Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more ...rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation-sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type-specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Neurons born in the embryo can undergo a protracted period of maturation lasting well into postnatal life. How gene expression changes are regulated during maturation and whether they can be ...recapitulated in cultured neurons remains poorly understood. Here, we show that mouse motor neurons exhibit pervasive changes in gene expression and accessibility of associated regulatory regions from embryonic till juvenile age. While motifs of selector transcription factors, ISL1 and LHX3, are enriched in nascent regulatory regions, motifs of NFI factors, activity-dependent factors, and hormone receptors become more prominent in maturation-dependent enhancers. Notably, stem cell-derived motor neurons recapitulate ~40% of the maturation expression program in vitro, with neural activity playing only a modest role as a late-stage modulator. Thus, the genetic maturation program consists of a core hardwired subprogram that is correctly executed in vitro and an extrinsically-controlled subprogram that is dependent on the in vivo context of the maturing organism.
RNA Polymerase II ChIA-PET data has revealed enhancers that are active in a profiled cell type and the genes that the enhancers regulate through chromatin interactions. The most commonly used ...computational method for analyzing ChIA-PET data, the ChIA-PET Tool, discovers interaction anchors at a spatial resolution that is insufficient to accurately identify individual enhancers. We introduce Germ, a computational method that estimates the likelihood that any two narrowly defined genomic locations are jointly occupied by RNA Polymerase II. Germ takes a blind deconvolution approach to simultaneously estimate the likelihood of RNA Polymerase II occupation as well as a model of the arrangement of read alignments relative to locations occupied by RNA Polymerase II. Both types of information are utilized to estimate the likelihood that RNA Polymerase II jointly occupies any two genomic locations. We apply Germ to RNA Polymerase II ChIA-PET data from embryonic stem cells to identify the genomic locations that are jointly occupied along with transcription start sites. We show that these genomic locations align more closely with features of active enhancers measured by ChIP-Seq than the locations identified using the ChIA-PET Tool. We also apply Germ to RNA Polymerase II ChIA-PET data from motor neuron progenitors. Based on the Germ results, we observe that a combination of cell type specific and cell type independent regulatory interactions are utilized by cells to regulate gene expression.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Transcription factor over-expression is a proven method for reprogramming cells to a desired cell type for regenerative medicine and therapeutic discovery. However, a general method for the ...identification of reprogramming factors to create an arbitrary cell type is an open problem. Here we examine the success rate of methods and data for differentiation by testing the ability of nine computational methods (CellNet, GarNet, EBseq, AME, DREME, HOMER, KMAC, diffTF and DeepAccess) to discover and rank candidate factors for eight target cell types with known reprogramming solutions. We compare methods that use gene expression, biological networks and chromatin accessibility data, and comprehensively test parameter and preprocessing of input data to optimize performance. We find the best factor identification methods can identify an average of 50-60% of reprogramming factors within the top ten candidates, and methods that use chromatin accessibility perform the best. Among the chromatin accessibility methods, complex methods DeepAccess and diffTF have higher correlation with the ranked significance of transcription factor candidates within reprogramming protocols for differentiation. We provide evidence that AME and diffTF are optimal methods for transcription factor recovery that will allow for systematic prioritization of transcription factor candidates to aid in the design of new reprogramming protocols.
Transcription factor over-expression is a proven method for reprogramming cells to a desired cell type for regenerative medicine and therapeutic discovery. However, a general method for the ...identification of reprogramming factors to create an arbitrary cell type is an open problem. Here we examine the success rate of methods and data for directed differentiation by testing the ability of nine computational methods (CellNet, GarNet, EBSeq, AME, DREME, HOMER, KMAC, diffTF, and DeepAccess) to discover and rank candidate factors for eight target cell types with known reprogramming solutions. We compare methods that utilize gene expression, biological networks, and chromatin accessibility data and comprehensively test parameter and pre-processing of input data to optimize performance. We find the best factor identification methods can identify an average of 50–60% of reprogramming factors within the top 10 candidates, and methods that use chromatin accessibility perform the best. Among the chromatin accessibility methods, complex methods DeepAccess and diffTF have higher correlation with the ranked significance of transcription factor candidates within reprogramming protocols for differentiation. We provide evidence that AME and diffTF are optimal methods for transcription factor recovery which will allow for systematic prioritization of transcription factor candidates to aid in the design of novel reprogramming protocols.
Generic spinal motor neuron identity is established by cooperative binding of programming transcription factors (TFs), Isl1 and Lhx3, to motor-neuron-specific enhancers. How expression of effector ...genes is maintained following downregulation of programming TFs in maturing neurons remains unknown. High-resolution exonuclease (ChIP-exo) mapping revealed that the majority of enhancers established by programming TFs are rapidly deactivated following Lhx3 downregulation in stem-cell-derived hypaxial motor neurons. Isl1 is released from nascent motor neuron enhancers and recruited to new enhancers bound by clusters of Onecut1 in maturing neurons. Synthetic enhancer reporter assays revealed that Isl1 operates as an integrator factor, translating the density of Lhx3 or Onecut1 binding sites into transient enhancer activity. Importantly, independent Isl1/Lhx3- and Isl1/Onecut1-bound enhancers contribute to sustained expression of motor neuron effector genes, demonstrating that outwardly stable expression of terminal effector genes in postmitotic neurons is controlled by a dynamic relay of stage-specific enhancers.
•Enhancer landscape in stem-cell-derived motor neurons remains highly dynamic•Motor neuron effector gene expression is maintained by stage-specific enhancers•Isl1 is recruited to transient enhancers via stage-specific programming TFs•Isl1 translates the density of clustered TFs into enhancer activity
Rhee et al. performed high-resolution genome-wide analysis of TF binding in nascent and maturing ESC-derived motor neurons. The study revealed mechanisms by which Isl1 integrates stage-specific TFs at dynamic enhancers to maintain neuronal cell identity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a method for the genome-wide de novo discovery of chromatin interactions. Existing computational methods typically ...fail to detect weak or dynamic interactions because they use a peak-calling step that ignores paired-end linkage information. We have developed a novel computational method called Chromatin Interaction Discovery (CID) to overcome this limitation with an unbiased clustering approach for interaction discovery. CID outperforms existing chromatin interaction detection methods with improved sensitivity, replicate consistency, and concordance with other chromatin interaction datasets. In addition, CID also outperforms other methods in discovering chromatin interactions from HiChIP data. We expect that the CID method will be valuable in characterizing 3D chromatin interactions and in understanding the functional consequences of disease-associated distal genetic variations.
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.
•Neuronal genes have expanded intergenic domain size and regulatory complexity•Neuronal genes are controlled by distinct cell- and stage-specific enhancers•Neuronal enhancers are not clustered but distributed across expanded non-coding DNA•Neuronal gene regulation expanded during transition from invertebrates to vertebrates
Closser et al. show that neuronal genes are associated with highly complex regulatory systems in expanded non-coding genomic domains. Neuronal enhancers are distributed broadly and utilized sparsely in cell-type- and cell-stage-specific patterns. Acquisition of new enhancers might be a fundamental process underlying the evolutionary increase in cellular complexity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
During nervous system development, thousands of distinct cell types are generated and assembled into complex circuits that control all aspects of animal cognition and behavior. Understanding what ...these diverse cells are, how they are generated, and what they do in the context of circuits and behavior form the fundamental efforts of the field of neuroscience. In this thesis, I investigate how the genomic organization of regulatory elements informs specific patterns of gene expression in the nervous system. In particular, I examine how distinct combinations of transcription factors interpret information encoded in the genome to control global gene expression programs in a cell-type-specific manner. In Chapter Two, I describe the establishment of a developmentally inspired transcriptional programming system to generate spinal and cranial motor neurons directly from mouse embryonic stem cells. Programmed motor neurons acquire general characteristics that mirror their in vivo counterparts, providing a robust system for studying cell fate specification in the nervous system. Combinatorial expression of cell-type-specific programming factors informs context-dependent enhancer binding and acquisition of appropriate cell-type-specific molecular and functional properties. In Chapter Three, I take advantage of this robust, experimentally accessible system to probe the chromatin-level organization and regulatory principles controlling specificity of motor neuron gene expression programs. Motor neuron genes are controlled by multiple distantly distributed enhancer constellations stretched across large regulatory domains. Using this motor neuron specification model, I discovered a unique regulatory organization controlling gene expression in the nervous system, whereby neuronal genes are controlled from uniquely complex regulatory domains acting over large distances. In Chapter Four, I extrapolate on the insights gained from studying motor neurons at a single point in time to investigate the dynamics of the regulatory environment during neuronal maturation. We demonstrate that enhancers are highly dynamic even after postmitotic specification. The dynamic nature of enhancers is dependent on combinatorial binding with new transcriptional cofactors. Overall, my results suggest that neuronal gene expression programs within a single cell type are regulated in a highly dynamic fashion by a complex set of enhancers. I propose that during development the immense cellular complexity of the nervous system is established and maintained by correspondingly complex repertoire of enhancers.
Loss of RNA homeostasis underlies numerous neurodegenerative and neuroinflammatory diseases. However, the molecular mechanisms that trigger neuroinflammation are poorly understood. Viral ...double-stranded RNA (dsRNA) triggers innate immune responses when sensed by host pattern recognition receptors (PRRs) present in all cell types. Here, we report that human neurons intrinsically carry exceptionally high levels of immunostimulatory dsRNAs and identify long 3'UTRs as giving rise to neuronal dsRNA structures. We found that the neuron-enriched ELAVL family of genes (
,
, and
) can increase (i) 3'UTR length, (ii) dsRNA load, and (iii) activation of dsRNA-sensing PRRs such as MDA5, PKR, and TLR3. In wild-type neurons, neuronal dsRNAs signaled through PRRs to induce tonic production of the antiviral type I interferon. Depleting
in WT neurons led to global shortening of 3'UTR length, reduced immunostimulatory dsRNA levels, and rendered WT neurons susceptible to herpes simplex virus and Zika virus infection. Neurons deficient in ADAR1, a dsRNA-editing enzyme mutated in the neuroinflammatory disorder Aicardi-Goutières syndrome, exhibited intolerably high levels of dsRNA that triggered PRR-mediated toxic inflammation and neuronal death. Depleting
in ADAR1 knockout neurons led to prolonged neuron survival by reducing immunostimulatory dsRNA levels. In summary, neurons are specialized cells where PRRs constantly sense "self" dsRNAs to preemptively induce protective antiviral immunity, but maintaining RNA homeostasis is paramount to prevent pathological neuroinflammation.
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