Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome.

Short tandem repeat (STR) instability causes transcriptional silencing in several repeat expansion disorders. In fragile X syndrome (FXS), mutation-length expansion of a CGG STR represses FMR1 via local DNA methylation. Here, we find megabase-scale H3K9me3 domains on autosomes and encompassing FMR1 on the X chromosome in FXS patient-derived iPSCs, iPSC-derived neural progenitors, EBV-transformed lymphoblasts, and brain tissue with mutation-length CGG expansion. H3K9me3 domains connect via inter-chromosomal interactions and demarcate severe misfolding of TADs and loops. They harbor long synaptic genes replicating at the end of S phase, replication-stress-induced double-strand breaks, and STRs prone to stepwise somatic instability. CRISPR engineering of the mutation-length CGG to premutation length reverses H3K9me3 on the X chromosome and multiple autosomes, refolds TADs, and restores gene expression. H3K9me3 domains can also arise in normal-length iPSCs created with perturbations linked to genome instability, suggesting their relevance beyond FXS. Our results reveal Mb-scale heterochromatinization and trans interactions among loci susceptible to instability.

Cu-Catalyzed Tandem C-N and C-C Bond Formation Leading to 4(1H)-Quinolones: A Scaffold with Diverse Biological Properties from Totally New Raw Materials in a Single Step.

A novel Cu-catalyzed tandem C-N and C-C bond-formation reaction has been developed to furnish 2-substituted-4-(1H)-quinolones. 4-(1H)-quinolones play an important role in medicinal chemistry. Many 2-aryl(alkyl)-4(1H)-quinolones are found to exhibit diverse biological properties. While traditional methods have inherent issues [like starting materials with incompatible functional groups (NH2 and keto groups)], many modern methods either require activated starting materials (like Ynones) or employ expensive metals (Pd, Rh, Au, etc.) involving carbonylation using CO or metal complexes. Our protocol presents an environmentally friendly one-step method for the construction of these useful 2-substituted-4-(1H)-quinolones from easily available aryl boronic acid (or pinacolate ester) and nitriles as new raw materials, using a cheap Cu-catalyst and O2 (air) as a green oxidant. We further extended its application to the synthesis of various natural products, including the first formal total synthesis of punarnavine. A plausible mechanism involving an aryl nitrilium ion (formed due to the intermolecular C-N bond-forming coupling between aryl boron species and the nitrile group) followed by tandem intramolecular C-C bond formation has been proposed.

One-Pot Synthesis of Chromone-2-carboxylate Scaffold: An Important Pharmacophore with Diverse Biological Properties.

Chromone-2-carboxylate scaffold is growing as an important pharmacophore in medicinal chemistry with diverse biological properties. We have developed a facile one-pot transformation of 2-fluoroacetophenone directly to chromone-2-carboxylate scaffold in a single step via a tandem C-C and C-O bond formation. The majority of the previously reported medicinal chemistry synthetic protocols primarily used only one procedure which follows a two-step strategy that needs to start with "2-hydroxyacetophenone". Our methodology not only serves as an alternative one-pot methodology but also allows chemists to start from different raw materials (2-fluoroacetophenone) other than the traditional ortho-hydroxyacetophenone for maintaining the regioselectivity in the cyclization step. We further demonstrated the utility of our protocol by successfully extending the application to the synthesis of two natural products (Halenic acids A and B), various bis-chromones including drug molecules (DSCG, cromoglicic acid), and potent anti-Alzheimer compound (F-cromolyn). This methodology can serve as a promising alternative tool for finding new bioactive chromones with diverse modifications due to the opportunity to use new raw materials in the synthesis of chromones.

Developmentally regulated higher-order chromatin interactions orchestrate B cell fate commitment.

Genome organization in 3D nuclear-space is important for regulation of gene expression. However, the alterations of chromatin architecture that impinge on the B cell-fate choice of multi-potent progenitors are still unclear. By integrating in situ Hi-C analyses with epigenetic landscapes and genome-wide expression profiles, we tracked the changes in genome architecture as the cells transit from a progenitor to a committed state. We identified the genomic loci that undergo developmental switch between A and B compartments during B-cell fate determination. Furthermore, although, topologically associating domains (TADs) are stable, a significant number of TADs display structural alterations that are associated with changes in cis-regulatory interaction landscape. Finally, we demonstrate the potential roles for Ebf1 and its downstream factor, Pax5, in chromatin reorganization and transcription regulation. Collectively, our studies provide a general paradigm of the dynamic relationship between chromatin reorganization and lineage-specific gene expression pattern that dictates cell-fate determination.

MASTR-seq: Multiplexed Analysis of Short Tandem Repeats with sequencing.

More than 60 human disorders have been linked to unstable expansion of short tandem repeat (STR) tracts. STR length and the extent of DNA methylation is linked to disease pathology and can be mosaic in a cell type-specific manner in several repeat expansion disorders. Mosaic phenomenon have been difficult to study to date due to technical bias intrinsic to repeat sequences and the need for multi-modal measurements at single-allele resolution. Nanopore long-read sequencing accurately measures STR length and DNA methylation in the same single molecule but is cost prohibitive for studies assessing a target locus across multiple experimental conditions or patient samples. Here, we describe MASTR-seq, M ultiplexed A nalysis of S hort T andem R epeats, for cost-effective, high-throughput, accurate, multi-modal measurements of DNA methylation and STR genotype at single-allele resolution. MASTR-seq couples long-read sequencing, Cas9-mediated target enrichment, and PCR-free multiplexed barcoding to achieve a >ten-fold increase in on-target read mapping for 8-12 pooled samples in a single MinION flow cell. We provide a detailed experimental protocol and computational tools and present evidence that MASTR-seq quantifies tract length and DNA methylation status for CGG and CAG STR loci in normal-length and mutation-length human cell lines. The MASTR-seq protocol takes approximately eight days for experiments and one additional day for data processing and analyses. Key points: We provide a protocol for MASTR-seq: M ultiplexed A nalysis of S hort T andem R epeats using Cas9-mediated target enrichment and PCR-free, multiplexed nanopore sequencing. MASTR-seq achieves a >10-fold increase in on-target read proportion for highly repetitive, technically inaccessible regions of the genome relevant for human health and disease.MASTR-seq allows for high-throughput, efficient, accurate, and cost-effective measurement of STR length and DNA methylation in the same single allele for up to 8-12 samples in parallel in one Nanopore MinION flow cell.