The relationship between neurodevelopmental transcriptional programs and insomnia: From Rubinstein-Taybi syndrome into energy metabolism.

Neurodevelopmental disorders (NDD) are characterized by cognitive, emotional, and/or motor skills impairment since childhood, and sleep disturbances are a common comorbidity. Rubinstein-Taybi syndrome (RSTS), a rare genetic syndrome associated with NDD, is caused by CREBBP haploinsufficiency. This gene encodes an acetyltransferase with crucial role on the establishment of transcriptional programs during neurodevelopment. Although insomnia has been reported in RSTS patients, the convergent mechanisms between this sleep disturbance and CREBBP loss-of-function are not fully understood. We tested weather the genetic architecture underlying CREBBP regulatory targets and insomnia-associated genes is significantly shared. We then identified the biological pathways enriched among these shared genes. The intersection between CREBBP regulatory targets and genes associated with insomnia included 7 overlapping genes, indicating significantly more overlap than expected by chance. An over-representation analysis on these intersect genes identified pathways related to mitochondrial activity. This finding indicates that the transcriptional programs established by CREBBP might impact insomnia-related biological pathways through the modulation of energy metabolism. The overlapping gene set and biological pathways highlighted by this study may serve as a primer for new functional investigations of shared molecular mechanisms between insomnia and CREBBP regulatory targets.

EP300 facilitates human trophoblast stem cell differentiation.

Early placenta development involves cytotrophoblast differentiation into extravillous trophoblast (EVT) and syncytiotrophoblast (STB). Defective trophoblast development and function may result in severe pregnancy complications, including fetal growth restriction and pre-eclampsia. The incidence of these complications is increased in pregnancies of fetuses affected by Rubinstein-Taybi syndrome, a developmental disorder predominantly caused by heterozygous mutations in CREB-binding protein (CREBBP) or E1A-binding protein p300 (EP300). Although the acetyltransferases CREBBP and EP300 are paralogs with many overlapping functions, the increased incidence of pregnancy complications is specific for EP300 mutations. We hypothesized that these complications have their origin in early placentation and that EP300 is involved in that process. Therefore, we investigated the role of EP300 and CREBBP in trophoblast differentiation, using human trophoblast stem cells (TSCs) and trophoblast organoids. We found that pharmacological CREBBP/EP300 inhibition blocks differentiation of TSCs into both EVT and STB lineages, and results in an expansion of TSC-like cells under differentiation-inducing conditions. Specific targeting by RNA interference or CRISPR/Cas9-mediated mutagenesis demonstrated that knockdown of EP300 but not CREBBP, inhibits trophoblast differentiation, consistent with the complications seen in Rubinstein-Taybi syndrome pregnancies. By transcriptome sequencing, we identified transforming growth factor alpha (TGFA, encoding TGF-α) as being strongly upregulated upon EP300 knockdown. Moreover, supplementing differentiation medium with TGF-α, which is a ligand for the epidermal growth factor receptor (EGFR), likewise affected trophoblast differentiation and resulted in increased TSC-like cell proliferation. These findings suggest that EP300 facilitates trophoblast differentiation by interfering with at least EGFR signaling, pointing towards a crucial role for EP300 in early human placentation.

Leveraging the Mendelian disorders of the epigenetic machinery to systematically map functional epigenetic variation.

Although each Mendelian Disorder of the Epigenetic Machinery (MDEM) has a different causative gene, there are shared disease manifestations. We hypothesize that this phenotypic convergence is a consequence of shared epigenetic alterations. To identify such shared alterations, we interrogate chromatin (ATAC-seq) and expression (RNA-seq) states in B cells from three MDEM mouse models (Kabuki [KS] type 1 and 2 and Rubinstein-Taybi type 1 [RT1] syndromes). We develop a new approach for the overlap analysis and find extensive overlap primarily localized in gene promoters. We show that disruption of chromatin accessibility at promoters often disrupts downstream gene expression, and identify 587 loci and 264 genes with shared disruption across all three MDEMs. Subtle expression alterations of multiple, IgA-relevant genes, collectively contribute to IgA deficiency in KS1 and RT1, but not in KS2. We propose that the joint study of MDEMs offers a principled approach for systematically mapping functional epigenetic variation in mammals.

