Simultaneous epigenetic perturbation and genome imaging reveal distinct roles of H3K9me3 in chromatin architecture and transcription

INTRODUCTION: Despite the long-observed correlation between H3K9me3, chromatin architecture, and transcriptional repression, how H3K9me3 regulates genome higher-order organization and transcriptional activity in living cells remains unclear. RESULT: Here, we develop EpiGo (Epigenetic perturbation induced Genome organization)-KRAB to introduce H3K9me3 at hundreds of loci spanning megabases on human chromosome 19 and simultaneously track genome organization. EpiGo-KRAB is sufficient to induce genomic clustering and de novo heterochromatin-like domain formation, which requires SETDB1, a methyltransferase of H3K9me3. Unexpectedly, EpiGo-KRAB-induced heterochromatin-like domain does not result in widespread gene repression except a small set of genes with concurrent loss of H3K4me3 and H3K27ac. Ectopic H3K9me3 appears to spread in inactive regions but is largely restricted from transcriptional initiation sites in active regions. Finally, Hi-C analysis showed that EpiGo-KRAB reshapes existing compartments mainly at compartment boundaries. CONCLUSIONS: These results reveal the role of H3K9me3 in genome organization could be partially separated from its function in gene repression

Simultaneous Epigenetic Perturbation and Genome Imaging Reveal Distinct Roles of H3K9me3 in Chromatin Architecture and Transcription [preprint]

Despite the long-observed correlation between H3K9me3, chromatin architecture and transcriptional repression, how H3K9me3 regulates genome higher-order organization and transcriptional activity in living cells remains unclear. Here we develop EpiGo (Epigenetic perturbation induced Genome organization)-KRAB to introduce H3K9me3 at hundreds of loci spanning megabases on human chromosome 19 and simultaneously track genome organization. EpiGo-KRAB is sufficient to induce de novo heterochromatin-like domain formation, which requires SETDB1, a methyltransferase of H3K9me3. Unexpectedly, EpiGo-KRAB induced heterochromatin-like domain does not result in widespread gene repression except a small set of genes with concurrent loss of H3K4me3 and H3K27ac. Ectopic H3K9me3 appears to spread in inactive regions but is largely restricted to transcriptional initiation sites in active regions. Finally, Hi-C analysis showed that EpiGo-KRAB induced to reshape existing compartments. These results reveal the role of H3K9me3 in genome organization could be partially separated from its function in gene repression

Intermediate Phases, structural variance and network demixing in chalcogenides: the unusual case of group V sulfides

We review Intermediate Phases (IPs) in chalcogenide glasses and provide a structural interpretation of these phases. In binary group IV selenides, IPs reside in the 2.40 < r < 2.54 range, and in binary group V selenides they shift to a lower r, in the 2.29< r < 2.40 range. Here r represents the mean coordination number of glasses. In ternary alloys containing equal proportions of group IV and V selenides, IPs are wider and encompass ranges of respective binary glasses. These data suggest that the local structural variance contributing to IP widths largely derives from four isostatic local structures of varying connectivity r; two include group V based quasi-tetrahedral (r = 2.29) and pyramidal (r = 2.40) units, and the other two are group IV based corner-sharing (r = 2.40) and edge-sharing (r = 2.67) tetrahedral units. Remarkably, binary group V (P, As) sulfides exhibit IPs that are shifted to even a lower r than their selenide counterparts; a result that we trace to excess Sn chains either partially (As-S) or completely (P-S) demixing from network backbone, in contrast to excess Sen chains forming part of the backbone in corresponding selenide glasses. In ternary chalcogenides of Ge with the group V elements (As, P), IPs of the sulfides are similar to their selenide counterparts, suggesting that presence of Ge serves to reign in the excess Sn chain fragments back in the backbone as in their selenide counterparts

Bone tissue formation with human mesenchymal stem cells and biphasic calcium phosphate ceramics: The local implication of osteoclasts and macrophages

