Long non-coding RNAs as regulators of the endocrine system

Long non-coding RNAs (lncRNAs) are a large and diverse group of RNAs that are often lineage-specific and that regulate multiple biological functions. Many are nuclear and are essential parts of ribonucleoprotein complexes that modify chromatin segments and establish active or repressive chromatin states; others are cytosolic and regulate the stability of mRNA or act as microRNA sponges. This Review summarizes the current knowledge of lncRNAs as regulators of the endocrine system, with a focus on the identification and mode of action of several endocrine-important lncRNAs. We highlight lncRNAs that have a role in the development and function of pancreatic β cells, white and brown adipose tissue, and other endocrine organs, and discuss the involvement of these molecules in endocrine dysfunction (for example, diabetes mellitus). We also address the associations of lncRNAs with nuclear receptors involved in major hormonal signalling pathways, such as estrogen and androgen receptors, and the relevance of these associations in certain endocrine cancers.National Science Foundation (U.S.) (Grant NRF-2011NRF-NRFF 001-025

Do Femtonewton Forces Affect Genetic Function? A Review

Protein-Mediated DNA looping is intricately related to gene expression. Therefore any mechanical constraint that disrupts loop formation can play a significant role in gene regulation. Polymer physics models predict that less than a piconewton of force may be sufficient to prevent the formation of DNA loops. Thus, it appears that tension can act as a molecular switch that controls the much larger forces associated with the processive motion of RNA polymerase. Since RNAP can exert forces over 20 pN before it stalls, a ‘substrate tension switch’ could offer a force advantage of two orders of magnitude. Evidence for such a mechanism is seen in recent in vitro micromanipulation experiments. In this article we provide new perspective on existing theory and experimental data on DNA looping in vitro and in vivo . We elaborate on the connection between tension and a variety of other intracellular mechanical constraints including sequence specific curvature and supercoiling. In the process, we emphasize that the richness and versatility of DNA mechanics opens up a whole new paradigm of gene regulation to explore.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41816/1/10867_2005_Article_9002.pd

MicroRNA-221 und die nicht-Ig-Teile der Ersatz leichten Kette als molekulare Kontrollpunkte in der B Zell Entwicklung