Insights into the Role of the Microbiota and of Short-Chain Fatty Acids in Rubinstein-Taybi Syndrome.

The short-chain fatty acid butyrate, produced by the gut microbiota, acts as a potent histone deacetylase (HDAC) inhibitor. We assessed possible ameliorative effects of butyrate, relative to other HDAC inhibitors, in in vitro and in vivo models of Rubinstein-Taybi syndrome (RSTS), a severe neurodevelopmental disorder caused by variants in the genes encoding the histone acetyltransferases CBP and p300. In RSTS cell lines, butyrate led to the patient-specific rescue of acetylation defects at subtoxic concentrations. Remarkably, we observed that the commensal gut microbiota composition in a cohort of RSTS patients is significantly depleted in butyrate-producing bacteria compared to healthy siblings. We demonstrate that the effects of butyrate and the differences in microbiota composition are conserved in a Drosophila melanogaster mutant for CBP, enabling future dissection of the gut-host interactions in an in vivo RSTS model. This study sheds light on microbiota composition in a chromatinopathy, paving the way for novel therapeutic interventions.

Update and Potential Opportunities in CBP [Cyclic Adenosine Monophosphate (cAMP) Response Element-Binding Protein (CREB)-Binding Protein] Research Using Computational Techniques.

CBP [cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)-binding protein] is one of the most researched proteins for its therapeutic function. Several studies have identified its vast functions and interactions with other transcription factors to initiate cellular signals of survival. In cancer and other diseases such as Alzheimer's, Rubinstein-taybi syndrome, and inflammatory diseases, CBP has been implicated and hence an attractive target in drug design and development. In this review, we explore the various computational techniques that have been used in CBP research, furthermore we identified computational gaps that could be explored to facilitate the development of highly therapeutic CBP inhibitors.

Introduction of a de novo Creb-binding protein gene mutation in sperm to produce a Rubinstein-Taybi syndrome model using inbred C57BL/6 mice.

Neurodevelopmental disorders, including intellectual disability and autism spectrum disorder, are often caused by de novo autosomal dominant mutations. While mouse models are frequently used to investigate these disorders, the genetic background sometimes affects the appearance or severity of mutant phenotypes. In a previous report, we developed a system to produce de novo heterozygous mutant mice using the Cre-LoxP system without the need to maintain the heterozygous mutant line itself (Takagi et al. 2015). To further verify the applicability of the de novo mutation system in sperm, we used this system to produce a mouse model for Rubinstein-Taybi syndrome, using a Cbp heterozygous mutant, which has been reported to be difficult to maintain on a C57BL/6 background. Here, we show that de novo Cbp- loss-of-function heterozygous mutant mice with a C57BL/6 background, present with a clear craniofacial phenotype and reduced locomotor activity in the open field test, which was not observed in the loss-of-function of Cbp heterozygous mutant line mice with a mixed genetic background, but was observed in the dominant negative Cbp heterozygous mutant line with a mixed genetic background. Meanwhile, the de novo heterozygous Cbp mutant mice still showed great variability in survival rates despite their inbred background. These results further confirmed that the de novo mutation system used in germ cells is effective for stable production and analysis of an autosomal dominant disorder mouse model, which is often difficult to maintain as a mutant mouse line.

The transcriptional coactivator and histone acetyltransferase CBP regulates neural precursor cell development and migration.

