Solid phase chemistry to covalently and reversibly capture thiolated RNA.

Here, we describe an approach to enrich newly transcribed RNAs from primary mouse neurons using 4-thiouridine (s4U) metabolic labeling and solid phase chemistry. This one-step enrichment procedure captures s4U-RNA by using highly efficient methane thiosulfonate (MTS) chemistry in an immobilized format. Like solution-based methods, this solid-phase enrichment can distinguish mature RNAs (mRNA) with differential stability, and can be used to reveal transient RNAs such as enhancer RNAs (eRNAs) and primary microRNAs (pri-miRNAs) from short metabolic labeling. Most importantly, the efficiency of this solid-phase chemistry made possible the first large scale measurements of RNA polymerase II (RNAPII) elongation rates in mouse cortical neurons. Thus, our approach provides the means to study regulation of RNA metabolism in specific tissue contexts as a means to better understand gene expression in vivo

Writing, Reading, and Translating the Clustered Protocadherin Cell Surface Recognition Code for Neural Circuit Assembly

The ability of neurites of individual neurons to distinguish between themselves and neurites from other neurons and to avoid self (self-avoidance) plays a key role in neural circuit assembly in both invertebrates and vertebrates. Similarly, when individual neurons of the same type project into receptive fields of the brain, they must avoid each other to maximize target coverage (tiling). Counterintuitively, these processes are driven by highly specific homophilic interactions between cell surface proteins that lead to neurite repulsion rather than adhesion. Among these proteins in vertebrates are the clustered protocadherins (Pcdhs), and key to their function is the generation of enormous cell surface structural diversity. Here we review recent advances in understanding how a Pcdh cell surface code is generated by stochastic promoter choice; how this code is amplified and read by homophilic interactions between Pcdh complexes at the surface of neurons; and, finally, how the Pcdh code is translated to cellular function, which mediates self-avoidance and tiling and thus plays a central role in the development of complex neural circuits. Not surprisingly, Pcdh mutations that diminish homophilic interactions lead to wiring defects and abnormal behavior in mice, and sequence variants in the Pcdh gene cluster are associated with autism spectrum disorders in family-based genetic studies in humans

Writing, Reading, and Translating the Clustered Protocadherin Cell Surface Recognition Code for Neural Circuit Assembly

The ability of neurites of individual neurons to distinguish between themselves and neurites from other neurons and to avoid self (self-avoidance) plays a key role in neural circuit assembly in both invertebrates and vertebrates. Similarly, when individual neurons of the same type project into receptive fields of the brain, they must avoid each other to maximize target coverage (tiling). Counterintuitively, these processes are driven by highly specific homophilic interactions between cell surface proteins that lead to neurite repulsion rather than adhesion. Among these proteins in vertebrates are the clustered protocadherins (Pcdhs), and key to their function is the generation of enormous cell surface structural diversity. Here we review recent advances in understanding how a Pcdh cell surface code is generated by stochastic promoter choice; how this code is amplified and read by homophilic interactions between Pcdh complexes at the surface of neurons; and, finally, how the Pcdh code is translated to cellular function, which mediates self-avoidance and tiling and thus plays a central role in the development of complex neural circuits. Not surprisingly, Pcdh mutations that diminish homophilic interactions lead to wiring defects and abnormal behavior in mice, and sequence variants in the Pcdh gene cluster are associated with autism spectrum disorders in family-based genetic studies in humans

Histone mimicry in HP1 is required for a conformational switch that regulates assembly of a minimal heterochromatin unit necessary for silencing in vivo

Long-term silencing of large regions of the genome is achieved through the formation of heterochromatin. From yeast to humans, heterochromatin is characterized by two key molecular signatures: (i) di or tri-methylation of lysine 9 of histone H3 (H3K9me2/3), and (ii) heterochromatin protein 1 (HP1). The association of HP1 with H3K9-methylated chromatin drives heterochromatin assembly and spread. Yet, how HP1 assembles on methylated nucleosomal templates and how the HP1-nucleosome complex is regulated are poorly understood. Using S. pombe as a model system, we show that two dimers of the HP1 protein, Swi6, binds to one nucleosome: each dimer contains one chromodomain (CD) that engages one copy of the H3K9-methyl mark, while the other CD is unoccupied. This HP1-nucleosome complex acts as a scaffold for the addition of other HP1 molecules that self-associate through a novel CD-CD interface nucleating from the unoccupied CDs. Chromodomain-mediated polymerization of HP1 on chromatin appears to (1) increase its association with methylated nucleosomes in vitro, (2) bridge neighboring methylated nucleosomes, and (3) increase heterochromatin assembly in vivo. Our data suggests that H3K9-methyl recognition and chromatin coating by HP1 are intrinsic to the fundamental architecture of the HP1-nucleosome complex. But they also raise the question of how methylated chromatin templates HP1 assembly. We found that two key features of heterochromatin, the H3K9me3 and the nucleosomal DNA, promote a conformational change in Swi6 that drives its association with nucleosomes. By binding to methylated nucleosomes, unbound Swi6 dimers switch from an autoinhibited state that is refractory to both methyl mark recognition and higher-order oligomerization to a state that is competent for spreading. Cryo-EM studies of the Swi6-nucleosome complex reveal the architecture of the spreading competent state. In vivo, mutants that disrupt such a switch also result in disruption of heterochromatin. The coupling of a conformational switch in HP1 to the recognition of specific features of methylated chromatin provides a mechanism for how HP1 can specifically target H3K9-methylated chromatin, thus preventing its aberrant spread into euchromatin. Finally, our discovery of these different HP1 conformational states provides a basic starting point for understanding how HP1 can switch between alternative functions in heterochromatin

