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Rana sylvatica (also known as Boreorana sylvatica) is one of the few vertebrates that spend extreme winters showing no physiological signs of life. Up to 70% of the total body water of the wood frog freezes as extracellular ice. Survival in extreme conditions requires regulation at transcriptional and translational levels to activate prosurvival pathways. N6-methyladenosine ([m.sup.6]A) methylation is one of the most common RNA modifications, regulating transcript processing and translation by executing important functions that affect regulatory pathways in stress conditions. In the study, regulation of [m.sup.6]A-related proteins in the liver of R. sylvatica was analyzed during 24 h frozen and 8 h thaw conditions. Decreases in the activity of demethylases of 28.44 [+ or -] 0.4% and 24.1 [+ or -] 0.9% of control values in frozen and thaw tissues, respectively, were observed. Total protein levels of [m.sup.6]A methyltransferase complex components methyltransferase-like 14 and Wilm's tumor associated protein were increased by 1.28-fold and 1.42-fold, respectively, during freezing. Demethylase fat mass and obesity, however, showed a decreasing trend, with a significant decrease in abundance during recovery from frozen conditions. Levels of mRNA degraders YTHDF2 and YTHDC2 also decreased under stress. Overall, increased levels of [m.sup.6]A methylation complex components, and suppressed levels of readers/erasers, provide evidence for the potential role of RNA methylation in freezing survival and its regulation in a hypometabolic state. Key words: epigenetics, freeze tolerance, RNA methylation, metabolic rate depression, Rana sylvatica (Boreorana sylvatica), Introduction The wood frog, Rana sylvatica (Boreorana sylvatica), is one of the freeze-tolerant model organisms that survive sub-zero temperatures for several months. During freezing, the wood frog employs multiple adaptation [...]

The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function.sup.1-3. Methods based on chromatin conformation capture have mapped chromatin structures in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi.sup.4-6. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation.sup.7, are invisible with such approaches.sup.8. Here we developed immunoGAM, an extension of genome architecture mapping (GAM).sup.2,9, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation.sup.2,10. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive 'melting' of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions. A new technique called immunoGAM, which combines genome architecture mapping (GAM) with immunoselection, enabled the discovery of specialized chromatin conformations linked to gene expression in specific cell populations from mouse brain tissues., Author(s): Warren Winick-Ng [sup.1] , Alexander Kukalev [sup.1] , Izabela Harabula [sup.1] [sup.2] , Luna Zea-Redondo [sup.1] [sup.2] , Dominik Szabó [sup.1] [sup.2] , Mandy Meijer [sup.3] , Leonid Serebreni [...]

Interconnectivity between neocortical areas is critical for sensory integration and sensorimotor transformations.sup.1-6. These functions are mediated by heterogeneous inter-areal cortical projection neurons (ICPN), which send axon branches across cortical areas as well as to subcortical targets.sup.7-9. Although ICPN are anatomically diverse.sup.10-14, they are molecularly homogeneous.sup.15, and how the diversity of their anatomical and functional features emerge during development remains largely unknown. Here we address this question by linking the connectome and transcriptome in developing single ICPN of the mouse neocortex using a combination of multiplexed analysis of projections by sequencing.sup.16,17 (MAPseq, to identify single-neuron axonal projections) and single-cell RNA sequencing (to identify corresponding gene expression). Focusing on neurons of the primary somatosensory cortex (S1), we reveal a protracted unfolding of the molecular and functional differentiation of motor cortex-projecting ([Formula omitted]) ICPN compared with secondary somatosensory cortex-projecting ([Formula omitted]) ICPN. We identify SOX11 as a temporally differentially expressed transcription factor in [Formula omitted] versus [Formula omitted] ICPN. Postnatal manipulation of SOX11 expression in S1 impaired sensorimotor connectivity and disrupted selective exploratory behaviours in mice. Together, our results reveal that within a single cortical area, different subtypes of ICPN have distinct postnatal paces of molecular differentiation, which are subsequently reflected in distinct circuit connectivities and functions. Dynamic differences in the expression levels of a largely generic set of genes, rather than fundamental differences in the identity of developmental genetic programs, may thus account for the emergence of intra-type diversity in cortical neurons. Combined analysis of the connectome and transcriptome in the mouse cortex indicates that dynamic differences in expression levels of largely generic sets of genes regulate differential targeting within neuronal subtypes., Author(s): Esther Klingler [sup.1] , Ugo Tomasello [sup.1] , Julien Prados [sup.2] , Justus M. Kebschull [sup.3] [sup.6] , Alessandro Contestabile [sup.1] , Gregorio L. Galiñanes [sup.1] , Sabine Fièvre [...]

Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth.sup.1-3. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes. Using time-controlled CRISPR screens, the authors identify AKIRIN2 as a factor involved in the nuclear import of the proteasome., Author(s): Melanie de Almeida [sup.1] [sup.2] , Matthias Hinterndorfer [sup.1] [sup.2] , Hanna Brunner [sup.1] [sup.2] , Irina Grishkovskaya [sup.1] , Kashish Singh [sup.1] [sup.5] , Alexander Schleiffer [sup.1] , [...]

