ChAHP2 and ChAHP control diverse retrotransposons by complementary activities.

Retrotransposon control in mammals is an intricate process that is effectuated by a broad network of chromatin regulatory pathways. We previously discovered ChAHP, a protein complex with repressive activity against short interspersed element (SINE) retrotransposons that is composed of the transcription factor ADNP, chromatin remodeler CHD4, and HP1 proteins. Here we identify ChAHP2, a protein complex homologous to ChAHP, in which ADNP is replaced by ADNP2. ChAHP2 is predominantly targeted to endogenous retroviruses (ERVs) and long interspersed elements (LINEs) via HP1ß-mediated binding of H3K9 trimethylated histones. We further demonstrate that ChAHP also binds these elements in a manner mechanistically equivalent to that of ChAHP2 and distinct from DNA sequence-specific recruitment at SINEs. Genetic ablation of ADNP2 alleviates ERV and LINE1 repression, which is synthetically exacerbated by additional depletion of ADNP. Together, our results reveal that the ChAHP and ChAHP2 complexes function to control both nonautonomous and autonomous retrotransposons by complementary activities, further adding to the complexity of mammalian transposon control.

Convergence of multiple RNA-silencing pathways on GW182/TNRC6.

The RNA-binding protein TRIM71/LIN-41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development, and cancer. TRIM71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Here, we uncover that TRIM71 represses its targets through RNA-supported interaction with TNRC6/GW182, a core component of the miRNA-induced silencing complex (miRISC). We demonstrate that AGO2, TRIM71, and UPF1 each recruit TNRC6 to specific sets of transcripts to silence them. As cellular TNRC6 levels are limiting, competition occurs among the silencing pathways, such that the loss of AGO proteins or of AGO binding to TNRC6 enhances the activities of the other pathways. We conclude that a miRNA-like silencing activity is shared among different mRNA silencing pathways and that the use of TNRC6 as a central hub provides a means to integrate their activities.

Protease-mediated processing of Argonaute proteins controls small RNA association.

Small RNA pathways defend the germlines of animals against selfish genetic elements, yet pathway activities need to be contained to prevent silencing of self genes. Here, we reveal a proteolytic mechanism that controls endogenous small interfering (22G) RNA activity in the Caenorhabditis elegans germline to protect genome integrity and maintain fertility. We find that DPF-3, a P-granule-localized N-terminal dipeptidase orthologous to mammalian dipeptidyl peptidase (DPP) 8/9, processes the unusually proline-rich N termini of WAGO-1 and WAGO-3 Argonaute (Ago) proteins. Without DPF-3 activity, these WAGO proteins lose their proper complement of 22G RNAs. Desilencing of repeat-containing and transposon-derived transcripts, DNA damage, and acute sterility ensue. These phenotypes are recapitulated when WAGO-1 and WAGO-3 are rendered resistant to DPF-3-mediated processing, identifying them as critical substrates of DPF-3. We conclude that N-terminal processing of Ago proteins regulates their activity and promotes silencing of selfish genetic elements by ensuring Ago association with appropriate small RNAs.

Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.

RNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5'-3') mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3'-5') is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.

BANP opens chromatin and activates CpG-island-regulated genes.

The majority of gene transcripts generated by RNA polymerase II in mammalian genomes initiate at CpG island (CGI) promoters1,2, yet our understanding of their regulation remains limited. This is in part due to the incomplete information that we have on transcription factors, their DNA-binding motifs and which genomic binding sites are functional in any given cell type3-5. In addition, there are orphan motifs without known binders, such as the CGCG element, which is associated with highly expressed genes across human tissues and enriched near the transcription start site of a subset of CGI promoters6-8. Here we combine single-molecule footprinting with interaction proteomics to identify BTG3-associated nuclear protein (BANP) as the transcription factor that binds this element in the mouse and human genome. We show that BANP is a strong CGI activator that controls essential metabolic genes in pluripotent stem and terminally differentiated neuronal cells. BANP binding is repelled by DNA methylation of its motif in vitro and in vivo, which epigenetically restricts most binding to CGIs and accounts for differential binding at aberrantly methylated CGI promoters in cancer cells. Upon binding to an unmethylated motif, BANP opens chromatin and phases nucleosomes. These findings establish BANP as a critical activator of a set of essential genes and suggest a model in which the activity of CGI promoters relies on methylation-sensitive transcription factors that are capable of chromatin opening.

Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d.

N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m6A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. Here we identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m6A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m6A levels and is involved in sex determination in Drosophila We demonstrate that Flacc promotes m6A deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, our work advances the molecular understanding of conservation and regulation of the m6A machinery.

Cooperation between HDAC3 and DAX1 mediates lineage restriction of embryonic stem cells.

Mouse embryonic stem cells (mESCs) are biased toward producing embryonic rather than extraembryonic endoderm fates. Here, we identify the mechanism of this barrier and report that the histone deacetylase Hdac3 and the transcriptional corepressor Dax1 cooperatively limit the lineage repertoire of mESCs by silencing an enhancer of the extraembryonic endoderm-specifying transcription factor Gata6. This restriction is opposed by the pluripotency transcription factors Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC transcriptional network.

Mouse nuclear RNAi-defective 2 promotes splicing of weak 5' splice sites.

Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5' splice site (5'SS). In mammals, many introns contain weak 5'SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (nuclear RNAi-defective 2), and CCDC174 (coiled-coil domain-containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and 5'SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP-specific proteins, and they are required for the selection and effective processing of weak 5'SSs. Our results reveal that mammalian cells use noncanonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5'SS sequences in hundreds of genes, promoting proper splice site choice, and accurate pre-mRNA splicing.

Differential phosphorylation of Clr4SUV39H by Cdk1 accompanies a histone H3 methylation switch that is essential for gametogenesis.

Methylation of histone H3 at lysine 9 (H3K9) is a hallmark of heterochromatin that plays crucial roles in gene silencing, genome stability, and chromosome segregation. In Schizosaccharomyces pombe, Clr4 mediates both di- and tri-methylation of H3K9. Although H3K9 methylation has been intensely studied in mitotic cells, its role during sexual differentiation remains unclear. Here, we map H3K9 methylation genome-wide during meiosis and show that constitutive heterochromatin temporarily loses H3K9me2 and becomes H3K9me3 when cells commit to meiosis. Cells lacking the ability to tri-methylate H3K9 exhibit meiotic chromosome segregation defects. Finally, the H3K9 methylation switch is accompanied by differential phosphorylation of Clr4 by the cyclin-dependent kinase Cdk1. Our results suggest that a conserved master regulator of the cell cycle controls the specificity of an H3K9 methyltransferase to prevent ectopic H3K9 methylation and to ensure faithful gametogenesis.

Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2.

Developmental cell fate specification is a unidirectional process that can be reverted in response to injury or experimental reprogramming. Whether differentiation and de-differentiation trajectories intersect mechanistically is unclear. Here, we performed comparative screening in lineage-related mouse naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), and identified the constitutively expressed zinc finger transcription factor (TF) Zfp281 as a bidirectional regulator of cell state interconversion. We showed that subtle chromatin binding changes in differentiated cells translate into activation of the histone H3 lysine 9 (H3K9) methyltransferase Ehmt1 and stabilization of the zinc finger TF Zic2 at enhancers and promoters. Genetic gain-of-function and loss-of-function experiments confirmed a critical role of Ehmt1 and Zic2 downstream of Zfp281 both in driving exit from the ESC state and in restricting reprogramming of EpiSCs. Our study reveals that cell type-invariant chromatin association of Zfp281 provides an interaction platform for remodeling the cis-regulatory network underlying cellular plasticity.

Structure of the human C9orf72-SMCR8 complex reveals a multivalent protein interaction architecture.

