Lineage-specific 3D genome organization is assembled at multiple scales by IKAROS.

A generic level of chromatin organization generated by the interplay between cohesin and CTCF suffices to limit promiscuous interactions between regulatory elements, but a lineage-specific chromatin assembly that supersedes these constraints is required to configure the genome to guide gene expression changes that drive faithful lineage progression. Loss-of-function approaches in B cell precursors show that IKAROS assembles interactions across megabase distances in preparation for lymphoid development. Interactions emanating from IKAROS-bound enhancers override CTCF-imposed boundaries to assemble lineage-specific regulatory units built on a backbone of smaller invariant topological domains. Gain of function in epithelial cells confirms IKAROS' ability to reconfigure chromatin architecture at multiple scales. Although the compaction of the Igκ locus required for genome editing represents a function of IKAROS unique to lymphocytes, the more general function to preconfigure the genome to support lineage-specific gene expression and suppress activation of extra-lineage genes provides a paradigm for lineage restriction.

A double-negative thymocyte-specific enhancer augments Notch1 signaling to direct early T cell progenitor expansion, lineage restriction and ß-selection.

T cell differentiation requires Notch1 signaling. In the present study, we show that an enhancer upstream of Notch1 active in double-negative (DN) mouse thymocytes is responsible for raising Notch1 signaling intrathymically. This enhancer is required to expand multipotent progenitors intrathymically while delaying early differentiation until lineage restrictions have been established. Early thymic progenitors lacking the enhancer show accelerated differentiation through the DN stages and increased frequency of B, innate lymphoid (IL) and natural killer (NK) cell differentiation. Transcription regulators for T cell lineage restriction and commitment are expressed normally, but IL and NK cell gene expression persists after T cell lineage commitment and T cell receptor ß VDJ recombination, Cd3 expression and ß-selection have been impaired. This Notch1 enhancer is inactive in double-positive (DP) thymocytes. Its aberrant reactivation at this stage in Ikaros mutants is required for leukemogenesis. Thus, the DN-specific Notch1 enhancer harnesses the regulatory architecture of DN and DP thymocytes to achieve carefully orchestrated changes in Notch1 signaling required for early lineage restrictions and normal T cell differentiation.

Functional interactions between Mi-2ß and AP1 complexes control response and recovery from skin barrier disruption.

Keratinocytes respond to environmental signals by eliciting induction of genes that preserve skin's integrity. Here we show that the transcriptional response to stress signaling is supported by short-lived epigenetic changes. Comparison of chromatin accessibility and transcriptional changes induced by barrier disruption or by loss of the nucleosome remodeler Mi-2ß identified their striking convergence in mouse and human keratinocytes. Mi-2ß directly repressed genes induced by barrier disruption by restricting AP1-enriched promoter-distal sites, occupied by Mi-2ß and JUNB at steady state and by c-JUN after Mi-2ß depletion or stress signaling. Barrier disruption led to a modest reduction in Mi-2ß expression and a further selective reduction of Mi-2ß localization at stress response genes, possibly through competition with activated c-JUN. Consistent with a repressive role at stress response genes, genetic ablation of Mi-2ß did not prevent reestablishment of barrier integrity but was required for return to homeostasis. Thus, a competition between Mi-2ß-repressive and activating AP1 complexes may permit rapid transcriptional response to and resolution from stress signaling.

Comparative evidence for the independent evolution of hair and sweat gland traits in primates.

Humans differ in many respects from other primates, but perhaps no derived human feature is more striking than our naked skin. Long purported to be adaptive, humans' unique external appearance is characterized by changes in both the patterning of hair follicles and eccrine sweat glands, producing decreased hair cover and increased sweat gland density. Despite the conspicuousness of these features and their potential evolutionary importance, there is a lack of clarity regarding how they evolved within the primate lineage. We thus collected and quantified the density of hair follicles and eccrine sweat glands from five regions of the skin in three species of primates: macaque, chimpanzee and human. Although human hair cover is greatly attenuated relative to that of our close relatives, we find that humans have a chimpanzee-like hair density that is significantly lower than that of macaques. In contrast, eccrine gland density is on average 10-fold higher in humans compared to chimpanzees and macaques, whose density is strikingly similar. Our findings suggest that a decrease in hair density in the ancestors of humans and apes was followed by an increase in eccrine gland density and a reduction in fur cover in humans. This work answers long-standing questions about the traits that make human skin unique and substantiates a model in which the evolution of expanded eccrine gland density was exclusive to the human lineage.

Direct control of regulatory T cells by keratinocytes.