Human mesenchymal stem cells (hMSC) have immunomodulative properties and, associated with calcium phosphate (CaP) ceramics, induce bone tissue repair. However, the mechanisms of osteoinduction by hMSC with CaP are not clearly established, in particular the role of osteoclasts and macrophages. Biphasic calcium phosphate (BCP) particles were implanted with or without hMSC in the paratibial muscles of nude mice. hMSC increased osteoblastic gene expression at 1 week, the presence of macrophages at 2 and 4 weeks, osteoclastogenesis at 4 and 8 weeks, and osteogenesis at 4 and 8 weeks. hMSC disappeared from the implantation site after 2 weeks, indicating that hMSC were inducers rather than effectors of bone formation. Induced blockage of osteoclastogenesis by anti-Rankl treatment significantly impaired bone formation, revealing the pivotal role of osteoclasts in bone formation. In summary, hMSC positively influence the body foreign reaction by attracting circulating haematopoietic stem cells and inducing their differentiation into macrophages M1 and osteoclasts, thus favouring bone formation

The Extrachromosomal EAST Protein of Drosophila Can Associate with Polytene Chromosomes and Regulate Gene Expression

The EAST protein of Drosophila is a component of an expandable extrachromosomal domain of the nucleus. To better understand its function, we studied the dynamics and localization of GFP-tagged EAST. In live larval salivary glands, EAST-GFP is highly mobile and localizes to the extrachromosomal nucleoplasm. When these cells are permeabilized, EAST-GFP rapidly associated with polytene chromosomes. The affinity to chromatin increases and mobility decreases with decreasing salt concentration. Deleting the C-terminal residues 1535 to 2301 of EAST strongly reduces the affinity to polytene chromosomes. The bulk of EAST-GFP co-localizes with heterochromatin and is absent from transcriptionally active chromosomal regions. The predominantly chromosomal localization of EAST-GFP can be detected in non-detergent treated salivary glands of pupae as they undergo apoptosis, however not in earlier stages of development. Consistent with this chromosomal pattern of localization, genetic evidence indicates a role for EAST in the repression of gene expression, since a lethal east mutation is allelic to the viable mutation suppressor of white-spotted. We propose that EAST acts as an ion sensor that modulates gene expression in response to changing intracellular ion concentrations

Continuous Elevation of PTH Increases the Number of Osteoblasts via Both Osteoclast-Dependent and -Independent Mechanisms

Sustained parathyroid hormone (PTH) elevation stimulates bone remodeling (ie, both resorption and formation). The former results from increased RANKL synthesis, but the cause of the latter has not been established. Current hypotheses include release of osteoblastogenic factors from osteoclasts or from the bone matrix during resorption, modulation of the production and activity of osteoblastogenic factors from cells of the osteoblast lineage, and increased angiogenesis. To dissect the contribution of these mechanisms, 6-month-old Swiss-Webster mice were infused for 5 days with 470 ng/h PTH(1-84) or 525 ng/h soluble RANKL (sRANKL). Both agents increased osteoclasts and osteoblasts in vertebral cancellous bone, but the ratio of osteoblasts to osteoclasts and the increase in bone formation was greater in PTH-treated mice. Cancellous bone mass was maintained in mice receiving PTH but lost in mice receiving sRANKL, indicating that maintenance of balanced remodeling requires osteoblastogenic effects beyond those mediated by osteoclasts. Consistent with this contention, PTH, but not sRANKL, decreased the level of the Wnt antagonist sclerostin and increased the expression of the Wnt target genes Nkd2, Wisp1, and Twist1. Furthermore, PTH, but not sRANKL, increased the number of blood vessels in the bone marrow. Weekly injections of the RANKL antagonist osteoprotegerin at 10 µg/g for 2 weeks prior to PTH infusion eliminated osteoclasts and osteoblasts and prevented the PTH-induced increase in osteoclasts, osteoblasts, and blood vessels. These results indicate that PTH stimulates osteoclast-dependent as well as osteoclast-independent (Wnt signaling) pro-osteoblastogenic pathways, both of which are required for balanced focal bone remodeling in cancellous bone. © 2010 American Society for Bone and Mineral Research

