KLF15 promotes transcription of KLF3 gene in bovine adipocytes
Hongfang GuoRajwali KhanSayed Haidar Abbas RazaYue NingDawei WeiSen WuSeyed Mahdi HosseiniIrfan UllahMatthew GarciaLinsen Zan
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Chromatin immunoprecipitation
Abstract Background Long non-coding RNAs (lncRNAs) regulate numerous biological processes, including adipogenesis. Research on adipogenesis will assist in the treatment of human metabolic diseases and improve meat quality in livestock, such as the content of intramuscular fat (IMF). However, the significance of lncRNAs in intramuscular adipogenesis remains unclear. This research aimed to reveal the lncRNAs transcriptomic profiles in the process of bovine intramuscular adipogenesis and to identify the lncRNAs involved in the adipogenesis of bovine intramuscular adipocytes. Results In this research, a landscape of lncRNAs was identified with RNA-seq in bovine intramuscular adipocytes at four adipogenesis stages (0 d, 3 d, 6 d, and 9 d after differentiation). A total of 7035 lncRNAs were detected, including 3396 novel lncRNAs. Based on the results of differential analysis, co-expression analysis, and functional prediction, we focused on the bovine intramuscular adipogenesis-associated long non-coding RNA ( BIANCR ), a novel lncRNA that may have an important regulatory function. The knockdown of BIANCR inhibited proliferation and promoted apoptosis of intramuscular preadipocytes. Moreover, BIANCR knockdown inhibited intramuscular adipogenesis by regulating the ERK1/2 signaling pathway. Conclusion This study obtained the landscape of lncRNAs during adipogenesis in bovine intramuscular adipocytes. BIANCR plays a crucial role in adipogenesis through the ERK1/2 signaling pathway. The results are noteworthy for improving beef meat quality, molecular breeding, and metabolic disease research. Graphical Abstract
Pathway Analysis
RNA-Seq
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Adipogenesis was defined as a differentiation process of preadipocytes into adipocytes. It is a key process in determining the number of mature adipocytes in the development of obesity. Thus, to regulation of adipogenesis is crucial target in obesity prevention. Mussel (Mytilus corscus ) has been reported to possess abundant dietary proteins and savory amino acids. However, antiadipogenic role and function in obesity prevention remains unknown. Here, we employed green-shelled mussel and its water extract (GME) was prepared. To elucidate the potential role of GME in 3T3-L1 adipogenesis, 3T3-L1 cells were treated with GME between day –2 to day 6. We found that GME dose-dependently suppressed adipogenesis and inhibited formation of lipid droplets accompanied by reduced level of adipogenic transcriptional factors such as C/EBPα and FAS. Our study further revealed that inhibitory function of GME wa largely limited to the adipogenesis day 0 to day 6. Taken together, our evidences provide new insights into the molecular basis underlying the antiadipogenic function of GME.
Lipid droplet
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Arl4D is a developmentally-regulated member of the ADP-ribosylation factor/ARF-like protein (ARF/Arl) family of Ras-related GTPases. Although Arl4 protein is reported to be expressed in adipose tissue, the function of Arl4D is unknown. To investigate the potential role of Arl4D in adipogenesis, we examined Arl4D expression during adipocyte differentiation and the effects of Arl4D overexpression on adipogenesis. Arl4D protein increased early in adipogenesis, with the highest expression at 4 h after adipogenesis initiation, followed by a decrease thereafter. Overexpression of Arl4D in 3T3-L1 cells potently inhibited their ability to differentiate and accumulate lipid, and reduced the expression of adipogenic genes. Furthermore, treatment with valproic acid, an Arl4D inducer, suppressed adipogenesis. These results suggest that rapid reduction of Arl4D is required for adipogenesis to proceed.
