Clock genes rescue nphp mutations in zebrafish.

The zebrafish pronephros model, using morpholino oligonucleotides (MO) to deplete target genes, has been extensively used to characterize human ciliopathy phenotypes. Recently, discrepancies between MO and genetically defined mutants have questioned this approach. We analyzed zebrafish with mutations in the nphp1-4-8 module to determine the validity of MO-based results. While MO-mediated depletion resulted in glomerular cyst and cloaca malformation, these ciliopathy-typical manifestations were observed at a much lower frequency in zebrafish embryos with defined nphp mutations. All nphp1-4-8 mutant zebrafish were viable and displayed decreased manifestations in the next (F2) generation, lacking maternal RNA contribution. While genetic compensation was further supported by the observation that nphp4-deficient mutants became partially refractory to MO-based nphp4 depletion, zebrafish embryos, lacking one nphp gene, became more sensitive to MO-based depletion of additional nphp genes. Transcriptome analysis of nphp8 mutant embryos revealed an upregulation of the circadian clock genes cry1a and cry5. MO-mediated depletion of cry1a and cry5 caused ciliopathy phenotypes in wild-type embryos, while cry1a and cry5 depletion in maternal zygotic nphp8 mutant embryos increased the frequency of glomerular cysts compared to controls. Importantly, cry1a and cry5 rescued the nephropathy-related phenotypes in nphp1, nphp4 or nphp8-depleted zebrafish embryos. Our results reveal that nphp mutant zebrafish resemble the MO-based phenotypes, albeit at a much lower frequency. Rapid adaption through upregulation of circadian clock genes seems to ameliorate the loss of nphp genes, contributing to phenotypic differences.

DOT1L deletion impairs the development of cortical parvalbumin-expressing interneurons.

The cortical plate (CP) is composed of excitatory and inhibitory neurons, the latter of which originate in the ganglionic eminences. From their origin in the ventral telencephalon, maturing postmitotic interneurons migrate during embryonic development over some distance to reach their final destination in the CP. The histone methyltransferase Disruptor of Telomeric Silencing 1-like (DOT1L) is necessary for proper CP development and layer distribution of glutamatergic neurons. However, its specific role on cortical interneuron development has not yet been explored. Here, we demonstrate that DOT1L affects interneuron development in a cell autonomous manner. Deletion of Dot1l in Nkx2.1-expressing interneuron precursor cells results in an overall reduction and altered distribution of GABAergic interneurons in the CP from postnatal day 0 onwards. We observed an altered proportion of GABAergic interneurons in the cortex, with a significant decrease in parvalbumin-expressing interneurons. Moreover, a decreased number of mitotic cells at the embryonic day E14.5 was observed upon Dot1l deletion. Altogether, our results indicate that reduced numbers of cortical interneurons upon DOT1L deletion result from premature cell cycle exit, but effects on postmitotic differentiation, maturation, and migration are likely at play as well.

Corpuscles of Stannius development requires FGF signaling.

The corpuscles of Stannius (CS) represent a unique endocrine organ of teleostean fish that secrets stanniocalcin-1 (Stc1) to maintain calcium homeostasis. Appearing at 20-25 somite stage in the distal zebrafish pronephros, stc1-expressing cells undergo apical constriction, and are subsequently extruded to form a distinct gland on top of the distal pronephric tubules at 50 â€‹h post fertilization (hpf). Several transcription factors (e.g. Hnf1b, Irx3b, Tbx2a/b) and signaling pathways (e.g. Notch) control CS development. We report now that Fgf signaling is required to commit tubular epithelial cells to differentiate into stc1-expressing CS cells. Inhibition of Fgf signaling by SU5402, dominant-negative Fgfr1, or depletion of fgf8a prevented CS formation and stc1 expression. Ablation experiments revealed that CS have the ability to partially regenerate via active cell migration involving extensive filopodia and lamellipodia formation. Activation of Wnt signaling curtailed stc1 expression, but had no effect on CS formation. Thus, our observations identify Fgf signaling as a crucial component of CS cell fate commitment.

