Antisense lncRNA Transcription Mediates DNA Demethylation to Drive Stochastic Protocadherin α Promoter Choice.

Stochastic activation of clustered Protocadherin (Pcdh) α, ß, and γ genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdhα gene choice involves the activation of an antisense promoter located in the first exon of each Pcdhα alternate gene. Transcription of an antisense long noncoding RNA (lncRNA) from this antisense promoter extends through the sense promoter, leading to DNA demethylation of the CTCF binding sites proximal to each promoter. Demethylation-dependent CTCF binding to both promoters facilitates cohesin-mediated DNA looping with a distal enhancer (HS5-1), locking in the transcriptional state of the chosen Pcdhα gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdhα promoters, resulting in proximity-biased DNA looping of the HS5-1 enhancer. Thus, antisense transcription-mediated promoter demethylation functions as a mechanism for distance-independent enhancer/promoter DNA looping to ensure stochastic Pcdhα promoter choice.

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.

CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function.

CTCF and the associated cohesin complex play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and ß-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context, the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. These findings reveal how 3D chromosome architecture can be encoded by linear genome sequences.

STL-seq reveals pause-release and termination kinetics for promoter-proximal paused RNA polymerase II transcripts.

Despite the critical regulatory function of promoter-proximal pausing, the influence of pausing kinetics on transcriptional control remains an active area of investigation. Here, we present Start-TimeLapse-seq (STL-seq), a method that captures the genome-wide kinetics of short, capped RNA turnover and reveals principles of regulation at the pause site. By measuring the rates of release into elongation and premature termination through the inhibition of pause release, we determine that pause-release rates are highly variable, and most promoter-proximal paused RNA polymerase II molecules prematurely terminate (∼80%). The preferred regulatory mechanism upon a hormonal stimulus (20-hydroxyecdysone) is to influence pause-release rather than termination rates. Transcriptional shutdown occurs concurrently with the induction of promoter-proximal termination under hyperosmotic stress, but paused transcripts from TATA box-containing promoters remain stable, demonstrating an important role for cis-acting DNA elements in pausing. STL-seq dissects the kinetics of pause release and termination, providing an opportunity to identify mechanisms of transcriptional regulation.

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.

A conformational switch in HP1 releases auto-inhibition to drive heterochromatin assembly.

A hallmark of histone H3 lysine 9 (H3K9)-methylated heterochromatin, conserved from the fission yeast Schizosaccharomyces pombe to humans, is its ability to spread to adjacent genomic regions. Central to heterochromatin spread is heterochromatin protein 1 (HP1), which recognizes H3K9-methylated chromatin, oligomerizes and forms a versatile platform that participates in diverse nuclear functions, ranging from gene silencing to chromosome segregation. How HP1 proteins assemble on methylated nucleosomal templates and how the HP1-nucleosome complex achieves functional versatility remain poorly understood. Here we show that binding of the key S. pombe HP1 protein, Swi6, to methylated nucleosomes drives a switch from an auto-inhibited state to a spreading-competent state. In the auto-inhibited state, a histone-mimic sequence in one Swi6 monomer blocks methyl-mark recognition by the chromodomain of another monomer. Auto-inhibition is relieved by recognition of two template features, the H3K9 methyl mark and nucleosomal DNA. Cryo-electron-microscopy-based reconstruction of the Swi6-nucleosome complex provides the overall architecture of the spreading-competent state in which two unbound chromodomain sticky ends appear exposed. Disruption of the switch between the auto-inhibited and spreading-competent states disrupts heterochromatin assembly and gene silencing in vivo. These findings are reminiscent of other conditionally activated polymerization processes, such as actin nucleation, and open up a new class of regulatory mechanisms that operate on chromatin in vivo.

Chromodomain-mediated oligomerization of HP1 suggests a nucleosome-bridging mechanism for heterochromatin assembly.

HP1 proteins are central to the assembly and spread of heterochromatin containing histone H3K9 methylation. The chromodomain (CD) of HP1 proteins specifically recognizes the methyl mark on H3 peptides, but the same extent of specificity is not observed within chromatin. The chromoshadow domain of HP1 proteins promotes homodimerization, but this alone cannot explain heterochromatin spread. Using the S. pombe HP1 protein, Swi6, we show that recognition of H3K9-methylated chromatin in vitro relies on an interface between two CDs. This interaction causes Swi6 to tetramerize on a nucleosome, generating two vacant CD sticky ends. On nucleosomal arrays, methyl mark recognition is highly sensitive to internucleosomal distance, suggesting that the CD sticky ends bridge nearby methylated nucleosomes. Strengthening the CD-CD interaction enhances silencing and heterochromatin spread in vivo. Our findings suggest that recognition of methylated nucleosomes and HP1 spread on chromatin are structurally coupled and imply that methylation and nucleosome arrangement synergistically regulate HP1 function.

Tuning cohesin trajectories enables differential readout of the Pcdhα cluster across neurons.

Expression of Protocadherin (Pcdh) genes is critical to the generation of neuron identity and wiring of the nervous system. Pcdhα genes are arranged in clusters and exhibit a range of expression profiles, from stochastic to deterministic. Because Pcdhα promoters have high sequence identity and share distal enhancers, how distinct neurons choose which gene to express remains unclear. We show that the interplay between multiple enhancers, epigenetics, and genome folding orchestrates differential readouts of the locus across neurons. The probability of Pcdhα promoter choice depends on enhancer/promoter encounters catalyzed by cohesin, whose extrusion trajectories determine the likelihood that an individual promoter can "escape" heterochromatin-mediated silencing. We propose that tunable locus-specific regulatory elements and cell type-specific cohesin activity underlie the generation of cellular diversity by Pcdh genes.

