BTB and CNC homology 1 deficiency disrupts intestinal IgA secretion through regulation of polymeric immunoglobulin receptor expression.

Immunoglobulin A (IgA)-mediated mucosal immunity is important for the host because it contributes to reducing infection risk and to establishing host-microbe symbiosis. BTB and CNC homology 1 (Bach1) is a transcriptional repressor with physiological and pathophysiological functions that are of particular interest for their relation to gastrointestinal diseases. However, Bach1 effects on IgA-mediated mucosal immunity remain unknown. For this study using Bach1-deficient (Bach1-/-) mice, we investigated the function of Bach1 in IgA-mediated mucosal immunity. Intestinal mucosa, feces, and plasma IgA were examined using immunosorbent assay. After cell suspensions were prepared from Peyer's patches and colonic lamina propria, they were examined using flow cytometry. The expression level of polymeric immunoglobulin receptor (pIgR), which plays an important role in the transepithelial transport of IgA, was evaluated using Western blotting, quantitative real-time PCR, and immunohistochemistry. Although no changes in the proportions of IgA-producing cells were observed, the amounts of IgA in the intestinal mucosa were increased in Bach1-/- mice. Furthermore, plasma IgA was increased in Bach1-/- mice, but fecal IgA was decreased, indicating that Bach1-/- mice have abnormal secretion of IgA into the intestinal lumen. In fact, Bach1 deficiency reduced pIgR expression in colonic mucosa at both the protein and mRNA levels. In the human intestinal epithelial cell line LS174T, suppression of Bach1 reduced pIgR mRNA stability. In contrast, the overexpression of Bach1 increased pIgR mRNA stability. These results demonstrate that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen via suppression of pIgR expression.NEW & NOTEWORTHY The transcriptional repressor Bach1 has been implicated in diverse intestinal functions, but the effects of Bach1 on IgA-mediated mucosal immunity remain unclear. We demonstrate here that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen, although the proportions of IgA-producing cells were not altered. Furthermore, Bach1 regulates the expression of pIgR, which plays an important role in the transepithelial transport of IgA, at the posttranscriptional level.

TANK Binding Kinase 1 Promotes BACH1 Degradation through Both Phosphorylation-Dependent and -Independent Mechanisms without Relying on Heme and FBXO22.

BTB and CNC homology 1 (BACH1) represses the expression of genes involved in the metabolism of iron, heme and reactive oxygen species. While BACH1 is rapidly degraded when it is bound to heme, it remains unclear how BACH1 degradation is regulated under other conditions. We found that FBXO22, a ubiquitin ligase previously reported to promote BACH1 degradation, polyubiquitinated BACH1 only in the presence of heme in a highly purified reconstitution assay. In parallel to this regulatory mechanism, TANK binding kinase 1 (TBK1), a protein kinase that activates innate immune response and regulates iron metabolism via ferritinophagy, was found to promote BACH1 degradation when overexpressed in 293T cells. While TBK1 phosphorylated BACH1 at multiple serine and threonine residues, BACH1 degradation was observed with not only the wild-type TBK1 but also catalytically impaired TBK1. The BACH1 degradation in response to catalytically impaired TBK1 was not dependent on FBXO22 but involved both autophagy-lysosome and ubiquitin-proteasome pathways judging from its suppression by using inhibitors of lysosome and proteasome. Chemical inhibition of TBK1 in hepatoma Hepa1 cells showed that TBK1 was not required for the heme-induced BACH1 degradation. Its inhibition in Namalwa B lymphoma cells increased endogenous BACH1 protein. These results suggest that TBK1 promotes BACH1 degradation in parallel to the FBXO22- and heme-dependent pathway, placing BACH1 as a downstream effector of TBK1 in iron metabolism or innate immune response.

Mecom mutation related to radioulnar synostosis with amegakaryocytic thrombocytopenia reduces HSPCs in mice.

