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Keratinocytes, the epithelial cells of the skin, reprogram their gene expression and produce immune effector molecules when exposed to environmental and endogenous triggers of inflammation. It remains unclear how keratinocytes process physiological signals generated during skin irritation and switch from a homeostatic to an inflammatory state. In this article, we show that the stress-activated protein kinase p38α is crucial for keratinocytes to prompt changes in their transcriptome upon cytokine stimulation and drive inflammation in allergen-exposed skin. p38α serves this function by phosphorylating p63, a transcription factor essential for the lineage identity and stemness of the skin epithelium. Phosphorylation by p38α alters the activity of p63 and redeploys this developmental transcription factor to a gene expression program linked to inflammation. Genetic ablation and pharmacological inhibition of p38α or the p38α–p63 target gene product MMP13 attenuate atopic dermatitis–like disease in mice. Our study reveals an epithelial molecular pathway promoting skin inflammation and actionable through treatment with topical small-molecule therapeutics.

Coordinated induction, but also repression, of genes are key to normal differentiation. Although the role of lineage-specific transcription regulators has been studied extensively, their functional integration with chromatin remodelers, one of the key enzymatic machineries that control chromatin accessibility, remains ill-defined. Here we investigate the role of Mi-2β, a SNF-2-like nucleosome remodeler and key component of the nucleosome remodeling and histone deacetylase (NuRD) complex in early B cells. Inactivation of Mi-2β arrested differentiation at the large pre-B-cell stage and caused derepression of cell adhesion and cell migration signaling factors by increasing chromatin access at poised enhancers and chromosome architectural elements. Mi-2β also supported IL-7R signaling, survival, and proliferation by repressing negative effectors of this pathway. Importantly, overexpression of Bcl2, a mitochondrial prosurvival gene and target of IL-7R signaling, partly rescued the differentiation block caused by Mi-2β loss. Mi-2β stably associated with chromatin sites that harbor binding motifs for IKAROS and EBF1 and physically associated with these transcription factors both on and off chromatin. Notably, Mi-2β shared loss-of-function cellular and molecular phenotypes with IKAROS and EBF1, albeit in a distinct fashion. Thus, the nucleosome remodeler Mi-2β promotes pre-B-cell differentiation by providing repression capabilities to distinct lineage-specific transcription factor-based regulatory networks.

IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem-epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD-YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem epithelial B-cell phenotype that underlies high-risk B-ALL. Funding Agencies|National Institutes of Health [R01CA162092, R21AI124326, R01CA090576, R01CA190964]

Purpose of review Loss of IKAROS in committed B cell precursors causes a block in differentiation while at the same time augments aberrant cellular properties, such as bone marrow stromal adhesion, self-renewal and resistance to glucocorticoid-mediated cell death. B cell acute lymphoblastic leukaemias originating from these early stages of B cell differentiation and associated with IKAROS mutations share a high-risk cellular phenotype suggesting that deregulation of IKAROS-based mechanisms cause a highly malignant disease process. Recent studies Recent studies show that IKAROS is critical for the activity of super-enhancers at genes required for pre-B cell receptor (BCR) signalling and differentiation, working either downstream of or in parallel with B cell master regulators such as EBF1 and PAX5. IKAROS also directly represses a cryptic regulatory network of transcription factors prevalent in mesenchymal and epithelial precursors that includes YAP1, TEAD1/2, LHX2 and LMO2, and their targets, which are not normally expressed in lymphocytes. IKAROS prevents not only expression of these 'extra-lineage' transcription factors but also their cooperation with endogenous B cell master regulators, such as EBF1 and PAX5, leading to the formation of a de novo for lymphocytes super-enhancer network. IKAROS coordinates with the Polycomb repression complex (PRC2) to provide stable repression of associated genes during B cell development. However, induction of regulatory factors normally repressed by IKAROS starts a feed-forward loop that activates de-novo enhancers and elevates them to super-enhancer status, thereby diminishing PRC2 repression and awakening aberrant epithelial-like cell properties in B cell precursors. Summary Insight into IKAROS-based transcriptional circuits not only sets new paradigms for cell differentiation but also provides new approaches for classifying and treating high-risk human B-ALL that originates from these early stages of B cell differentiation.

