Screening for gestational diabetes is too late to prevent ill health effects on offspring, study finds

Obese women who develop gestational diabetes are five times more likely to carry a baby with excessive fetal growth by six months of pregnancy than women who do not develop impaired glucose tolerance...

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α-SNAP Enhances SNARE Zippering by Stabilizing the SNARE Four-Helix Bundle

Publication date: Available online 7 April 2016
Source:Cell Reports
Author(s): Lu Ma, Yuhao Kang, Junyi Jiao, Aleksander A. Rebane, Hyo Keun Cha, Zhiqun Xi, Hong Qu, Yongli Zhang
Intracellular membrane fusion is mediated by dynamic assembly and disassembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs). α-SNAP guides NSF to disassemble SNARE complexes after membrane fusion. Recent experiments showed that α-SNAP also dramatically enhances SNARE assembly and membrane fusion. How α-SNAP is involved in these opposing activities is not known. Here, we examine the effect of α-SNAP on the stepwise assembly of the synaptic SNARE complex using optical tweezers. We found that α-SNAP destabilized the linker domain (LD) of the SNARE complex but stabilized its C-terminal domain (CTD) through a conformational selection mechanism. In contrast, α-SNAP minimally affected assembly of the SNARE N-terminal domain (NTD), indicating that α-SNAP barely bound the partially assembled trans-SNARE complex. Thus, α-SNAP recognizes the folded CTD for SNARE disassembly with NSF and subtly modulates membrane fusion by altering the stabilities of the SNARE CTD and LD.

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Using a single-molecule approach, Ma et al. find that α-SNAP plays distinct roles in stepwise SNARE assembly: it destabilizes the SNARE linker domain, stabilizes the C-terminal domain, but hardly affects the N-terminal domain. The findings shed light on membrane fusion as well as α-SNAP’s opposing functions in SNARE assembly and disassembly.


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HDAC3 Is a Master Regulator of mTEC Development

Publication date: Available online 7 April 2016
Source:Cell Reports
Author(s): Yael Goldfarb, Noam Kadouri, Ben Levi, Asaf Sela, Yonatan Herzig, Ronald N. Cohen, Anthony N. Hollenberg, Jakub Abramson
The thymus provides a unique microenvironment enabling development and selection of T lymphocytes. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by facilitating negative selection of self-reactive thymocytes and the generation of Foxp3⁺ regulatory T cells. Although studies have highlighted the non-canonical nuclear factor κB (NF-κB) pathway as the key regulator of mTEC development, comprehensive understanding of the molecular pathways regulating this process still remains incomplete. Here, we demonstrate that the development of functionally competent mTECs is regulated by the histone deacetylase 3 (Hdac3). Although histone deacetylases are global transcriptional regulators, this effect is highly specific only to Hdac3, as neither Hdac1 nor Hdac2 inactivation caused mTEC ablation. Interestingly, Hdac3 induces an mTEC-specific transcriptional program independently of the previously recognized RANK-NFκB signaling pathway. Thus, our findings uncover yet another layer of complexity of TEC lineage divergence and highlight Hdac3 as a major and specific molecular switch crucial for mTEC differentiation.

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Goldfarb et al. show that Hdac3 is essential for normal development and function of medullary thymic epithelial cells (mTECs) independently of non-canonical NFκB signaling. Their findings highlight Hdac3 as a master switch inducing the mTEC transcriptional program in immature TECs.


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The Mitochondrial Respiratory Chain Is Required for Organismal Adaptation to Hypoxia

Publication date: Available online 7 April 2016
Source:Cell Reports
Author(s): Robert B. Hamanaka, Samuel E. Weinberg, Colleen R. Reczek, Navdeep S. Chandel
Hypoxia-inducible factors (HIFs) are crucial for cellular and organismal adaptation to hypoxia. The mitochondrial respiratory chain is the largest consumer of oxygen in most mammalian cells; however, it is unknown whether the respiratory chain is necessary for in vivo activation of HIFs and organismal adaptation to hypoxia. HIF-1 activation in the epidermis has been shown to be a key regulator of the organismal response to hypoxic conditions, including renal production of erythropoietin (Epo). Therefore, we conditionally deleted expression of TFAM in mouse epidermal keratinocytes. TFAM is required for maintenance of the mitochondrial genome, and TFAM-null cells are respiratory deficient. TFAM loss in epidermal keratinocytes reduced epidermal levels of HIF-1α protein and diminished the hypoxic induction of HIF-dependent transcription in epidermis. Furthermore, epidermal TFAM deficiency impaired hypoxic induction of renal Epo expression. Our results demonstrate that the mitochondrial respiratory chain is essential for in vivo HIF activation and organismal adaptation to hypoxia.