Radioulnar synostosis with amegakaryocytic thrombocytopenia (RUSAT) is an inherited bone marrow failure syndrome characterized by the congenital fusion of the forearm bones. RUSAT is largely caused by missense mutations that are clustered in a specific region of the MDS1 and EVI1 complex locus (MECOM). EVI1, a transcript variant encoded by MECOM, is a zinc finger transcription factor involved in hematopoietic stem cell maintenance that induce leukemic transformation when overexpressed. Mice with exonic deletions in Mecom show reduced hematopoietic stem and progenitor cells (HSPCs). However, the pathogenic roles of RUSAT-associated MECOM mutations in vivo have not yet been elucidated. To investigate the impact of the RUSAT-associated MECOM mutation on the phenotype, we generated knockin mice harboring a point mutation (translated into EVI1 p.H752R and MDS1-EVI1 p.H942R), which corresponds to an EVI1 p.H751R and MDS1-EVI1 p.H939R mutation identified in a patient with RUSAT. Homozygous mutant mice died at embryonic day 10.5 to 11.5. Heterozygous mutant mice (Evi1KI/+ mice) grew normally without radioulnar synostosis. Male Evi1KI/+ mice, aged between 5 and 15 weeks, exhibited lower body weight, and those aged ≥16 weeks showed low platelet counts. Flow cytometric analysis of bone marrow cells revealed a decrease in HSPCs in Evi1KI/+ mice between 8 and 12 weeks. Moreover, Evi1KI/+ mice showed delayed leukocyte and platelet recovery after 5-fluorouracil-induced myelosuppression. These findings suggest that Evi1KI/+ mice recapitulate the bone marrow dysfunction in RUSAT, similar to that caused by loss-of-function Mecom alleles.

Heme-dependent induction of mitophagy program during differentiation of murine erythroid cells.

Although establishment and maintenance of mitochondria are essential for the production of massive amounts of heme in erythroblasts, mitochondria must be degraded upon terminal differentiation to red blood cells (RBCs), thus creating a biphasic regulatory process. Previously, we reported that iron deficiency in mice promotes mitochondrial retention in RBCs, suggesting that a proper amount of iron and/or heme is necessary for the degradation of mitochondria during erythroblast maturation. Because the transcription factor GATA1 regulates autophagy in erythroid cells, which involves mitochondrial clearance (mitophagy), we investigated the relationship between iron or heme and mitophagy by analyzing the expression of genes related to GATA1 and autophagy and the impact of iron or heme restriction on the amount of mitochondria. We found that heme promotes the expression of GATA1-regulated mitophagy-related genes and the induction of mitophagy. GATA1 might induce the expression of the autophagy-related genes Atg4d and Stk11 for mitophagy through a heme-dependent mechanism in murine erythroleukemia (MEL) cells and a genetic rescue system with G1E-ER-GATA1 erythroblast cells derived from Gata1-null murine embryonic stem cells. These results provide evidence for a biphasic mechanism in which mitochondria are essential for heme generation, and the heme generated during differentiation promotes mitophagy and mitochondrial disposal. This mechanism provides a molecular framework for understanding this fundamentally important cell biological process.

Lipid peroxidation and the subsequent cell death transmitting from ferroptotic cells to neighboring cells.

Ferroptosis regulated cell death due to the iron-dependent accumulation of lipid peroxide. Ferroptosis is known to constitute the pathology of ischemic diseases, neurodegenerative diseases, and steatohepatitis and also works as a suppressing mechanism against cancer. However, how ferroptotic cells affect surrounding cells remains elusive. We herein report the transfer phenomenon of lipid peroxidation and cell death from ferroptotic cells to nearby cells that are not exposed to ferroptotic inducers (FINs). While primary mouse embryonic fibroblasts (MEFs) and NIH3T3 cells contained senescence-associated ß-galactosidase (SA-ß-gal)-positive cells, they were decreased upon induction of ferroptosis with FINs. The SA-ß-gal decrease was inhibited by ferroptotic inhibitors and knockdown of Atg7, pointing to the involvement of lipid peroxidation and activated autophagosome formation during ferroptosis. A transfer of cell culture medium of cells treated with FINs, type 1 or 2, caused the reduction in SA-ß-gal-positive cells in recipient cells that had not been exposed to FINs. Real-time imaging of Kusabira Orange-marked reporter MEFs cocultured with ferroptotic cells showed the generation of lipid peroxide and deaths of the reporter cells. These results indicate that lipid peroxidation and its aftereffects propagate from ferroptotic cells to surrounding cells, even when the surrounding cells are not exposed to FINs. Ferroptotic cells are not merely dying cells but also work as signal transmitters inducing a chain of further ferroptosis.