Environmental challenges to epithelial cells trigger gene expression changes that elicit context-appropriate immune responses. We found that the chromatin remodeler Mi-2ß controls epidermal homeostasis by regulating the genes involved in keratinocyte and immune-cell activation to maintain an inactive state. Mi-2ß depletion resulted in rapid deployment of both a pro-inflammatory and an immunosuppressive response in the skin. A key target of Mi-2ß in keratinocytes is the pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP). Loss of TSLP receptor (TSLPR) signaling specifically in regulatory T (Treg) cells prevented their activation and permitted rapid progression from a skin pro-inflammatory response to a lethal systemic condition. Thus, in addition to their well-characterized role in pro-inflammatory responses, keratinocytes also directly support immune-suppressive responses that are critical for re-establishing organismal homeostasis.

A genetic basis of variation in eccrine sweat gland and hair follicle density.

Among the unique features of humans, one of the most salient is the ability to effectively cool the body during extreme prolonged activity through the evapotranspiration of water on the skin's surface. The evolution of this novel physiological ability required a dramatic increase in the density and distribution of eccrine sweat glands relative to other mammals and a concomitant reduction of body hair cover. Elucidation of the genetic underpinnings for these adaptive changes is confounded by a lack of knowledge about how eccrine gland fate and density are specified during development. Moreover, although reciprocal changes in hair cover and eccrine gland density are required for efficient thermoregulation, it is unclear if these changes are linked by a common genetic regulation. To identify pathways controlling the relative patterning of eccrine glands and hair follicles, we exploited natural variation in the density of these organs between different strains of mice. Quantitative trait locus mapping identified a large region on mouse Chromosome 1 that controls both hair and eccrine gland densities. Differential and allelic expression analysis of the genes within this interval coupled with subsequent functional studies demonstrated that the level of En1 activity directs the relative numbers of eccrine glands and hair follicles. These findings implicate En1 as a newly identified and reciprocal determinant of hair follicle and eccrine gland density and identify a pathway that could have contributed to the evolution of the unique features of human skin.

Eos Is Redundant for Regulatory T Cell Function but Plays an Important Role in IL-2 and Th17 Production by CD4+ Conventional T Cells.

Eos belongs to the Ikaros family of transcription factors. It was reported to be a regulatory T cell (Treg) signature gene, to play a critical role in Treg suppressor functions, and to maintain Treg stability. We used mice with a global deficiency in Eos to re-examine the role of Eos expression in both Tregs and conventional T cells (Tconvs). Tregs from Eos-deficient (Eos(-/-)) mice developed normally, displayed a normal Treg phenotype, and exhibited normal suppressor function in vitro. Eos(-/-) Tregs were as effective as Tregs from wild-type (WT) mice in suppressing inflammation in a model of inflammatory bowel disease. Bone marrow (BM) from Eos(-/-) mice was as effective as that from WT mice in controlling T cell activation when used to reconstitute immunodeficient mice in the presence of scurfy fetal liver cells. Surprisingly, Eos was expressed in activated Tconvs and was required for IL-2 production, CD25 expression, and proliferation in vitro by CD4(+) Tconvs. Eos(-/-) mice developed more severe experimental autoimmune encephalomyelitis than WT mice, displayed increased numbers of effector T cells in the periphery and CNS, and amplified IL-17 production. In conclusion, our studies are not consistent with a role for Eos in Treg development and function but demonstrate that Eos plays an important role in the activation and differentiation of Tconvs.

Earlier-born secondary hair follicles exhibit phenotypic plasticity.

The mouse pelage is composed of four distinct hair types. The fact that the follicles that generate these hair types form in successive waves during late embryonic development suggested the model that distinct epigenetic states of the inductive mesenchyme fixed when the follicles are formed specify the distinctive hair morphologies. This model is inconsistent with the observation that many follicles produce different hair types in successive hair cycles. In this study, the characteristics of the hair follicles that switch between the production of different hair types were examined. These follicles were born earlier than those that do not switch between hair types and made longer hairs. They also expressed a higher level of Sox2 in the dermal papilla and had more DP cells per follicle. These observations are consistent with the hypothesis that different birthdates specify the potential of different follicles. However, rather than directly specifying hair type, birthdate correlates with three types: guard hairs, a plastic population that can make awl, auchene or zigzag hairs, and a population that normally makes only zigzag hairs. Although Sox2 expression levels in the DP identify this subset during the morphogenetic cycle, Sox2 expression is not a fixed epigenetic state specified when the follicle is first formed.

