BIND&MODIFY: Long-range single-molecule mapping of chromatin modification in eukaryotes v1

Here we describe a powerful method, BIND&MODIFY, for probing histone modifications and transcription factors at single molecular level. Our approach used the recombinant fused protein A-M.EcoGII, which tethers the methyltransferase M.EcoGII to the protein binding sites and locally labels the neighboring DNA regions via artificial methylations. This method could reveal ingle-molecule heterogenous histone modification status and CpG methylation at the same time, and could enable quantify the correlation between the distal elements. Further applications based on this method's concept could be applied to probe multiple protein binding events on the same single molecular DNA. The method proposed herein may soon become an essential tool for third-generation sequencing in the future.

Conserved Structure Modules within the lncRNA SChLAP1 Mediate Protein Recognition Implicated in Aggressive Prostate Cancer

The lncRNA Second Chromosome Locus Associated with Prostate 1 (SChLAP1) was previously identified as a predictive biomarker and driver of aggressive prostate cancer. Recent work suggests that SChLAP1 may bind the SWI/SNF chromatin remodeling complex to promote prostate cancer metastasis, though the exact role of SWI/SNF recognition is debated. To date, there are no detailed biochemical studies of apo SChLAP1 or the SChLAP1:SWI/SNF complex. Herein, we report the first secondary structure model of SChLAP1 utilizing SHAPE-MaP both in vitro and in cellulo. Comparison of the in vitro and in cellulo data via ΔSHAPE identified putative protein binding sites within SChLAP1, specifically to evolutionarily conserved exons of the transcript. We also demonstrate that global SChLAP1 secondary structure is sensitive to both purification method and magnesium concentration. Further, we identified a 3'-fragment of SChLAP1 (SChLAP1Frag) that harbors multiple potential protein binding sites and presents a robustly folded secondary structure, supporting a functional role for this region. This work lays the foundation for future efforts in selective targeting and disruption of the SChLAP1:protein interface and the development of new therapeutic avenues in prostate cancer treatment.

Heavy Metals and Human Health: Possible Exposure Pathways and the Competition for Protein Binding Sites

Heavy metals enter the human body through the gastrointestinal tract, skin, or via inhalation. Toxic metals have proven to be a major threat to human health, mostly because of their ability to cause membrane and DNA damage, and to perturb protein function and enzyme activity. These metals disturb native proteins’ functions by binding to free thiols or other functional groups, catalyzing the oxidation of amino acid side chains, perturbing protein folding, and/or displacing essential metal ions in enzymes. The review shows the physiological and biochemical effects of selected toxic metals interactions with proteins and enzymes. As environmental contamination by heavy metals is one of the most significant global problems, some detoxification strategies are also mentioned.

Genome-wide identification of protein binding sites in mammalian cells

We present GWPBS-Cap, a method to capture genome-wide protein binding sites (PBSs) without using antibodies. Using this technique, we identified many protein binding sites with different binding strengths between proteins and DNA. The PBSs can be useful to predict transcription binding sites and the co-localization of multiple transcription factors in the genome. The results also revealed that active promoters contained more protein binding sites with lower NaCl tolerances. Taken together, GWPBS-Cap can be used to efficiently identify protein binding sites and reveal genome-wide landscape of DNA-protein interactions.

Long-range single-molecule mapping of chromatin modification in eukaryotes

The epigenetic modifications of histones are essential marks related to the development and disease pathogenesis, including human cancers. Mapping histone modification has emerged as the widely used tool for studying epigenetic regulation. However, existing approaches limited by fragmentation and short-read sequencing cannot provide information about the long-range chromatin states and represent the average chromatin status in samples. We leveraged the advantage of long read sequencing to develop a method "BIND&MODIFY" for profiling the histone modification of individual DNA fiber. Our approach is based on the recombinant fused protein A-EcoGII, which tethers the methyltransferase EcoGII to the protein binding sites and locally labels the neighboring DNA regions through artificial methylations. We demonstrate that the aggregated BIND&MODIFY signal matches the bulk-level ChIP-seq and CUT&TAG, observe the single-molecule heterogenous histone modification status, and quantify the correlation between distal elements. This method could be an essential tool in the future third-generation sequencing ages.