Infection perturbs Bach2- and Bach1-dependent erythroid lineage 'choice' to cause anemia.

Elucidation of how the differentiation of hematopoietic stem and progenitor cells (HSPCs) is reconfigured in response to the environment is critical for understanding the biology and disorder of hematopoiesis. Here we found that the transcription factors (TFs) Bach2 and Bach1 promoted erythropoiesis by regulating heme metabolism in committed erythroid cells to sustain erythroblast maturation and by reinforcing erythroid commitment at the erythro-myeloid bifurcation step. Bach TFs repressed expression of the gene encoding the transcription factor C/EBPß, as well as that of its target genes encoding molecules important for myelopoiesis and inflammation; they achieved the latter by binding to their regulatory regions also bound by C/EBPß. Lipopolysaccharide diminished the expression of Bach TFs in progenitor cells and promoted myeloid differentiation. Overexpression of Bach2 in HSPCs promoted erythroid development and inhibited myelopoiesis. Knockdown of BACH1 or BACH2 in human CD34+ HSPCs impaired erythroid differentiation in vitro. Thus, Bach TFs accelerate erythroid commitment by suppressing the myeloid program at steady state. Anemia of inflammation and myelodysplastic syndrome might involve reduced activity of Bach TFs.

Leucine/glutamine and v-ATPase/lysosomal acidification via mTORC1 activation are required for position-dependent regeneration.

In animal regeneration, control of position-dependent cell proliferation is crucial for the complete restoration of patterned appendages in terms of both, shape and size. However, detailed mechanisms of this process are largely unknown. In this study, we identified leucine/glutamine and v-ATPase/lysosomal acidification, via mechanistic target of rapamycin complex 1 (mTORC1) activation, as effectors of amputation plane-dependent zebrafish caudal fin regeneration. mTORC1 activation, which functions in cell proliferation, was regulated by lysosomal acidification possibly via v-ATPase activity at 3 h post amputation (hpa). Inhibition of lysosomal acidification resulted in reduced growth factor-related gene expression and suppression of blastema formation at 24 and 48 hpa, respectively. Along the proximal-distal axis, position-dependent lysosomal acidification and mTORC1 activation were observed from 3 hpa. We also report that Slc7a5 (L-type amino acid transporter), whose gene expression is position-dependent, is necessary for mTORC1 activation upstream of lysosomal acidification during fin regeneration. Furthermore, treatment with leucine and glutamine, for both proximal and distal fin stumps, led to an up-regulation in cell proliferation via mTORC1 activation, indicating that leucine/glutamine signaling possesses the ability to change the position-dependent regeneration. Our findings reveal that leucine/glutamine and v-ATPase/lysosomal acidification via mTORC1 activation are required for position-dependent zebrafish fin regeneration.

Bach2 Promotes B Cell Receptor-Induced Proliferation of B Lymphocytes and Represses Cyclin-Dependent Kinase Inhibitors.