The dermal papilla: an instructive niche for epithelial stem and progenitor cells in development and regeneration of the hair follicle.

The dermal papilla (DP) of the hair follicle is both a chemical and physical niche for epithelial progenitor cells that regenerate the cycling portion of the hair follicle and generate the hair shaft. Here, we review experiments that revealed the importance of the DP in regulating the characteristics of the hair shaft and frequency of hair follicle regeneration. More recent work showed that the size of this niche is dynamic and actively regulated and reduction in DP cell number per follicle is sufficient to cause hair thinning and loss. The formation of the DP during follicle neogenesis provides a context to contemplate the mechanisms that maintain DP size and the potential to exploit these processes for hair preservation or restoration.

Harmonic Scalpel versus electrocautery and surgical clips in head and neck free-flap harvesting.

We sought to determine the safety and utility of Harmonic Scalpel-assisted free-flap harvesting as an alternative to a combined electrocautery and surgical clip technique. The medical records of 103 patients undergoing radial forearm free-flap reconstruction (105 free flaps) for head and neck surgical defects between 2006 and 2008 were reviewed. The use of bipolar electrocautery and surgical clips for division of small perforating vessels (n = 53) was compared to ultrasonic energy (Harmonic Scalpel; Ethicon Endo-Surgery, Inc., Cincinnati, Ohio) (n = 52) free-tissue harvesting techniques. Flap-harvesting time was reduced with the use of the Harmonic Scalpel when compared with electrocautery and surgical clip harvest (31.4 vs. 36.9 minutes, respectively; p = 0.06). Two patients who underwent flap harvest with electrocautery and surgical clips developed postoperative donor site hematomas, whereas no donor site complications were noted in the Harmonic Scalpel group. Recipient site complication rates for infection, fistula, and hematoma were similar for both harvesting techniques (p = 0.77). Two flap failures occurred in the clip-assisted radial forearm free-flap harvest group, and none in the Harmonic Scalpel group. Median length of hospitalization was significantly reduced for patients who underwent free-flap harvest with the Harmonic Scalpel when compared with the other technique (7 vs. 8 days; p = 0.01). The Harmonic Scalpel is safe, and its use is feasible for radial forearm free-flap harvest.

The "skinny" on Wnt signaling in stem cells.

Although previous reports suggested that canonical Wnt signaling has opposing effects on epidermal and hair follicle stem cells, two recent papers (Choi et al., 2013; Lim et al., 2013) now show that Wnt signaling promotes proliferation in both stem cell populations, revealing new insights into regeneration of both skin compartments.

Transcriptional regulation of the Ikzf1 locus.

Ikaros is a critical regulator of lymphocyte development and homeostasis; thus, understanding its transcriptional regulation is important from both developmental and clinical perspectives. Using a mouse transgenic reporter approach, we functionally characterized a network of highly conserved cis-acting elements at the Ikzf1 locus. We attribute B-cell and myeloid but not T-cell specificity to the main Ikzf1 promoter. Although this promoter was unable to counter local chromatin silencing effects, each of the 6 highly conserved Ikzf1 intronic enhancers alleviated silencing. Working together, the Ikzf1 enhancers provided locus control region activity, allowing reporter expression in a position and copy-independent manner. Only 1 of the Ikzf1 enhancers was responsible for the progressive upregulation of Ikaros expression from hematopoietic stem cells to lymphoid-primed multipotent progenitors to T-cell precursors, which are stages of differentiation dependent on Ikaros for normal outcome. Thus, Ikzf1 is regulated by both epigenetic and transcriptional factors that target its enhancers in both redundant and specific fashions to provide an expression profile supportive of normal lymphoid lineage progression and homeostasis. Mutations in the Ikzf1 regulatory elements and their interacting factors are likely to have adverse effects on lymphopoiesis and contribute to leukemogenesis.

Dermal papilla cell number specifies hair size, shape and cycling and its reduction causes follicular decline.

Although the hair shaft is derived from the progeny of keratinocyte stem cells in the follicular epithelium, the growth and differentiation of follicular keratinocytes is guided by a specialized mesenchymal population, the dermal papilla (DP), that is embedded in the hair bulb. Here we show that the number of DP cells in the follicle correlates with the size and shape of the hair produced in the mouse pelage. The same stem cell pool gives rise to hairs of different sizes or types in successive hair cycles, and this shift is accompanied by a corresponding change in DP cell number. Using a mouse model that allows selective ablation of DP cells in vivo, we show that DP cell number dictates the size and shape of the hair. Furthermore, we confirm the hypothesis that the DP plays a crucial role in activating stem cells to initiate the formation of a new hair shaft. When DP cell number falls below a critical threshold, hair follicles with a normal keratinocyte compartment fail to generate new hairs. However, neighbouring follicles with a few more DP cells can re-enter the growth phase, and those that do exploit an intrinsic mechanism to restore both DP cell number and normal hair growth. These results demonstrate that the mesenchymal niche directs stem and progenitor cell behaviour to initiate regeneration and specify hair morphology. Degeneration of the DP population in mice leads to the types of hair thinning and loss observed during human aging, and the results reported here suggest novel approaches to reversing hair loss.