The Ikaros family of DNA-binding proteins are critical regulators of lymphocyte differentiation. In multipotent, hematopoietic progenitors, Ikaros supports transcriptional priming of genes promoting lymphocyte differentiation. Ikaros targets the Nucleosome Remodeling Deacetylase (NuRD) complex to lymphoid lineage genes, thereby increasing chromatin accessibility and transcriptional priming. After lymphoid lineage specification, Ikaros expression is raised to levels characteristic of intermediate B cell and T cell precursors, which is necessary to support maturation and prevent leukemogenesis. Loss of Ikaros in T cell precursors allows the NuRD complex to repress lymphocyte genes and extends its targeting to genes that support growth and proliferation, causing their activation and triggering a cascade of events that leads to leukemogenesis. Loss of Ikaros in B cell precursors blocks differentiation and perpetuates stromal adhesion by enhancing integrin signaling. The combination of integrin and cytokine signaling in Ikaros-deficient pre-B cells promotes their survival and self-renewal. The stages of lymphocyte differentiation that are highly dependent on Ikaros are underscored by changes in Ikaros transcription, supported by a complex network of stage-specific regulatory networks that converge upon the Ikzf1 locus. It is increasingly apparent that understanding the regulatory networks that operate upstream and downstream of Ikaros is critical not only for our understanding of normal lymphopoiesis, but also in placing the right finger on the mechanisms that support hematopoietic malignancies in mouse and human.

The ability of somatic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that keep stem cell-specific genes active and key differentiation factors repressed but poised for activation. The epigenetic factors that provide this type of regulation remain ill-defined. Here we provide the first evidence that the SNF2-like ATPase Mi-2β of the Nucleosome Remodeling Deacetylase (NuRD) complex is required for maintenance of and multilineage differentiation in the early hematopoietic hierarchy. Shortly after conditional inactivation of Mi-2β, there is an increase in cycling and a decrease in quiescence in an HSC (hematopoietic stem cell)-enriched bone marrow population. These cycling mutant cells readily differentiate into the erythroid lineage but not into the myeloid and lymphoid lineages. Together, these effects result in an initial expansion of mutant HSC and erythroid progenitors that are later depleted as more differentiated proerythroblasts accumulate at hematopoietic sites exhibiting features of erythroid leukemia. Examination of gene expression in the mutant HSC reveals changes in the expression of genes associated with self-renewal and lineage priming and a pivotal role of Mi-2β in their regulation. Thus, Mi-2β provides the hematopoietic system with immune cell capabilities as well as with an extensive regenerative capacity.

Ikaros DNA binding factor plays critical roles in lymphocyte development. Changes in Ikaros expression levels during lymphopoiesis are controlled by redundant but also unique regulatory elements of its locus that are critical for this developmental process. We have recently shown that Ikaros binds its own locus in thymocytes in vivo. Here, we evaluated the role of an Ikaros binding site within its major lympho-myeloid promoter. We identified an Ikaros/Ets binding site within a promoter sub-region that was highly conserved in mouse and human. Deletion of this binding site increased the percentage of the reporter-expressing mouse lines, indicating that its loss provided a more permissive chromatin environment. However, once transcription was established, the lack of this site decreased transcriptional activity. These findings implicate a dual role for Ikaros/Ets1 binding on Ikzf1 expression that is exerted at least through its promoter.