Defective Desmosomal Adhesion Causes Arrhythmogenic Cardiomyopathy by Involving an Integrin-αVß6/TGF-ß Signaling Cascade.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrofatty tissue replacement, systolic dysfunction, and life-threatening arrhythmias. A substantial proportion of ACM is caused by mutations in genes of the desmosomal cell-cell adhesion complex, but the underlying mechanisms are not well understood. In the current study, we investigated the relevance of defective desmosomal adhesion for ACM development and progression. METHODS: We mutated the binding site of DSG2 (desmoglein-2), a crucial desmosomal adhesion molecule in cardiomyocytes. This DSG2-W2A mutation abrogates the tryptophan swap, a central interaction mechanism of DSG2 on the basis of structural data. Impaired adhesive function of DSG2-W2A was confirmed by cell-cell dissociation assays and force spectroscopy measurements by atomic force microscopy. The DSG2-W2A knock-in mouse model was analyzed by echocardiography, ECG, and histologic and biomolecular techniques including RNA sequencing and transmission electron and superresolution microscopy. The results were compared with ACM patient samples, and their relevance was confirmed in vivo and in cardiac slice cultures by inhibitor studies applying the small molecule EMD527040 or an inhibitory integrin-αVß6 antibody. RESULTS: The DSG2-W2A mutation impaired binding on molecular level and compromised intercellular adhesive function. Mice bearing this mutation develop a severe cardiac phenotype recalling the characteristics of ACM, including cardiac fibrosis, impaired systolic function, and arrhythmia. A comparison of the transcriptome of mutant mice with ACM patient data suggested deregulated integrin-αVß6 and subsequent transforming growth factor-ß signaling as driver of cardiac fibrosis. Blocking integrin-αVß6 led to reduced expression of profibrotic markers and reduced fibrosis formation in mutant animals in vivo. CONCLUSIONS: We show that disruption of desmosomal adhesion is sufficient to induce a phenotype that fulfils the clinical criteria to establish the diagnosis of ACM, confirming the dysfunctional adhesion hypothesis. Deregulation of integrin-αVß6 and transforming growth factor-ß signaling was identified as a central step toward fibrosis. A pilot in vivo drug test revealed this pathway as a promising target to ameliorate fibrosis. This highlights the value of this model to discern mechanisms of cardiac fibrosis and to identify and test novel treatment options for ACM.

[Life events related to the German reunification and their associations with psychosocial health in a sample of northeast Germany]. / Nach-Wende-Ereignisse und deren Assoziation mit psychosozialer Gesundheit in einer nordostdeutschen Stichprobe.

Objectives: The aim of the present study was to investigate (1) the type and frequency of reported life events of the East German population related to the German reunification and (2) their associations with psychosocial health. Methods: Data of 2247 participants of the Study of Health in Pomerania was used.These qualitative responses were analysed using quantitative content analysis. Their associations with subjective physical and mental health, optimism, social support, depressive symptoms, and chronic stress were examined. Results: Eight life event categories were identified (education, employment-related changes, material changes, new opportunities, personal life events, politics, separations, reunifications). Especially, experiencing new opportunities was associated with a higher level of optimism as well as a lower level of depressive symptoms and chronic stress. Conclusions: In this study, events frequently described in the literature (e.g., employment-related and social changes) were confirmed and systematized.The observed associations of these events with psychosocial factors should be examined further in future studies.

Single-cell mRNA profiling reveals changes in solute carrier expression and suggests a metabolic switch during zebrafish pronephros development.

