DIDO is necessary for the adipogenesis that promotes diet-induced obesity.

The prevalence of overweight and obesity continues to rise in the population worldwide. Because it is an important predisposing factor for cancer, cardiovascular diseases, diabetes mellitus, and COVID-19, obesity reduces life expectancy. Adipose tissue (AT), the main fat storage organ with endocrine capacity, plays fundamental roles in systemic metabolism and obesity-related diseases. Dysfunctional AT can induce excess or reduced body fat (lipodystrophy). Dido1 is a marker gene for stemness; gene-targeting experiments compromised several functions ranging from cell division to embryonic stem cell differentiation, both in vivo and in vitro. We report that mutant mice lacking the DIDO N terminus show a lean phenotype. This consists of reduced AT and hypolipidemia, even when mice are fed a high-nutrient diet. DIDO mutation caused hypothermia due to lipoatrophy of white adipose tissue (WAT) and dermal fat thinning. Deep sequencing of the epididymal white fat (Epi WAT) transcriptome supported Dido1 control of the cellular lipid metabolic process. We found that, by controlling the expression of transcription factors such as C/EBPα or PPARγ, Dido1 is necessary for adipocyte differentiation, and that restoring their expression reestablished adipogenesis capacity in Dido1 mutants. Our model differs from other lipodystrophic mice and could constitute a new system for the development of therapeutic intervention in obesity.

Hepatitis, testicular degeneration, and ataxia in DIDO3-deficient mice with altered mRNA processing.

BACKGROUND: mRNA processing is an essential step of gene expression; its malfunction can lead to different degrees of physiological disorder from subclinical disease to death. We previously identified Dido1 as a stemness marker and a gene involved in embryonic stem cell differentiation. DIDO3, the largest protein encoded by the Dido1 gene, is necessary for accurate mRNA splicing and correct transcription termination. The deletion of Dido1 exon16, which encodes the carboxy-terminal half of DIDO3, results in early embryonic lethality in mouse. RESULTS: We obtained mice bearing a Cre-LoxP conditional version of that deletion and studied the effects of inducing it ubiquitously in adult stages. DIDO3-deficient mice survive the deletion but suffer mild hepatitis, testicular degeneration, and progressive ataxia, in association with systemic alterations in mRNA splicing and transcriptional readthrough. CONCLUSIONS: These results offer insight into the distinct vulnerabilities in mouse organs following impairment of the mRNA processing machinery, and could aid understanding of human health dependence on accurate mRNA metabolism.

Interplay between splicing and transcriptional pausing exerts genome-wide control over alternative polyadenylation.

Recent studies have identified multiple polyadenylation sites in nearly all mammalian genes. Although these are interpreted as evidence for alternative polyadenylation, our knowledge of the underlying mechanisms is still limited. Most studies only consider the immediate surroundings of gene ends, even though in vitro experiments have uncovered the involvement of external factors such as splicing. Whereas in vivo splicing manipulation was impracticable until recently, we now used mutants in the Death Inducer Obliterator (DIDO) gene to study their impact on 3' end processing. We observe multiple rounds of readthrough and gene fusions, suggesting that no arbitration between polyadenylation sites occurs. Instead, a window of opportunity seems to control end processing. Through the identification of T-rich sequence motifs, our data indicate that splicing and transcriptional pausing interact to regulate alternative polyadenylation. We propose that 3' splice site activation comprises a variable timer, which determines how long transcription proceeds before polyadenylation signals are recognized. Thus, the role of core polyadenylation signals could be more passive than commonly believed. Our results provide new insights into the mechanisms of alternative polyadenylation and expand the catalog of related aberrations.Abbreviations APA: alternative polyadenylation; bp: basepair; MEF: mouse embryonic fibroblasts; PA: polyadenylation; PAS: polyadenylation site; Pol II: (RNA) polymerase II ; RT-PCR:reverse-transcriptase PCR; SF:splicing factor; SFPQ:splicing factor rich in proline and glutamine; SS:splice site; TRSM:Thymidine rich sequence motif; UTR:untranslated terminal region.

