Dissecting the Causal Mechanism of X-Linked Dystonia-Parkinsonism by Integrating Genome and Transcriptome Assembly.

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders.

Genesis of chromatin and transcription dynamics in the origin of species.

Histone proteins compact and stabilize the genomes of Eukarya and Archaea. By forming nucleosome(-like) structures they restrict access of DNA-binding transcription regulators to cis-regulatory DNA elements. Dynamic competition between histones and transcription factors is facilitated by different classes of proteins including ATP-dependent remodeling enzymes that control assembly, access, and editing of chromatin. Here, we summarize the knowledge on dynamics underlying transcriptional regulation across the domains of life with a focus on ATP-dependent enzymes in chromatin structure or in TATA-binding protein activity. These insights suggest directions for future studies on the evolution of transcription regulation and chromatin dynamics.

Epigenetics and transcription regulation during eukaryotic diversification: the saga of TFIID.

The basal transcription factor TFIID is central for RNA polymerase II-dependent transcription. Human TFIID is endowed with chromatin reader and DNA-binding domains and protein interaction surfaces. Fourteen TFIID TATA-binding protein (TBP)-associated factor (TAF) subunits assemble into the holocomplex, which shares subunits with the Spt-Ada-Gcn5-acetyltransferase (SAGA) coactivator. Here, we discuss the structural and functional evolution of TFIID and its divergence from SAGA. Our orthologous tree and domain analyses reveal dynamic gains and losses of epigenetic readers, plant-specific functions of TAF1 and TAF4, the HEAT2-like repeat in TAF2, and, importantly, the pre-LECA origin of TFIID and SAGA. TFIID evolution exemplifies the dynamic plasticity in transcription complexes in the eukaryotic lineage.

Transcript Buffering: A Balancing Act between mRNA Synthesis and mRNA Degradation.

Transcript buffering involves reciprocal adjustments between overall rates in mRNA synthesis and degradation to maintain similar cellular concentrations of mRNAs. This phenomenon was first discovered in yeast and encompasses coordination between the nuclear and cytoplasmic compartments. Transcript buffering was revealed by novel methods for pulse labeling of RNA to determine in vivo synthesis and degradation rates. In this Perspective, we discuss the current knowledge of transcript buffering. Emphasis is placed on the future challenges to determine the nature and directionality of the buffering signals, the generality of transcript buffering beyond yeast, and the molecular mechanisms responsible for this balancing.

greenPipes: an integrated data analysis pipeline for greenCUT&RUN and CUT&RUN genome-localization datasets.

MOTIVATION: To study gene regulation through transcription factors and chromatin modifiers, a variety of genome-wide techniques are used. Recently, CUT&RUN-based technologies have become popular, but a pipeline for the comprehensive analysis of CUT&RUN datasets is currently lacking. Here, we present the "greenPipes" package, which includes fine-tuned parameters specifically for bioinformatic analyses of greenCUT&RUN and CUT&RUN datasets. greenPipes provides additional functionalities for data analysis and data integration with other -omics technologies, which are either not available in other pipelines developed for CUT&RUN datasets or scattered in the literature as individual packages. AVAILABILITY AND IMPLEMENTATION: Source code and a manual of the greenPipes are freely available on GitHub website (https://github.com/snizam001/greenPipes). The test datasets, comprehensive annotation files, and other datasets are available at https://osf.io/ruhj9/. CONTACT: n.sheikh@dkfz-heidelberg.de or m.timmers@dkfz-heidelberg.de. SUPPLEMENTARY INFORMATION: The handbook of greenPipes is available online at Bioinformatics as Supplementary text.

SAGA Is a General Cofactor for RNA Polymerase II Transcription.

Prior studies suggested that SAGA and TFIID are alternative factors that promote RNA polymerase II transcription, with about 10% of genes in S. cerevisiae dependent on SAGA. We reassessed the role of SAGA by mapping its genome-wide location and role in global transcription in budding yeast. We find that SAGA maps to the UAS elements of most genes, overlapping with Mediator binding and irrespective of previous designations of SAGA- or TFIID-dominated genes. Disruption of SAGA through mutation or rapid subunit depletion reduces transcription from nearly all genes, measured by newly synthesized RNA. We also find that the acetyltransferase Gcn5 synergizes with Spt3 to promote global transcription and that Spt3 functions to stimulate TBP recruitment at all tested genes. Our data demonstrate that SAGA acts as a general cofactor required for essentially all RNA polymerase II transcription and is not consistent with the previous classification of SAGA- and TFIID-dominated genes.

