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Background The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles. Results Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A. Conclusions Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.

Despite major progress in defining the functional roles of genes, a complete understanding of their influences is far from being realized, even in relatively simple organisms. A major milestone in this direction arose via the completion of the yeast Saccharomyces cerevisiae gene-knockout collection (YKOC), which has enabled high-throughput reverse genetics, phenotypic screenings and analyses of synthetic-genetic interactions1–3. Ensuing experimental work has also highlighted some inconsistencies and mistakes in the YKOC, or genome instability events that rebalance the effects of specific knockouts4–6, but a complete overview of these is lacking. The identification and analysis of genes that are required for maintaining genomic stability have traditionally relied on reporter assays and on the study of deletions of individual genes, but whole-genome-sequencing technologies now enable—in principle—the direct observation of genome instability globally and at scale. To exploit this opportunity, we sequenced the whole genomes of nearly all of the 4,732 strains comprising the homozygous diploid YKOC. Here, by extracting information on copy-number variation of tandem and interspersed repetitive DNA elements, we describe—for almost every single non-essential gene—the genomic alterations that are induced by its loss. Analysis of this dataset reveals genes that affect the maintenance of various genomic elements, highlights cross-talks between nuclear and mitochondrial genome stability, and shows how strains have genetically adapted to life in the absence of individual non-essential genes. Whole-genome sequencing of the strains of the Saccharomyces cerevisiae gene-knockout collection reveals the effects of the deletion of non-essential genes on genome stability.

To maintain genome stability, organisms depend on faithful chromosome segregation, a process affected by diverse genetic pathways, some of which are not directly linked to mitosis. In this study, we set out to explore one such pathway represented by an undercharacterized gene, SNO1, identified previously in screens of the yeast knockout (YKO) library for mitotic fidelity genes. We found that the causative factor increasing mitotic error rate in the sno1Δ mutant is not loss of the Sno1 protein, but rather perturbation to the mRNA of the neighboring convergent gene, CTF13, encoding an essential component for forming the yeast kinetochore. This is caused by a combination of the Kanamycin resistance gene and the transcriptional terminator used in the YKO library affecting the CTF13 mRNA level and quality . We further provide a list of gene pairs potentially subjected to this artifact, which may be useful for accurate phenotypic interpretation of YKO mutants.

The nuclear lamina provides structure to the nucleus and serves as an interface between the cytoskeleton and large heterochromatin domains called LADs. This study describes the microproteome of this LAD/lamina interface., The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, the so-called lamina-associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2β to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin-directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify both LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina, identified putative LAD proximal proteins and found several proteins that appear to interface with both micro-proteomes. Importantly, several proteins essential for LAD function, including heterochromatin regulating proteins related to H3K9 methylation, were identified in this study.

The dynamic 3D organization of the genome is central to the regulation of gene expression and developmental progression, with its disruption being implicated in various diseases. The nuclear lamina, a proteinaceous meshwork underlying the nuclear envelope (NE), provides both structural and regulatory influences on genome organization through the tethering of large inactive genomic regions, called Lamina Associated Domains (LADs), to the nuclear periphery. Evidence suggests that the A type lamins, lamins A and C, are the predominant lamins involved in the peripheral association of LADs, with these two isotypes forming distinct networks and potentially involved in different cellular processes. Here we tested whether lamins A and C have distinct roles in genome organization by examining chromosome architecture in cells in which lamin C or lamin A are specifically down-regulated. We find that lamin C (not lamin A) is required for the 3D organization of LADs and overall chromosome organization in the cell nucleus. Striking differences in the localization of lamin A and lamin C are present as cells exit mitosis that persist through early G1. Whereas lamin A associates with the nascent NE during telophase, lamin C remains in the interior surrounding nucleoplasmic LAD clusters. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and consequently global 3D genome organization, is perturbed only in cells depleted of lamin C, and not in cells depleted of lamin A. We conclude that lamin C regulates LAD dynamics after mitosis and is a key regulator of genome organization in mammalian cells. These findings reveal an unexpectedly central role for lamin C in genome organization, including both inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE.