Dido3-dependent SFPQ recruitment maintains efficiency in mammalian alternative splicing.

Alternative splicing is facilitated by accessory proteins that guide spliceosome subunits to the primary transcript. Many of these splicing factors recognize the RNA polymerase II tail, but SFPQ is a notable exception even though essential for mammalian RNA processing. This study reveals a novel role for Dido3, one of three Dido gene products, in alternative splicing. Binding of the Dido3 amino terminus to histones and to the polymerase jaw domain was previously reported, and here we show interaction between its carboxy terminus and SFPQ. We generated a mutant that eliminates Dido3 but preserves other Dido gene products, mimicking reduced Dido3 levels in myeloid neoplasms. Dido mutation suppressed SFPQ binding to RNA and increased skipping for a large group of exons. Exons bearing recognition sequences for alternative splicing factors were nonetheless included more efficiently. Reduced SFPQ recruitment may thus account for increased skipping of SFPQ-dependent exons, but could also generate a splicing factor surplus that becomes available to competing splice sites. Taken together, our data indicate that Dido3 is an adaptor that controls SFPQ utilization in RNA splicing. Distributing splicing factor recruitment over parallel pathways provides mammals with a simple mechanism to regulate exon usage while maintaining RNA splicing efficiency.

A Unique pH-Dependent Recognition of Methylated Histone H3K4 by PPS and DIDO.

The protein partner of Sans-fille (PPS) and its human homolog DIDO mediate diverse chromatin activities, including the regulation of stemness genes in embryonic stem cells and splicing in Drosophila. Here, we show that the PHD fingers of PPS and DIDO recognize the histone mark H3K4me3 in a pH-dependent manner: the binding is enhanced at high pH values but is decreased at low pH. Structural analysis reveals that the pH dependency is due to the presence of a histidine residue in the K4me3-binding aromatic cage of PPS. The pH-dependent mechanism is conserved in DIDO but is lost in yeast Bye1. Acidification of cells leads to the accelerated efflux of endogenous DIDO, indicating the pH-dependent sensing of H3K4me3 in vivo. This novel mode for the recognition of H3K4me3 establishes the PHD fingers of PPS and DIDO as unique epigenetic readers and high pH sensors and suggests a role for the histidine switch during mitosis.

Epigenetic countermarks in mitotic chromosome condensation.

Mitosis in metazoans is characterized by abundant phosphorylation of histone H3 and involves the recruitment of condensin complexes to chromatin. The relationship between the 2 phenomena and their respective contributions to chromosome condensation in vivo remain poorly understood. Recent studies have shown that H3T3 phosphorylation decreases binding of histone readers to methylated H3K4 in vitro and is essential to displace the corresponding proteins from mitotic chromatin in vivo. Together with previous observations, these data provide further evidence for a role of mitotic histone H3 phosphorylation in blocking transcriptional programs or preserving the 'memory' PTMs. Mitotic protein exclusion can also have a role in depopulating the chromatin template for subsequent condensin loading. H3 phosphorylation thus serves as an integral step in the condensation of chromosome arms.

Dido3-dependent HDAC6 targeting controls cilium size.

Primary cilia are involved in a variety of physiological processes such as sensing of the environment, cell growth and development. Numerous developmental disorders and pathologies arise from defects in these organelles. Multiple proteins that promote formation and disassembly of the primary cilium have been identified, but little is known about the mechanisms that control steady-state cilium size. Here, we show that death inducer obliterator (Dido3)-dependent targeting of histone deacetylase 6 (HDAC6) is a key determinant of cilium size in growth-arrested cells. The amount of either protein negatively correlates with cilium size. Dido3 availability at the centrosome governs ciliary HDAC6 levels, and redistribution of the two proteins controls tubulin acetylation. In turn, basal body localization of Dido3 and HDAC6 depends on the actin network, previously shown to limit cilium size independent of the cell cycle. These results show that not only kinase-dependent activation of a deacetylase but also its subcellular distribution controls substrate selection.

Dido3 PHD modulates cell differentiation and division.

