Authors: Gu C, Nguyen HN, Ganini D, Chen Z, Jessen HJ, Gu Z, Wang H, Shears SB
Journal: Proc Natl Acad Sci U S A. 2017 Nov 7;114(45):11968-11973. doi: 10.1073/pnas.1702370114. Epub 2017 Oct 25.
The inositol pyrophosphates 5-InsP7 (diphosphoinositol pentakisphosphate) and 1,5-InsP8 (bis-diphosphoinositol tetrakisphosphate) are highly energetic cellular signals interconverted by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks). Here, we used CRISPR to KO PPIP5Ks in the HCT116 colon cancer cell line. This procedure eliminates 1,5-InsP8 and raises 5-InsP7 levels threefold. Expression of p53 and p21 was up-regulated; proliferation and G1/S cell-cycle transition slowed. Thus, PPIP5Ks are potential targets for tumor therapy. Deletion of the PPIP5Ks elevated [ATP] by 35%; both [ATP] and [5-InsP7] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP7] provides direct support for an energy-sensing attribute (i.e., 1 mM Km for ATP) of the 5-InsP7-generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP7 levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in PPIP5K-/- HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP8 and 5-InsP7 as drivers of the hypermetabolic and p53-elevated phenotypes, we used IP6K2 RNAi and the pan-IP6K inhibitor, N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine (TNP), to return 5-InsP7 levels in PPIP5K-/- cells to those of WT cells without rescuing 1,5-InsP8 levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP7 regulates p53 expression, whereas 1,5-InsP8 regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP8 to bioenergetic homeostasis.
Authors: Tang S1, Fang Y1, Huang G2,3, Xu X4, Padilla-Banks E5, Fan W1, Xu Q1, Sanderson SM6, Foley JF7, Dowdy S7, McBurney MW8, Fargo DC4, Williams CJ5, Locasale JW6, Guan Z9, Li X
Journal: EMBO J. 2017 Nov 2;36(21):3175-3193. doi: 10.15252/embj.201796708. Epub 2017 Oct 11.
Methionine metabolism is critical for epigenetic maintenance, redox homeostasis, and animal development. However, the regulation of methionine metabolism remains unclear. Here, we provide evidence that SIRT1, the most conserved mammalian NAD+-dependent protein deacetylase, is critically involved in modulating methionine metabolism, thereby impacting maintenance of mouse embryonic stem cells (mESCs) and subsequent embryogenesis. We demonstrate that SIRT1-deficient mESCs are hypersensitive to methioninerestriction/depletion-induced differentiation and apoptosis, primarily due to a reduced conversion of methionine to S-adenosylmethionine. This reduction markedly decreases methylation levels of histones, resulting in dramatic alterations in gene expression profiles. Mechanistically, we discover that the enzyme converting methionine to S-adenosylmethionine in mESCs, methionine adenosyltransferase 2a (MAT2a), is under control of Myc and SIRT1. Consistently, SIRT1 KO embryos display reduced Mat2a expression and histone methylation and are sensitive to maternal methionine restriction-induced lethality, whereas maternal methionine supplementation increases the survival of SIRT1 KO newborn mice. Our findings uncover a novel regulatory mechanism for methionine metabolism and highlight the importance of methionine metabolismin SIRT1-mediated mESC maintenance and embryonic development.
Authors: Chen W, Tian R, Xu C, Yung BC, Wang G, Liu Y, Ni Q, Zhang F, Zhou Z, Wang J, Niu G, Ma Y, Fu L, Chen X
Journal: Nat Commun. 2017 Nov 24;8(1):1777. doi: 10.1038/s41467-017-01764-1
The delivery of therapeutic peptides for diabetes therapy is compromised by short half-lives of drugs with the consequent need for multiple daily injections that reduce patient compliance and increase treatment cost. In this study, we demonstrate a smart exendin-4 (Ex4) delivery device based on microneedle (MN)-array patches integrated with dual mineralized particles separately containing Ex4 and glucose oxidase (GOx). The dual mineralized particle-based system can specifically release Ex4 while immobilizing GOx as a result of the differential response to the microenvironment induced by biological stimuli. In this manner, the system enables glucose-responsive and closed-loop release to significantly improve Ex4 therapeutic performance. Moreover, integration of mineralized particles can enhance the mechanical strength of alginate-based MN by crosslinking to facilitate skin penetration, thus supporting painless and non-invasive transdermal administration. We believe this smart glucose-responsive Ex4 delivery holds great promise for type 2 diabetes therapy by providing safe, long-term, and on-demand Ex4 therapy.