A major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G4C2 repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain-of-function by repeat G4C2 RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy. Here, we report the structure of the C9orf72-SMCR8 complex at 3.8 Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals 2 distinct dimerization interfaces between C9orf72 and SMCR8 that involves an extensive network of interactions. Homology between C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2), a GTPase activating protein (GAP) complex, enabled identification of a key residue within the active site of SMCR8. Further structural analysis suggested that a coiled-coil region within the uDenn domain of SMCR8 could act as an interaction platform for other coiled-coil proteins, and its deletion reduced the interaction of the C9orf72-SMCR8 complex with FIP200 upon starvation. In summary, this study contributes toward our understanding of the biological function of the C9orf72-SMCR8 complex.

The Effect of a Temporary Stoma on Long-term Functional Outcomes Following Surgery for Rectal Cancer.

BACKGROUND: Patients with rectal cancer may undergo surgical resection with or without a temporary stoma. OBJECTIVE: This study primarily aimed to compare long-term functional outcomes between patients with and without a temporary stoma after surgery for rectal cancer. The secondary aim was to investigate the effect of time to stoma reversal on functional outcomes. DESIGN: This was a multicenter, cross-sectional study. SETTINGS: This study was conducted at 7 Dutch hospitals. PATIENTS: Included were patients who had undergone rectal cancer surgery (2009-2015). Excluded were deceased patients, who were deceased, had a permanent stoma, or had intellectual disability. MAIN OUTCOME MEASURES: Functional outcomes were measured using the Rome IV criteria for constipation and fecal incontinence and the low anterior resection syndrome score. RESULTS: Of 656 patients, 32% received a temporary ileostomy and 20% a temporary colostomy (86% response). Follow-up was at 56 (interquartile range, 38.5-79) months. Patients who had a temporary ileostomy experienced less constipation, more fecal incontinence, and more major low anterior resection syndrome than those without a temporary stoma. Patients who had a temporary colostomy experienced more major low anterior resection syndrome than those without a temporary stoma. A temporary ileostomy or colostomy was not associated with constipation or fecal incontinence after correction for confounding factors (eg, anastomotic height, anastomotic leakage, radiotherapy). Time to stoma reversal was not associated with constipation, fecal incontinence, or major low anterior resection syndrome. LIMITATIONS: Cross-sectional design. CONCLUSIONS: Although patients with a temporary ileostomy or colostomy have worse functional outcomes in the long term, it seems that the reason for creating a temporary stoma, rather than the stoma itself, underlies this phenomenon. Time to reversal of a temporary stoma does not influence functional outcomes. See Video Abstract . EL EFECTO DEL ESTOMA TEMPORAL SOBRE LOS RESULTADOS FUNCIONALES A LARGO PLAZO DESPUS DE LA CIRUGA POR CNCER DE RECTO: ANTECEDENTES:Los pacientes con cáncer de recto pueden someterse a resección quirúrgica con o sin un estoma temporal.OBJETIVO:El objetivo principal de este estudio fue comparar los resultados funcionales a largo plazo entre pacientes con y sin estoma temporal después de cirugía por cáncer de recto. El objetivo secundario fue investigar el efecto del tiempo transcurrido hasta la reversión del estoma sobre los resultados funcionales.DISEÑO:Este fue un estudio transversal multicéntrico.ESCENARIO:Este estudio se llevó a cabo en siete hospitales holandeses.PACIENTES:Se incluyeron pacientes sometidos a cirugía de cáncer de recto (2009-2015). Se excluyeron pacientes fallecidos, pacientes con estoma permanente o discapacidad intelectual.PRINCIPALES MEDIDAS DE RESULTADO:Los resultados funcionales se midieron utilizando los criterios de Roma IV para el estreñimiento y la incontinencia fecal y la puntuación del síndrome de resección anterior baja (LARS).RESULTADOS:De 656 pacientes, el 32% recibió una ileostomía temporal y el 20% una colostomía temporal (respuesta del 86%). El seguimiento fue de 56.0 (RIQ 38.5-79.0) meses. Los pacientes a los que se les realizó una ileostomía temporal experimentaron menos estreñimiento, más incontinencia fecal y más LARS mayor que los pacientes sin un estoma temporal. Los pacientes que tuvieron una colostomía temporal experimentaron más LARS mayor que los pacientes sin un estoma temporal. Una ileostomía o colostomía temporal no se asoció con estreñimiento o incontinencia fecal después de la corrección de factores de confusión (p. ej., altura anastomótica, fuga anastomótica, radioterapia). El tiempo hasta la reversión del estoma no se asoció con estreñimiento, incontinencia fecal o LARS mayor.LIMITACIONES:El presente estudio está limitado por su diseño transversal.CONCLUSIONES:Aunque los pacientes con una ileostomía o colostomía temporal tienen peores resultados funcionales a largo plazo, parece que la razón para crear un estoma temporal, más que el estoma en sí, se asocia a este fenómeno. El tiempo hasta la reversión de un estoma temporal no influye en los resultados funcionales. (Traducción-Dr. Jorge Silva Velazco ).