BTB and CNC homology 2 (Bach2) is a transcriptional repressor that is required for the formation of the germinal center (GC) and reactions, including class switch recombination and somatic hypermutation of Ig genes in B cells, within the GC. Although BCR-induced proliferation is essential for GC reactions, the function of Bach2 in regulating B cell proliferation has not been elucidated. In this study, we demonstrate that Bach2 is required to sustain high levels of B cell proliferation in response to BCR signaling. Following BCR engagement in vitro, B cells from Bach2-deficient (Bach2-/-) mice showed lower incorporation of BrdU and reduced cell cycle progression compared with wild-type cells. Bach2-/- B cells also underwent increased apoptosis, as evidenced by an elevated frequency of sub-G1 cells and early apoptotic cells. Transcriptome analysis of BCR-engaged B cells from Bach2-/- mice revealed reduced expression of the antiapoptotic gene Bcl2l1 encoding Bcl-xL and elevated expression of cyclin-dependent kinase inhibitor (CKI) family genes, including Cdkn1a, Cdkn2a, and Cdkn2b Reconstitution of Bcl-xL expression partially rescued the proliferation defect of Bach2-/- B cells. Chromatin immunoprecipitation experiments showed that Bach2 bound to the CKI family genes, indicating that these genes are direct repression targets of Bach2. These findings identify Bach2 as a requisite factor for sustaining high levels of BCR-induced proliferation, survival, and cell cycle progression, and it promotes expression of Bcl-xL and repression of CKI genes. BCR-induced proliferation defects may contribute to the impaired GC formation observed in Bach2-/- mice.

S-Adenosylmethionine Synthesis Is Regulated by Selective N6-Adenosine Methylation and mRNA Degradation Involving METTL16 and YTHDC1.

S-adenosylmethionine (SAM) is an important metabolite as a methyl-group donor in DNA and histone methylation, tuning regulation of gene expression. Appropriate intracellular SAM levels must be maintained, because methyltransferase reaction rates can be limited by SAM availability. In response to SAM depletion, MAT2A, which encodes a ubiquitous mammalian methionine adenosyltransferase isozyme, was upregulated through mRNA stabilization. SAM-depletion reduced N6-methyladenosine (m6A) in the 3' UTR of MAT2A. In vitro reactions using recombinant METTL16 revealed multiple, conserved methylation targets in the 3' UTR. Knockdown of METTL16 and the m6A reader YTHDC1 abolished SAM-responsive regulation of MAT2A. Mutations of the target adenine sites of METTL16 within the 3' UTR revealed that these m6As were redundantly required for regulation. MAT2A mRNA methylation by METTL16 is read by YTHDC1, and we suggest that this allows cells to monitor and maintain intracellular SAM levels.

The mTOR-Bach2 Cascade Controls Cell Cycle and Class Switch Recombination during B Cell Differentiation.

The transcription factor Bach2 regulates both acquired and innate immunity at multiple steps, including antibody class switching and regulatory T cell development in activated B and T cells, respectively. However, little is known about the molecular mechanisms of Bach2 regulation in response to signaling of cytokines and antigen. We show here that mammalian target of rapamycin (mTOR) controls Bach2 along B cell differentiation with two distinct mechanisms in pre-B cells. First, mTOR complex 1 (mTORC1) inhibited accumulation of Bach2 protein in nuclei and reduced its stability. Second, mTOR complex 2 (mTORC2) inhibited FoxO1 to reduce Bach2 mRNA expression. Using expression profiling and chromatin immunoprecipitation assay, the Ccnd3 gene, encoding cyclin D3, was identified as a new direct target of Bach2. A proper cell cycle was lost at pre-B and mature B cell stages in Bach2-deficient mice. Furthermore, AZD8055, an mTOR inhibitor, increased class switch recombination in wild-type mature B cells but not in Bach2-deficient cells. These results suggest that the mTOR-Bach2 cascade regulates proper cell cycle arrest in B cells as well as immunoglobulin gene rearrangement.