Regulated expression of Ikaros is critical for normal hemopoiesis and lymphocyte development. To elucidate the mechanisms underlying transcription of Ikaros, tissue-specific DNase I-hypersensitive sites (DHS) were mapped throughout the Ikaros locus, and several promoters were identified. The activity of these regulatory regions was elucidated using an enhanced green fluorescent protein (EGFP) reporter in transgenic mice. Two genomic fragments, each containing a distinct promoter and its associated DHS cluster, were found to be active in the myeloid (DHS-C2 and DHS-C3) and B-cell (DHS-C3) lineages. Although neither of these regulatory regions was active within the majority of differentiating thymocytes and mature T cells, the DHS-C3 region was active at the earliest stages (DN1–DN3) of T-cell differentiation. However, when the DHS-C3 region was combined with the downstream intronic DHS-C6 cluster, its activity was maintained and raised to higher levels at subsequent stages of T-cell differentiation. This combination of regulatory elements provided reporter expression that closely resembles that of endogenous Ikaros during hemo-lymphopoiesis, and it decreased (but did not alleviate) position effect variegation within the expressing cell types.

Precise control of gene expression plays fundamental roles in brain development, but the roles of chromatin regulators in neuronal connectivity have remained poorly understood. We report that depletion of the NuRD complex by invivo RNAi and conditional knockout of the core NuRD subunit Chd4 profoundly impairs the establishment of granule neuron parallel fiber/Purkinje cell synapses in the rodent cerebellar cortex invivo. By interfacing genome-wide sequencing of transcripts and ChIP-seq analyses, we uncover a network of repressed genes and distinct histone modifications at target gene promoters that are developmentally regulated by the NuRD complex in the cerebellum invivo. Finally, in a targeted invivo RNAi screen of NuRD target genes, we identify a program of NuRD-repressed genes that operate as critical regulators of presynaptic differentiation in the cerebellar cortex. Our findings define NuRD-dependent promoter decommissioning as a developmentally regulated programming mechanism that drives synaptic connectivity in the mammalian brain.

Deletion of the DNA-binding domain of the transcription factor Ikaros generates dominant-negative isoforms that interfere with its activity and correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemia (B-ALL). Here we found that conditional inactivation of the Ikaros DNA-binding domain in early pre-B cells arrested their differentiation at a stage at which integrin-dependent adhesion to niches augmented signaling via mitogen-activated protein kinases, proliferation and self-renewal and attenuated signaling via the pre-B cell signaling complex (pre-BCR) and the differentiation of pre-B cells. Transplantation of polyclonal Ikaros-mutant pre-B cells resulted in long-latency oligoclonal pre-B-ALL, which demonstrates that loss of Ikaros contributes to multistep B cell leukemogenesis. Our results explain how normal pre-B cells transit from a highly proliferative and stroma-dependent phase to a stroma-independent phase during which differentiation is enabled, and suggest potential therapeutic strategies for Ikaros-mutant B-ALL.

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., link_to_OA_fulltext

Cell fate depends on the interplay between chromatin regulators and transcription factors. Here we show that activity of the Mi-2β nucleosome-remodeling and histone-deacetylase (NuRD) complex was controlled by the Ikaros family of lymphoid lineage-determining proteins. Ikaros, an integral component of the NuRD complex in lymphocytes, tethered this complex to active genes encoding molecules involved in lymphoid differentiation. Loss of Ikaros DNA-binding activity caused a local increase in chromatin remodeling and histone deacetylation and suppression of lymphoid cell-specific gene expression. Without Ikaros, the NuRD complex also redistributed to transcriptionally poised genes that were not targets of Ikaros (encoding molecules involved in proliferation and metabolism), which induced their reactivation. Thus, release of NuRD from Ikaros regulation blocks lymphocyte maturation and mediates progression to a leukemic state by engaging functionally opposing epigenetic and genetic networks.

