Topoisomerase I inhibition and peripheral nerve injury induce DNA breaks and ATF3-associated axon regeneration in sensory neurons.

Although axonal damage induces rapid changes in gene expression in primary sensory neurons, it remains unclear how this process is initiated. The transcription factor ATF3, one of the earliest genes responding to nerve injury, regulates expression of downstream genes that enable axon regeneration. By exploiting ATF3 reporter systems, we identify topoisomerase inhibitors as ATF3 inducers, including camptothecin.

Direct analysis of brain phenotypes via neural blastocyst complementation.

We provide a protocol for generating forebrain structures in vivo from mouse embryonic stem cells (ESCs) via neural blastocyst complementation (NBC). We developed this protocol for studies of development and function of specific forebrain regions, including the cerebral cortex and hippocampus. We describe a complete workflow, from methods for modifying a given genomic locus in ESCs via CRISPR-Cas9-mediated editing to the generation of mouse chimeras with ESC-reconstituted forebrain regions that can be directly analyzed.

Neural blastocyst complementation enables mouse forebrain organogenesis.

Genetically modified mice are commonly generated by the microinjection of pluripotent embryonic stem (ES) cells into wild-type host blastocysts, producing chimeric progeny that require breeding for germline transmission and homozygosity of modified alleles. As an alternative approach and to facilitate studies of the immune system, we previously developed RAG2-deficient blastocyst complementation. Because RAG2-deficient mice cannot undergo V(D)J recombination, they do not develop B or T lineage cells beyond the progenitor stage: injecting RAG2-sufficient donor ES cells into RAG2-deficient blastocysts generates somatic chimaeras in which all mature lymphocytes derive from donor ES cells.

Intrinsically Disordered Proteins Drive Emergence and Inheritance of Biological Traits.

Prions are a paradigm-shifting mechanism of inheritance in which phenotypes are encoded by self-templating protein conformations rather than nucleic acids. Here, we examine the breadth of protein-based inheritance across the yeast proteome by assessing the ability of nearly every open reading frame (ORF; ∼5,300 ORFs) to induce heritable traits. Transient overexpression of nearly 50 proteins created traits that remained heritable long after their expression returned to normal.

Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells.

High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global run-on sequencing analyses of NSPCs.

Long Neural Genes Harbor Recurrent DNA Break Clusters in Neural Stem/Progenitor Cells.

Repair of DNA double-strand breaks (DSBs) by non-homologous end joining is critical for neural development, and brain cells frequently contain somatic genomic variations that might involve DSB intermediates. We now use an unbiased, high-throughput approach to identify genomic regions harboring recurrent DSBs in primary neural stem/progenitor cells (NSPCs). We identify 27 recurrent DSB clusters (RDCs), and remarkably, all occur within gene bodies.

Cross-kingdom chemical communication drives a heritable, mutually beneficial prion-based transformation of metabolism.

In experimental science, organisms are usually studied in isolation, but in the wild, they compete and cooperate in complex communities. We report a system for cross-kingdom communication by which bacteria heritably transform yeast metabolism. An ancient biological circuit blocks yeast from using other carbon sources in the presence of glucose.

IgH class switching exploits a general property of two DNA breaks to be joined in cis over long chromosomal distances.

Antibody class switch recombination (CSR) in B lymphocytes joins two DNA double-strand breaks (DSBs) lying 100-200 kb apart within switch (S) regions in the immunoglobulin heavy-chain locus (IgH). CSR-activated B lymphocytes generate multiple S-region DSBs in the donor Sμ and in a downstream acceptor S region, with a DSB in Sμ being joined to a DSB in the acceptor S region at sufficient frequency to drive CSR in a large fraction of activated B cells. Such frequent joining of widely separated CSR DSBs could be promoted by IgH-specific or B-cell-specific processes or by general aspects of chromosome architecture and DSB repair.

Prions are a common mechanism for phenotypic inheritance in wild yeasts.

The self-templating conformations of yeast prion proteins act as epigenetic elements of inheritance. Yeast prions might provide a mechanism for generating heritable phenotypic diversity that promotes survival in fluctuating environments and the evolution of new traits. However, this hypothesis is highly controversial.

Activation of miR-31 function in already-established metastases elicits metastatic regression.

Distant metastases, rather than the primary tumors from which these lesions arise, are responsible for >90% of carcinoma-associated mortality. Many patients already harbor disseminated tumor cells in their bloodstream, bone marrow, and distant organs when they initially present with cancer. Hence, truly effective anti-metastatic therapeutics must impair the proliferation and survival of already-established metastases.

Concurrent suppression of integrin alpha5, radixin, and RhoA phenocopies the effects of miR-31 on metastasis.

miR-31 inhibits breast cancer metastasis via the pleiotropic suppression of a cohort of prometastatic target genes that include integrin alpha(5) (ITGA5), radixin (RDX), and RhoA. We previously showed that the concomitant overexpression of ITGA5, RDX, and RhoA was capable of overriding the antimetastatic effects of ectopically expressed miR-31 in vivo. However, these prior studies failed to investigate whether the combined suppression of the endogenous mRNAs encoding these three proteins recapitulated the in vivo consequences of miR-31 expression on metastasis.

Concomitant suppression of three target genes can explain the impact of a microRNA on metastasis.

It remains unclear whether a microRNA (miRNA) affects a given phenotype via concomitant down-regulation of its entire repertoire of targets or instead by suppression of only a modest subset of effectors. We demonstrate that inhibition of breast cancer metastasis by miR-31-a miRNA predicted to modulate >200 mRNAs-can be entirely explained by miR-31's pleiotropic regulation of three targets. Thus, concurrent re-expression of integrin-alpha5, radixin, and RhoA abrogates miR-31-imposed metastasis suppression.

System-wide peripheral biomarker discovery using information theory.

The identification of reliable peripheral biomarkers for clinical diagnosis, patient prognosis, and biological functional studies would allow for access to biological information currently available only through invasive methods. Traditional approaches have so far considered aspects of tissues and biofluid markers independently. Here we introduce an information theoretic framework for biomarker discovery, integrating biofluid and tissue information.