Authors: Shalaby KH1, Lyons-Cohen MR1, Whitehead GS, Thomas SY, Prinz I, Nakano H, Cook DN
Journal: J Allergy Clin Immunol. 2017 Nov 14. pii: S0091-6749(17)31753-0. doi: 10.1016/j.jaci.2017.10.023. [Epub ahead of print]
BACKGROUND: Mechanisms that elicit mucosal Th17 cell responses have been described, yet how these cells are sustained in chronically inflamed tissues remains unclear.
OBJECTIVE: We sought to understand whether the maintenance of lung Th17 inflammation requires environmental agents in addition to antigen, and to identify the lung antigen-presenting cell types (APCs) that sustain the self-renewal of Th17 cells.
METHODS: Animals were repeatedly exposed to aspiration of ovalbumin alone, or together with environmental adjuvants, including extracts of common house dust (HDE), to test their role in maintaining lung inflammation. Alternatively, antigen-specific effector/memory Th17 cells, generated in culture using CD4+ T cells from Il17a fate-mapping mice, were adoptively transferred to assess their persistence in genetically modified animals lacking distinct lung APC subsets or cell-specific TLR4 signaling. Th17 cells were also co-cultured with lung APC subsets to determine which of these could revive their expansion and activation.
RESULTS: Th17 cells and consequent neutrophilic inflammation were poorly sustained by inhaled antigen alone, but were augmented by inhalation of antigen together with HDE. This was associated with weight loss and changes in lung physiology consistent with interstitial lung disease. The effect of HDE required TLR4 signaling predominantly in lung hematopoietic cells, including CD11c+ cells. CD103+ and CD11b+conventional dendritic cells (cDCs) directly interacted with Th17 cells in situ and revived the clonal expansion of Th17 cells ex vivo and in vivo, whereas lung macrophages and B cells could not.
CONCLUSION: Th17-dependent inflammation in the lungs can be sustained by persistent TLR4-mediated activation of lung cDCs.
Authors: Yang, Kohler ME, Chien CD, Sauter CT, Jacoby E, Yan C, Hu Y, Wanhainen K, Qin H, Fry TJ
Journal: Sci Transl Med. 2017 Nov 22;9(417). pii: eaag1209. doi: 10.1126/scitranslmed.aag1209
Chimeric antigen receptor (CAR)-expressing T cells induce durable remissions in patients with relapsed/refractory B cell malignancies. CARs are synthetic constructs that, when introduced into mature T cells, confer a second, non-major histocompatibility complex-restricted specificity in addition to the endogenous T cell receptor (TCR). The implications of TCR activation on CAR T cell efficacy has not been well defined. Using an immunocompetent, syngeneic murine model of CD19-targeted CAR T cell therapy for pre-B cell acute lymphoblastic leukemia in which the CAR is introduced into T cells with known TCR specificity, we demonstrate loss of CD8 CAR T cell efficacy associated with T cell exhaustion and apoptosis when TCR antigen is present. CD4 CAR T cells demonstrate equivalent cytotoxicity to CD8 CAR T cells and, in contrast, retain in vivo efficacy despite TCR stimulation. Gene expression profiles confirm increased exhaustion and apoptosis of CD8 CAR T cells upon dual receptor stimulation compared to CD4 CAR T cells and indicate inherent differences between CD4 and CD8 CAR T cells in the use of T cell-associated signaling pathways. These results provide insights into important aspects of CAR T cell immune biology and indicate opportunities to rationally design CAR constructs to optimize clinical efficacy.