Activity-dependent neuroprotective protein recruits HP1 and CHD4 to control lineage-specifying genes.

De novo mutations in ADNP, which encodes activity-dependent neuroprotective protein (ADNP), have recently been found to underlie Helsmoortel-Van der Aa syndrome, a complex neurological developmental disorder that also affects several other organ functions 1 . ADNP is a putative transcription factor that is essential for embryonic development 2 . However, its precise roles in transcriptional regulation and development are not understood. Here we show that ADNP interacts with the chromatin remodeller CHD4 and the chromatin architectural protein HP1 to form a stable complex, which we refer to as ChAHP. Besides mediating complex assembly, ADNP recognizes DNA motifs that specify binding of ChAHP to euchromatin. Genetic ablation of ChAHP components in mouse embryonic stem cells results in spontaneous differentiation concomitant with premature activation of lineage-specific genes and in a failure to differentiate towards the neuronal lineage. Molecularly, ChAHP-mediated repression is fundamentally different from canonical HP1-mediated silencing: HP1 proteins, in conjunction with histone H3 lysine 9 trimethylation (H3K9me3), are thought to assemble broad heterochromatin domains that are refractory to transcription. ChAHP-mediated repression, however, acts in a locally restricted manner by establishing inaccessible chromatin around its DNA-binding sites and does not depend on H3K9me3-modified nucleosomes. Together, our results reveal that ADNP, via the recruitment of HP1 and CHD4, regulates the expression of genes that are crucial for maintaining distinct cellular states and assures accurate cell fate decisions upon external cues. Such a general role of ChAHP in governing cell fate plasticity may explain why ADNP mutations affect several organs and body functions and contribute to cancer progression1,3,4. Notably, we found that the integrity of the ChAHP complex is disrupted by nonsense mutations identified in patients with Helsmoortel-Van der Aa syndrome, and this could be rescued by aminoglycosides that suppress translation termination 5 . Therefore, patients might benefit from therapeutic agents that are being developed to promote ribosomal read-through of premature stop codons6,7.

Kinematic Investigation of Healthy, Arthritic, and Postsurgery Thumbs: Is the Metacarpophalangeal Joint the Gateway to Carpometacarpal Arthritis?

The thumb carpometacarpal (CMC) joint is one of the most likely joints to develop osteoarthritis (OA). If conservative treatments fail to alleviate symptoms, surgery may be pursued. Kinematic outcomes of CMC surgery techniques have been described, but current tools have limitations in capturing motion abilities. The goals of this study were (1) develop a new and robust set of kinematic outcome measures, and apply them to (2) a cohort of younger and older control individuals without CMC OA to determine age and sex-related changes, and (3) a cohort of participants with CMC OA before, 3 months, and 6 months after undergoing thumb ligament reconstruction with tendon interposition surgery to detect the impacts of surgery. 52 (26 males, 26 females) control and 18 (3 males, 15 females) surgical participants were tested. Kinematics were investigated using motion capture by mapping the three-dimensional motion space of the whole thumb, and two-dimensional motion boundaries of the metacarpal (MC) and proximal phalange (PP). Visual analog pain score was recorded. Older control participants had shifted regions of motion compared to younger participants (p ≤ 0.027), suggesting asymptomatic CMC wear. Control females had 31% more metacarpophalangeal (MCP) motion than control males (p = 0.013), which could alter loading paths through the CMC joint and increase OA risk. Pain at 6 months postsurgery was 72% less than presurgery (p < 0.001), but motion abilities were 20-28% less than presurgery (p ≤ 0.074) and 24-40% less than control participants (p ≤ 0.066). These techniques have the possibility of identifying presymptomatic motion changes, including those at the metacarpophalangeal joint in CMC OA progression.