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Sestrin-3, together with the other two members Sestrin-1 and Sestrin-2, belongs to the Sestrin family. The Sestrin protein family has been demonstrated to be involved in antioxidative, metabolic homeostasis, and even the development of nonalcoholic steatohepatitis (NASH). However, the adipogenic regulatory role of SESN3 in adipogenesis still needs to be further explored. In this study, we demonstrated SESN3 inhibited porcine pre-adipocyte proliferation, thus suppressing its adipogenesis. Meanwhile, SESN3 has been demonstrated to inhibit Smad3 thus protecting against NASH. Further, for our previous study, we found mmu-miR-124 involved in 3T3-L1 cell adipogenesis regulation. In this study, we also identified that ssc-miR-124 inhibited porcine pre-adipocyte proliferation, thus suppressing its adipogenesis, and the SMAD3 was an inhibitor of ssc-miR-124 by binding to its promoter. Furthermore, the ssc-miR-124 targeted porcine C/EBPα and GR and thus inhibited pre-adipocyte adipogenesis. In conclusion, SESN3 inhibited SMAD3, thus improving ssc-miR124, and then suppressed C/EBPα and GR to regulate pre-adipocytes adipogenesis.
Nonalcoholic steatohepatitis
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Abstract Objective Extracts of Artemisia scoparia (SCO) have antidiabetic properties in mice and enhance adipogenesis in vitro , but the underlying mechanisms are unknown. Thiazolidinediones, including rosiglitazone (ROSI), are pharmacological activators of peroxisome proliferator‐activated receptor gamma that also promote adipogenesis. The aim of this study was to examine adipogenic pathways responsible for SCO‐mediated adipogenesis and identify potential differences between SCO and ROSI in the ability to promote adipocyte development. Methods The ability of SCO or ROSI to promote adipogenesis in 3T3‐L1 cells following systematic omission of the common triad of adipogenic effectors dexamethasone, 1‐methyl‐3‐isobutylxanthine (MIX), and insulin was examined. Adipogenesis was assessed by both neutral lipid quantitation and adipocyte marker gene expression. Results The results demonstrate that SCO and ROSI promote adipogenesis and increase the expression of several peroxisome proliferator‐activated receptor gamma target genes involved in lipid accumulation in the absence of MIX. However, ROSI can enhance adipogenesis in the absence of MIX and insulin and differentially regulates adipogenic and lipid metabolism genes as compared with SCO. Conclusions These data demonstrate the adipogenic capabilities of SCO are similar but not identical to ROSI, thereby warranting further research into SCO as a promising source of therapeutic compounds in the treatment of metabolic disease states.
Rosiglitazone
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A reporter assay system is an essential tool for investigating gene expression mechanisms. In the case of bifidobacteria, several convenient and sensitive reporter systems have been developed. Here, we developed a new reporter system for bifidobacteria using the chloramphenicol acetyltransferase gene (cat) from Staphylococcus aureus. This enzyme stoichiometrically produced free CoA-SH, which was analyzed quantitatively with Ellman’s test using 2-nitrobenzoic acid (DTNB). The 2-nitro-5-thiobenzoate (TNB2-) produced showed a strong yellowish color with maximum absorbance at 412 nm. We also constructed a new pBCMAT plasmid series for CAT assays in bifidobacteria to evaluate promoters and terminators. Analyses using promoters from Bifidobacterium longum NCC2705 indicated that the CAT assay using these promoters is quantitative, has a wide measurement range, and is stable. In addition, this assay was useful for several bifidobacterial species, including B. longum, Bifidobacterium breve, and Bifidobacterium adolescentis. Compared with evoglow-Bs2, a fluorescent protein used under anaerobic conditions, the CAT assay showed about 0.25% background activity. In analyses using this CAT assay, we identified 11 promoters and 12 terminators of B. longum NCC2705. The genes encoding ribosomal proteins, elongation factors, and transfer RNAs possessed strong promoters, and terminators that include strong stem-loops and poly-U tails structures tended to show high activities. Although the abovementioned promoters made stronger contributions to expression activities than the terminators, the maximum fold difference in the activities among the tested terminators was approximately 17-fold. Modification of the -10 box and 5’-UTR in the promoters and the structure around the stem-loop in the terminators affected expression levels. These results suggest that the CAT assay is useful for various analyses of bifidobacterial gene expression.