Authors: Steiner AZ, Pritchard D, Stanczyk FZ, Kesner JS, Meadows JW, Herring AH, Baird DD
Journal: JAMA. 2017 Oct 10;318(14):1367-1376. doi: 10.1001/jama.2017.14588.
IMPORTANCE: Despite lack of evidence of their utility, biomarkers of ovarian reserve are being promoted as potential markers of reproductivepotential.
OBJECTIVE: To determine the associations between biomarkers of ovarian reserve and reproductive potential among women of late reproductive age.
DESIGN, SETTING, AND PARTICIPANTS: Prospective time-to-pregnancy cohort study (2008 to date of last follow-up in March 2016) of women (N = 981) aged 30 to 44 years without a history of infertility who had been trying to conceive for 3 months or less, recruited from the community in the Raleigh-Durham, North Carolina, area.
EXPOSURES: Early-follicular-phase serum level of antimüllerian hormone (AMH), follicle-stimulating hormone (FSH), and inhibin B and urinary level of FSH.
MAIN OUTCOMES AND MEASURES: The primary outcomes were the cumulative probability of conception by 6 and 12 cycles of attempt and relative fecundability (probability of conception in a given menstrual cycle). Conception was defined as a positive pregnancy test result.
RESULTS: A total of 750 women (mean age, 33.3 [SD, 3.2] years; 77% white; 36% overweight or obese) provided a blood and urine sample and were included in the analysis. After adjusting for age, body mass index, race, current smoking status, and recent hormonal contraceptive use, women with low AMH values (<0.7 ng/mL [n = 84]) did not have a significantly different predicted probability of conceiving by 6 cycles of attempt (65%; 95% CI, 50%-75%) compared with women (n = 579) with normal values (62%; 95% CI, 57%-66%) or by 12 cycles of attempt (84% [95% CI, 70%-91%] vs 75% [95% CI, 70%-79%], respectively). Women with high serum FSH values (>10 mIU/mL [n = 83]) did not have a significantly different predicted probability of conceiving after 6 cycles of attempt (63%; 95% CI, 50%-73%) compared with women (n = 654) with normal values (62%; 95% CI, 57%-66%) or after 12 cycles of attempt (82% [95% CI, 70%-89%] vs 75% [95% CI, 70%-78%], respectively). Women with high urinary FSH values (>11.5 mIU/mg creatinine [n = 69]) did not have a significantly different predicted probability of conceiving after 6 cycles of attempt (61%; 95% CI, 46%-74%) compared with women (n = 660) with normal values (62%; 95% CI, 58%-66%) or after 12 cycles of attempt (70% [95% CI, 54%-80%] vs 76% [95% CI, 72%-80%], respectively). Inhibin B levels (n = 737) were not associated with the probability of conceiving in a given cycle (hazard ratio per 1-pg/mL increase, 0.999; 95% CI, 0.997-1.001).
CONCLUSIONS AND RELEVANCE: Among women aged 30 to 44 years without a history of infertility who had been trying to conceive for 3 months or less, biomarkers indicating diminished ovarian reserve compared with normal ovarian reserve were not associated with reduced fertility. These findings do not support the use of urinary or blood follicle-stimulating hormone tests or antimüllerian hormone levels to assess natural fertility for women with these characteristics.
Authors: Guo TW, Bartesaghi A, Yang H, Falconieri V, Rao P, Merk A, Eng ET, Raczkowski AM, Fox T, Earl LA, Patel DJ, Subramaniam S
Journal: Cell. 2017 Oct 5;171(2):414-426.e12. doi: 10.1016/j.cell.2017.09.006.
Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.
