Significance Calcium is an important intracellular second messenger that regulates many biological processes. Many extracellular environmental cues lead to cellular calcium-level changes, which impact on the output of gene expression. In cardiomyocytes, calcium is known to control gene expression at the level of transcription, whereas its role in regulating alternative splicing has not been explored. Our studies demonstrate that in these cells a network of alternatively spliced exons exists, which responds to the altered calcium levels by changing their splicing patterns. Our studies further elucidate an epigenetic regulatory mechanism, triggered by calcium signaling pathways, that leads to histone hyperacetylation along gene bodies, which increases the transcriptional elongation rate of RNA polymerase II and impacts alternative splicing.
The activation of NF-κB has emerged as an important mechanism for the modulation of the response to DNA double-strand breaks (DSBs). The concomitant SUMOylation and phosphorylation of IKKγ by PIASy and ATM, respectively, is a key event in this mechanism. However, the mechanism through which mammalian cells are able to accomplish these IKKγ modifications in a timely and lesion-specific manner remains unclear. In this study, we demonstrate that LRP16 constitutively interacts with PARP1 and IKKγ. This interaction is essential for efficient interactions among PARP1, IKKγ, and PIASy, the modifications of IKKγ, and the activation of NF-κB following DSB induction. The regulation of LRP16 in NF-κB activation is dependent on the DSB-specific sensors Ku70/Ku80. These data strongly suggest that LRP16, through its constitutive interactions with PARP1 and IKKγ, functions to facilitate the lesion-specific recruitment of PARP1 and IKKγ and, ultimately, the concomitant recruitment of PIASy to IKKγ in response to DSB damage. Therefore, the study has provided important new mechanistic insights concerning DSB-induced NF-κB activation.
Abstract Metabolic engineering for hyperaccumulation of lipids in vegetative tissues of high biomass crops promises a step change in oil yields for the production of advanced biofuels. Energycane is the ideal feedstock for this approach due to its exceptional biomass production and persistence under marginal conditions. Here, we evaluated metabolically engineered energycane with constitutive expression of the lipogenic factors WRINKLED 1 ( WRI 1), DIACYLGLYCEROL ACYLTRANSFERASE 1 ( DGAT 1), and OLEOSIN 1 ( OLE 1) for the accumulation of triacylglycerol (TAG), total fatty acid (TFA), and biomass under field conditions at the University of Florida‐IFAS experiment station near Citra, Florida. TAG and TFA accumulation were highest in leaves (up to 9.9% and 12.9% of DW, respectively), followed by juice from crushed stems, stems, and roots. TAG and TFA accumulation increased up to harvest time and correlated highest with OLE 1 and DGAT 1 expression. Biomass dry weight, TAG, and TFA content differed greatly depending on DGAT 1 and OLE 1 expression in transgenic lines with similar WRI 1 expression. Biomass did not significantly differ between WT and line L2 with DAGT 1 and OLE 1 expressed at low levels and TAG and TFA accumulating to 12‐ and 1.6‐fold that of WT leaves, respectively. In contrast, line L13, with intron‐mediated enhancement of DGAT 1 expression, displayed a 245‐ to 330‐fold increase in TAG and a 4.75‐ to 6.45‐fold increase in TFA content compared with WT leaves and a biomass reduction of 52%. These results provide the basis for developing novel feedstocks for expanding plant lipid production and point to new prospects for advanced biofuels.
Multiple familial trichoepithelioma (MFT) and familial cylindromatosis are two clinically distinct cancer syndromes. MFT patients developed mostly trichoepithelioma in the face while cylindromatosis patients developed cylindromas predominantly (approximately 90%) on the head and neck. However, multiple familial trichoepithelioma is occasionally associated with familial cylindromatosis while cylindromatosis patients can also develop trichoepithelioma. This has led to the speculation that the 2 types of dermatoses may be caused by dysfunction of a common pathway. Previously, a candidate MTF locus has been mapped to 9p21 while disease gene for familial cylindromatosis, the CYLD gene located on 16q21-13 has been identified. Here, we show that mutations in the CYLD gene are also the genetic basis for three different Chinese families with MFT. Sequence analysis reveal a single nucleotide deletion, c.1462delA (P.Ile488fsX9) in exon 9, a nonsense mutation, c.2128C>T (p. Gln710X) in exon 17, and a missense mutation, c.2822A>T (p. Asp941Val) in exon 21 in each of the three families respectively. This provides direct evidence that the mutations in CYLD can cause two clinically distinct cancer syndromes.
