Biology subjects

LaBaer, J.

Publications and source records attributed to LaBaer, J..

3 recordsLinked to original sources

bioRxiv2025

RAD18 Methylation by the Methyltransferase SETD6 Attenuates DNA Breaks

This study investigated the interaction between the SETD6 lysine methyltransferase and RAD18, a key protein in the DNA damage repair pathway. SETD6 belongs to the SET-domain-containing family of proteins, which are known to catalyze protein methylation, a post-translational modification that plays a critical role in regulating protein function, stability, and interactions. Using protein microarray technology, we identified RAD18 as an interactor and substrate of SETD6. We confirmed this interaction through ELISA and immunoprecipitation assays, demonstrating that SETD6 directly binds and methylates RAD18. Using mass spectrometry and site-directed mutagenesis, we identified that RAD18 undergoes mono-methylation at the K73 and K406 residues. Furthermore, we found that RAD18 methylation affects its nuclear localization. Specifically, SETD6 KO cells exhibited increased nuclear RAD18 levels, suggesting that methylation status influences RAD18s shuttling between the cytoplasm and nucleus. Notably, depletion of SETD6 led to elevated markers of DNA damage ({gamma}H2AX) and increased DNA breaks, as evidenced by comet assays. Restoring SETD6 activity significantly reduced DNA damage, while a catalytic inactive mutant did not have this effect, underscoring the importance of SETD6s enzymatic function. Overall, our results demonstrate that SETD6-mediated methylation of RAD18 is essential for attenuating DNA breaks, thereby regulating its cellular localization and function in maintaining genomic integrity.

molecular biology↗

bioRxiv2023

Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq) to Study DNA Methylation Using Low Amounts of DNA

DNA methylation is an epigenetic mark that has a crucial role in regulating gene expression. Aberrant DNA methylation results in severe diseases in humans, such as cancer, autoimmune disease, atherosclerosis, and cardiovascular diseases. Whole-genome bisulfite sequencing and methylated DNA immunoprecipitation are available to study DNA methylation changes, but they are typically used on a few samples at a time. Here, we developed a novel method called Multiplexed Methylated DNA Immunoprecipitation Sequencing (Mx-MeDIP-Seq), that can be used to analyze many DNA samples in parallel, requiring only small amounts of input DNA. In this method, 10 different DNA samples were fragmented, purified, barcoded, and pooled prior to immunoprecipitation. In a head-to-head comparison, we observed 99% correlation between MeDIP-Seq performed individually or combined as Mx-MeDIP-Seq. Moreover, multiplexed MeDIP led to more than 95% normalized percent recovery and a 25-fold enrichment ratio by qPCR, like the enrichment of the conventional method. This technique was successfully performed with as little as 25 ng of DNA, equivalent to 3400 to 6200 cells. Up to 10 different samples were processed simultaneously in a single run. Overall, the Mx-MeDIP-Seq method is cost-effective with faster processing to analyze DNA methylome, making this technique more suitable for high-throughput DNA methylome analysis.

genomics↗

bioRxiv2017

Tandem histone-binding domains enhance the activity of a synthetic chromatin effector

Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than DNA sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. We have previously reported the development and validation of the \"Polycomb-based transcription factor\" (PcTF), a fusion protein that recognizes histone modifications through a protein-protein interaction between its polycomb chromodomain (PCD) motif and trimethylated lysine 27 of histone H3 (H3K27me3) at genomic sites. We demonstrated that PcTF activates genes at methyl-histone-enriched loci in cancer-derived cell lines. However, PcTF induces modest activation of a methyl-histone associated reporter compared to a DNA-binding activator. Therefore, we modified PcTF to enhance its target affinity. Here, we demonstrate the activity of a modified regulator called Pc2TF, which has two tandem copies of the H3K27me3-binding PCD at the N-terminus. Pc2TF shows higher affinity for H3K27me3 in vitro and shows enhanced gene activation in HEK293 cells compared to PcTF. These results provide compelling evidence that the intrinsic histone-binding activity of the PCD motif can be used to tune the activity of synthetic histone-binding transcriptional regulators.

synthetic biology↗