Developing organisms need to adapt to environmental variations as well as to rapid changes in substrate availability and energy demands imposed by fast-growing tissues and organs. Little is known about the adjustments that kidneys undergo in response to these challenges. We performed single-cell RNA sequencing of zebrafish pronephric duct cells to understand how the developing kidney responds to changes in filtered substrates and intrinsic energy requirements. We found high levels of glucose transporters early in development and increased expression of monocarboxylate transporters at later times. This indicates that the zebrafish embryonic kidney displays a high glucose transporting capacity during early development, which is replaced by the ability to absorb monocarboxylates and amino acids at later stages. This change in transport capacity was accompanied by the upregulation of mitochondrial carriers, indicating a switch to increased oxidative phosphorylation to meet the increasing energy demand of a developing kidney.NEW & NOTEWORTHY The zebrafish embryonic kidney has high levels of glucose transporters during early development, which are replaced by monocarboxylate and amino acid transporters later on. Inhibition of Na+-glucose cotransporter-dependent glucose transport by sotagliflozin also increased slc2a1a expression, supporting the idea that the glucose transport capacity is dynamically adjusted during zebrafish pronephros development. Concurrent upregulation of mitochondrial SCL25 transporters at later stages supports the idea that the pronephros adjusts to changing substrate supplies and/or energy demands during embryonic development.

DOT1L promotes progenitor proliferation and primes neuronal layer identity in the developing cerebral cortex.

Cortical development is controlled by transcriptional programs, which are orchestrated by transcription factors. Yet, stable inheritance of spatio-temporal activity of factors influencing cell fate and localization in different layers is only partly understood. Here we find that deletion of Dot1l in the murine telencephalon leads to cortical layering defects, indicating DOT1L activity and chromatin methylation at H3K79 impact on the cell cycle, and influence transcriptional programs conferring upper layer identity in early progenitors. Specifically, DOT1L prevents premature differentiation by increasing expression of genes that regulate asymmetric cell division (Vangl2, Cenpj). Loss of DOT1L results in reduced numbers of progenitors expressing genes including SoxB1 gene family members. Loss of DOT1L also leads to altered cortical distribution of deep layer neurons that express either TBR1, CTIP2 or SOX5, and less activation of transcriptional programs that are characteristic for upper layer neurons (Satb2, Pou3f3, Cux2, SoxC family members). Data from three different mouse models suggest that DOT1L balances transcriptional programs necessary for proper neuronal composition and distribution in the six cortical layers. Furthermore, because loss of DOT1L in the pre-neurogenic phase of development impairs specifically generation of SATB2-expressing upper layer neurons, our data suggest that DOT1L primes upper layer identity in cortical progenitors.

Neuronal Activity, TGFß-Signaling and Unpredictable Chronic Stress Modulate Transcription of Gadd45 Family Members and DNA Methylation in the Hippocampus.

Neuronal activity is altered in several neurological and psychiatric diseases. Upon depolarization not only neurotransmitters are released but also cytokines and other activators of signaling cascades. Unraveling their complex implication in transcriptional control in receiving cells will contribute to understand specific central nervous system (CNS) pathologies and will be of therapeutically interest. In this study we depolarized mature hippocampal neurons in vitro using KCl and revealed increased release not only of brain-derived neurotrophic factor (BDNF) but also of transforming growth factor beta (TGFB). Neuronal activity together with BDNF and TGFB controls transcription of DNA modifying enzymes specifically members of the DNA-damage-inducible (Gadd) family, Gadd45a, Gadd45b, and Gadd45g. MeDIP followed by massive parallel sequencing and transcriptome analyses revealed less DNA methylation upon KCl treatment. Psychiatric disorder-related genes, namely Tshz1, Foxn3, Jarid2, Per1, Map3k5, and Arc are transcriptionally activated and demethylated upon neuronal activation. To analyze whether misexpression of Gadd45 family members are associated with psychiatric diseases, we applied unpredictable chronic mild stress (UCMS) as established model for depression to mice. UCMS led to reduced expression of Gadd45 family members. Taken together, our data demonstrate that Gadd45 family members are new putative targets for UCMS treatments.

Wnt3a-dependent and -independent protein interaction networks of chromatin-bound ß-catenin in mouse embryonic stem cells.