Glassy State Lead Tellurite Nanobelts: Synthesis and Properties

The lead tellurite nanobelts have been first synthesized in the composite molten salts (KNO3/LiNO3) method, which is cost-effective, one-step, easy to control, and performed at low-temperature and in ambient atmosphere. Scanning electron microscopy, X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectrum, energy dispersive X-ray spectroscopy and FT-IR spectrum are used to characterize the structure, morphology, and composition of the samples. The results show that the as-synthesized products are amorphous and glassy nanobelts with widths of 200–300 nm and lengths up to tens of microns and the atomic ratio of Pb:Te:O is close to 1:1.5:4. Thermo-gravimetric analysis (TGA) and differential scanning calorimetry (DSC) and investigations of the corresponding structure and morphology change confirm that the nanobelts have low glass transition temperature and thermal stability. Optical diffuse reflectance spectrum indicates that the lead tellurite nanobelts have two optical gaps at ca. 3.72 eV and 4.12 eV. Photoluminescence (PL) spectrum and fluorescence imaging of the products exhibit a blue emission (round 480 nm)

C1-Ten Is a Protein Tyrosine Phosphatase of Insulin Receptor Substrate 1 (IRS-1), Regulating IRS-1 Stability and Muscle Atrophy

Muscle atrophy occurs under various catabolic conditions, including insulin deficiency, insulin resistance, or increased levels of glucocorticoids. This results from reduced levels of insulin receptor substrate 1 (IRS-1), leading to decreased phosphatidylinositol 3-kinase activity and thereby activation of FoxO transcription factors. However, the precise mechanism of reduced IRS-1 under a catabolic condition is unknown. Here, we report that C1-Ten is a novel protein tyrosine phosphatase (PTPase) of IRS-1 that acts as a mediator to reduce IRS-1 under a catabolic condition, resulting in muscle atrophy. C1-Ten preferentially dephosphorylated Y612 of IRS-1, which accelerated IRS-1 degradation. These findings suggest a novel type of IRS-1 degradation mechanism which is dependent on C1-Ten and extends our understanding of the molecular mechanism of muscle atrophy under catabolic conditions. C1-Ten expression is increased by catabolic glucocorticoid and decreased by anabolic insulin. Reflecting these hormonal regulations, the muscle C1-Ten is upregulated in atrophy but downregulated in hypertrophy. This reveals a previously unidentified role of C1-Ten as a relevant PTPase contributing to skeletal muscle atrophy.open2

Biogenesis of the Signal Recognition Particle (Srp) Involves Import of Srp Proteins into the Nucleolus, Assembly with the Srp-Rna, and Xpo1p-Mediated Export

The signal recognition particle (SRP) targets nascent secretory proteins to the ER, but how and where the SRP assembles is largely unknown. Here we analyze the biogenesis of yeast SRP, which consists of an RNA molecule (scR1) and six proteins, by localizing all its components. Although scR1 is cytoplasmic in wild-type cells, nuclear localization was observed in cells lacking any one of the four SRP “core proteins” Srp14p, Srp21p, Srp68p, or Srp72p. Consistently, a major nucleolar pool was detected for these proteins. Sec65p, on the other hand, was found in both the nucleoplasm and the nucleolus, whereas Srp54p was predominantly cytoplasmic. Import of the core proteins into the nucleolus requires the ribosomal protein import receptors Pse1p and Kap123p/Yrb4p, which might, thus, constitute a nucleolar import pathway. Nuclear export of scR1 is mediated by the nuclear export signal receptor Xpo1p, is distinct from mRNA transport, and requires, as evidenced by the nucleolar accumulation of scR1 in a dis3/rrp44 exosome component mutant, an intact scR1 3′ end. A subset of nucleoporins, including Nsp1p and Nup159p (Rat7p), are also necessary for efficient translocation of scR1 from the nucleus to the cytoplasm. We propose that assembly of the SRP requires import of all SRP core proteins into the nucleolus, where they assemble into a pre-SRP with scR1. This particle can then be targeted to the nuclear pores and is subsequently exported to the cytoplasm in an Xpo1p-dependent way

Actin: its cumbersome pilgrimage through cellular compartments

In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days’ knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin