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A key mechanism in cellular regulation is the ability of the transcriptional machinery to physically access DNA. Pioneer transcription factors interact with DNA to open chromatin, which subsequently enables changes to gene expression during development, disease, or as a response to environmental stimuli. However, the regulation of DNA accessibility via the recruitment of transcription factors is difficult to understand in the context of the native genome because every genomic site is distinct in multiple ways. Here we introduce the Multiplexed Integrated Accessibility Assay (MIAA), a multiplexed parallel reporter assay which measures changes to genome accessibility as a result of the integration of synthetic oligonucleotide phrase libraries into a controlled, natively inaccessible genomic context. We apply MIAA to measure the effects of sequence motifs on cell type-specific DNA accessibility between mouse embryonic stem cells and embryonic stem cell-derived definitive endoderm cells, screening a total of 7,905 distinct phrases. MIAA is able to recapitulate differential accessibility patterns of 100-nt sequences derived from natively differential genomic regions, identifying the presence of E-box motifs common to epithelial-mesenchymal transition driver transcription factors in stem cell-specific accessible regions that become repressed during differentiation to endoderm. We further present causal evidence that a single binding motif for a key regulatory transcription factor is sufficient to open chromatin, and classify sets of stem cell-specific, endoderm-specific, and shared pioneer factor motifs. We also demonstrate that over-expression of two definitive endoderm transcription factors, Brachyury and FoxA2, results in changes to accessibility in phrases containing their respective DNA-binding motifs. Finally, we use MIAA results to explore the order of motif interactions and identify preferential motif ordering arrangements that appear to have an effect on accessibility.

Genomic interactions provide important context to our understanding of the state of the genome. One question is whether specific transcription factor interactions give rise to genome organization. We introduce spatzie, an R package and a website that implements statistical tests for significant transcription factor motif cooperativity between enhancer-promoter interactions. We conducted controlled experiments under realistic simulated data from ChIP-seq to confirm spatzie is capable of discovering co-enriched motif interactions even in noisy conditions. We then use spatzie to investigate cell type specific transcription factor cooperativity within recent human ChIA-PET enhancer-promoter interaction data. The method is available online at https://spatzie.mit.edu.

Summary Proper assembly and function of the nervous system requires the generation of a uniquely diverse population of neurons expressing a cell-type-specific combination of effector genes that collectively define neuronal morphology, connectivity, and function. How countless partially overlapping but cell-type-specific patterns of gene expression are controlled at the genomic level remains poorly understood. Here we show that neuronal genes are associated with highly complex gene regulatory systems composed of independent cell-type- and cell-stage-specific regulatory elements that reside in expanded non-coding genomic domains. Mapping enhancer-promoter interactions revealed that motor neuron enhancers are broadly distributed across the large chromatin domains. This distributed regulatory architecture is not a unique property of motor neurons but is employed throughout the nervous system. The number of regulatory elements increased dramatically during the transition from invertebrates to vertebrates, suggesting that acquisition of new enhancers might be a fundamental process underlying the evolutionary increase in cellular complexity.

SummaryBuilding a nervous system is a protracted process that starts with the specification of individual neuron types and ends with the formation of mature neural circuits. The molecular mechanisms that regulate the temporal progression of maturation in individual cell types remain poorly understood. In this work, we have mapped the gene expression and chromatin accessibility changes in mouse spinal motor neurons throughout their lifetimes. We found that both motor neuron gene expression and putative regulatory elements are dynamic during the first three weeks of postnatal life, when motor circuits are maturing. Genes that are up-regulated during this time contribute to adult motor neuron diversity and function. Almost all of the chromatin regions that gain accessibility during maturation are motor neuron specific, yet a majority of the transcription factor binding motifs enriched in these regions are shared with other mature neurons. Collectively, these findings suggest that a core transcriptional program operates in a context-dependent manner to access cell-type-specific cis-regulatory systems associated with maturation genes. Discovery of general principles governing neuronal maturation might inform methods for transcriptional reprogramming of neuronal age and for improved modelling of age-related neurodegenerative diseases.

Planaria are complex metazoans that repair damage to their bodies and cease remodeling when a correct anatomy has been achieved. This model system offers a unique opportunity to understand how large‐scale anatomical homeostasis emerges from the activities of individual cells. Much progress has been made on the molecular genetics of stem cell activity in planaria. However, recent data also indicate that the global pattern is regulated by physiological circuits composed of ionic and neurotransmitter signaling. Here, we overview the multi‐scale problem of understanding pattern regulation in planaria, with specific focus on bioelectric signaling via ion channels and gap junctions (electrical synapses), and computational efforts to extract explanatory models from functional and molecular data on regeneration. We present a perspective that interprets results in this fascinating field using concepts from dynamical systems theory and computational neuroscience. Serving as a tractable nexus between genetic, physiological, and computational approaches to pattern regulation, planarian pattern homeostasis harbors many deep insights for regenerative medicine, evolutionary biology, and engineering.

The shape of an animal body plan is constructed from protein components encoded by the genome. However, bioelectric networks composed of many cell types have their own intrinsic dynamics, and can drive distinct morphological outcomes during embryogenesis and regeneration. Planarian flatworms are a popular system for exploring body plan patterning due to their regenerative capacity, but despite considerable molecular information regarding stem cell differentiation and basic axial patterning, very little is known about how distinct head shapes are produced. Here, we show that after decapitation in G. dorotocephala, a transient perturbation of physiological connectivity among cells (using the gap junction blocker octanol) can result in regenerated heads with quite different shapes, stochastically matching other known species of planaria (S. mediterranea, D. japonica, and P. felina). We use morphometric analysis to quantify the ability of physiological network perturbations to induce different species-specific head shapes from the same genome. Moreover, we present a computational agent-based model of cell and physical dynamics during regeneration that quantitatively reproduces the observed shape changes. Morphological alterations induced in a genomically wild-type G. dorotocephala during regeneration include not only the shape of the head but also the morphology of the brain, the characteristic distribution of adult stem cells (neoblasts), and the bioelectric gradients of resting potential within the anterior tissues. Interestingly, the shape change is not permanent, after regeneration is complete, intact animals remodel back to G. dorotocephala-appropriate head shape within several weeks in a secondary phase of remodeling following initial complete regeneration. We present a conceptual model to guide future work to delineate the molecular mechanisms by which bioelectric networks stochastically select among a small set of discrete head morphologies. Taken together, these data and analyses shed light on important physiological modifiers of morphological information in dictating species-specific shape, and reveal them to be a novel instructive input into head patterning in regenerating planaria.