Canonical Wnt signaling is repeatedly used during development to control cell fate, and it is often implicated in human cancer. ß-catenin, the effector of Wnt signaling, has a dual function in the cell and is involved in both cell adhesion and transcription. Nuclear ß-catenin controls transcription through association with transcription factors of the TCF family and the recruitment of epigenetic modifiers. In this study, we used a strategy combining the genetic manipulation of mouse embryonic stem cells with affinity purification and quantitative mass spectroscopy utilizing stable isotope labeling with amino acids in cell culture to study the interactome of chromatin-bound ß-catenin with and without Wnt3a stimulation. We uncovered previously unknown interactions of ß-catenin with transcription factors and chromatin-modifying complexes. Our proof-of-principle experiments show that ß-catenin can recruit the H3K4me2/1 demethylase LSD1 to regulate the expression of the tumor suppressor Lefty1 in mouse embryonic stem cells. The mRNA levels of LSD1 and ß-catenin are inversely correlated with the levels of Lefty1 in pancreas and breast tumors, implying that this mechanism is common to mouse embryonic stem cells and cancer cells.

DPM1 modulates desmosomal adhesion and epidermal differentiation through SERPINB5.

Glycosylation is essential to facilitate cell-cell adhesion and differentiation. We determined the role of the dolichol phosphate mannosyltransferase (DPM) complex, a central regulator for glycosylation, for desmosomal adhesive function and epidermal differentiation. Deletion of the key molecule of the DPM complex, DPM1, in human keratinocytes resulted in weakened cell-cell adhesion, impaired localization of the desmosomal components desmoplakin and desmoglein-2, and led to cytoskeletal organization defects in human keratinocytes. In a 3D organotypic human epidermis model, loss of DPM1 caused impaired differentiation with abnormally increased cornification, reduced thickness of non-corneal layers, and formation of intercellular gaps in the epidermis. Using proteomic approaches, SERPINB5 was identified as a DPM1-dependent interaction partner of desmoplakin. Mechanistically, SERPINB5 reduced desmoplakin phosphorylation at serine 176, which was required for strong intercellular adhesion. These results uncover a novel role of the DPM complex in connecting desmosomal adhesion with epidermal differentiation.

Unbiased screening identifies regulators of cell-cell adhesion and treatment options in pemphigus.

Cell-cell junctions, and specifically desmosomes, are crucial for robust intercellular adhesion. Desmosomal function is compromised in the autoimmune blistering skin disease pemphigus vulgaris. We combine whole-genome knockout screening and a promotor screen of the desmosomal gene desmoglein 3 in human keratinocytes to identify novel regulators of intercellular adhesion. Kruppel-like-factor 5 (KLF5) directly binds to the desmoglein 3 regulatory region and promotes adhesion. Reduced levels of KLF5 in patient tissue indicate a role in pemphigus vulgaris. Autoantibody fractions from patients impair intercellular adhesion and reduce KLF5 levels in in vitro and in vivo disease models. These effects were dependent on increased activity of histone deacetylase 3, leading to transcriptional repression of KLF5. Inhibiting histone deacetylase 3 increases KLF5 levels and protects against the deleterious effects of autoantibodies in murine and human pemphigus vulgaris models. Together, KLF5 and histone deacetylase 3 are regulators of desmoglein 3 gene expression and intercellular adhesion and represent potential therapeutic targets in pemphigus vulgaris.

Transcriptional neoteny in the human brain.

In development, timing is of the utmost importance, and the timing of developmental processes often changes as organisms evolve. In human evolution, developmental retardation, or neoteny, has been proposed as a possible mechanism that contributed to the rise of many human-specific features, including an increase in brain size and the emergence of human-specific cognitive traits. We analyzed mRNA expression in the prefrontal cortex of humans, chimpanzees, and rhesus macaques to determine whether human-specific neotenic changes are present at the gene expression level. We show that the brain transcriptome is dramatically remodeled during postnatal development and that developmental changes in the human brain are indeed delayed relative to other primates. This delay is not uniform across the human transcriptome but affects a specific subset of genes that play a potential role in neural development.

Multimerization and H3K9me3 binding are required for CDYL1b heterochromatin association.