Mot1, Ino80C, and NC2 Function Coordinately to Regulate Pervasive Transcription in Yeast and Mammals.

Pervasive transcription initiates from cryptic promoters and is observed in eukaryotes ranging from yeast to mammals. The Set2-Rpd3 regulatory system prevents cryptic promoter function within expressed genes. However, conserved systems that control pervasive transcription within intergenic regions have not been well established. Here we show that Mot1, Ino80 chromatin remodeling complex (Ino80C), and NC2 co-localize on chromatin and coordinately suppress pervasive transcription in S. cerevisiae and murine embryonic stem cells (mESCs). In yeast, all three proteins bind subtelomeric heterochromatin through a Sir3-stimulated mechanism and to euchromatin via a TBP-stimulated mechanism. In mESCs, the proteins bind to active and poised TBP-bound promoters along with promoters of polycomb-silenced genes apparently lacking TBP. Depletion of Mot1, Ino80C, or NC2 by anchor away in yeast or RNAi in mESCs leads to near-identical transcriptome phenotypes, with new subtelomeric transcription in yeast, and greatly increased pervasive transcription in both yeast and mESCs.

Epigenetic-focused CRISPR/Cas9 screen identifies (absent, small, or homeotic)2-like protein (ASH2L) as a regulator of glioblastoma cell survival.

BACKGROUND: Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers. METHOD: In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. RESULTS: Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. CONCLUSION: Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. Video Abstract.

De Novo Heterozygous POLR2A Variants Cause a Neurodevelopmental Syndrome with Profound Infantile-Onset Hypotonia.

The RNA polymerase II complex (pol II) is responsible for transcription of all ∼21,000 human protein-encoding genes. Here, we describe sixteen individuals harboring de novo heterozygous variants in POLR2A, encoding RPB1, the largest subunit of pol II. An iterative approach combining structural evaluation and mass spectrometry analyses, the use of S. cerevisiae as a model system, and the assessment of cell viability in HeLa cells allowed us to classify eleven variants as probably disease-causing and four variants as possibly disease-causing. The significance of one variant remains unresolved. By quantification of phenotypic severity, we could distinguish mild and severe phenotypic consequences of the disease-causing variants. Missense variants expected to exert only mild structural effects led to a malfunctioning pol II enzyme, thereby inducing a dominant-negative effect on gene transcription. Intriguingly, individuals carrying these variants presented with a severe phenotype dominated by profound infantile-onset hypotonia and developmental delay. Conversely, individuals carrying variants expected to result in complete loss of function, thus reduced levels of functional pol II from the normal allele, exhibited the mildest phenotypes. We conclude that subtle variants that are central in functionally important domains of POLR2A cause a neurodevelopmental syndrome characterized by profound infantile-onset hypotonia and developmental delay through a dominant-negative effect on pol-II-mediated transcription of DNA.

What can clinical immunology learn from inborn errors of epigenetic regulators?

The epigenome is at the interface between environmental factors and the genome, regulating gene transcription, DNA repair, and replication. Epigenetic modifications play a crucial role in establishing and maintaining cell identity and are especially crucial for neurology, musculoskeletal integrity, and the function of the immune system. Mutations in genes encoding for the components of the epigenetic machinery lead to the development of distinct disorders, especially involving the central nervous system and host defense. In this review, we focus on the role of epigenetic modifications for the function of the immune system. By studying the immune phenotype of patients with monogenic mutations in components of the epigenetic machinery (inborn errors of epigenetic regulators), we demonstrate the importance of DNA methylation, histone modifications, chromatin remodeling, noncoding RNAs, and mRNA processing for immunity. Moreover, we give a short overview on therapeutic strategies targeting the epigenome.

Volumetric evaluation of CT images of adrenal glands in primary aldosteronism.

OBJECTIVES: To investigate whether adrenal volumetry provides better agreement with adrenal vein sampling (AVS) than conventional CT for subtyping PA. Furthermore, we evaluated whether the size of this contralateral adrenal was a prognostic factor for clinical outcome after unilateral adrenalectomy. METHODS: We retrospectively analyzed volumes of both adrenal glands of the 180 CT-scans (88/180 with unilateral and 92/180 with bilateral disease) of the patients with PA included in the SPARTACUS trial of which 85 also had undergone an AVS. In addition, we examined CT-scans of 20 healthy individuals to compare adrenal volumes with published normal values. RESULTS: Adrenal volume was higher for the left than the right adrenal (mean and SD: 6.49 ± 2.77 ml versus 5.25 ± 1.87 ml for the right adrenal; p < 0.001). Concordance between volumetry and AVS in subtyping was 58.8%, versus 51.8% between conventional CT results and AVS (p = NS). The volumes of the contralateral adrenals in the patients with unilateral disease (right 4.78 ± 1.37 ml; left 6.00 ± 2.73 ml) were higher than those of healthy controls reported in the literature (right 3.62 ± 1.23 ml p < 0.001; left 4.84 ± 1.67 ml p = 0.02). In a multivariable analysis the contralateral volume was not associated with biochemical or clinical success, nor with the defined daily doses of antihypertensive agents at 1 year follow-up. CONCLUSIONS: Volumetry of the adrenal glands is not superior to current assessment of adrenal size by CT for subtyping patients with PA. Furthermore, in patients with unilateral disease the size of the contralateral adrenal is enlarged but its size is not associated with outcome.