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Hamanaka et al. show that loss of mitochondrial respiratory activity in the mouse epidermis impairs hypoxic activation of HIF-1. Since epidermal HIF-1 promotes cutaneous vasodilation and blood flow during hypoxia and potentiates the hypoxic response in the internal organs, mice with loss of TFAM in the epidermis are unable to induce renal erythropoietin expression in response to hypoxia.


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The Biogenesis of Nascent Circular RNAs

Publication date: Available online 7 April 2016
Source:Cell Reports
Author(s): Yang Zhang, Wei Xue, Xiang Li, Jun Zhang, Siye Chen, Jia-Lin Zhang, Li Yang, Ling-Ling Chen
Steady-state circular RNAs (circRNAs) have been mapped to thousands of genomic loci in mammals. We studied circRNA processing using metabolic tagging of nascent RNAs with 4-thiouridine (4sU). Strikingly, the efficiency of circRNA processing from pre-mRNA is extremely low endogenously. Additional studies revealed that back-splicing outcomes correlate with fast RNA Polymerase II elongation rate and are tightly controlled by cis-elements in vivo. Additionally, prolonged 4sU labeling in cells shows that circRNAs are largely processed post-transcriptionally and that circRNAs are stable. Circular RNAs that are abundant at a steady-state level tend to accumulate. This is particularly true in cells, such as neurons, that have slow division rates. This study uncovers features of circRNA biogenesis by investigating the link between nascent circRNA processing and transcription.

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Zhang et al. study the link between circRNA processing and transcription using 4sUDRB-seq. They find that circRNA production from pre-mRNA back-splicing is slow and largely occurs post-transcriptionally. The authors argue that circRNAs that are abundant at a steady-state level tend to be transcribed quickly and accumulate.


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BET Bromodomain Inhibition Releases the Mediator Complex from Select cis-Regulatory Elements

Publication date: Available online 7 April 2016
Source:Cell Reports
Author(s): Anand S. Bhagwat, Jae-Seok Roe, Beverly Y.L. Mok, Anja F. Hohmann, Junwei Shi, Christopher R. Vakoc
The bromodomain and extraterminal (BET) protein BRD4 can physically interact with the Mediator complex, but the relevance of this association to the therapeutic effects of BET inhibitors in cancer is unclear. Here, we show that BET inhibition causes a rapid release of Mediator from a subset of cis-regulatory elements in the genome of acute myeloid leukemia (AML) cells. These sites of Mediator eviction were highly correlated with transcriptional suppression of neighboring genes, which are enriched for targets of the transcription factor MYB and for functions related to leukemogenesis. A shRNA screen of Mediator in AML cells identified the MED12, MED13, MED23, and MED24 subunits as performing a similar regulatory function to BRD4 in this context, including a shared role in sustaining a block in myeloid maturation. These findings suggest that the interaction between BRD4 and Mediator has functional importance for gene-specific transcriptional activation and for AML maintenance.

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In this study, Bhagwat et al. show that the Mediator complex and BRD4 are linked coactivators that support gene-specific transcriptional activation in leukemia cells. They provide evidence that small-molecule inhibitors of BRD4 exert anti-leukemia effects by interfering with Mediator function to suppress transcription.


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CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape

Publication date: Available online 7 April 2016
Source:Cell Reports
Author(s): Zhen Wang, Qinghua Pan, Patrick Gendron, Weijun Zhu, Fei Guo, Shan Cen, Mark A. Wainberg, Chen Liang
Cas9 cleaves specific DNA sequences with the assistance of a programmable single guide RNA (sgRNA). Repairing this broken DNA by the cell’s error-prone non-homologous end joining (NHEJ) machinery leads to insertions and deletions (indels) that often impair DNA function. Using HIV-1, we have now demonstrated that many of these indels are indeed lethal for the virus, but that others lead to the emergence of replication competent viruses that are resistant to Cas9/sgRNA. This unexpected contribution of Cas9 to the development of viral resistance is facilitated by some indels that are not deleterious for viral replication, but that are refractory to recognition by the same sgRNA as a result of changing the target DNA sequences. This observation illustrates two opposite outcomes of Cas9/sgRNA action, i.e., inactivation of HIV-1 and acceleration of viral escape, thereby potentially limiting the use of Cas9/sgRNA in HIV-1 therapy.

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Wang et al. report that HIV-1 can escape Cas9/sgRNA-mediated inhibition. They reveal that the NHEJ repair machinery generates mutations in the HIV-1 Cas9 cleavage site that result in two outcomes: viral replication suppression and viral escape.


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