Death Inducer Obliterator 3 (Dido3) is implicated in the maintenance of stem cell genomic stability and tumorigenesis. Here, we show that Dido3 regulates the expression of stemness genes in embryonic stem cells through its plant homeodomain (PHD) finger. Binding of Dido3 PHD to histone H3K4me3 is disrupted by threonine phosphorylation that triggers Dido3 translocation from chromatin to the mitotic spindle. The crystal structure of Dido3 PHD in complex with H3K4me3 reveals an atypical aromatic-cage-like binding site that contains a histidine residue. Biochemical, structural, and mutational analyses of the binding mechanism identified the determinants of specificity and affinity and explained the inability of homologous PHF3 to bind H3K4me3. Together, our findings reveal a link between the transcriptional control in embryonic development and regulation of cell division.

Chromothripsis: breakage-fusion-bridge over and over again.

The acquisition of massive but localized chromosome translocations, a phenomenon termed chromothripsis, has received widespread attention since its discovery over a year ago. Until recently, chromothripsis was believed to originate from a single catastrophic event, but the molecular mechanisms leading to this event are yet to be uncovered. Because a thorough interpretation of the data are missing, the phenomenon itself has wrongly acquired the status of a mechanism used to justify many kinds of complex rearrangements. Although the assumption that all translocations in chromothripsis originate from a single event has met with criticism, satisfactory explanations for the intense but localized nature of this phenomenon are still missing. Here, we show why the data used to describe massive catastrophic rearrangements are incompatible with a model comprising a single event only and propose a molecular mechanism in which a combination of known cellular pathways accounts for chromothripsis. Instead of a single traumatic event, the protection of undamaged chromosomes by telomeres can limit repetitive breakage-fusion-bridge events to a single chromosome arm. Ultimately, common properties of chromosomal instability, such as aneuploidy and centromere fission, might establish the complex genetic pattern observed in this genomic state.

The leukemia-associated fusion protein MN1-TEL blocks TEL-specific recognition sequences.

The leukemia-associated fusion protein MN1-TEL combines the transcription-activating domains of MN1 with the DNA-binding domain of the transcriptional repressor TEL. Quantitative photobleaching experiments revealed that ∼20% of GFP-tagged MN1 and TEL is transiently immobilised, likely due to indirect or direct DNA binding, since transcription inhibition abolished immobilisation. Interestingly, ∼50% of the MN1-TEL fusion protein was immobile with much longer binding times than unfused MN1 and TEL. MN1-TEL immobilisation was not observed when the TEL DNA-binding domain was disrupted, suggesting that MN1-TEL stably occupies TEL recognition sequences, preventing binding of factors required for proper transcription regulation, which may contribute to leukemogenesis.

Linking stem cells to chromosomal instability.

The discovery of a stem cell population in human neoplasias has given a new impulse to the study of the origins of cancer. The tissue compartment in which transformation first occurs likely comprises stem cells, since these cells need to consolidate the short-term and long-term requisites of tissue renewal. Because of their unique role, stem cells have a combination of characteristics that makes them susceptible to genetic damage, transformation, and tumor initiation. One type of genetic damage in particular, chromosomal instability, might affect the stem cell compartment, because it induces an ongoing cycle of DNA damage and alters cellular programming. Here, we will discuss some of the recently described links between SC, chromosomal instability, and carcinogenesis, and outline some of the consequences for oncoimmunology.

The ETS family member TEL binds to nuclear receptors RAR and RXR and represses gene activation.

Retinoic acid receptor (RAR) signaling is important for regulating transcriptional activity of genes involved in growth, differentiation, metabolism and reproduction. Defects in RAR signaling have been implicated in cancer. TEL, a member of the ETS family of transcription factors, is a DNA-binding transcriptional repressor. Here, we identify TEL as a transcriptional repressor of RAR signaling by its direct binding to both RAR and its dimerisation partner, the retinoid x receptor (RXR) in a ligand-independent fashion. TEL is found in two isoforms, created by the use of an alternative startcodon at amino acid 43. Although both isoforms bind to RAR and RXR in vitro and in vivo, the shorter form of TEL represses RAR signaling much more efficiently. Binding studies revealed that TEL binds closely to the DNA binding domain of RAR and that both Helix Loop Helix (HLH) and DNA binding domains of TEL are mandatory for interaction. We have shown that repression by TEL does not involve recruitment of histone deacetylases and suggest that polycomb group proteins participate in the process.