The distribution of CCK-immunoreactive cells was investigated by the indirect fluorescence method in the cerebral cortex of LEC (Long-Evans Cinnamon) rats which have recently been introduced as a model of jaundice and hepatic encephalopathy. Reduction of CCK-immunoreactive nerve cells was observed in the brains of LEC rats with stage III to V hepatic encephalopathy in comparison with the rats without hepatic encephalopathy. These observations were confirmed by counting the CCK-positive nerve cells at magnification x 125. 3 microscopic fields per animal were immunostained and CCK-immunoreactive nerve cells counted. The number of CCK-immunoreactive cells per field was 28.5 +/- 0.7 (mean +/- SEM, n = 5) in rats without hepatic encephalopathy, and 14.1 +/- 2.0 (n = 3) in rats with stage III to V hepatic encephalopathy. Thus, the number of CCK-immunoreactive nerve cells was significantly less in rats with stage III to V hepatic encephalopathy (p less than 0.05). A decrease in CCK-immunoreactive nerve cells was not observed in the rats with stage I or II hepatic encephalopathy. This study shows that there is a relationship between the severity of hepatic encephalopathy and the number of CCK-immunoreactive nerve cells in the cerebral cortex.

Hematopoiesis is the developmental process by which all blood and immune cells are generated. A decade-old scheme has supported an early and complete separation of the erythro-myeloid from the lymphoid lineages. Recent advances have re-drawn this map, separating lymphoid and myeloid from erythroid programs early in development. Subsequently, the fate restriction of both the lympho-myeloid and the erythro-megakaryocyte progenitors is dependent on Ikaros and its associated chromatin regulators. Genetic studies of this family of nuclear factors are now providing unique insight into the functional molecular signatures that bestow plasticity to the hematopoietic stem cell and its early progeny.

Ikaros is expressed in early hematopoietic progenitors and is required for lymphoid differentiation. Analysis of Ikaros null populations revealed a lack of defining markers for early fate-restricted progenitors, but it was difficult to discern whether Ikaros was required for formation of these populations, or for expression of these markers. Here we use a GFP reporter based on Ikaros regulatory elements to identify the HSC and separate early progenitors in both wild-type and Ikaros-null mice. The presence of lympho-myeloid progenitors is revealed in Ikaros-null mice, which lack the defining factor Flt3 and are capable of myeloid, but not lymphoid differentiation. In contrast, lack of Ikaros in the common myeloid progenitor results in increased formation of erythro-megakaryocyte at the expense of myeloid progenitors and influences their subsequent differentiation. By this approach, pivotal roles for Ikaros in distinct fate decisions in the early hematopoietic hierarchy are revealed.

We infected interleukin-5 (IL-5)-transgenic (IL-5-Tg) and IL-5 receptor alpha knockout (IL-5R alpha -/-) mice with Angiostrongylus cantonensis to determine the possible roles of IL-5 and eosinophils in A. cantonensis infection in mice. IL-5-Tg mice demonstrated significantly higher eosinophilia in bone marrow, blood and cerebrospinal fluid (CSF), lower intracranial worm recovery and smaller female worms than naive C3H/HeN mice. Both IL-5-Tg and C3H/HeN mice evoked antigen-specific serum and CSF IgA antibody responses as early as days 5 and 7 postinfection, respectively. Prominent eosinophil infiltration was noted around intracranial worms in the subarachnoid spaces of the mouse brains; eosinophils adhering to the worm surface were degranulated. In contrast, IL-5R alpha -/- mice yielded a higher worm recovery than wild-type or heterozygous mice at day 20 postinfection and failed to provoke CSF eosinophilia. These findings indicate that A. cantonensis infection in the mouse causes IL-5 production and subsequent CSF eosinophilia, the latter probably being involved in the killing of intracranial worms.

We generated interleukin-5 receptor alpha chain (IL-5R alpha)-deficient (IL-5R alpha-/-) mice by gene targeting. The IL-5R alpha-/- mice showed decreased numbers of B-1 cells concomitant with low serum concentrations of IgM and IgG3. They showed no IL-5-induced enhancement of B cell responses to T-independent antigens. The number of alpha beta T cell receptor-positive thymocytes tended to decrease in 3-week-old IL-5R alpha-/- mice, returning to normal by 6 weeks of age. The IL-5R alpha-/- mice produced basal levels of eosinophils, while their bone marrow cells failed to form eosinophilic colonies in response to IL-5. Impaired eosinophilopoiesis in IL-5R alpha-/-mice enhanced the survival of Angiostrongylus cantonensis. These results indicate that IL-5-induced eosinophils serve as potent effector cells in the killing of Angiostrongylus cantonensis in mice.