Wild diploid wheat, Triticum urartu (T. urartu) is the progenitor of bread wheat, and understanding its genetic diversity and genome function will provide considerable reference for dissecting genomic information of common wheat. In this study, we investigated the morphological and genetic diversity and population structure of 238 T. urartu accessions collected from different geographic regions. This collection had 19.37 alleles per SSR locus and its polymorphic information content (PIC) value was 0.76, and the PIC and Nei's gene diversity (GD) of high-molecular-weight glutenin subunits (HMW-GSs) were 0.86 and 0.88, respectively. UPGMA clustering analysis indicated that the 238 T. urartu accessions could be classified into two subpopulations, of which Cluster I contained accessions from Eastern Mediterranean coast and those from Mesopotamia and Transcaucasia belonged to Cluster II. The wide range of genetic diversity along with the manageable number of accessions makes it one of the best collections for mining valuable genes based on marker-trait association. Significant associations were observed between simple sequence repeats (SSR) or HMW-GSs and six morphological traits: heading date (HD), plant height (PH), spike length (SPL), spikelet number per spike (SPLN), tiller angle (TA) and grain length (GL). Our data demonstrated that SSRs and HMW-GSs were useful markers for identification of beneficial genes controlling important traits in T. urartu, and subsequently for their conservation and future utilization, which may be useful for genetic improvement of the cultivated hexaploid wheat.
Summary Starch in wheat grain provides humans with carbohydrates and influences the quality of wheaten food. However, no transcriptional regulator of starch synthesis has been identified first in common wheat ( Triticum aestivum ) due to the complex genome. Here, a novel basic leucine zipper (bZIP) family transcription factor TubZIP28 was found to be preferentially expressed in the endosperm throughout grain‐filling stages in Triticum urartu , the A genome donor of common wheat. When TubZIP28 was overexpressed in common wheat, the total starch content increased by c. 4%, which contributed to c. 5% increase in the thousand kernel weight. The grain weight per plant of overexpression wheat was also elevated by c. 9%. Both in vitro and in vivo assays showed that TubZIP28 bound to the promoter of cytosolic AGPase and enhanced both the transcription and activity of the latter. Knockout of the homologue TabZIP28 in common wheat resulted in declines of both the transcription and activity of cytosolic AGPase in developing endosperms and c. 4% reduction of the total starch in mature grains. To the best of our knowledge, TubZIP28 and TabZIP28 are transcriptional activators of starch synthesis first identified in wheat, and they could be superior targets to improve the starch content and yield potential of wheat.