Neuronal-specific microexon splicing of TAF1 mRNA is directly regulated by SRRM4/nSR100.

Neuronal microexons represent the most highly conserved class of alternative splicing events and their timed expression shapes neuronal biology, including neuronal commitment and differentiation. The six-nt microexon 34' is included in the neuronal form of TAF1 mRNA, which encodes the largest subunit of the basal transcription factor TFIID. In this study, we investigate the tissue distribution of TAF1-34' mRNA and protein and the mechanism responsible for its neuronal-specific splicing. Using isoform-specific RNA probes and antibodies, we observe that canonical TAF1 and TAF1-34' have different distributions in the brain, which distinguish proliferating from post-mitotic neurons. Knockdown and ectopic expression experiments demonstrate that the neuronal-specific splicing factor SRRM4/nSR100 promotes the inclusion of microexon 34' into TAF1 mRNA, through the recognition of UGC sequences in the poly-pyrimidine tract upstream of the regulated microexon. These results show that SRRM4 regulates temporal and spatial expression of alternative TAF1 mRNAs to generate a neuronal-specific TFIID complex.

ATAC-king the complexity of SAGA during evolution.

The yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) coactivator complex exerts functions in gene expression, including activator interaction, histone acetylation, histone deubiquitination, mRNA export, chromatin recognition, and regulation of the basal transcription machinery. These diverse functions involve distinct modules within this multiprotein complex. It has now become clear that yeast SAGA has diverged during metazoan evolution into two related complexes, SAGA and ATAC, which exist in two flavors in vertebrates. The compositions of metazoan ATAC and SAGA complexes have been characterized, and functional analyses indicate that these complexes have important but distinct roles in transcription, histone modification, signaling pathways, and cell cycle regulation.

A central role for TFIID in the pluripotent transcription circuitry.

Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states.

Genomic integrity of ground-state pluripotency.

Pluripotent cells appear to be in a transient state during early development. These cells have the capability to transition into embryonic stem cells (ESCs). It has been reported that mouse pluripotent cells cultivated in chemically defined media sustain the ground state of pluripotency. Because the epigenetic pattern of pluripotent cells reflects their environment, culture under different conditions causes epigenetic changes, which could lead to genomic instability. This study focused on the DNA methylation pattern of repetitive elements (REs) and their activation levels under two ground-state conditions and assessed the genomic integrity of ESCs. We measured the methylation and expression level of REs in different media. The results indicated that although the ground-state conditions show higher REs activity, they did not lead to DNA damage; therefore, the level of genomic instability is lower under the ground-state compared with the conventional condition. Our results indicated that when choosing an optimum condition, different features of the condition must be considered to have epigenetically and genomically stable stem cells.

Soft X-ray refractive index by reconciling total electron yield with specular reflection: experimental determination of the optical constants of graphite.

The complex refractive index of many materials is poorly known in the soft X-ray range across absorption edges. This is due to saturation effects that occur there in total-electron-yield and fluorescence-yield spectroscopy and that are strongest at resonance energies. Aiming to obtain reliable optical constants, a procedure that reconciles electron-yield measurements and reflection spectroscopy by correcting these saturation effects is presented. The procedure takes into account the energy- and polarization-dependence of the photon penetration depth as well as the creation efficiency for secondary electrons and their escape length. From corrected electron-yield spectra the absorption constants and the imaginary parts of the refractive index of the material are determined. The real parts of the index are subsequently obtained through a Kramers-Kronig transformation. These preliminary optical constants are refined by simulating reflection spectra and adapting them, so that measured reflection spectra are reproduced best. The efficacy of the new procedure is demonstrated for graphite. The optical constants that have been determined for linearly polarized synchrotron light incident with p- and s-geometry provide a detailed and reliable representation of the complex refractive index of the material near π- and σ-resonances. They are also suitable for allotropes of graphite such as graphene.