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel--the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway.sup.1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis. AMBRA1-mediated degradation of cyclin D through CRL4-DDB1 regulates cell proliferation and prevents replication stress in neurodevelopment and cancer., Author(s): Emiliano Maiani [sup.1] [sup.2] , Giacomo Milletti [sup.3] [sup.4] , Francesca Nazio [sup.3] , Søs Grønbæk Holdgaard [sup.1] , Jirina Bartkova [sup.5] [sup.6] , Salvatore Rizza [sup.7] , Valentina [...]

BRCA1-associated protein 1 (BAP1) regulates calcium flux in the endoplasmic reticulum to facilitate the execution of apoptosis, unveiling a new facet of the role of BAP1 as an environmental tumour suppressor. A new role for BAP1 in tumour suppression BAP1 is a tumour suppressor associated with germline mutations in several malignancies including uveal melanoma and mesothelioma. BAP1 tumour suppressor activity has previously been linked to its nuclear role in maintaining genome integrity. Here, the authors reveal a new role for BAP1 in the endoplasmic reticulum, where it regulates calcium flux to facilitate the execution of apoptosis. Loss of BAP1 function prevents apoptosis in transformed cells with accumulated DNA damage. The results unveil a new facet of the role of BAP1 as an environmental tumour suppressor. BRCA1-associated protein 1 (BAP1) is a potent tumour suppressor gene that modulates environmental carcinogenesis.sup.1,2,3. All carriers of inherited heterozygous germline BAP1-inactivating mutations (BAP1.sup.+/-) developed one and often several BAP1.sup.-/- malignancies in their lifetime.sup.4, mostly malignant mesothelioma, uveal melanoma.sup.2,5, and so on.sup.6,7,8,9,10. Moreover, BAP1-acquired biallelic mutations are frequent in human cancers.sup.8,11,12,13,14. BAP1 tumour suppressor activity has been attributed to its nuclear localization, where it helps to maintain genome integrity.sup.15,16,17. The possible activity of BAP1 in the cytoplasm is unknown. Cells with reduced levels of BAP1 exhibit chromosomal abnormalities and decreased DNA repair by homologous recombination.sup.18, indicating that BAP1 dosage is critical. Cells with extensive DNA damage should die and not grow into malignancies. Here we discover that BAP1 localizes at the endoplasmic reticulum. Here, it binds, deubiquitylates, and stabilizes type 3 inositol-1,4,5-trisphosphate receptor (IP3R3), modulating calcium (Ca.sup.2+) release from the endoplasmic reticulum into the cytosol and mitochondria, promoting apoptosis. Reduced levels of BAP1 in BAP1.sup.+/- carriers cause reduction both of IP3R3 levels and of Ca.sup.2+ flux, preventing BAP1.sup.+/- cells that accumulate DNA damage from executing apoptosis. A higher fraction of cells exposed to either ionizing or ultraviolet radiation, or to asbestos, survive genotoxic stress, resulting in a higher rate of cellular transformation. We propose that the high incidence of cancers in BAP1.sup.+/- carriers results from the combined reduced nuclear and cytoplasmic activities of BAP1. Our data provide a mechanistic rationale for the powerful ability of BAP1 to regulate gene-environment interaction in human carcinogenesis., Author(s): Angela Bononi [sup.1] , Carlotta Giorgi [sup.2] , Simone Patergnani [sup.2] , David Larson [sup.1] , Kaitlyn Verbruggen [sup.1] , Mika Tanji [sup.1] , Laura Pellegrini [sup.1] , Valentina [...]

Eukaryotic cells coordinately control anabolic and catabolic processes to maintain cell and tissue homeostasis. Mechanistic target of rapamycin complex 1 (mTORC1) promotes nutrient-consuming-anabolic processes, such as protein synthesis (1). Here we show that as well as increasing protein synthesis, mTORC1 activation in mouse and human cells also promotes an increased capacity for protein degradation. Cells with activated mTORC1 exhibited elevated levels of intact and active proteasomes through a global increase in the expression of genes encoding proteasome subunits. The increase in proteasome gene expression, cellular proteasome content, and rates of protein turnover downstream of mTORC1 were all dependent on induction of the transcription factor nuclear factor erythroid-derived 2-related factor 1 (NRF1; also known as NFE2L1). Genetic activation of mTORC1 through loss of the tuberous sclerosis complex tumour suppressors, TSC1 or TSC2, or physiological activation of mTORC1 in response to growth factors or feeding resulted in increased NRF1 expression in cells and tissues. We find that this NRF1-dependent elevation in proteasome levels serves to increase the intracellular pool of amino acids, which thereby influences rates of new protein synthesis. Therefore, mTORC1 signalling increases the efficiency of proteasome-mediated protein degradation for both quality control and as a mechanism to supply substrate for sustained protein synthesis., In response to growth signals, mTORC1 promotes anabolic processes, such as protein synthesis, and its chronic activation is believed to underlie a variety of complex human diseases, including cancer and [...]