Proteins containing defined recognition modules mediate readout and translation of histone modifications. These factors are thought to initiate downstream signaling events regulating chromatin structure and function. We identified CDYL1 as an interaction partner of histone H3 trimethylated on lysine 9 (H3K9me3). CDYL1 belongs to a family of chromodomain factors found in vertebrates. We show that three different splicing variants of CDYL1, a, b, and c, are differentially expressed in various tissues with CDYL1b being the most abundant variant. Although all three splicing variants share a common C-terminal enoyl-CoA hydratase-like domain, only CDYL1b contains a functional chromodomain implicated in H3K9me3 binding. A splicing event introducing an N-terminal extension right at the beginning of the chromodomain of CDYL1a inactivates its chromodomain. CDYL1c does not contain a chromodomain at all. Although CDYL1b displays binding affinity to methyl-lysine residues in different sequence context similar to chromodomains in other chromatin factors, we demonstrate that the CDYL1b chromodomain/H3K9me3 interaction is necessary but not sufficient for association of the factor with heterochromatin. Indeed, multimerization of the protein via the enoyl-CoA hydratase-like domain is essential for H3K9me3 chromatin binding in vitro and heterochromatin localization in vivo. In agreement, overexpression of CDYL1c that can multimerize, but does not interact with H3K9me3 can displace CDYL1b from heterochromatin. Our results imply that multimeric binding to H3K9me3 by CDYL1b homomeric complexes is essential for efficient chromatin targeting. We suggest that similar multivalent binding stably anchors other histone modification binding factors on their target chromatin regions.

DOT1L-mediated murine neuronal differentiation associates with H3K79me2 accumulation and preserves SOX2-enhancer accessibility.

During neuronal differentiation, the transcriptional profile and the epigenetic context of neural committed cells is subject to significant rearrangements, but a systematic quantification of global histone modification changes is still missing. Here, we show that H3K79me2 increases and H3K27ac decreases globally during in-vitro neuronal differentiation of murine embryonic stem cells. DOT1L mediates all three degrees of methylation of H3K79 and its enzymatic activity is critical to modulate cellular differentiation and reprogramming. In this context, we find that inhibition of DOT1L in neural progenitor cells biases the transcriptional state towards neuronal differentiation, resulting in transcriptional upregulation of genes marked with H3K27me3 on the promoter region. We further show that DOT1L inhibition affects accessibility of SOX2-bound enhancers and impairs SOX2 binding in neural progenitors. Our work provides evidence that DOT1L activity gates differentiation of progenitors by allowing SOX2-dependent transcription of stemness programs.

Functionality of intergenic transcription: an evolutionary comparison.

Although a large proportion of human transcription occurs outside the boundaries of known genes, the functional significance of this transcription remains unknown. We have compared the expression patterns of known genes as well as intergenic transcripts within the ENCODE regions between humans and chimpanzees in brain, heart, testis, and lymphoblastoid cell lines. We find that intergenic transcripts show patterns of tissue-specific conservation of their expression, which are comparable to exonic transcripts of known genes. This suggests that intergenic transcripts are subject to functional constraints that restrict their rate of evolutionary change as well as putative positive selection to an extent comparable to that of classical protein-coding genes. In brain and testis, we find that part of this intergenic transcription is caused by widespread use of alternative promoters. Further, we find that about half of the expression differences between humans and chimpanzees are due to intergenic transcripts.

Differential Methylation of H3K79 Reveals DOT1L Target Genes and Function in the Cerebellum In Vivo.