Modeling recent human evolution in mice by expression of a selected EDAR variant.

An adaptive variant of the human Ectodysplasin receptor, EDARV370A, is one of the strongest candidates of recent positive selection from genome-wide scans. We have modeled EDAR370A in mice and characterized its phenotype and evolutionary origins in humans. Our computational analysis suggests the allele arose in central China approximately 30,000 years ago. Although EDAR370A has been associated with increased scalp hair thickness and changed tooth morphology in humans, its direct biological significance and potential adaptive role remain unclear. We generated a knockin mouse model and find that, as in humans, hair thickness is increased in EDAR370A mice. We identify new biological targets affected by the mutation, including mammary and eccrine glands. Building on these results, we find that EDAR370A is associated with an increased number of active eccrine glands in the Han Chinese. This interdisciplinary approach yields unique insight into the generation of adaptive variation among modern humans.

Dynasplint for the management of trismus after treatment of upper aerodigestive tract cancer: a retrospective study.

In order to evaluate the Dynasplint Trismus System (DTS) for the relief of trismus secondary to the treatment of head and neck cancer, we conducted a retrospective chart review of patients who had undergone DTS therapy during a 1-year period. Our inclusion criteria were cancer of the upper aerodigestive tract; treatment with radiation, chemotherapy, and/or surgery; and a maximal incisal opening (MIO) of less than 30 mm. MIO and the rate of improvement of trismus ("gain") were measured at selected intervals. Twenty-six patients met our study criteria; their pretherapy mean MIO was 19.3 mm. At the time of their most recent measurement, the mean MIO had increased to 25.5 mm-a measured gain of 32%. Although the initial rate of gain was 0.36 mm/day during the first 6 weeks, improvement leveled off over time, and the overall rate of gain was 0.16 mm/day. We conclude that the DTS is effective in increasing the mandibular range of motion at a rate of change that is maximized during initial treatment.

Beta-catenin activity in the dermal papilla of the hair follicle regulates pigment-type switching.

The switch between black and yellow pigment is mediated by the interaction between Melanocortin receptor 1 (Mc1r) and its antagonist Agouti, but the genetic and developmental mechanisms that modify this interaction to obtain different coat color in distinct environments are poorly understood. Here, the role of Wnt/ß-catenin signaling in the regulation of pigment-type switching was studied. Loss and gain of function of ß-catenin in the dermal papilla (DP) of the hair follicle results in yellow and black animals, respectively. ß-Catenin activity in the DP suppresses Agouti expression and activates Corin, a negative regulator of Agouti activity. In addition, ß-catenin activity in the DP regulates melanocyte activity by a mechanism that is independent of both Agouti and Corin. The coordinate and inverse regulation of Agouti and Corin renders pelage pigmentation sensitive to changes in ß-catenin activity in the DP that do not alter pelage structure. As a result, the signals that specify two biologically distinct quantitative traits are partially uncoupled despite their common regulation by the ß-catenin pathway in the same cells.

beta-catenin activity in the dermal papilla regulates morphogenesis and regeneration of hair.

The activity of keratinocytes in the hair follicle is regulated by signals from a specialized mesenchymal niche, the dermal papilla (DP). Here, mice expressing cre recombinase in the DP were developed to probe the interaction between follicular keratinocytes and the DP in vivo. Inactivation of the beta-catenin gene within DP of fully developed hair follicles results in dramatically reduced proliferation of the progenitors and their progeny that generate the hair shaft, and, subsequently, premature induction of the destructive phase of the hair cycle. It also prevents regeneration of the cycling follicle from stem cells. Gene expression analysis reveals that beta-catenin activity in the DP regulates signaling pathways, including FGF and IGF, that can mediate the DP's inductive effects. This study reveals a signaling loop that employs Wnt/beta-catenin signaling in both epithelial progenitor cells and their mesenchymal niche to govern and coordinate the interactions between these compartments to guide hair morphogenesis.