The penetrance of paraganglioma and pheochromocytoma in SDHB germline mutation carriers.

Germline mutations in succinate dehydrogenase B (SDHB) predispose to hereditary paraganglioma (PGL) syndrome type 4. The risk of developing PGL or pheochromocytoma (PHEO) in SDHB mutation carriers is subject of recent debate. In the present nationwide cohort study of SDHB mutation carriers identified by the clinical genetics centers of the Netherlands, we have calculated the penetrance of SDHB associated tumors using a novel maximum likelihood estimator. This estimator addresses ascertainment bias and missing data on pedigree size and structure. A total of 195 SDHB mutation carriers were included, carrying 27 different SDHB mutations. The 2 most prevalent SDHB mutations were Dutch founder mutations: a deletion in exon 3 (31% of mutation carriers) and the c.423+1G>A mutation (24% of mutation carriers). One hundred and twelve carriers (57%) displayed no physical, radiological or biochemical evidence of PGL or PHEO. Fifty-four patients had a head and neck PGL (28%), 4 patients had a PHEO (2%), 26 patients an extra-adrenal PGL (13%). The overall penetrance of SDHB mutations is estimated to be 21% at age 50 and 42% at age 70 when adequately corrected for ascertainment. These estimates are lower than previously reported penetrance estimates of SDHB-linked cohorts. Similar disease risks are found for different SDHB germline mutations as well as for male and female SDHB mutation carriers.

Expression of p27Kip1 and p18Ink4c in human multiple endocrine neoplasia type 1-related pancreatic neuroendocrine tumors.

PURPOSE: Pancreatic neuroendocrine tumors are a major manifestation of multiple endocrine neoplasia type 1 (MEN1). This tumor syndrome is caused by germline mutations in MEN1, encoding menin. Insight into pathogenesis of these tumors might lead to new biomarkers and therapeutic targets for these patients. Several lines of evidence point towards a role for p27Kip1 and p18Ink4c in MEN1-related tumor development in animal models for MEN1, but their contribution to human MEN1-related pancreatic neuroendocrine tumor development is not known. METHODS: In this study, we characterized protein expression of p27Kip1 and p18Ink4c in human MEN1-related PanNETs by immunohistochemistry. From the nationwide DutchMEN1 Study Group database including > 90% of the Dutch MEN1 population, MEN1-patients, who underwent pancreatic surgery, were selected. A tissue micro-array was constructed with available paraffin tissue blocks, and PanNETs from 61 MEN1 patients were eligible for analysis. RESULTS: Expression of p27Kip1 was high in 57 (93%) PanNETs and 67% of the tumors showed low expression of p18Ink4c (67.3%). No association was found between expression of either p27Kip1 or p18Ink4c and clinic-pathological characteristics. CONCLUSIONS: These findings indicate that loss of p18Ink4c, but not p27Kip1, is a common event in the development of MEN1-related PanNETs. Restoration of p18Ink4c function through CDK4/6 inhibitors could be a therapeutic option for MEN1-related PanNETs.

Results of a systematic literature review of treatment modalities for jugulotympanic paraganglioma, stratified per Fisch class.

OBJECTIVE: Key for successful jugulotympanic paraganglioma management is a personalised approach aiming for the best practice for each individual patient. To this end, a systematic review is performed, evaluating the local control and complication rates for the different treatment modalities stratified by the broadly accepted Fisch classification. DESIGN: A systematic literature review according to the PRISMA statement was performed. A detailed overview of individual treatment outcomes per Fisch class is provided. MAIN OUTCOME MEASURES: Local control, cranial nerve damage, complications, function recovery. RESULTS: Eighteen studies were selected, resembling 83 patients treated with radiotherapy and 299 with surgery. Excellent local control was found post-surgery for class A and B tumours, and risk of cranial nerve damage was <1%. For class C1-4 tumours, local control was 80%-95% post-surgery (84% post-radiotherapy), and cranial nerve damage was found in 71%-76% (none post-radiotherapy; P < .05). There was no difference in treatment outcomes between tumours of different C class. For class C1-4De/Di tumours, local control was 38%-86% (98% post-radiotherapy; P < .05) and cranial nerve damage/complication rates were 67%-100% (3% post-radiotherapy; P < .05). C1-4DeDi tumours showed lesser local control and cranial nerve damage rates when compared to C1-4De tumours. CONCLUSIONS: An individual risk is constituted for surgery and radiotherapy, stratified per Fisch class. For class A and B tumours, surgery is a suitable treatment option. For class C and D tumours, radiotherapy results in lower complication rates and similar or better local control rates when compared to the surgical group.