The disruptor of telomeric silencing 1-like (DOT1L) mediates methylation of histone H3 at position lysine 79 (H3K79). Conditional knockout of Dot1l in mouse cerebellar granule cells (Dot1l-cKOAtoh1) led to a smaller external granular layer with fewer precursors of granule neurons. Dot1l-cKOAtoh1 mice had impaired proliferation and differentiation of granular progenitors, which resulted in a smaller cerebellum. Mutant mice showed mild ataxia in motor behavior tests. In contrast, Purkinje cell-specific conditional knockout mice showed no obvious phenotype. Genome-wide transcription analysis of Dot1l-cKOAtoh1 cerebella using microarrays revealed changes in genes that function in cell cycle, cell migration, axon guidance, and metabolism. To identify direct DOT1L target genes, we used genome-wide profiling of H3K79me2 and transcriptional analysis. Analysis of differentially methylated regions (DR) and differentially expressed genes (DE) revealed in total 12 putative DOT1L target genes in Dot1l-cKOAtoh1 affecting signaling (Tnfaip8l3, B3galt5), transcription (Otx1), cell migration and axon guidance (Sema4a, Sema5a, Robo1), cholesterol and lipid metabolism (Lss, Cyp51), cell cycle (Cdkn1a), calcium-dependent cell-adhesion or exocytosis (Pcdh17, Cadps2), and unknown function (Fam174b). Dysregulated expression of these target genes might be implicated in the ataxia phenotype observed in Dot1l-cKOAtoh1.

The Clinically Used Iron Chelator Deferasirox Is an Inhibitor of Epigenetic JumonjiC Domain-Containing Histone Demethylases.

Fe(II)- and 2-oxoglutarate (2OG)-dependent JumonjiC domain-containing histone demethylases (JmjC KDMs) are "epigenetic eraser" enzymes involved in the regulation of gene expression and are emerging drug targets in oncology. We screened a set of clinically used iron chelators and report that they potently inhibit JMJD2A (KDM4A) in vitro. Mode of action investigations revealed that one compound, deferasirox, is a bona fide active site-binding inhibitor as shown by kinetic and spectroscopic studies. Synthesis of derivatives with improved cell permeability resulted in significant upregulation of histone trimethylation and potent cancer cell growth inhibition. Deferasirox was also found to inhibit human 2OG-dependent hypoxia inducible factor prolyl hydroxylase activity. Therapeutic effects of clinically used deferasirox may thus involve transcriptional regulation through 2OG oxygenase inhibition. Deferasirox might provide a useful starting point for the development of novel anticancer drugs targeting 2OG oxygenases and a valuable tool compound for investigations of KDM function.

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark.

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

4-Biphenylalanine- and 3-Phenyltyrosine-Derived Hydroxamic Acids as Inhibitors of the JumonjiC-Domain-Containing Histone Demethylase KDM4A.

Overexpression of the histone lysine demethylase KDM4A, which regulates H3K9 and H3K36 methylation states, has been related to the pathology of several human cancers. We found that a previously reported hydroxamate-based histone deacetylase (HDAC) inhibitor (SW55) was also able to weakly inhibit this demethylase with an IC50 value of 25.4 µm. Herein we report the synthesis and biochemical evaluations, with two orthogonal in vitro assays, of a series of derivatives of this lead structure. With extensive chemical modifications on the lead structure, also by exploiting the versatility of the radical arylation with aryldiazonium salts, we were able to increase the potency of the derivatives against KDM4A to the low-micromolar range and, more importantly, to obtain demethylase selectivity with respect to HDACs. Cell-permeable derivatives clearly showed a demethylase-inhibition-dependent antiproliferative effect against HL-60 human promyelocytic leukemia cells.

Substituted 2-(2-aminopyrimidin-4-yl)pyridine-4-carboxylates as potent inhibitors of JumonjiC domain-containing histone demethylases.

BACKGROUND: Aberrant expression of iron(II)- and 2-oxoglutarate-dependent JumonjiC histone demethylases has been linked to cancer. Potent demethylase inhibitors are drug candidates and biochemical tools to elucidate the functional impact of demethylase inhibition. METHODS & RESULTS: Virtual screening identified a novel lead scaffold against JMJD2A with low-micromolar potency in vitro. Analogs were acquired from commercial sources respectively synthesized in feedback with biological testing. Optimized compounds were transformed into cell-permeable prodrugs. A cocrystal x-ray structure revealed the mode of binding of these compounds as competitive to 2-oxoglutarate and confirmed kinetic experiments. Selectivity studies revealed a preference for JMJD2A and JARID1A over JMJD3. CONCLUSION: Virtual screening and rational structural optimization led to a novel scaffold for highly potent and selective JMJD2A inhibitors.