Authors: Kang HS1, Kumar D2, Liao G1, Lichti-Kaiser K1, Gerrish K3, Liao XH4, Refetoff S4,5, Jothi R2, Jetten AM
Journal: J Clin Invest. 2017 Oct 30. pii: 94417. doi: 10.1172/JCI94417. [Epub ahead of print]
Deficiency in Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in humans is associated with the development of congenital hypothyroidism. However, the functions of GLIS3 in the thyroid gland and the mechanism by which GLIS3 dysfunction causes hypothyroidism are unknown. In the current study, we demonstrate that GLIS3 acts downstream of thyroid-stimulating hormone (TSH) and TSH receptor (TSHR) and is indispensable for TSH/TSHR-mediated proliferation of thyroid follicular cells and biosynthesis of thyroid hormone. Using ChIP-Seq and promoter analysis, we demonstrate that GLIS3 is critical for the transcriptional activation of several genes required for thyroidhormone biosynthesis, including the iodide transporters Nis and Pds, both of which showed enhanced GLIS3 binding at their promoters. The repression of cell proliferation of GLIS3-deficient thyroid follicular cells was due to the inhibition of TSH-mediated activation of the mTOR complex 1/ribosomal protein S6 (mTORC1/RPS6) pathway as well as the reduced expression of several cell division-related genes regulated directly by GLIS3. Consequently, GLIS3 deficiency in a murine model prevented the development of goiter as well as the induction of inflammatory and fibrotic genes during chronic elevation of circulating TSH. Our study identifies GLIS3 as a key regulator of TSH/TSHR-mediated thyroid hormone biosynthesis and proliferation of thyroid follicular cells and uncovers a mechanism by which GLIS3 deficiency causes neonatal hypothyroidism and prevents goiter development.
Authors: Cinghu S, Yang P, Kosak JP, Conway AE, Kumar D, Oldfield AJ, Adelman K, Jothi R
Journal: Mol Cell. 2017 Oct 5;68(1):104-117.e6. doi: 10.1016/j.molcel.2017.09.010
Eukaryotic gene transcription is regulated at many steps, including RNA polymerase II (Pol II) recruitment, transcription initiation, promoter-proximal Pol II pause release, and transcription termination; however, mechanisms regulating transcription during productive elongation remain poorly understood. Enhancers, which activate gene transcription, themselves undergo Pol II-mediated transcription, but our understanding of enhancer transcription and enhancer RNAs (eRNAs) remains incomplete. Here we show that transcription at intragenic enhancers interferes with and attenuates host gene transcription during productive elongation. While the extent of attenuation correlates positively with nascent eRNA expression, the act of intragenic enhancer transcription alone, but not eRNAs, explains the attenuation. Through CRISPR/Cas9-mediated deletions, we demonstrate a physiological role for intragenic enhancer-mediated transcription attenuation in cell fate determination. We propose that intragenic enhancers not only enhance transcription of one or more genes from a distance but also fine-tune transcription of their host gene through transcription interference, facilitating differential utilization of the same regulatory element for disparate functions.
Authors: Li P, Wang L, Bennett BD, Wang J, Li J, Qin Y, Takaku M, Wade PA, Wong J, Hu G
Journal: Nucleic Acids Res. 2017 Oct 11. doi: 10.1093/nar/gkx884. [Epub ahead of print]
Transposable elements, including endogenous retroviruses (ERVs), constitute a large fraction of the mammalian genome. They are transcriptionally silenced during early development to protect genome integrity and aberrant transcription. However, the mechanisms that control their repression are not fully understood. To systematically study ERV repression, we carried out an RNAi screen in mouse embryonic stem cells (ESCs) and identified a list of novel regulators. Among them, Rif1 displays the strongest effect. Rif1 depletion by RNAi or gene deletion led to increased transcription and increased chromatin accessibility at ERV regions and their neighboring genes. This transcriptional de-repression becomes more severe when DNA methylation is lost. On the mechanistic level, Rif1 directly occupies ERVs and is required for repressive histone mark H3K9me3 and H3K27me3 assembly and DNA methylation. It interacts with histone methyltransferases and facilitates their recruitment to ERV regions. Importantly, Rif1 represses ERVs in human ESCs as well, and the evolutionally-conserved HEAT-like domain is essential for its function. Finally, Rif1 acts as a barrier during somatic cell reprogramming, and its depletion significantly enhances reprogramming efficiency. Together, our study uncovered many previously uncharacterized repressors of ERVs, and defined an essential role of Rif1 in the epigenetic defense against ERV activation.