cell-cycle-analysis-c-elegans-germline-with-thymidine-analog

Cell Cycle Analysis in the <em>C. elegans</em> Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

Washington University School of Medicine, St. Louis, Missouri

Zinc is essential for biological systems, and aberrant zinc metabolism is implicated in a broad range of human diseases. To maintain homeostasis in response to fluctuating levels of dietary zinc, animals regulate gene expression; however, mechanisms that mediate the transcriptional response to fluctuating levels of zinc have not been fully defined. Here, we identified DNA enhancer elements that mediate intestine-specific transcriptional activation in response to high levels of dietary zinc in C. elegans. Using bioinformatics, we characterized an evolutionarily conserved enhancer element present in multiple zinc-inducible genes, the high zinc activation (HZA) element. The HZA was consistently adjacent to a GATA element that mediates expression in intestinal cells. Functional studies using transgenic animals demonstrated that this modular system of DNA enhancers mediates tissue-specific transcriptional activation in response to high levels of dietary zinc. We used this information to search the genome and successfully identified novel zinc-inducible genes. To characterize the mechanism of enhancer function, we demonstrated that the GATA transcription factor ELT-2 and the mediator subunit MDT-15 are necessary for zinc-responsive transcriptional activation. These findings define new mechanisms of zinc homeostasis and tissue-specific regulation of transcription. 

A modular system of DNA enhancer elements mediates tissue-specific activation of transcription by high dietary zinc in C. elegans.

The LIN-1 ETS transcription factor plays a pivotal role in controlling cell fate decisions during development of the Caenorhabditis elegans vulva. Prior to activation of the RTK/Ras/ERK-signaling pathway, LIN-1 functions as a SUMOylated transcriptional repressor that inhibits vulval cell fate. Here we demonstrate using the yeast two-hybrid system that SUMOylation of LIN-1 mediates interactions with a protein predicted to be involved in transcriptional repression: the RAD-26 Mi-2β/CHD4 component of the nucleosome remodeling and histone deacetylation (NuRD) transcriptional repression complex. Genetic studies indicated that rad-26 functions to inhibit vulval cell fates in worms. Using the yeast two-hybrid system, we showed that the EGL-27/MTA1 component of the NuRD complex binds the carboxy-terminus of LIN-1 independently of LIN-1 SUMOylation. EGL-27 also binds UBC-9, an enzyme involved in SUMOylation, and MEP-1, a zinc-finger protein previously shown to bind LIN-1. Genetic studies indicate that egl-27 inhibits vulval cell fates in worms. These results suggest that LIN-1 recruits multiple proteins that repress transcription via both the SUMOylated amino-terminus and the unSUMOylated carboxy-terminus. Assays in cultured cells showed that the carboxy-terminus of LIN-1 was converted to a potent transcriptional activator in response to active ERK. We propose a model in which LIN-1 recruits multiple transcriptional repressors to inhibit the 1° vulval cell fate, and phosphorylation by ERK converts LIN-1 to a transcriptional activator that promotes the 1° vulval cell fate. 

Conversion of the LIN-1 ETS protein of Caenorhabditis elegans from a SUMOylated transcriptional repressor to a phosphorylated transcriptional activator.

The proliferating cell nuclear antigen (PCNA or PCN-1 in C. elegans), an essential processivity factor for DNA polymerase δ, has been widely used as a marker of S-phase. In C. elegans early embryos, PCN-1 accumulation is cyclic, localizing to the nucleus during S-phase and the cytoplasm during the rest of the cell cycle. The C. elegans larval and adult germline is an important model systems for studying cell cycle regulation, and it was observed that the cell cycle regulator cyclin E (CYE-1 in C. elegans) displays a non-cyclic, continuous accumulation pattern in this tissue. The accumulation pattern of PCN-1 has not been well defined in the larval and adult germline, and the objective of this study was to determine if the accumulation pattern is cyclic, as in other cells and organisms, or continuous, similar to cyclin E.

Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from continuous in the adult germline to intermittent in the early embryo of C. elegans.

Kerry Kornfeld

Department of Developmental Biology

Washington University School of Medicine, St. Louis, Missouri

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Kerry Kornfeld

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Developmental Biology

Washington University School of Medicine, St. Louis, Missouri

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Department of Developmental Biology