Levels of cholecystokinin (CCK) immunoreactivity and distribution of CCK immunoreactive cells were studied in the cerebral cortex of LEC (Long Evans Cinnamon) rats with hepatic encephalopathy. CCK immunoreactivity in water extract of cerebral cortex of LEC rats with hepatic encephalopathy ( n = 7 ) was 41.5 ± 2.6 (mean ± S.E.M. pmol/g wet wt.) and that of LEC rats without encephalopathy ( n = 8 ) was 67.1 ± 6.9 , the difference being significant ( P ). CCK immunoreactive cells assessed by immunohistochemistry were also markedly decreased in the cortex of LEC rats with hepatic encephalopathy of stage IV. Thus, CCK reduction was observed in the cerebral cortex of LEC rats with hepatic encephalopathy which are provided as a model for analysis of the pathogenesis of acute hepatic encephalopathy.

Here we investigate the mechanisms that underlie the induction of developmental potential and establishment of cell fate during early hematopoiesis. A cascade of lineage-affiliated gene expression signatures, primed in hematopoietic stem cells (HSC) and differentially propagated in lineage-restricted progenitors, is identified. First evidence is provided for a stochastic sampling of lymphoid, erythroid and myeloid transcripts in HSC and multipotent progenitors (MPP). Multi-lineage priming is subsequently resolved upon lineage restrictions. Nonetheless, an unexpected association of lymphoid and myeloid signatures is detected past a nominal myeloid restriction point and a previously unappreciated lymphoid potential is revealed for this stage in development. New insight is provided into Ikaros' role as a bivalent regulator of multi-lineage priming during early hematopoiesis. Whereas Ikaros is responsible for activation of a cascade of lymphoid signatures in the HSC, at subsequent restriction points it is also involved in the repression of lineage-inappropriate signatures including stem cell-specific genes.

Rare individuals with inactivating mutations in the Huntington’s disease gene (HTT) exhibit variable abnormalities that imply essential HTT roles during organ development. Here we report phenotypes produced when increasingly severe hypomorphic mutations in the murine HTT orthologue Htt, (HdhneoQ20, HdhneoQ50, HdhneoQ111), were placed over a null allele (Hdhex4/5). The most severe hypomorphic allele failed to rescue null lethality at gastrulation, while the intermediate, though still severe, alleles yielded recessive perinatal lethality and a variety of fetal abnormalities affecting body size, skin, skeletal and ear formation, and transient defects in hematopoiesis. Comparative molecular analysis of wild-type and Htt-null retinoic acid-differentiated cells revealed gene network dysregulation associated with organ development that nominate polycomb repressive complexes and miRNAs as molecular mediators. Together these findings demonstrate that Htt is required both pre- and post-gastrulation to support normal development., Author summary The HTT gene mutated in Huntington’s Disease (HD) has essential roles during normal development. However, still not fully understood are the functional consequences of its partial inactivation. Our genetic study provides a comprehensive description of the effects of progressively more severe decreases in expression of Htt, the murine HTT counterpart. The most severe Htt decrease leads to lethality of early embryos, while intermediate but still severely reduced Htt dosages yield a variety of recessively inherited developmental abnormalities affecting body size, skin, skeletal and ear formation, and hematopoiesis. Comparative molecular analysis of differentiating wild-type cells and cells lacking Htt function further elucidate genes networks dysregulated during organ development. These nominate chromatin regulators and short non-coding RNAs as key molecular mediators. Together these findings demonstrate that Htt is required from conception to support normal embryonic and fetal development.