizing hormone gene, Lhb We demonstrate that poorly differentiated gonadotropes express a TET1 isoform lacking the N-terminal CXXC-domain, which represses Lhb gene expression directly and does not catalyze 5hmC at the gene promoter. We show that this isoform is also expressed in other differentiated tissues, and that it is regulated by an alternative promoter whose activity is repressed by the liganded estrogen and androgen receptors, and by the hypothalamic gonadotropin-releasing hormone through activation of PKA. Its expression is also regulated by DNA methylation, including at an upstream enhancer that is protected by TET2, to allow Tet1 expression. The down-regulation of TET1 relieves its repression of the methylated Lhb gene promoter, which is then hydroxymethylated and activated by TET2 for full reproductive competence.

Isolation of Stem Cells and Progenitors from Mouse Epidermis.

The epidermis consists of several distinct compartments including the interfollicular epidermis (IFE), sweat glands, sebaceous glands (SGs), and the hair follicle (HF). While the IFE and SGs are in a constant state of self-renewal, the HF cycles between phases of growth, destruction, and rest. The hair follicle stem cells (HFSCs) that fuel this perpetual cycle have been well described and are located in a niche termed the bulge. These bulge SCs express markers such as CD34 and Keratin 15 (K15), enabling the isolation of these cells. Here, we describe a powerful method for isolating HFSCs and epidermal progenitors from mouse skin utilizing fluorescence activated cell-sorting (FACS). Upon isolation, cells can be expanded and utilized in various in vivo and in vitro models aimed at studying the function of these unique cells. © 2017 by John Wiley & Sons, Inc.

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin.

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

Transcriptional enhancers: Transcription, function and flexibility.

Active transcriptional enhancers are often transcribed to eRNAs, whose changing levels mirror those of the target gene mRNA. We discuss some of the reported functions of these eRNAs and their likely diversity to allow utilization of distinct cis regulatory regions to enhance transcription in diverse developmental and cellular contexts.

RNA transcribed from a distal enhancer is required for activating the chromatin at the promoter of the gonadotropin α-subunit gene.

Since the discovery that many transcriptional enhancers are transcribed into long noncoding RNAs termed "enhancer RNAs" (eRNAs), their putative role in enhancer function has been debated. Very recent evidence has indicted that some eRNAs play a role in initiating or activating transcription, possibly by helping recruit and/or stabilize binding of the general transcription machinery to the proximal promoter of their target genes. The distal enhancer of the gonadotropin hormone α-subunit gene, chorionic gonadotropin alpha (Cga), is responsible for Cga cell-specific expression in gonadotropes and thyrotropes, and we show here that it encodes two bidirectional nonpolyadenylated RNAs whose levels are increased somewhat by exposure to gonadotropin-releasing hormone but are not necessarily linked to Cga transcriptional activity. Knockdown of the more distal eRNA led to a drop in Cga mRNA levels, initially without effect on the forward eRNA levels. With time, however, the repression on the Cga increased, and the forward eRNA levels were suppressed also. We demonstrate that the interaction of the enhancer with the promoter is lost after eRNA knockdown. Dramatic changes also were seen in the chromatin, with an increase in total histone H3 occupancy throughout this region and a virtual loss of histone H3 Lys 4 trimethylation at the promoter following the eRNA knockdown. Moreover, histone H3 Lys 27 (H3K27) acetylation, which was found at both enhancer and promoter in wild-type cells, appeared to have been replaced by H3K27 trimethylation at the enhancer. Thus, the Cga eRNA mediates the physical interaction between these genomic regions and determines the chromatin structure of the proximal promoter to allow gene expression.

Divergent RNA binding specificity of yeast Puf2p.

PUF proteins bind mRNAs and regulate their translation, stability, and localization. Each PUF protein binds a selective group of mRNAs, enabling their coordinate control. We focus here on the specificity of Puf2p and Puf1p of Saccharomyces cerevisiae, which copurify with overlapping groups of mRNAs. We applied an RNA-adapted version of the DRIM algorithm to identify putative binding sequences for both proteins. We first identified a novel motif in the 3' UTRs of mRNAs previously shown to associate with Puf2p. This motif consisted of two UAAU tetranucleotides separated by a 3-nt linker sequence, which we refer to as the dual UAAU motif. The dual UAAU motif was necessary for binding to Puf2p, as judged by gel shift, yeast three-hybrid, and coimmunoprecipitation from yeast lysates. The UAAU tetranucleotides are required for optimal binding, while the identity and length of the linker sequences are less critical. Puf1p also binds the dual UAAU sequence, consistent with the prior observation that it associates with similar populations of mRNAs. In contrast, three other canonical yeast PUF proteins fail to bind the Puf2p recognition site. The dual UAAU motif is distinct from previously known PUF protein binding sites, which invariably possess a UGU trinucleotide. This study expands the repertoire of cis elements bound by PUF proteins and suggests new modes by which PUF proteins recognize their mRNA targets.

Identification and characterization of extensive intra-molecular associations between 3'-UTRs and their ORFs.

During eukaryotic translation, mRNAs may form intra-molecular interactions between distant domains. The 5'-cap and the polyA tail were shown to interact through their associated proteins, and this can induce physical compaction of the mRNA in vitro. However, the stability of this intra-molecular association in translating mRNAs and whether additional contacts exist in vivo are largely unknown. To explore this, we applied a novel approach in which several endogenous polysomal mRNAs from Saccharomyces cerevisiae were cleaved near their stop codon and the resulting 3'-UTR fragments were tested either for co-sedimentation or co-immunoprecipitation (co-IP) with their ORFs. In all cases a significant fraction of the 3'-UTR fragments sedimented similarly to their ORF-containing fragments, yet the extent of co-sedimentation differed between mRNAs. Similar observations were obtained by a co-IP assay. Interestingly, various treatments that are expected to interfere with the cap to polyA interactions had no effect on the co-sedimentation pattern. Moreover, the 3'-UTR appeared to co-sediment with different regions from within the ORF. Taken together, these results indicate extensive physical associations between 3'-UTRs and their ORFs that vary between genes. This implies that polyribosomal mRNAs are in a compact configuration in vivo.

The 3'-UTR mediates the cellular localization of an mRNA encoding a short plasma membrane protein.

Cotranslational synthesis of proteins into the endoplasmic reticulum is preceded by targeting of the translating mRNA once a signal peptide emerges from the ribosome exit tunnel. Many mRNAs, however, are unlikely to be targeted by this process because they encode proteins that do not contain a signal peptide or because they are too short to be recognized by the signal recognition particle. Herein we tested the possible involvement of the 3'-UTR in the localization of an mRNA that encodes a very short Saccharomyces cerevisiae protein (Pmp1). We found by ribosome density mapping, sedimentation analysis, differential centrifugation, and fluorescent in situ hybridization that the 3'-UTR is essential for the association of the transcript with membrane compartments. Fusion of the 3'-UTR to heterologous open reading frames conferred on them a sedimentation and cellular localization pattern resembling that of PMP1. Mutation analysis revealed that a repeating UG-rich sequence within the 3'-UTR is important for membrane association. Taken together, our results reveal an essential role for elements within the 3'-UTR in the localization of an mRNA that is likely to be ignored by the standard signal-dependant mechanism.