Bifidobacterium longum
Chloramphenicol acetyltransferase
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Abstract Adipogenesis is a complex and tightly regulated process that plays a vital role in multiple aspects of human health. The molecular mechanisms regulating adipogenesis are incompletely understood, although key facets of the signaling and regulatory pathways have been defined. ADP-ribosylation – the process whereby ADP-ribose moieties are covalently transferred from NAD+ to substrate proteins – has been shown to control multiple components of the adipogenic regulatory machinery, including the transcription factor C/EBPb. In order to explore the role of mono(ADP-ribosyl)transferases (MARTs) during adipogenesis, we used an siRNA-mediated knockdown screen in mouse 3T3-L1 preadipocytes to determine which MARTs are required for the differentiation of the preadipocytes into mature adipocytes. This screen identified PARP7 the primary candidate. Using a variety of cell-based and biochemical assays, we have identified key aspects of PARP7's involvement throughout adipogenesis. We observed dynamic localization of PARP7 during adipogenesis, moving from the nucleus in early adipogenesis to the cytosol during mid to late adipogenesis. The requirement for PARP7, as determined by knockdown, was most evident in early stages of adipogenesis. Knockdown of PARP7 was rescued by a PPAR g agonist, suggesting that PARP7 acts before the onset of the later stages of adipogenesis leading to mature adipocytes. Together, these findings led us to hypothesize that PARP7 plays a critical role in the nucleus during early adipogenesis. Preliminary data have revealed that PARP7 binds to C/EBPb, a key "first wave" transcription factor that drives adipogenesis, during a time when C/EBPb is poly(ADP-ribosyl)ated by PARP1 in the nucleus. Ongoing investigations are exploring: (1) the mechanisms by which PARP7 binds to C/EBPb; (2) the impact of PARP7 binding on C/EBPb stability; and (3) the biological outcomes of PARP7-C/EBPb interactions. Collectively, these studies provide an avenue to better understand the role of PARP7 in the regulation of adipogenesis. This work is supported by a grant from the NIH/NIDDK (R01 DK069710) and funds from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment to W.L.K., and a predoctoral fellowship from the American Heart Association to M.S.S. Presentation: Monday, June 13, 2022 12:15 p.m. - 12:30 p.m.
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Chloramphenicol acetyltransferase
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SUMMARY The leap from simple unicellularity to complex multicellularity remains one of life's major enigmas. The origins of metazoan developmental gene regulatory mechanisms are sought by analyzing gene regulation in extant eumetazoans, sponges, and unicellular organisms. The main hypothesis of this manuscript is that, developmental enhancers evolved from unicellular inducible promoters that diversified the expression of regulatory genes during metazoan evolution. Promoters and enhancers are functionally similar; both can regulate the transcription of distal promoters and both direct local transcription. Additionally, enhancers have experimentally characterized structural features that reveal their origin from inducible promoters. The distal co‐operative regulation among promoters identified in unicellular opisthokonts possibly represents the precursor of distal regulation of promoters by enhancers. During metazoan evolution, constitutive‐type promoters of regulatory genes would have acquired novel receptivity to distal regulatory inputs from promoters of inducible genes that eventually specialized as enhancers. The novel regulatory interactions would have caused constitutively expressed genes controlling differential gene expression in unicellular organisms to become themselves differentially expressed. The consequence of the novel regulatory interactions was that regulatory pathways of unicellular organisms became interlaced and ultimately evolved into the intricate developmental gene regulatory networks (GRNs) of extant metazoans.
Multicellular organism
Gene regulatory network
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