CREB (cyclic AMP response element binding protein) binding protein (CBP, CREBBP) is a ubiquitously expressed transcription coactivator with intrinsic histone acetyltransferase (KAT) activity. Germline mutations within the CBP gene are known to cause Rubinstein-Taybi syndrome (RSTS), a developmental disorder characterized by intellectual disability, specific facial features and physical anomalies. Here, we investigate mechanisms of CBP function during brain development in order to elucidate morphological and functional mechanisms underlying the development of RSTS. Due to the embryonic lethality of conventional CBP knockout mice, we employed a tissue specific knockout mouse model (hGFAP-cre::CBPFl/Fl, mutant mouse) to achieve a homozygous deletion of CBP in neural precursor cells of the central nervous system.Our findings suggest that CBP plays a central role in brain size regulation, correct neural cell differentiation and neural precursor cell migration. We provide evidence that CBP is both important for stem cell viability within the ventricular germinal zone during embryonic development and for unhindered establishment of adult neurogenesis. Prominent histological findings in adult animals include a significantly smaller hippocampus with fewer neural stem cells. In the subventricular zone, we observe large cell aggregations at the beginning of the rostral migratory stream due to a migration deficit caused by impaired attraction from the CBP-deficient olfactory bulb. The cerebral cortex of mutant mice is characterized by a shorter dendrite length, a diminished spine number, and a relatively decreased number of mature spines as well as a reduced number of synapses.In conclusion, we provide evidence that CBP is important for neurogenesis, shaping neuronal morphology, neural connectivity and that it is involved in neuronal cell migration. These findings may help to understand the molecular basis of intellectual disability in RSTS patients and may be employed to establish treatment options to improve patients' quality of life.

Generation of three iPSC lines (IAIi002, IAIi004, IAIi003) from Rubinstein-Taybi syndrome 1 patients carrying CREBBP non sense c.4435G>T, p.(Gly1479*) and c.3474G>A, p.(Trp1158*) and missense c.4627G>T, p.(Asp1543Tyr) mutations.

Rubinstein-Taybi syndrome (RSTS) is a neurodevelopmental disorder characterized by growth retardation, skeletal anomalies and intellectual disability, caused by heterozygous mutations in either CREBBP (RSTS1) or EP300 (RSTS2) genes. We characterized 3 iPSC lines generated by Sendai from blood of RSTS1 patients with unique non sense c.4435G > T, p.(Gly1479*), c.3474G > A, p.(Trp1158*) and missense c.4627G > T, p.(Asp1543Tyr) CREBBP mutations. All lines displayed iPSC morphology, pluripotency markers, trilineage differentiation potential, stable karyotype and specific mutations. Western-blot using a CREB-Binding Protein N-terminus antibody demonstrated the same amount of full length protein as control in the missense mutation line and reduced amount in lines with stop mutations.

iPSC-derived neurons of CREBBP- and EP300-mutated Rubinstein-Taybi syndrome patients show morphological alterations and hypoexcitability.

Rubinstein-Taybi syndrome (RSTS) is a rare neurodevelopmental disorder characterized by distinctive facial features, growth retardation, broad thumbs and toes and mild to severe intellectual disability, caused by heterozygous mutations in either CREBBP or EP300 genes, encoding the homologous CBP and p300 lysine-acetyltransferases and transcriptional coactivators. No RSTS in vitro induced Pluripotent Stem Cell (iPSC)-neuronal model is available yet to achieve mechanistic insights on cognitive impairment of RSTS patients. We established iPSC-derived neurons (i-neurons) from peripheral blood cells of three CREBBP- and two EP300-mutated patients displaying different levels of intellectual disability, and four unaffected controls. Pan neuronal and cortical-specific markers were expressed by all patients' i-neurons. Altered morphology of patients' differentiating neurons, showing reduced branch length and increased branch number, and hypoexcitability of differentiated neurons emerged as potential disease biomarkers. Anomalous neuronal morphology and reduced excitability varied across different RSTS patients' i-neurons. Further studies are needed to validate these markers and assess whether they reflect cognitive and behavioural impairment of the donor patients.

Generation of the Rubinstein-Taybi syndrome type 2 patient-derived induced pluripotent stem cell line (IAIi001-A) carrying the EP300 exon 23 stop mutation c.3829A > T, p.(Lys1277*).