The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, so-called lamin associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2β to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify both LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina, identified putative LAD proximal proteins and found several proteins that appear to interface with both micro-proteomes. Importantly, several proteins essential for LAD function, including heterochromatin regulating proteins related to H3K9 methylation, were identified in this study.

Recent reports highlight that human decision-making is influenced by the time of day and whether one is a morning or evening person (i.e., chronotype). Here, we test whether these behavioral effects are associated with endogenous biological rhythms. We asked participants to complete two well-established decision-making tasks in the morning or evening: the matrix task (an ethical decision task) and the balloon analog risk task (BART; a risk-taking task), and we measured their chronotype in two ways. First, participants completed a self-report measure, the Horne-Östberg Morningness-Eveningness Questionnaire (MEQ). Second, we measured the expression of two circadian clock-regulated genes—Per3 and Nr1d2—from peripheral clock cells in participants’ hair follicle samples. Using a cosinor model, we estimated the phase of the peripheral clock and assigned RNA chronotypes to participants with advanced (larks) or delayed (owls) phases. The behavioral data were analyzed independently for self-reported (MEQ) and RNA-based chronotypes. We find that significant chronotype and/or time-of-day effects between larks and owls in decision-making tasks occur only in RNA-based chronotypes. Our results provide evidence that time-of-day effects on decision-making can be explained by phase differences in oscillating clock genes and suggest that variation in the molecular clockwork may influence inter-individual differences in decision-making behavior.

Medulloblastoma (MB) is the most common primary pediatric malignant brain tumor. Genetic classification and distinct histologic subtypes defines MB into 4 groups: classic (WNT), sonic hedgehog (Shh), group 3 and group 4. In addition, recent studies have provided evidence that p53 abnormalities is seen in various sub-groups of MB and that p53-mutated SHH MBs commonly harbor genetic anomalies including MYCN and GLI2 amplifications, which confers drug resistance. Here, we test the hypothesis that specific immunohistological markers, their correlation with the amplification of the oncogene MYC, and abnormalities in tumor suppressor gene p53, may define their metastatic potential. Materials and Methods: Immunohistological analysis of MB tumors (n=41) was evaluated for the expression of glioma transcription factor 1 (GLI-1), Grb2-associated binding protein 1 (GAB-1), natriuretic peptide receptor (NPR), voltage-gated potassium channel (KV1) and mutant p53. FISH analysis was performed to determine MYC amplification or iso- P53. p53-mutant MB cell line was used to investigate the signaling pathway leading to proliferation, migration, and drug resistance using HDAC (LBH-589) and PI3K/mTOR (BKM-120/rapamycin) inhibitors. Results: we showed that: 1) GAB-1 was highly expressed in the Shh group (82%) and KV1 expression was evenly distributed in all subtypes; 2) No obvious correlation with expression of GLI-1, GAB-1, NPR, or KV1 with metastasis was seen; 3) Analysis of loss of p53 and overexpression of MYC varied in each subtype; 4) Combined Treatment with LBH-589 and BKM-120 reduced cell proliferation, migration and S-phase entry, however, MB cells were resistant to BKM -120 treatments, while LBH-589 caused massive apoptosis; 5) Tumor formation was suppressed by BKM-120 given with mTOR inhibitors. Conclusion: the result of this study suggests that expression of GLI-1, GAB-1, NPR, KV1 and p53 was important in defining the subgroups of MB, although their metastatic potential remains to be understood. MB cells shows reduced cell proliferation, cell cycle and migration, when treated with HDAC inhibitor LBH-589, however these cells display complete drug resistance to PI3K inhibitors. Citation Format: Samuel Gelnick, Anubhav G. Amin, Molly Gordon, Raphael Salles Scortegagna de Medeiros, Nelci Zanon, Raj Murali, Meena Jhanwar-Uniyal. Histological subtypes correlate with distinct genetic abnormalities in defining therapeutic targets in medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3118. doi:10.1158/1538-7445.AM2017-3118

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