During starvation, organisms modify both gene expression and metabolism to adjust to the energy stress. We previously reported that Caenorhabditis elegans lacing AMP-activated protein kinase (AMPK) exhibit transgenerational reproductive defects associated with abnormally elevated trimethylated histone H3 at lysine 4 (H3K4me3) levels in the germ line following recovery from acute starvation. Here, we show that these H3K4me3 marks are significantly increased at promoters, driving aberrant transcription elongation resulting in the accumulation of R-loops in starved AMPK mutants. DNA-RNA immunoprecipitation followed by high-throughput sequencing (DRIP-seq) analysis demonstrated that a significant proportion of the genome was affected by R-loop formation. This was most pronounced in the promoter–transcription start site regions of genes, in which the chromatin was modified by H3K4me3. Like H3K4me3, the R-loops were also found to be heritable, likely contributing to the transgenerational reproductive defects typical of these mutants following starvation. Strikingly, AMPK mutant germ lines show considerably more RAD-51 (the RecA recombinase) foci at sites of R-loop formation, potentially sequestering them from their roles at meiotic breaks or at sites of induced DNA damage. Our study reveals a previously unforeseen role of AMPK in maintaining genome stability following starvation. The downstream effects of R-loops on DNA damage sensitivity and germline stem cell integrity may account for inappropriate epigenetic modification that occurs in numerous human disorders, including various cancers.
During periods of starvation organisms must modify both gene expression and metabolic pathways to adjust to the energy stress. We previously reported that C. elegans that lack AMPK have transgenerational reproductive defects that result from abnormally elevated H3K4me3 levels in the germ line following recovery from acute starvation1. Here we show that H3K4me3 is dramatically increased at promoters, driving aberrant transcription elongation that results in the accumulation of R-loops in the starved AMPK mutants. DRIP-seq analysis demonstrated that a significant proportion of the genome was affected by R-loop formation with a dramatic expansion in the number of R-loops at numerous loci, most pronounced at the promoter-TSS regions of genes in the starved AMPK mutants. The R-loops are transmissible into subsequent generations, likely contributing to the transgenerational reproductive defects typical of these mutants following starvation. Strikingly, AMPK null germ lines show considerably more RAD-51 foci at sites of R-loop formation, potentially sequestering it from its critical role at meiotic breaks and/or at sites of induced DNA damage. Our study reveals a previously unforeseen role of AMPK in maintaining genome stability following starvation, where in its absence R-loops accumulate, resulting in reproductive compromise and DNA damage hypersensitivity.
Galectin‐1 (Gal‐1), a protein that impacts the fate and function of immune cells known to fight infection, eliminates cancer, and promotes inflammation, is found in most mammalian tissues at low levels. A small 130 amino acid “jelly‐roll” shaped ß‐galactoside‐binding lectin with a hydrophobic core, Gal‐1 plays a role in controlling intracellular processes, such as cell cycle progression and cell proliferation. Gal‐1 binds with high affinity to glycoconjugates galactose (Gal) and N‐acetylglucosamine (GlcNAc) by van der Waals forces and hydrogen bonding via a highly conserved carbohydrate recognition domain. Because native Gal‐1 oxidizes rapidly and loses its carbohydrate‐binding activity, studying the effect of Gal‐1 has been difficult. The Dimitroff laboratory engineered a Gal‐1 – human immunoglobulin Fc chimeric molecule (Gal‐1hFc), which facilitates dimerization while preventing oxidation‐induced multimerization. Experimental evidence has demonstrated that Gal‐1hFc behaves like native Gal‐1, enabling the use of the chimera to study Gal‐1’s effect on immune responses. The Governor’s Academy SMART (Students Modeling A Research Topic) Team designed a model using 3D printing technology to provide further evidence of Gal‐1hFc’s structure and binding function. Grant Funding Source: Supported by grants from NIH‐CTSA UL1RR031973 and NIH/NCI RO1CA118124
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