Rubinstein-Taybi syndrome (RSTS) is a neurodevelopmental disorder characterized by growth retardation, skeletal anomalies and intellectual disability, caused by heterozygous mutation in either the CREBBP (RSTS1) or EP300 (RSTS2) genes. We generated an induced pluripotent stem cell line from an RSTS2 patient's blood mononuclear cells by Sendai virus non integrative reprogramming method. The iPSC line (IAIi001RSTS2-65-A) displayed iPSC morphology, expressed pluripotency markers, possessed trilineage differentiation potential and was stable by karyotyping. Mutation and western blot analyses demonstrated in IAIi001RSTS2-65-A the patient's specific non sense mutation in exon 23 c.3829A > T, p.(Lys 1277*) and showed reduced quantity of wild type p300 protein.

Opposing Effects of CREBBP Mutations Govern the Phenotype of Rubinstein-Taybi Syndrome and Adult SHH Medulloblastoma.

Recurrent mutations in chromatin modifiers are specifically prevalent in adolescent or adult patients with Sonic hedgehog-associated medulloblastoma (SHH MB). Here, we report that mutations in the acetyltransferase CREBBP have opposing effects during the development of the cerebellum, the primary site of origin of SHH MB. Our data reveal that loss of Crebbp in cerebellar granule neuron progenitors (GNPs) during embryonic development of mice compromises GNP development, in part by downregulation of brain-derived neurotrophic factor (Bdnf). Interestingly, concomitant cerebellar hypoplasia was also observed in patients with Rubinstein-Taybi syndrome, a congenital disorder caused by germline mutations of CREBBP. By contrast, loss of Crebbp in GNPs during postnatal development synergizes with oncogenic activation of SHH signaling to drive MB growth, thereby explaining the enrichment of somatic CREBBP mutations in SHH MB of adult patients. Together, our data provide insights into time-sensitive consequences of CREBBP mutations and corresponding associations with human diseases.

Rubinstein-Taybi Syndrome and Epigenetic Alterations.

Rubinstein-Taybi syndrome (RSTS) is a rare genetic disorder in humans characterized by growth and psychomotor delay, abnormal gross anatomy, and mild to severe mental retardation (Rubinstein and Taybi, Am J Dis Child 105:588-608, 1963, Hennekam et al., Am J Med Genet Suppl 6:56-64, 1990). RSTS is caused by de novo mutations in epigenetics-associated genes, including the cAMP response element-binding protein (CREBBP), the gene-encoding protein referred to as CBP, and the EP300 gene, which encodes the p300 protein, a CBP homologue. Recent studies of the epigenetic mechanisms underlying cognitive functions in mice provide direct evidence for the involvement of nuclear factors (e.g., CBP) in the control of higher cognitive functions. In fact, a role for CBP in higher cognitive function is suggested by the finding that RSTS is caused by heterozygous mutations at the CBP locus (Petrij et al., Nature 376:348-351, 1995). CBP was demonstrated to possess an intrinsic histone acetyltransferase activity (Ogryzko et al., Cell 87:953-959, 1996) that is required for CREB-mediated gene expression (Korzus et al., Science 279:703-707, 1998). The intrinsic protein acetyltransferase activity in CBP might directly destabilize promoter-bound nucleosomes, facilitating the activation of transcription. Due to the complexity of developmental abnormalities and the possible genetic compensation associated with this congenital disorder, however, it is difficult to establish a direct role for CBP in cognitive function in the adult brain. Although aspects of the clinical presentation in RSTS cases have been extensively studied, a spectrum of symptoms found in RSTS patients can be accessed only after birth, and, thus, prenatal genetic tests for this extremely rare genetic disorder are seldom considered. Even though there has been intensive research on the genetic and epigenetic function of the CREBBP gene in rodents, the etiology of this devastating congenital human disorder is largely unknown.

Rubinstein-Taybi syndrome.

Rubinstein-Taybi syndrome (RTS) is characterized by moderate to severe intellectual disability, distinctive facial features, and broad thumbs and great toes. RTS is caused by haploinsufficiency of CREBBP or EP300 gene.

Rubinstein-Taybi Syndrome in a 19-years old boy.