Anastomotic Height Is a Valuable Indicator of Long-term Bowel Function Following Surgery for Rectal Cancer.

BACKGROUND: The exact relation between anastomotic height after rectal cancer surgery and postoperative bowel function problems has not been investigated in the long term, resulting in ineffective treatment. OBJECTIVE: The goal of this study was to determine the effect of anastomotic height on long-term bowel function and generic quality of life. DESIGN: This was a multicenter, cross-sectional study. SETTINGS: Seven hospitals in the north of the Netherlands participated. PATIENTS: All patients who underwent rectal cancer surgery between 2009 and 2015 in participating hospitals received the validated Defecation and Fecal Continence and Short-Form 36 questionnaires. Deceased patients, patients with a permanent stoma or an anastomosis >15 cm from the anal verge, patients with intellectual disability, and patients living abroad were excluded. MAIN OUTCOME MEASURES: Primary outcomes were constipation (Rome IV), fecal incontinence (Rome IV), and major low anterior resection syndrome. Secondary outcomes were the generic quality of life scores. RESULTS: The study population ( n = 630) had a median follow-up of 58.0 months. In multivariable analysis, constipation (OR = 1.08; 95% CI, 1.02-1.15; p = 0.011), fecal incontinence (OR = 0.91; 95% CI, 0.84-0.97; p = 0.006), and major low anterior resection syndrome (OR = 0.93; 95% CI, 0.87-0.99; p = 0.027), were significantly associated with anastomotic height. The curves illustrating the probability of constipation and fecal incontinence crossed at an anastomotic height of 7 cm, with 95% CIs overlapping between 4.5 and 9.5 cm. There was no relation between quality-of-life scores and anastomotic height. LIMITATIONS: The study is limited by its cross-sectional design. CONCLUSIONS: This study might serve as a guide for the clinician to effectively screen and treat fecal incontinence and constipation during patient follow-up after rectal cancer surgery. More attention should be paid to fecal incontinence in patients with an anastomosis below 4.5 cm and toward constipation in patients with an anastomosis above 9.5 cm. See Video Abstract at http://links.lww.com/DCR/B858 . LA ALTURA ANASTOMTICA ES UN INDICADOR VALIOSO DE LA FUNCIN INTESTINAL A LARGO PLAZO DESPUS DE LA CIRUGA PARA EL CNCER DE RECTO: ANTECEDENTES:La relación exacta entre la altura anastomótica después de la cirugía de cáncer de recto y los problemas posoperatorios de la función intestinal no se ha investigado a largo plazo, lo que causa un tratamiento ineficaz.OBJETIVO:Determinar el efecto de la altura anastomótica sobre la función intestinal a largo plazo y la calidad de vida genérica.DISEÑO:Estudio multicéntrico transversal.DISEÑO DEL ESTUDIO:Participaron siete hospitales holandeses en el norte de los Países Bajos.PACIENTES:Todos los pacientes que se sometieron a cirugía de cáncer de recto entre 2009 y 2015 en los hospitales participantes recibieron los cuestionarios validados de Defecación y Continencia Fecal y Short-Form 36. Se excluyeron pacientes fallecidos, pacientes con estoma permanente o anastomosis > 15 cm del borde anal, discapacidad intelectual o residentes en el extranjero.PRINCIPALES MEDIDAS DE RESULTADO:Los resultados primarios fueron estreñimiento (Roma IV), incontinencia fecal (Roma IV) y síndrome de resección anterior baja mayor. Los resultados secundarios fueron las puntuaciones genéricas de calidad de vida.RESULTADOS:La población de estudio (N = 630) tuvo una mediana de seguimiento de 58.0 meses. En el análisis multivariable el estreñimiento (OR = 1,08, IC del 95%, 1,02-1,15, p = 0,011), incontinencia fecal (OR = 0,91, IC del 95%, 0,84-0,97, p = 0,006) y síndrome de resección anterior baja mayor (OR = 0,93, IC del 95%, 0,87-0,99, p = 0,027) se asociaron significativamente con la altura anastomótica. Las curvas que ilustran la probabilidad de estreñimiento e incontinencia fecal se cruzaron a una altura anastomótica de 7 cm, con IC del 95% superpuestos entre 4,5 y 9,5 cm. No hubo relación entre las puntuaciones de calidad de vida y la altura anastomótica.LIMITACIONES:El estudio está limitado por su diseño transversal.CONCLUSIONES:Este estudio podría servir como una guía para que el médico evalúe y trate eficazmente la incontinencia fecal y el estreñimiento durante el seguimiento de los pacientes después de la cirugía de cáncer de recto. Se debe prestar más atención a la incontinencia fecal en pacientes con anastomosis por debajo de 4,5 cm y al estreñimiento en pacientes con anastomosis por encima de 9,5 cm. Consulte Video Resumen en http://links.lww.com/DCR/B858 . (Traducción-Dr. Yazmin Berrones-Medina ).