Inflammatory responses induce an identity crisis of alveolar macrophages, leading to pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a severe respiratory disease characterized by dyspnea caused by accumulation of surfactant protein. Dysfunction of alveolar macrophages (AMs), which regulate the homeostasis of surfactant protein, leads to the development of PAP; for example, in mice lacking BTB and CNC homology 2 (Bach2). However, how Bach2 helps prevent PAP is unknown, and the cell-specific effects of Bach2 are undefined. Using mice lacking Bach2 in specific cell types, we found that the PAP phenotype of Bach2-deficient mice is due to Bach2 deficiency in more than two types of immune cells. Depletion of hyperactivated T cells in Bach2-deficient mice restored normal function of AMs and ameliorated PAP. We also found that, in Bach2-deficient mice, hyperactivated T cells induced gene expression patterns that are specific to other tissue-resident macrophages and dendritic cells. Moreover, Bach2-deficient AMs exhibited a reduction in cell cycle progression. IFN-γ released from T cells induced Bach2 expression in AMs, in which Bach2 then bound to regulatory regions of inflammation-associated genes in myeloid cells. Of note, in AMs, Bach2 restricted aberrant responses to excessive T cell-induced inflammation, whereas, in T cells, Bach2 puts a brake on T cell activation. Moreover, Bach2 stimulated the expression of multiple histone genes in AMs, suggesting a role of Bach2 in proper histone expression. We conclude that Bach2 is critical for the maintenance of AM identity and self-renewal in inflammatory environments. Treatments targeting T cells may offer new therapeutic strategies for managing secondary PAP.

A Bach2-Cebp Gene Regulatory Network for the Commitment of Multipotent Hematopoietic Progenitors.

Hematopoietic stem cell and multipotent progenitor (MPP) commitment can be tuned in response to an infection so that their differentiation is biased toward myeloid cells. Here, we find that Bach2, which inhibits myeloid differentiation in common lymphoid progenitors, represses a cohort of myeloid genes and activates those linked to lymphoid function. Bach2 repressed both Cebpb and its target Csf1r, encoding C/EBPß and macrophage colony-stimulating factor receptor (M-CSFr), respectively, whereas C/EBPß repressed Bach2 and activated Csf1r. Bach2 and C/EBPß further bound to overlapping regulatory regions at their myeloid target genes, suggesting the presence of a gene regulatory network (GRN) with mutual repression between these factors and a feedforward loop leading to myeloid gene regulation. Lipopolysaccharide reduced the expression of Bach2, resulting in enhanced myeloid differentiation. The Bach2-C/EBPß GRN pathway thus tunes MPP commitment to myeloid and lymphoid lineages both under normal conditions and after infection.

Histone H3 Lysine 27 Trimethylation Leads to Loss of Mesendodermal Competence During Gastrulation in Zebrafish Ectodermal Cells.

Previous studies in Xenopus have shown that forced expression of Nodal signaling can change ectodermal cells to a mesodermal fate by the early gastrula stage, suggesting mesodermal competence in early ectoderm cells. This mesodermal competence in ectodermal cells has been shown to be regulated at the level of nucleocytoplasmic localization of Smad2 in Xenopus. However, the regulation of mesodermal competence through epigenetic mechanisms has not been fully elucidated. Here, we used a constitutively active form of zebrafish Smad2 (Smad2ca) to overcome the inhibition of Nodal signaling via the nuclear exclusion of Smad2. While heat-shock-dependent expression of Smad2ca at 5 h post fertilization (hpf) induced ectopic expression of mesendodermal genes in zebrafish ectodermal cells, responsiveness to Smad2ca was lost by 7 hpf. Chromatin immunoprecipitation-quantitative PCR analyses revealed that in ectodermal cells, levels of H3K27me3, but not H3K9me3, at both transcriptional start site (TSS) and 3'-flanking regions of mesendodermal genes at 9 hpf were markedly higher than those at 5 hpf. In contrast to mesendodermal genes, the levels of H3K27me3 at the TSS, but not 3'-flanking regions, of ectodermal genes remained low in ectodermal cells even at 9 hpf. We also found that chemical inhibition of H3K27me3 modification was able to recover the mesendodermal competence in ectodermal cells at 7 hpf, but not at 10 hpf. Taken together, our results suggest that the mesendodermal competence in zebrafish ectodermal cells is restricted by multiple mechanisms, including upregulation of H3K27me3 levels at the TSS of mesendodermal genes during early gastrulation.

Zebrafish as a Model to Study Cohesin and Cohesinopathies.