INTRODUCTION: Rubinstein-Taybi syndrome is a rare genetic multisystem disorder comprising motor organ dysfunction, craniofacial dysmorphism and psychomotor retardation, frequently with the abnormalities of the thyroid gland. OBJECTIVE: Presentation of a case of a 19-year-old patient with Rubinstein-Taybi syndrome in whom serum TSH, fT3 and fT4 levels were assessed. CASE: Craniofacial abnormalities including: microcephaly, underdeveloped maxilla, micrognathia, high arched palate, malocclusion, down-slanting palpebral fissures, thick eyelashes and full eyebrows. Clinodactyly, broad thumbs and toes were observed in the musculoskeletal system. The patient presented with moderate mental retardation, short stature and obesity. Furthermore, I° thoracolumbar scoliosis, elbow joint deformation resulting from the radial head dislocation and limitation of the right hip motion as a consequence of Perthes disease were found. Genetic testing revealed a mutation affecting the CREBBP gene located on the short arm of chromosome 16. The measured serum TSH level was 1.510 µlU/ml (normal range 0.27-4.20), fT3 5.1 pmol/l (normal range 4.1-6.7), fT4 15.5 pmol/l (normal range 13.1-21.3). The patient is subjected to long-term rehabilitation. CONCLUSIONS: The obtained results of laboratory tests of serum TSH, fT3 and fT4 levels point to a lack of thyroid gland dysfunction in the patient with Rubinstein-Taybi syndrome. Rehabilitation treatment of patients with RTS is necessary to improve the patient's mobility.

Insights into genotype-phenotype correlations from CREBBP point mutation screening in a cohort of 46 Rubinstein-Taybi syndrome patients.

The genetic basis of Rubinstein-Taybi syndrome (RSTS), a rare, sporadic, clinically heterogeneous disorder characterized by cognitive impairment and a wide spectrum of multiple congenital anomalies, is primarily due to private mutations in CREBBP (approximately 55% of cases) or EP300 (approximately 8% of cases). Herein, we report the clinical and the genetic data taken from a cohort of 46 RSTS patients, all carriers of CREBBP point mutations. Molecular analysis revealed 45 different gene alterations including 31 inactivating (21 frameshift and 10 nonsense), 10 missense and 4 splicing mutations. Bioinformatic tools and transcript analyses were used to predict the functional effects of missense and splicing alterations. Of the 45 mutations, 42 are unreported and 3 were described previously. Recurrent mutations maybe a key tool in addressing genotype-phenotype correlations in patients sharing the same defects (at the genomic or transcript level) and specific clinical signs, demonstrated here in two cases. The clinical data of our cohort evidenced frequent signs such as arched eyebrows, epicanthus, synophrys and/or frontal hypertrichosis and broad phalanges that, previously overlooked in RSTS diagnosis, now could be considered. Some suggested correlations between organ-specific anomalies and affected CREB-binding protein domains broaden the RSTS clinical spectrum and perhaps will enhance patient follow-up and clinical care.

Válvula Aortica bicúspide en un paciente con síndorme de Rubinstein-Taybi / Bicuspid aortic valve in a patient with Rubinstein-Taybi syndrome

El síndrome de Rubinstein-Taybi es una enfermedad infrecuente que puede acompañarse de malformaciones cardiovasculares. Se presenta el caso de un paciente adulto con síndrome de Rubinstein-Taybi intervenido bajo circulación extracorpórea por presentar una válvula aórtica bicúspide

Rubinstein-Taybi syndrome is a rare disease that may be associated with cardiovascular malformations. We report the case of an adult patient with Rubinstein-Taybi syndrome who underwent an open heart procedure due to a bicuspid aortic valve

Epigenetic modifications in the nervous system and their impact upon cognitive impairments.

