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The sustained expression of the MAFB transcription factor in human islet β-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet β-cell-enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet β-cell population and that expression is postnatally restricted in mouse β-cells by de novo DNA methylation. To gain insight into how MAFB affects human β-cells, we developed a mouse model to ectopically express in adult mouse β-cells using transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse β-cells, suggesting that the human adult β-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet β-cell defects in a mouse mutant lacking MafA in β-cells. Of note, transgenic production of MafB in β-cells elevated tryptophan hydroxylase 1 mRNA production during pregnancy, which drives the serotonin biosynthesis critical for adaptive maternal β-cell responses. Together, these studies provide novel insight into the role of MAFB in human islet β-cells.
© 2018 by the American Diabetes Association.
Inactivation of the von Hippel-Lindau (VHL) E3 ubiquitin ligase protein is a hallmark of clear cell renal cell carcinoma (ccRCC). Identifying how pathways affected by VHL loss contribute to ccRCC remains challenging. We used a genome-wide in vitro expression strategy to identify proteins that bind VHL when hydroxylated. Zinc fingers and homeoboxes 2 (ZHX2) was found as a VHL target, and its hydroxylation allowed VHL to regulate its protein stability. Tumor cells from ccRCC patients with loss-of-function mutations usually had increased abundance and nuclear localization of ZHX2. Functionally, depletion of ZHX2 inhibited VHL-deficient ccRCC cell growth in vitro and in vivo. Mechanistically, integrated chromatin immunoprecipitation sequencing and microarray analysis showed that ZHX2 promoted nuclear factor κB activation. These studies reveal ZHX2 as a potential therapeutic target for ccRCC.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen Candida albicans is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of C. albicans HSF1 induces morphogenesis in the absence of external cues. HSF1 depletion compromises Hsp90 function, thereby driving filamentation. HSF1 overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.
Power/sample size (power) analysis estimates the likelihood of successfully finding the statistical significance in a data set. There has been a growing recognition of the importance of power analysis in the proper design of experiments. Power analysis is complex, yet necessary for the success of large studies. It is important to design a study that produces statistically accurate and reliable results. Power computation methods have been well established for both microarray-based gene expression studies and genotyping microarray-based genome-wide association studies. High-throughput sequencing (HTS) has greatly enhanced our ability to conduct biomedical studies at the highest possible resolution (per nucleotide). However, the complexity of power computations is much greater for sequencing data than for the simpler genotyping array data. Research on methods of power computations for HTS-based studies has been recently conducted but is not yet well known or widely used. In this article, we describe the power computation methods that are currently available for a range of HTS-based studies, including DNA sequencing, RNA-sequencing, microbiome sequencing and chromatin immunoprecipitation sequencing. Most importantly, we review the methods of power analysis for several types of sequencing data and guide the reader to the relevant methods for each data type.
Intensive efforts are focused on identifying regulators of human pancreatic islet cell growth and maturation to accelerate development of therapies for diabetes. After birth, islet cell growth and function are dynamically regulated; however, establishing these age-dependent changes in humans has been challenging. Here, we describe a multimodal strategy for isolating pancreatic endocrine and exocrine cells from children and adults to identify age-dependent gene expression and chromatin changes on a genomic scale. These profiles revealed distinct proliferative and functional states of islet α cells or β cells and histone modifications underlying age-dependent gene expression changes. Expression of SIX2 and SIX3, transcription factors without prior known functions in the pancreas and linked to fasting hyperglycemia risk, increased with age specifically in human islet β cells. SIX2 and SIX3 were sufficient to enhance insulin content or secretion in immature β cells. Our work provides a unique resource to study human-specific regulators of islet cell maturation and function.
Copyright © 2016 Elsevier Inc. All rights reserved.
BACKGROUND - In glioblastoma (GBM), Id1 serves as a functional marker for self-renewing cancer stem-like cells. We investigated the mechanism by which cyclooxygenase-2 (Cox-2)-derived prostaglandin E2 (PGE2) induces Id1 and increases GBM self-renewal and radiation resistance.
METHODS - Mouse and human GBM cells were stimulated with dimethyl-PGE2 (dmPGE2), a stabilized form of PGE2, to test for Id1 induction. To elucidate the signal transduction pathway governing the increase in Id1, a combination of short interfering RNA knockdown and small molecule inhibitors and activators of PGE2 signaling were used. Western blotting, quantitative real-time (qRT)-PCR, and chromatin immunoprecipitation assays were employed. Sphere formation and radiation resistance were measured in cultured primary cells. Immunohistochemical analyses were carried out to evaluate the Cox-2-Id1 axis in experimental GBM.
RESULTS - In GBM cells, dmPGE2 stimulates the EP4 receptor leading to activation of ERK1/2 MAPK. This leads, in turn, to upregulation of the early growth response1 (Egr1) transcription factor and enhanced Id1 expression. Activation of this pathway increases self-renewal capacity and resistance to radiation-induced DNA damage, which are dependent on Id1.
CONCLUSIONS - In GBM, Cox-2-derived PGE2 induces Id1 via EP4-dependent activation of MAPK signaling and the Egr1 transcription factor. PGE2-mediated induction of Id1 is required for optimal tumor cell self-renewal and radiation resistance. Collectively, these findings identify Id1 as a key mediator of PGE2-dependent modulation of radiation response and lend insight into the mechanisms underlying radiation resistance in GBM patients.
© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: email@example.com.
A functional complex consisting of androgen receptor (AR) and forkhead box A1 (FOXA1) proteins supports prostatic development, differentiation, and disease. In addition, the interaction of FOXA1 with cofactors such as nuclear factor I (NFI) family members modulates AR target gene expression. However, the global role of specific NFI family members has yet to be described in the prostate. In these studies, chromatin immunoprecipitation followed by DNA sequencing in androgen-dependent LNCaP prostate cancer cells demonstrated that 64.3% of NFIB binding sites are associated with AR and FOXA1 binding sites. Interrogation of published data revealed that genes associated with NFIB binding sites are predominantly induced after dihydrotestosterone treatment of LNCaP cells, whereas NFIB knockdown studies demonstrated that loss of NFIB drives increased AR expression and superinduction of a subset of AR target genes. Notably, genes bound by NFIB only are associated with cell division and cell cycle. To define the role of NFIB in vivo, mouse Nfib knockout prostatic tissue was rescued via renal capsule engraftment. Loss of Nfib expression resulted in prostatic hyperplasia, which did not resolve in response to castration, and an expansion of an intermediate cell population in a small subset of grafts. In human benign prostatic hyperplasia, luminal NFIB loss correlated with more severe disease. Finally, some areas of intermediate cell expansion were also associated with NFIB loss. Taken together, these results show a fundamental role for NFIB as a coregulator of AR action in the prostate and in controlling prostatic hyperplasia.
Transcription factor expression fluctuates during β-cell ontogeny, and disruptions in this pattern can affect the development or function of those cells. Here we uncovered that murine endocrine pancreatic progenitors express high levels of the homeodomain transcription factor Prox1, whereas both immature and mature β-cells scarcely express this protein. We also investigated if sustained Prox1 expression is incompatible with β-cell development or maintenance using transgenic mouse approaches. We discovered that Prox1 upregulation in mature β-cells has no functional consequences; in contrast, Prox1 overexpression in immature β-cells promotes acute fasting hyperglycemia. Using a combination of immunostaining and quantitative and comparative gene expression analyses, we determined that Prox1 upregulation reduces proliferation, impairs maturation, and enables apoptosis in postnatal β-cells. Also, we uncovered substantial deficiency in β-cells that overexpress Prox1 of the key regulator of β-cell maturation MafA, several MafA downstream targets required for glucose-stimulated insulin secretion, and genes encoding important components of FGF signaling. Moreover, knocking down PROX1 in human EndoC-βH1 β-cells caused increased expression of many of these same gene products. These and other results in our study indicate that reducing the expression of Prox1 is beneficial for the expansion and maturation of postnatal β-cells.
© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
Type 1 diabetes mellitus (T1DM) is associated with chronic systemic inflammation and enhanced susceptibility to systemic bacterial infection (sepsis). We hypothesized that low insulin concentrations in T1DM trigger the enzyme 5-lipoxygenase (5-LO) to produce the lipid mediator leukotriene B4 (LTB4), which triggers systemic inflammation that may increase susceptibility to polymicrobial sepsis. Consistent with chronic inflammation, peritoneal macrophages from two mouse models of T1DM had greater abundance of the adaptor MyD88 (myeloid differentiation factor 88) and its direct transcriptional effector STAT-1 (signal transducer and activator of transcription 1) than macrophages from nondiabetic mice. Expression of Alox5, which encodes 5-LO, and the concentration of the proinflammatory cytokine interleukin-1β (IL-1β) were also increased in peritoneal macrophages and serum from T1DM mice. Insulin treatment reduced LTB4 concentrations in the circulation and Myd88 and Stat1 expression in the macrophages from T1DM mice. T1DM mice treated with a 5-LO inhibitor had reduced Myd88 mRNA in macrophages and increased abundance of IL-1 receptor antagonist and reduced production of IL-β in the circulation. T1DM mice lacking 5-LO or the receptor for LTB4 also produced less proinflammatory cytokines. Compared to wild-type or untreated diabetic mice, T1DM mice lacking the receptor for LTB4 or treated with a 5-LO inhibitor survived polymicrobial sepsis, had reduced production of proinflammatory cytokines, and had decreased bacterial counts. These results uncover a role for LTB4 in promoting sterile inflammation in diabetes and the enhanced susceptibility to sepsis in T1DM.
Copyright © 2015, American Association for the Advancement of Science.
Regulatory T cells (Tregs) are a specialized subset of CD4(+) T cells that maintain self-tolerance by functionally suppressing autoreactive lymphocytes. The Treg compartment is composed of thymus-derived Tregs (tTregs) and peripheral Tregs (pTregs) that are generated in secondary lymphoid organs after exposure to antigen and specific cytokines, such as TGF-β. With regard to this latter lineage, pTregs [and their ex vivo generated counterparts, induced Tregs (iTregs)] offer particular therapeutic potential because these cells can be raised against specific antigens to limit autoimmunity. We now report that transcription factor Krüppel-like factor 2 (KLF2) is necessary for the generation of iTregs but not tTregs. Moreover, drugs that limit KLF2 proteolysis during T-cell activation enhance iTreg development. To the authors' knowledge, this study identifies the first transcription factor to distinguish between i/pTreg and tTreg ontogeny and demonstrates that KLF2 is a therapeutic target for the production of regulatory T cells.