Centromere fission, not telomere erosion, triggers chromosomal instability in human carcinomas.

The majority of sporadic carcinomas suffer from a kind of genetic instability in which chromosome number changes occur together with segmental defects. This means that changes involving intact chromosomes accompany breakage-induced alterations. Whereas the causes of aneuploidy are described in detail, the origins of chromosome breakage in sporadic carcinomas remain disputed. The three main pathways of chromosomal instability (CIN) proposed until now (random breakage, telomere fusion and centromere fission) are largely based on animal models and in vitro experiments, and recent studies revealed several discrepancies between animal models and human cancer. Here, we discuss how the experimental systems translate to human carcinomas and compare the theoretical breakage products to data from patient material and cancer cell lines. The majority of chromosomal defects in human carcinomas comprises pericentromeric breaks that are captured by healthy telomeres, and only a minor proportion of chromosome fusions can be attributed to telomere erosion or random breakage. Centromere fission, not telomere erosion, is therefore the most probably trigger of CIN and early carcinogenesis. Similar centromere-telomere fusions might drive a subset of congenital defects and evolutionary chromosome changes.

Are aneuploidy and chromosome breakage caused by a CINgle mechanism?

Genetic instability is a hallmark of cancer. Most tumors show complex patterns of translocations, amplifications, and deletions, which have occupied scientists for decades. A specific problem arises in carcinomas with a genetic defect termed chromosomal instability; these solid tumors undergo gains and losses of entire chromosomes, as well as segmental defects caused by chromosome breaks. To date, the apparent inconsistency between intact and broken chromosomes has precluded identification of an underlying mechanism. The recent identification of centromeric breaks alongside aneuploidy in cells with spindle defects indicates that a single mechanism could account for all genetic alterations characteristic of chromosomal instability. Since a poorly controlled spindle can cause merotelic attachments, kinetochore distortion, and subsequent chromosome breakage, spindle defects can generate the sticky ends necessary to start a breakage-fusion-bridge cycle. The characteristic breakpoint of spindle-generated damage, adjacent to the centromere, also explains the losses and gains of whole chromosome arms, which are especially prominent in low-grade tumors. The recent data indicate that spindle defects are an early event in tumor formation, and an important initiator of carcinogenesis.

Centromere-localized breaks indicate the generation of DNA damage by the mitotic spindle.

Most carcinomas present some form of chromosome instability in combination with spindle defects. Numerical instability is likely caused by spindle aberrations, but the origin of breaks and translocations remains elusive. To determine whether one mechanism can bring about both types of instability, we studied the relationship between DNA damage and spindle defects. Although lacking apparent repair defects, primary Dido mutant cells formed micronuclei containing damaged DNA. The presence of centromeres showed that micronuclei were caused by spindle defects, and cell cycle markers showed that DNA damage was generated during mitosis. Although the micronuclei themselves persisted, the DNA damage within was repaired during S and G2 phases. DNA breaks in Dido mutant cells regularly colocalized with centromeres, which were occasionally distorted. Comparable defects were found in APC mutant cell lines, an independent system for spindle defects. On the basis of these results, we propose a model for break formation in which spindle defects lead to centromere shearing.

Synaptonemal complex assembly and H3K4Me3 demethylation determine DIDO3 localization in meiosis.