MicroRNAs regulieren komplexe Genexpressions Programme von Zellen auf post-transkriptionaler Ebene. Ich habe gefunden, dass miR-128a, 221 und 222 in zwei nah ver-wandten Stadien der B-Zellentwicklung in der Maus unterschiedlich exprimiert sind: hoch in Pax5-/- CD19- pro/präB Zellen und niedrig in Pax5+/+ CD19+ präB-I Zellen. Die Ex-pression von miR-221 und 222, aber nicht die von 128a, wird durch Pax5 Expression kon-trolliert. Überexpression von diesen miRNAs in Wildtyp präB-I durch Transduktion mit LTR deletierten SIN-retroviralen Vektoren, kontrolliert durch einen tet-induzierbaren Promotor, ändert weder die CD19 Expression noch die Unfähigkeit sich in T Zellen zu entwickeln. Jedoch können, nach Transplantation in Immun-defiziente Rag1-/- Mäuse, miR-221, aber nicht miR-222 transduzierte und induzierte Zellen nun wie pro/präB Zellen ins Knochenmark wandern. Die transplantierten, miR-221 exprimierenden Zellen verlassen das Knochenmark, wenn die Expression durch die Entfernung vom Doxycyclin im Trink-wasser beendet wird. Zwei Gene, Integrin α4 und Syndecan 4, die bei Zelladhäsion und Zellwanderung eine Rolle spielen, konnten als potentielle direkte oder indirekte Ziele für die miR-221 identifiziert werden. Zusammen mit einer Immunglobulin (Ig) schweren μ Kette bilden die Proteine λ5 und VpreB den Prä-B-Zell Rezeptor. In Zusammenarbeit mit Kazuo Ohnishi und Yuki Yana-gisawa habe ich λ5-/- / VpreB1-/- / VpreB2-/- Abelson Virus transformierte präB Zelllinien etabliert und diese mit λ5 und VpreB Genen in Wildtyp form oder mit Nicht-Ig Teil dele-tierten Genen transduziert. Immer, wenn Prä-B-Zell Rezeptoren mit Nicht-Ig deletierten λ5 Genen transduziert wurden, war die Oberflächenablagerung von Prä-B-Zell Rezeptoren erhöht, während das Entfernen der Nicht-Ig Teile von VpreB die Ablagerung verringerten. Die durch anti-μ-schwere Ketten Antikörper stimulierte Phosphorylierung von Syk, SLP65 oder PLC-γ2 und die Calcium Mobilisierung aus intra-zellulären Speichern war abhängig von der Menge an Prä-B-Zell Rezeptoren auf der Oberfläche. Aus diesen Ergebnissen schliesse ich, dass die VpreB Komponente die Fitness von neu generierten schweren V Domänen von Ig-schweren Ketten für das spätere Paaren mit Ig-leichten Ketten sondiert, und der nicht-Ig Teil den Prä-B-Zell Rezeptor auf der Oberfläche der prä-B Zelle fixiert. Im Unterschied dazu vernetzt der Nicht-Ig Teil der λ5 Komponente den Prä-B-Zell Re-zeptor wodurch dieser internalisiert wird, und die Zellen zur Proliferation stimuliert wer-den.MicroRNAs regulate sets of gene expression programs of cells on posttranscriptional levels. I found miR-128a, 221 and 222 differentially expressed at two closely related stages of B-cell development in the mouse, high in Pax5-/- CD19- pro/preB cells and low in Pax5+/+ CD19+ preB-I cells. Expression of miR-221 and 222, but not of 128a, is controlled by Pax5 expression. Over-expression of either of these miRNAs in wild type preB-I cells by transduction with an LTR-defective SIN-retroviral vector under the control of a tet-inducible promotor does not change their CD19 expression, nor their inability to develop to T cells. However, upon transplantation into immune-deficient Rag1-/- mice preB-I cells transduced with miR-221 and induced with doxycycline, but not those transduced with miR-222, gain the capacity of pro/preB cells to home to the bone marrow. The transplanted, miR-221 expressing cells leave the bone marrow when the expression of miRNA is terminated by the removal of doxycycline. Two migration-related genes, encoding integrin α4 and syndecan 4 are potential direct or indirect targets for the miR-221 dependent regulation of retention of pro/preB cells in bone marrow. The VpreB and λ5 proteins, together with Igμ-H chains form preB cell receptors (preBCRs). In collaboration with Kazuo Ohnishi and Yuki Yanagisawa I established λ5-/- / VpreB1-/- / VpreB2-/- Abelson virus transformed cell lines and reconstituted these cells with λ5 and VpreB in wild type form or with deleted non-Ig parts. Whenever preBCRs had the non-Ig part of λ5 deleted, surface deposition was increased, while deletion of VpreB non-Ig part decreased it. The levels of phosphorylation of Syk, SLP65 or PLC-γ2, and of Ca2+ mobilization from intracellular stores, stimulated by μH chain cross-linking antibody, were dependent on the levels of surface-bound preBCRs. It appears that VpreB probes the fitness of newly generated VH-domains of IgH chains for later pairing with IgL chains and its non-Ig part fixes the preBCRs on the surface. By contrast the non-Ig part of λ5 cross-links preBCRs for down-regulation and stimulation

The Super-Enhancer-Derived alncRNA-EC7/Bloodlinc Potentiates Red Blood Cell Development in trans

Enhancer-derived RNAs are thought to act locally by contributing to their parent enhancer function. Whether large domains of clustered enhancers (super-enhancers) also produce cis-acting RNAs, however, remains unclear. Unlike typical enhancers, super-enhancers form large spans of robustly transcribed chromatin, amassing capped and polyadenylated RNAs that are sufficiently abundant to sustain trans functions. Here, we show that one such RNA, alncRNA-EC7/Bloodlinc, is transcribed from a super-enhancer of the erythroid membrane transporter SLC4A1/BAND3 but diffuses beyond this site. Bloodlinc localizes to trans-chromosomal loci encoding critical regulators and effectors of terminal erythropoiesis and directly binds chromatin-organizing and transcription factors, including the chromatin attachment factor HNRNPU. Inhibiting Bloodlinc or Hnrnpu compromises the terminal erythropoiesis gene program, blocking red cell production, whereas expressing Bloodlinc ectopically stimulates this program and can promote erythroblast proliferation and enucleation in the absence of differentiation stimuli. Thus, Bloodlinc is a trans-acting super-enhancer RNA that potentiates red blood cell development. Keywords enhancer RNA; super-enhancer; long non-coding RNA; erythropoiesis; red blood cellNational Institutes of Health (U.S.) (Grant DK068348)National Institutes of Health (U.S.) (Grant 5P01-HL066105