Insight into the nodal cells transcriptome of the streptophyte green alga Chara braunii S276.

Charophyceae are the most complex streptophyte algae, possessing tissue-like structures, rhizoids and a cellulose-pectin-based cell wall akin to embryophytes. Together with the Zygnematophyceae and the Coleochaetophycae, the Charophyceae form a grade in which the Zygnematophyceae share a last common ancestor with land plants. The availability of genomic data, its short life cycle, and the ease of non-sterile cultivation in the laboratory have made the species Chara braunii an emerging model system for streptophyte terrestrialization and early land plant evolution. In this study, tissue containing nodal cells was prepared under the stereomicroscope, and an RNA-seq dataset was generated and compared to transcriptome data from whole plantlets. In both samples, transcript coverage was high for genes encoding ribosomal proteins and a homolog of the putative PAX3- and PAX7-binding protein 1. Gene ontology was used to classify the putative functions of the differently expressed genes. In the nodal cell sample, main upregulated molecular functions were related to protein, nucleic acid, ATP- and DNA binding. Looking at specific genes, several signaling-related genes and genes encoding sugar-metabolizing enzymes were found to be expressed at a higher level in the nodal cell sample, while photosynthesis-and chloroplast-related genes were expressed at a comparatively lower level. We detected the transcription of 21 different genes encoding DUF4360-containing cysteine-rich proteins. The data contribute to the growing understanding of Charophyceae developmental biology by providing a first insight into the transcriptome composition of Chara nodal cells.

The impact of salt stress on the physiology and the transcriptome of the model streptophyte green alga Chara braunii.

Chara braunii is a model for early land plant evolution and terrestrialization. Salt stress has a profound effect on water and ion transport activities, thereby interacting with many other processes, including inorganic carbon acquisition for photosynthesis. In this study, we analyzed the impact of salt stress (5 practical salt units, PSU) on the physiology and gene expression in C. braunii. Photosynthesis was only slightly affected 6 h after salt addition and returned to control levels after 48 h. Several organic compounds such as proline, glutamate, sucrose, and 2-aminobutyrate accumulated in salt-treated thalli and might contribute to osmotic potential acclimation, whereas the amount of K+ decreased. We quantified transcript levels for 17,387 genes, of which 95 were up-regulated and 44 down-regulated after salt addition. Genes encoding proteins of the functional groups ion/solute transport and cell wall synthesis/modulation were enriched among the up-regulated genes 24-48 h after salt stress, indicating their role in osmotic acclimation. However, a homolog to land plant ERD4 osmosensors was transiently upregulated after 6 h, and phylogenetic analyses suggested that these sensors evolved in Charophyceae. Down-regulated genes were mainly related to photosynthesis and carbon metabolism/fixation, consistent with the observed lowered growth after extended cultivation. The changed expression of genes encoding proteins for inorganic carbon acquisition might be related to the impact of salt on ionic relations and inorganic carbon uptake. The results indicate that C. braunii can tolerate enhanced salt concentrations in a defined acclimation process, including distinct gene expression changes to achieve new metabolic homeostasis.

Extracellular regulated kinase phosphorylates mitofusin 1 to control mitochondrial morphology and apoptosis.

Controlled changes in mitochondrial morphology participate in cellular signaling cascades. However, the molecular mechanisms modifying mitochondrial shape are largely unknown. Here we show that the mitogen-activated protein (MAP) kinase cascade member extracellular-signal-regulated kinase (ERK) phosphorylates the pro-fusion protein mitofusin (MFN) 1, modulating its participation in apoptosis and mitochondrial fusion. Phosphoproteomic and biochemical analyses revealed that MFN1 is phosphorylated at an atypical ERK site in its heptad repeat (HR) 1 domain. This site proved essential to mediate MFN1-dependent mitochondrial elongation and apoptosis regulation by the MEK/ERK cascade. A mutant mimicking constitutive MFN1 phosphorylation was less efficient in oligomerizing and mitochondria tethering but bound more avidly to the proapoptotic BCL-2 family member BAK, facilitating its activation and cell death. Moreover, neuronal apoptosis following oxygen glucose deprivation and MEK/ERK activation required an intact MFN1(T562). Our data identify MFN1 as an ERK target to modulate mitochondrial shape and apoptosis.

Static and Dynamic Accuracy and Occlusion Robustness of SteamVR Tracking 2.0 in Multi-Base Station Setups.

The tracking of objects and person position, orientation, and movement is relevant for various medical use cases, e.g., practical training of medical staff or patient rehabilitation. However, these demand high tracking accuracy and occlusion robustness. Expensive professional tracking systems fulfill these demands, however, cost-efficient and potentially adequate alternatives can be found in the gaming industry, e.g., SteamVR Tracking. This work presents an evaluation of SteamVR Tracking in its latest version 2.0 in two experimental setups, involving two and four base stations. Tracking accuracy, both static and dynamic, and occlusion robustness are investigated using a VIVE Tracker (3.0). A dynamic analysis further compares three different velocities. An error evaluation is performed using a Universal Robots UR10 robotic arm as ground-truth system under nonlaboratory conditions. Results are presented using the Root Mean Square Error. For static experiments, tracking errors in the submillimeter and subdegree range are achieved by both setups. Dynamic experiments achieved errors in the submillimeter range as well, yet tracking accuracy suffers from increasing velocity. Four base stations enable generally higher accuracy and robustness, especially in the dynamic experiments. Both setups enable adequate accuracy for diverse medical use cases. However, use cases demanding very high accuracy should primarily rely on SteamVR Tracking 2.0 with four base stations.

The BTB-domain transcription factor ZBTB2 recruits chromatin remodelers and a histone chaperone during the exit from pluripotency.

Transcription factors (TFs) harboring broad-complex, tramtrack, and bric-a-brac (BTB) domains play important roles in development and disease. These BTB domains are thought to recruit transcriptional modulators to target DNA regions. However, a systematic molecular understanding of the mechanism of action of this TF family is lacking. Here, we identify the zinc finger BTB-TF Zbtb2 from a genetic screen for regulators of exit from pluripotency and demonstrate that its absence perturbs embryonic stem cell differentiation and the gene expression dynamics underlying peri-implantation development. We show that ZBTB2 binds the chromatin remodeler Ep400 to mediate downstream transcription. Independently, the BTB domain directly interacts with nucleosome remodeling and deacetylase and histone chaperone histone regulator A. Nucleosome remodeling and deacetylase recruitment is a common feature of BTB TFs, and based on phylogenetic analysis, we propose that this is a conserved evolutionary property. Binding to UBN2, in contrast, is specific to ZBTB2 and requires a C-terminal extension of the BTB domain. Taken together, this study identifies a BTB-domain TF that recruits chromatin modifiers and a histone chaperone during a developmental cell state transition and defines unique and shared molecular functions of the BTB-domain TF family.