Chromatin conformation,localization,and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin archite...Chromatin conformation,localization,and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin architectures in healthy and diseased states,the requirement for cell fix-ation precludes the comprehensive dynamic analysis necessary to fully understand chromatin activ-ities. This has spurred the development and application of a variety of imaging methodologies for visualizing single chromosomal loci in the native cellular context. In this review,we describe currently-available approaches for imaging single genomic loci in cells,with special focus on clus-tered regularly interspaced short palindromic repeats (CRISPR)-based imaging approaches. In addition,we discuss some of the challenges that limit the application of CRISPR-based genomic imaging approaches,and potential solutions to address these challenges. We anticipate that,with continued refinement of CRISPR-based imaging techniques,significant understanding can be gained to help decipher chromatin activities and their relevance to cellular physiology and pathogenesis.展开更多
We recently reported an unconventional mechanism by which miRNAs inhibit HIV-1 viral production. This occurs when miRNAs bind nonspecifically to the viral structural protein Gag, interfering with viral RNA-medi- ated ...We recently reported an unconventional mechanism by which miRNAs inhibit HIV-1 viral production. This occurs when miRNAs bind nonspecifically to the viral structural protein Gag, interfering with viral RNA-medi- ated Gag assembly at the plasma membrane. Conse- quently, misassembled viral complexes are redirected into the endocytic pathway where they are delivered to lysosomes for degradation. In this study, we demon- strate that autophagy is a critical mediator of the viral degradation pathway and that this pathway is not HIV-1 specific. Misassembled viral complexes were found to colocalize extensively with LC3 and p62 in late endo- somesllysosomes, demonstrating a convergence of autophagy with functional degradative compartments. Knocking down autophagosome formation machineries reduced this convergence, while treatment with autop- hagy-inducer rapamycin enhanced the convergence. Furthermore, similar autophagy-dependent nonspecific miRNA inhibition of murine leukemia virus （MLV） assembly was shown. Overall, these results reveal autophagy as a crucial regulator of the retroviral degradation pathway in host cells initiated by non- specific miRNA-Gag interactions. These findings could have significant implications for understanding how cells may regulate retroviral complex assembly by miRNA expression and autophagy, and raise the possi- bility that similar regulations can occur in other biolog- ical contexts.展开更多
基金supported by grants from the National Key R&D Program of China (Grant Nos. 2016YFA0501603 and 2016YFA0100702)National Natural Science Foundation of China (Grant No. 31771583)+1 种基金Beijing Natural Science Foundation (Grant No. 7162114)Beijing Municipal R&D Key Project (Grant No. Z151100003915081), China
文摘Chromatin conformation,localization,and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin architectures in healthy and diseased states,the requirement for cell fix-ation precludes the comprehensive dynamic analysis necessary to fully understand chromatin activ-ities. This has spurred the development and application of a variety of imaging methodologies for visualizing single chromosomal loci in the native cellular context. In this review,we describe currently-available approaches for imaging single genomic loci in cells,with special focus on clus-tered regularly interspaced short palindromic repeats (CRISPR)-based imaging approaches. In addition,we discuss some of the challenges that limit the application of CRISPR-based genomic imaging approaches,and potential solutions to address these challenges. We anticipate that,with continued refinement of CRISPR-based imaging techniques,significant understanding can be gained to help decipher chromatin activities and their relevance to cellular physiology and pathogenesis.
文摘We recently reported an unconventional mechanism by which miRNAs inhibit HIV-1 viral production. This occurs when miRNAs bind nonspecifically to the viral structural protein Gag, interfering with viral RNA-medi- ated Gag assembly at the plasma membrane. Conse- quently, misassembled viral complexes are redirected into the endocytic pathway where they are delivered to lysosomes for degradation. In this study, we demon- strate that autophagy is a critical mediator of the viral degradation pathway and that this pathway is not HIV-1 specific. Misassembled viral complexes were found to colocalize extensively with LC3 and p62 in late endo- somesllysosomes, demonstrating a convergence of autophagy with functional degradative compartments. Knocking down autophagosome formation machineries reduced this convergence, while treatment with autop- hagy-inducer rapamycin enhanced the convergence. Furthermore, similar autophagy-dependent nonspecific miRNA inhibition of murine leukemia virus （MLV） assembly was shown. Overall, these results reveal autophagy as a crucial regulator of the retroviral degradation pathway in host cells initiated by non- specific miRNA-Gag interactions. These findings could have significant implications for understanding how cells may regulate retroviral complex assembly by miRNA expression and autophagy, and raise the possi- bility that similar regulations can occur in other biolog- ical contexts.