Epigenetic regulation has been long considered to be a critical mechanism in the control of key aspects of cellular functions such as cell division, growth, and cell fate determination. Exciting recent developments have demonstrated that epigenetic mechanisms can also play necessary roles in the nervous system by regulating, for example, neuronal gene expression, DNA damage, and genome stability. Despite the fact that postmitotic neurons are developmentally less active then dividing cells, epigenetic regulation appears to provide means of both long-lasting and very dynamic regulation of neuronal function. Growing evidence indicates that epigenetic mechanisms in the central nervous system (CNS) are important for regulating not only specific aspects of individual neuronal metabolism but also for maintaining function of neuronal circuits and regulating their behavioral outputs. Multiple reports demonstrated that higher-level cognitive behaviors, such as learning and memory, are subject to a sophisticated epigenetic control, which includes interplay between multiple mechanisms of neuronal chromatin modification. Experiments with animal models have demonstrated that various epigenetic manipulations can affect cognition in different ways, from severe dysfunction to substantial improvement. In humans, epigenetic dysregulation has been known to underlie a number of disorders that are accompanied by mental impairment. Here, we review some of the epigenetic mechanisms that regulate cognition and how their disruption may contribute to cognitive dysfunctions. Due to the fact that histone acetylation and DNA methylation are some of the best-studied and critically important epigenomic modifications our research team has particularly strong expertise in, in this review, we are going to concentrate on histone acetylation, as well as DNA methylation/hydroxymethylation, in the mammalian CNS. Additional epigenetic modifications, not surveyed here, are being discussed in depth in the other review articles in this issue of Neuropharmacology.

The role of genetics in the establishment and maintenance of the epigenome.

Epigenetic mechanisms play an important role in gene regulation during development. DNA methylation, which is probably the most important and best-studied epigenetic mechanism, can be abnormally regulated in common pathologies, but the origin of altered DNA methylation remains unknown. Recent research suggests that these epigenetic alterations could depend, at least in part, on genetic mutations or polymorphisms in DNA methyltransferases and certain genes encoding enzymes of the one-carbon metabolism pathway. Indeed, the de novo methyltransferase 3B (DNMT3B) has been recently found to be mutated in several types of cancer and in the immunodeficiency, centromeric region instability and facial anomalies syndrome (ICF), in which these mutations could be related to the loss of global DNA methylation. In addition, mutations in glycine-N-methyltransferase (GNMT) could be associated with a higher risk of hepatocellular carcinoma and liver disease due to an unbalanced S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio, which leads to aberrant methylation reactions. Also, genetic variants of chromatin remodeling proteins and histone tail modifiers are involved in genetic disorders like α thalassemia X-linked mental retardation syndrome, CHARGE syndrome, Cockayne syndrome, Rett syndrome, systemic lupus erythematous, Rubinstein-Taybi syndrome, Coffin-Lowry syndrome, Sotos syndrome, and facioescapulohumeral syndrome, among others. Here, we review the potential genetic alterations with a possible role on epigenetic factors and discuss their contribution to human disease.

Histone acetylation deficits in lymphoblastoid cell lines from patients with Rubinstein-Taybi syndrome.

BACKGROUND: Rubinstein-Taybi syndrome (RSTS) is a congenital neurodevelopmental disorder defined by postnatal growth deficiency, characteristic skeletal abnormalities and mental retardation and caused by mutations in the genes encoding for the transcriptional co-activators with intrinsic lysine acetyltransferase (KAT) activity CBP and p300. Previous studies have shown that neuronal histone acetylation is reduced in mouse models of RSTS. METHODS: The authors identified different mutations at the CREBBP locus and generated lymphoblastoid cell lines derived from nine patients with RSTS carrying distinct CREBBP mutations that illustrate different grades of the clinical severity in the spectrum of the syndrome. They next assessed whether histone acetylation levels were altered in these cell lines. RESULTS: The comparison of CREBBP-mutated RSTS cell lines with cell lines derived from patients with an unrelated mental retardation syndrome or healthy controls revealed significant deficits in histone acetylation, affecting primarily histone H2B and histone H2A. The most severe defects were observed in the lines carrying the whole deletion of the CREBBP gene and the truncating mutation, both leading to a haploinsufficiency state. Interestingly, this deficit was rescued by treatment with an inhibitor of histone deacetylases (HDACi). CONCLUSIONS: The authors' results extend to humans the seminal observations in RSTS mouse models and point to histone acetylation defects, mainly involving H2B and H2A, as relevant molecular markers of the disease.