WAPL functions as a rheostat of Protocadherin isoform diversity that controls neural wiring.

Neural type-specific expression of clustered Protocadherin (Pcdh) proteins is essential for the establishment of connectivity patterns during brain development. In mammals, deterministic expression of the same Pcdh isoform promotes minimal overlap of tiled projections of serotonergic neuron axons throughout the brain, while stochastic expression of Pcdh genes allows for convergence of tightly packed, overlapping olfactory sensory neuron axons into targeted structures. How can the same gene locus generate opposite transcriptional programs that orchestrate distinct spatial arrangements of axonal patterns? Here, we reveal that cell type-specific Pcdh expression and axonal behavior depend on the activity of cohesin and its unloader, WAPL (wings apart-like protein homolog). While cohesin erases genomic-distance biases in Pcdh choice, WAPL functions as a rheostat of cohesin processivity that determines Pcdh isoform diversity.

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.

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'.

Clustered protocadherins methylation alterations in cancer.

BACKGROUND: Clustered protocadherins (PCDHs) map in tandem at human chromosome 5q31 and comprise three multi-genes clusters: α-, ß- and γ-PCDH. The expression of this cluster consists of a complex mechanism involving DNA hub formation through DNA-CCTC binding factor (CTCF) interaction. Methylation alterations can affect this interaction, leading to transcriptional dysregulation. In cancer, clustered PCDHs undergo a mechanism of long-range epigenetic silencing by hypermethylation. RESULTS: In this study, we detected frequent methylation alterations at CpG islands associated to these clustered PCDHs in all the solid tumours analysed (colorectal, gastric and biliary tract cancers, pilocytic astrocytoma), but not hematologic neoplasms such as chronic lymphocytic leukemia. Importantly, several altered CpG islands were associated with CTCF binding sites. Interestingly, our analysis revealed a hypomethylation event in pilocytic astrocytoma, suggesting that in neuronal tissue, where PCDHs are highly expressed, these genes become hypomethylated in this type of cancer. On the other hand, in tissues where PCDHs are lowly expressed, these CpG islands are targeted by DNA methylation. In fact, PCDH-associated CpG islands resulted hypermethylated in gastrointestinal tumours. CONCLUSIONS: Our study highlighted a strong alteration of the clustered PCDHs methylation pattern in the analysed solid cancers and suggested these methylation aberrations in the CpG islands associated with PCDH genes as powerful diagnostic biomarkers.

Incorporation of positively charged ribonucleic guanidine linkages into oligodeoxyribonucleotides: Development of potent antisense agents.

Oligodeoxynucleic acid (21-mer) containing both negatively charged phosphate and positively charged ribonucleic guanidine linkages (RNG/DNA chimera) have been synthesized. DNA binding characteristics and nuclease resistance of RNG/DNA chimeras have been evaluated. Using the bcr-abl oncogene (cause of chronic myeloid leukemia) as a target, the binding of a 21-mer RNG/DNA chimera that includes six RNG's is more than 103.5 stronger than the binding of 21-mer composed solely of DNA.

Mechanisms of functional promiscuity by HP1 proteins.

Heterochromatin protein 1 (HP1) proteins were originally identified as critical components in heterochromatin-mediated gene silencing and are now recognized to play essential roles in several other processes including gene activation. Several eukaryotes possess more than one HP1 paralog. Despite high sequence conservation, the HP1 paralogs achieve diverse functions. Further, in many cases, the same HP1 paralog is implicated in multiple functions. Recent biochemical studies have revealed interesting paralog-specific biophysical differences and unanticipated conformational versatility in HP1 proteins that may account for this functional promiscuity. Here we review these findings and describe a molecular framework that aims to link the conformational flexibility of HP1 proteins observed in vitro with their functional promiscuity observed in vivo.

Structural basis of silencing: Sir3 BAH domain in complex with a nucleosome at 3.0 Å resolution.

Gene silencing is essential for regulating cell fate in eukaryotes. Altered chromatin architectures contribute to maintaining the silenced state in a variety of species. The silent information regulator (Sir) proteins regulate mating type in Saccharomyces cerevisiae. One of these proteins, Sir3, interacts directly with the nucleosome to help generate silenced domains. We determined the crystal structure of a complex of the yeast Sir3 BAH (bromo-associated homology) domain and the nucleosome core particle at 3.0 angstrom resolution. We see multiple molecular interactions between the protein surfaces of the nucleosome and the BAH domain that explain numerous genetic mutations. These interactions are accompanied by structural rearrangements in both the nucleosome and the BAH domain. The structure explains how covalent modifications on H4K16 and H3K79 regulate formation of a silencing complex that contains the nucleosome as a central component.

DNA sequence recognition by Hoechst 33258 conjugates of hairpin pyrrole/imidazole polyamides.

A series of hairpin pyrrole/imidazole polyamides linked to a Hoechst 33258 (Ht) analogue (5-7) were synthesized on solid-phase by adopting an Fmoc technique using a series of PyBOP/HOBt mediated coupling reactions. The dsDNA binding properties of Ht-polyamides 5-7 were determined by thermal denaturation experiments. Hairpin Ht-polyamides 5-7 bound to dsDNA sequences 16 and 18 show DeltaTm values that are 14-18 degrees higher than linear Ht-polyamides bound to the same sequences. All three Ht-polyamides were found to be selective for their 9-bp match dsDNA sequences, supporting a relative stronger interaction of an Im/Py anti-parallel dimer with an appropriately positioned G/Cbp rather than sequences containing only A/Tbps. In addition, Ht-polyamides 5 and 7 showed a 20-fold preference for a properly placed G/Cbp over a C/Gbp, while 6 showed a 10-fold preference.