Purpose: A group of radiolabeled thymidine analogs were developed as radio-tracers for imaging herpes viral thymidine kinase (HSV1-tk) or its variants used as reporter gene.A transgenic mouse model was created to express tk upon liver injury or naturally occurring hepatocellular carcinoma (HCC).The purpose of this study was to use this unique animal model for initial testing with radio-labeled thymidine analogs, mainly a pair of newly emerging nucleoside analogs, D-FMAU and L-FMAU.Methods: A transgeneic mouse model was created by putting a fused reporter gene system, firefly luciferase (luc) and HSV1-tk, under the control of mouse alpha fetoprotein (Afp) promoter.Initial multimodal imaging, which was consisted of bioluminescent imaging (BLI) and planar gamma scintigraphy with [ 125 I]-FIAU, was used for examining the model creation in the new born and liver injury in the adult mice.Carcinogen diethylnitrosamine (DEN) was then administrated to induce HCC in these knock-in mice such that microPET imaging could be used to track the activity of Afp promoter during tumor development and progression by imaging tk expression first with [ 18 F]-FHBG.Dynamic PET scans with D-[ 18 F]-FMAU and L-[ 18 F]-FMAU were then performed to evaluate this pair of relatively new tracers.Cells were derived from these liver tumors for uptake assays using H-3 labeled version of PET tracers.Results: The mouse model with dual reporters: HSV1-tk and luc placed under the transcriptional control of an endogenous Afp promoter was used for imaging studies.The expression of the Afp gene was highly specific in proliferative hepatocytes, in regenerative liver, and in developing fetal liver, and thus provided an excellent indicator for liver injury and cancer development in adult mice.Both D-FMAU and L-FMAU showed stable liver tumor uptake where the tk gene was expressed under the Afp promoter.The performance of this pair of tracers was slightly different in terms of signal-to-background ratio as well as tracer clearance.Conclusion: The newly created knock-in mouse model was used to demonstrate the use of the dual-reporter genes driven by well-characterized cancer-specific transcriptional units in conjunction with in vivo imaging as a paradigm in studying naturally occurring cancer in live animals.While BLI is suitable for small animal imaging with luc expression, PET with L-FMAU seemed be the choice for liver injury or liver cancer imaging with this animal model for future investigations.
15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a prostaglandin-degrading enzyme that is highly expressed in normal colon mucosa but is ubiquitously lost in human colon cancers. Herein, we demonstrate that 15-PGDH is active in vivo as a highly potent suppressor of colon neoplasia development and acts in the colon as a required physiologic antagonist of the prostaglandin-synthesizing activity of the cyclooxygenase 2 (COX-2) oncogene. We first show that 15-PGDH gene knockout induces a marked 7.6-fold increase in colon tumors arising in the Min (multiple intestinal neoplasia) mouse model. Furthermore, 15-PGDH gene knockout abrogates the normal resistance of C57BL/6J mice to colon tumor induction by the carcinogen azoxymethane (AOM), conferring susceptibility to AOM-induced adenomas and carcinomas in situ . Susceptibility to AOM-induced tumorigenesis is mediated by a marked induction of dysplasia, proliferation, and cyclin D1 expression throughout microscopic aberrant crypt foci arising in 15-PGDH null colons and is concomitant with a doubling of prostaglandin E 2 in 15-PGDH null colonic mucosa. A parallel role for 15-PGDH loss in promoting the earliest steps of colon neoplasia in humans is supported by our finding of a universal loss of 15-PGDH expression in microscopic colon adenomas recovered from patients with familial adenomatous polyposis, including adenomas as small as a single crypt. These models thus delineate the in vivo significance of 15-PGDH-mediated negative regulation of the COX-2 pathway and moreover reveal the particular importance of 15-PGDH in opposing the neoplastic progression of colonic aberrant crypt foci.
CPTs (camptothecins) are an important class of effective anticancer agents that target type I topoisomerase in humans. Irinotecan and topotecan are currently used to treat various types of cancers and many CPT derivatives are being developed. However, these drugs are only effective in a small percentage of each type of cancer and the molecular underpinning for this individualized response to the drug has remained elusive. Thus, identification of the main determinants for cell survival in response to this unique class of drug should help to improve their clinical applications. In the present study, we examined whether RECQL5 constitutes an important determinant of CPT resistance in colon cancer cells. Specifically, RECQL5-deficient derivatives of both DDL1 and HCT116 cells, two colorectal cancer cell lines were generated by adenovirus-based somatic gene-targeting experiments and the CPT sensitivity between the RECQL5-proficient parental lines and their corresponding RECQL5-deficient derivatives were examined. We found that deletion of RECQL5 from DDL1 and HCT116 cells both resulted in a significant enhancement in CPT sensitivity under in vitro culture conditions. More importantly, xenograft tumours derived from RECQL5-deficient HCT116 cells, but not those from the parental line, could be cured by a CPT-based therapy in nude mice. Thus, the present study has identified RECQL5 as a major determinant for CPT resistance in colorectal cancer cells and a potential candidate as a biomarker for irinotecan-based treatment for colon cancer.
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