SYK kinase mediates brown fat differentiation and activation

Brown adipose tissue (BAT) metabolism influences glucose homeostasis and metabolic health in mice and humans. Sympathetic stimulation of β-adrenergic receptors in response to cold induces proliferation, differentiation, and UCP1 expression in pre-adipocytes and mature brown adipocytes. Here we show that spleen tyrosine kinase (SYK) is upregulated during brown adipocyte differentiation and activated by β-adrenergic stimulation. Deletion or inhibition of SYK, a kinase known for its essential roles in the immune system, blocks brown and white pre-adipocyte proliferation and differentiation in vitro, and results in diminished expression of Ucp1 and other genes regulating brown adipocyte function in response to β-adrenergic stimulation. Adipocyte-specific SYK deletion in mice reduces BAT mass and BAT that developed consisted of SYK-expressing brown adipocytes that had escaped homozygous Syk deletion. SYK inhibition in vivo represses β-agonist-induced thermogenesis and oxygen consumption. These results establish SYK as an essential mediator of brown fat formation and function

De Novo Reconstruction of Adipose Tissue Transcriptomes Reveals Long Non-coding RNA Regulators of Brown Adipocyte Development

Brown adipose tissue (BAT) protects against obesity by promoting energy expenditure via uncoupled respiration. To uncover BAT-specific long non-coding RNAs (lncRNAs), we used RNA-seq to reconstruct de novo transcriptomes of mouse brown, inguinal white, and epididymal white fat and identified ∼1,500 lncRNAs, including 127 BAT-restricted loci induced during differentiation and often targeted by key regulators PPARγ, C/EBPα, and C/EBPβ. One of them, lnc-BATE1, is required for establishment and maintenance of BAT identity and thermogenic capacity. lnc-BATE1 inhibition impairs concurrent activation of brown fat and repression of white fat genes and is partially rescued by exogenous lnc-BATE1 with mutated siRNA-targeting sites, demonstrating a function in trans. We show that lnc-BATE1 binds heterogeneous nuclear ribonucleoprotein U and that both are required for brown adipogenesis. Our work provides an annotated catalog for the study of fat depot-selective lncRNAs and establishes lnc-BATE1 as a regulator of BAT development and physiology.National Institutes of Health (U.S.) (Grants DK047618, DK068348 and 5P01 HL066105)Singapore. Ministry of Health (Singapore National Research Foundation. CBRG Grant NMRC/CBRG/0070/2014

PD-L1 is an activation-independent marker of brown adipocytes

Programmed death ligand 1 (PD-L1) is expressed on a number of immune and cancer cells, where it can downregulate antitumor immune responses. Its expression has been linked to metabolic changes in these cells. Here we develop a radiolabeled camelid single-domain antibody (anti-PD-L1 VHH) to track PD-L1 expression by immuno-positron emission tomography (PET). PET-CT imaging shows a robust and specific PD-L1 signal in brown adipose tissue (BAT). We confirm expression of PD-L1 on brown adipocytes and demonstrate that signal intensity does not change in response to cold exposure or β-adrenergic activation. This is the first robust method of visualizing murine brown fat independent of its activation state

A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling

Reactive oxygen species (ROS) such as hydrogen peroxide (H[subscript 2]O[subscript 2]) govern cellular homeostasis by inducing signaling. H[subscript 2]O[subscript 2] modulates the activity of phosphatases and many other signaling molecules through oxidation of critical cysteine residues, which led to the notion that initiation of ROS signaling is broad and nonspecific, and thus fundamentally distinct from other signaling pathways. Here, we report that H[subscript 2]O[subscript 2] signaling bears hallmarks of a regular signal transduction cascade. It is controlled by hierarchical signaling events resulting in a focused response as the results place the mitochondrial respiratory chain upstream of tyrosine-protein kinase Lyn, Lyn upstream of tyrosine-protein kinase SYK (Syk), and Syk upstream of numerous targets involved in signaling, transcription, translation, metabolism, and cell cycle regulation. The active mediators of H[subscript 2]O[subscript 2] signaling colocalize as H[subscript 2]O[subscript 2] induces mitochondria-associated Lyn and Syk phosphorylation, and a pool of Lyn and Syk reside in the mitochondrial intermembrane space. Finally, the same intermediaries control the signaling response in tissues and species responsive to H[subscript 2]O[subscript 2] as the respiratory chain, Lyn, and Syk were similarly required for H[subscript 2]O[subscript 2] signaling in mouse B cells, fibroblasts, and chicken DT40 B cells. Consistent with a broad role, the Syk pathway is coexpressed across tissues, is of early metazoan origin, and displays evidence of evolutionary constraint in the human. These results suggest that H[subscript 2]O[subscript 2] signaling is under control of a signal transduction pathway that links the respiratory chain to the mitochondrial intermembrane space-localized, ubiquitous, and ancient Syk pathway in hematopoietic and nonhematopoietic cells.Grant RO1 DK04761