Histone mimicry in HP1 is required for a conformational switch that regulates assembly of a minimal heterochromatin unit necessary for silencing in vivo

Long-term silencing of large regions of the genome is achieved through the formation of heterochromatin. From yeast to humans, heterochromatin is characterized by two key molecular signatures: (i) di or tri-methylation of lysine 9 of histone H3 (H3K9me2/3), and (ii) heterochromatin protein 1 (HP1). The association of HP1 with H3K9-methylated chromatin drives heterochromatin assembly and spread. Yet, how HP1 assembles on methylated nucleosomal templates and how the HP1-nucleosome complex is regulated are poorly understood. Using S. pombe as a model system, we show that two dimers of the HP1 protein, Swi6, binds to one nucleosome: each dimer contains one chromodomain (CD) that engages one copy of the H3K9-methyl mark, while the other CD is unoccupied. This HP1-nucleosome complex acts as a scaffold for the addition of other HP1 molecules that self-associate through a novel CD-CD interface nucleating from the unoccupied CDs. Chromodomain-mediated polymerization of HP1 on chromatin appears to (1) increase its association with methylated nucleosomes in vitro, (2) bridge neighboring methylated nucleosomes, and (3) increase heterochromatin assembly in vivo. Our data suggests that H3K9-methyl recognition and chromatin coating by HP1 are intrinsic to the fundamental architecture of the HP1-nucleosome complex. But they also raise the question of how methylated chromatin templates HP1 assembly. We found that two key features of heterochromatin, the H3K9me3 and the nucleosomal DNA, promote a conformational change in Swi6 that drives its association with nucleosomes. By binding to methylated nucleosomes, unbound Swi6 dimers switch from an autoinhibited state that is refractory to both methyl mark recognition and higher-order oligomerization to a state that is competent for spreading. Cryo-EM studies of the Swi6-nucleosome complex reveal the architecture of the spreading competent state. In vivo, mutants that disrupt such a switch also result in disruption of heterochromatin. The coupling of a conformational switch in HP1 to the recognition of specific features of methylated chromatin provides a mechanism for how HP1 can specifically target H3K9-methylated chromatin, thus preventing its aberrant spread into euchromatin. Finally, our discovery of these different HP1 conformational states provides a basic starting point for understanding how HP1 can switch between alternative functions in heterochromatin

The generation of a protocadherin cell-surface recognition code for neural circuit assembly

The assembly of functional neural circuits in vertebrate organisms requires complex mechanisms of self-recognition and self-avoidance. Neurites (axons and dendrites) from the same neuron recognize and avoid self, but engage in synaptic interactions with other neurons. Vertebrate neural self-avoidance requires the expression of distinct repertoires of clustered Protocadherin (Pcdh) cell-surface protein isoforms, which act as cell-surface molecular barcodes that mediate highly specific homophilic self-recognition, followed by repulsion. The generation of sufficiently diverse cell-surface barcodes is achieved by the stochastic and combinatorial activation of a subset of clustered Pcdh promoters in individual neurons. This remarkable mechanism leads to the generation of enormous molecular diversity at the cell surface. Here we review recent studies showing that stochastic expression of individual Pcdhα isoforms is accomplished through an extraordinary mechanism involving the activation of 'antisense strand' promoter within Pcdhα 'variable' exons, antisense transcription of a long non-coding RNA through the upstream 'sense strand' promoter, demethylation of this promoter, binding of the CTCF/cohesin complex and DNA looping to a distant enhancer through a mechanism of chromatin 'extrusion'

The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice.

DNA sequence variants in the TBK1 gene associate with or cause sporadic or familial amyotrophic lateral sclerosis (ALS). Here we show that mice bearing human ALS-associated TBK1 missense loss-of-function mutations, or mice in which the Tbk1 gene is selectively deleted in motor neurons, do not display a neurodegenerative disease phenotype. However, loss of TBK1 function in motor neurons of the SOD1G93A mouse model of ALS impairs autophagy, increases SOD1 aggregation, and accelerates early disease onset without affecting lifespan. By contrast, point mutations that decrease TBK1 kinase activity in all cells also accelerate disease onset but extend the lifespan of SOD1 mice. This difference correlates with the failure to activate high levels of expression of interferon-inducible genes in glia. We conclude that loss of TBK1 kinase activity impacts ALS disease progression through distinct pathways in different spinal cord cell types and further implicate the importance of glia in neurodegeneration