The cohesin protein complex regulates multiple cellular events including sister chromatid cohesion and gene expression. Several distinct human diseases called cohesinopathies have been associated with genetic mutations in cohesin subunit genes or genes encoding regulators of cohesin function. Studies in different model systems, from yeast to mouse have provided insights into the molecular mechanisms of action of cohesin/cohesin regulators and their implications in the pathogenesis of cohesinopathies. The zebrafish has unique advantages for embryonic analyses and quantitative gene knockdown with morpholinos during the first few days of development, in contrast to knockouts of cohesin regulators in flies or mammals, which are either lethal as homozygotes or dramatically compensated for in heterozygotes. This has been particularly informative for Rad21, where a role in gene expression was first shown in zebrafish, and Nipbl, where the fish work revealed tissue-specific functions in heart, gut, and limbs, and long-range enhancer-promoter interactions that control Hox gene expression in vivo. Here we discuss the utility of the zebrafish in studying the developmental and pathogenic roles of cohesin.

Iron-heme-Bach1 axis is involved in erythroblast adaptation to iron deficiency.

Iron plays the central role in oxygen transport by erythrocytes as a constituent of heme and hemoglobin. The importance of iron and heme is also to be found in their regulatory roles during erythroblast maturation. The transcription factor Bach1 may be involved in their regulatory roles since it is deactivated by direct binding of heme. To address whether Bach1 is involved in the responses of erythroblasts to iron status, low iron conditions that induced severe iron deficiency in mice were established. Under iron deficiency, extensive gene expression changes and mitophagy disorder were induced during maturation of erythroblasts. Bach1-/- mice showed more severe iron deficiency anemia in the developmental phase of mice and a retarded recovery once iron was replenished when compared with wild-type mice. In the absence of Bach1, the expression of globin genes and Hmox1 (encoding heme oxygenase-1) was de-repressed in erythroblasts under iron deficiency, suggesting that Bach1 represses these genes in erythroblasts under iron deficiency to balance the levels of heme and globin. Moreover, an increase in genome-wide DNA methylation was observed in erythroblasts of Bach1-/- mice under iron deficiency. These findings reveal the principle role of iron as a regulator of gene expression in erythroblast maturation and suggest that the iron-heme-Bach1 axis is important for a proper adaptation of erythroblast to iron deficiency to avoid toxic aggregates of non-heme globin.

The double knockout of Bach1 and Bach2 in mice reveals shared compensatory mechanisms in regulating alveolar macrophage function and lung surfactant homeostasis.

Pulmonary alveolar proteinosis (PAP) is a disease resulting from a dysfunction of the alveolar macrophages (AMs) where excess surfactant protein accumulates in the alveolar spaces. We previously reported that Bach2 KO mice developed PAP due to a defect in the handling of lipids by AMs. To investigate the functions of Bach1 and Bach2, which are regulated by oxidative stress, in the AMs and in lung homeostasis, we generated mice that lacked both Bach1 and Bach2 (Bach1/2 DKO mice). The Bach1/2 DKO mice showed more severe PAP phenotype than Bach2 KO mice with abnormal AMs, whereas the Bach1 KO mice did not develop any pulmonary disease. The PAP-like disease in the Bach1/2 DKO and Bach2 KO mice was not ameliorated by antioxidant, suggesting that ROS was not involved in the onset of PAP in the absence of Bach1 and Bach2. A microarray and a chromatin immunoprecipitation sequence analysis revealed that Bach1 and Bach2 directly repress the common set of genes involved in the inflammatory response, and that Bach2 is a major contributor to this repression. These results suggest that Bach1 and Bach2 work in a complementary manner to maintain the normal function of the AMs and surfactant homeostasis in the lung.

BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers.

T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.

Using mouse and zebrafish models to understand the etiology of developmental defects in Cornelia de Lange Syndrome.