Synapsis of homologous chromosomes is a key meiotic event, mediated by a large proteinaceous structure termed the synaptonemal complex. Here, we describe a role in meiosis for the murine death-inducer obliterator (Dido) gene. The Dido gene codes for three proteins that recognize trimethylated histone H3 lysine 4 through their amino-terminal plant homeodomain domain. DIDO3, the largest of the three isoforms, localizes to the central region of the synaptonemal complex in germ cells. DIDO3 follows the distribution of the central region protein SYCP1 in Sycp3-/- spermatocytes, which lack the axial elements of the synaptonemal complex. This indicates that synapsis is a requirement for DIDO3 incorporation. Interestingly, DIDO3 is missing from the synaptonemal complex in Atm mutant spermatocytes, which form synapses but show persistent trimethylation of histone H3 lysine 4. In order to further address a role of epigenetic modifications in DIDO3 localization, we made a mutant of the Dido gene that produces a truncated DIDO3 protein. This truncated protein, which lacks the histone-binding domain, is incorporated in the synaptonemal complex irrespective of histone trimethylation status. DIDO3 protein truncation in Dido mutant mice causes mild meiotic defects, visible as gaps in the synaptonemal complex, but allows for normal meiotic progression. Our results indicate that histone H3 lysine 4 demethylation modulates DIDO3 localization in meiosis and suggest epigenetic regulation of the synaptonemal complex.

Dido disruption leads to centrosome amplification and mitotic checkpoint defects compromising chromosome stability.

Numerical and/or structural centrosome abnormalities have been correlated with most solid tumors and hematological malignancies. Tumorigenesis also is linked to defects in the mitotic or spindle assembly checkpoint, a key control mechanism that ensures accurate segregation of chromosomes during mitosis. We have reported that targeted disruption of the Dido gene causes a transplantable myelodysplastic/myeloproliferative disease in mice. Here, we report that Dido3, the largest splice variant of the Dido gene, is a centrosome-associated protein whose disruption leads to supernumerary centrosomes, failure to maintain cellular mitotic arrest, and early degradation of the mitotic checkpoint protein BubR1. These aberrations result in enhanced aneuploidy in the Dido mutant cells. Dido gene malfunction thus is reported to be part of an impaired signaling cascade that results in a defective mitotic checkpoint, leading to chromosome instability.

Death inducer obliterator protein 1 in the context of DNA regulation. Sequence analyses of distant homologues point to a novel functional role.

Death inducer obliterator protein 1 [DIDO1; also termed DIO-1 and death-associated transcription factor 1 (DATF-1)] is encoded by a gene thus far described only in higher vertebrates. Current gene ontology descriptions for this gene assign its function to an apoptosis-related process. The protein presents distinct splice variants and is distributed ubiquitously. Exhaustive sequence analyses of all DIDO variants identify distant homologues in yeast and other organisms. These homologues have a role in DNA regulation and chromatin stability, and form part of higher complexes linked to active chromatin. Further domain composition analyses performed in the context of related homologues suggest that DIDO-induced apoptosis is a secondary effect. Gene-targeted mice show alterations that include lagging chromosomes, and overexpression of the gene generates asymmetric nuclear divisions. Here we describe the analysis of these eukaryote-restricted proteins and propose a novel, DNA regulatory function for the DIDO protein in mammals.

Targeted disruption of the Mn1 oncogene results in severe defects in development of membranous bones of the cranial skeleton.

Fusion of the MN1 gene to TEL (ETV6) results in myeloid leukemia. The fusion protein combines the transcription activating domain of MN1 and the DNA binding domain of TEL and is thought to act as a deranged transcription factor. In addition, disruption of the large first exon of the MN1 gene is thought to inactivate MN1 function in a meningioma. To further investigate the role of MN1 in cancer, we generated Mn1 knockout mice. Mn1(+/-) animals were followed for 30 months, but they had no higher incidence of tumor formation than wild-type littermates. Mn1 null mice, however, were found to die at birth or shortly thereafter as the result of a cleft palate. Investigation of newborn or embryonic day 15.5 (E15.5) to E17.5 null mice revealed that the development of several bones in the skull was abnormal. The affected bones are almost exclusively formed by intramembranous ossification. They are either completely agenic at birth (alisphenoid and squamosal bones and vomer), hypoplastic, deformed (basisphenoid, pterygoid, and presphenoid), or substantially thinner (frontal, parietal, and interparietal bones). In heterozygous mice hypoplastic membranous bones and incomplete penetrance of the cleft palate were observed. We conclude that Mn1 is an important factor in development of membranous bones.