Cornelia de Lange Syndrome (CdLS) is a multisystem birth defects disorder that affects every tissue and organ system in the body. Understanding the factors that contribute to the origins, prevalence, and severity of these developmental defects provides the most direct approach for developing screens and potential treatments for individuals with CdLS. Since the majority of cases of CdLS are caused by haploinsufficiency for NIPBL (Nipped-B-like, which encodes a cohesin-associated protein), we have developed mouse and zebrafish models of CdLS by using molecular genetic tools to create Nipbl-deficient mice and zebrafish (Nipbl(+/-) mice, zebrafish nipbl morphants). Studies of these vertebrate animal models have yielded novel insights into the developmental etiology and genes/gene pathways that contribute to CdLS-associated birth defects, particularly defects of the gut, heart, craniofacial structures, nervous system, and limbs. Studies of these mouse and zebrafish CdLS models have helped clarify how deficiency for NIPBL, a protein that associates with cohesin and other transcriptional regulators in the nucleus, affects processes important to the emergence of the structural and physiological birth defects observed in CdLS: NIPBL exerts chromosome position-specific effects on gene expression; it influences long-range interactions between different regulatory elements of genes; and it regulates combinatorial and synergistic actions of genes in developing tissues. Our current understanding is that CdLS should be considered as not only a cohesinopathy, but also a "transcriptomopathy," that is, a disease whose underlying etiology is the global dysregulation of gene expression throughout the organism. © 2016 Wiley Periodicals, Inc.

Temporal effects of Notch signaling and potential cooperation with multiple downstream effectors on adenohypophysis cell specification in zebrafish.

The adenohypophysis (AH) consists of six distinct types of hormone-secreting cells. In zebrafish, although proper differentiation of all AH cell types has been shown to require Notch signaling within a period of 14-16 h postfertilization (hpf), the mechanisms underlying this process remain to be elucidated. Herein, we observed using the Notch inhibitor dibenzazepine (DBZ) that Notch signaling also contributed to AH cell specification beyond 16 hpf. Specification of distinct cell types was perturbed by DBZ treatment for different time frames, suggesting that AH cells are specified by Notch-dependent and cell-type-specific mechanisms. We also found that two hes-family genes, her4.1 and hey1, were expressed in the developing AH under the influence of Notch signaling. her4.1 knockdown reduced expression of proopiomelanocortin a (pomca), growth hormone (gh), and prolactin, whereas hey1 was responsible only for gh expression. Simultaneous loss of both Her4.1 and Hey1 produced milder phenotypes than that of DBZ-treated embryos. Moreover, DBZ treatment from 18 hpf led to a significant down-regulation of both gh and pomca genes only when combined with injection of a subthreshold level of her4.1-morpholino. These observations suggest that multiple downstream effectors, including Her4.1 and Hey1, mediate Notch signaling during AH cell specification.

Genetic complementation analysis showed distinct contributions of the N-terminal tail of H2A.Z to epigenetic regulations.

H2A.Z is one of the most evolutionally conserved histone variants. In vertebrates, this histone variant has two isoforms, H2A.Z.1 and H2A.Z.2, each of which is coded by an individual gene. H2A.Z is involved in multiple epigenetic regulations, and in humans, it also has relevance to carcinogenesis. In this study, we used the H2A.Z DKO cells, in which both H2A.Z isoform genes could be inducibly knocked out, for the functional analysis of H2A.Z by a genetic complementation assay, as the first example of its kind in vertebrates. Ectopically expressed wild-type H2A.Z and two N-terminal mutants, a nonacetylable H2A.Z mutant and a chimera in which the N-terminal tail of H2A.Z.1 was replaced with that of the canonical H2A, complemented the mitotic defects of H2A.Z DKO cells similarly, suggesting that both acetylation and distinctive sequence of the N-terminal tail of H2A.Z are not required for mitotic progression. In contrast, each one of these three forms of H2A.Z complemented the transcriptional defects of H2A.Z DKO cells differently. These results suggest that the N-terminal tail of vertebrate H2A.Z makes distinctively different contributions to these epigenetic events. Our results also imply that this genetic complementation system is a novel and useful tool for the functional analysis of H2A.Z.