Chromatin-Associated RNA Sequencing (ChAR-seq).
Current protocols in molecular biology
RNA is a fundamental component of chromatin. Noncoding RNAs (ncRNAs) can associate with chromatin to influence gene expression and chromatin state; many also act at long distances from their transcriptional origin. Yet we know almost nothing about the functions or sites of action for most ncRNAs. Current methods to identify sites of RNA interaction with the genome are limited to the study of a single RNA at a time. Here we describe a protocol for ChAR-seq, a strategy to identify all chromatin-associated RNAs and map their DNA contacts genome-wide. In ChAR-seq, proximity ligation of RNA and DNA to a linker molecule is used to construct a chimeric RNA-DNA molecule that is converted to DNA for sequencing. In a single assay, ChAR-seq can discover de novo chromatin interactions of distinct RNAs, including nascent transcripts, splicing RNAs, and long noncoding RNAs (lncRNAs). Resulting "maps" of genome-bound RNAs should provide new insights into RNA biology. © 2019 by John Wiley & Sons, Inc.
View details for PubMedID 30786161
Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts
RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here, we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells, we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.
View details for PubMedID 29648534
Zygotic Genome Activation in Vertebrates.
2017; 42 (4): 316–32
The first major developmental transition in vertebrate embryos is the maternal-to-zygotic transition (MZT) when maternal mRNAs are degraded and zygotic transcription begins. During the MZT, the embryo takes charge of gene expression to control cell differentiation and further development. This spectacular organismal transition requires nuclear reprogramming and the initiation of RNAPII at thousands of promoters. Zygotic genome activation (ZGA) is mechanistically coordinated with other embryonic events, including changes in the cell cycle, chromatin state, and nuclear-to-cytoplasmic component ratios. Here, we review progress in understanding vertebrate ZGA dynamics in frogs, fish, mice, and humans to explore differences and emphasize common features.
View details for PubMedID 28829942
View details for PubMedCentralID PMC5714289
The insulator protein BEAF-32 is required for Hippo pathway activity in the terminal differentiation of neuronal subtypes
2016; 143 (13): 2389-2397
The Hippo pathway is crucial for not only normal growth and apoptosis but also cell fate specification during development. What controls Hippo pathway activity during cell fate specification is incompletely understood. In this article, we identify the insulator protein BEAF-32 as a regulator of Hippo pathway activity in Drosophila photoreceptor differentiation. Though morphologically uniform, the fly eye is composed of two subtypes of R8 photoreceptor neurons defined by expression of light-detecting Rhodopsin proteins. In one R8 subtype, active Hippo signaling induces Rhodopsin 6 (Rh6) and represses Rhodopsin 5 (Rh5), whereas in the other subtype, inactive Hippo signaling induces Rh5 and represses Rh6. The activity state of the Hippo pathway in R8 cells is determined by the expression of warts, a core pathway kinase, which interacts with the growth regulator melted in a double-negative feedback loop. We show that BEAF-32 is required for expression of warts and repression of melted Furthermore, BEAF-32 plays a second role downstream of Warts to induce Rh6 and prevent Rh5 fate. BEAF-32 is dispensable for Warts feedback, indicating that BEAF-32 differentially regulates warts and Rhodopsins. Loss of BEAF-32 does not noticeably impair the functions of the Hippo pathway in eye growth regulation. Our study identifies a context-specific regulator of Hippo pathway activity in post-mitotic neuronal fate, and reveals a developmentally specific role for a broadly expressed insulator protein.
View details for DOI 10.1242/dev.134700
View details for Web of Science ID 000392714600014
View details for PubMedID 27226322
Single-base pair differences in a shared motif determine differential Rhodopsin expression
2015; 350 (6265): 1258-1261
The final identity and functional properties of a neuron are specified by terminal differentiation genes, which are controlled by specific motifs in compact regulatory regions. To determine how these sequences integrate inputs from transcription factors that specify cell types, we compared the regulatory mechanism of Drosophila Rhodopsin genes that are expressed in subsets of photoreceptors to that of phototransduction genes that are expressed broadly, in all photoreceptors. Both sets of genes share an 11-base pair (bp) activator motif. Broadly expressed genes contain a palindromic version that mediates expression in all photoreceptors. In contrast, each Rhodopsin exhibits characteristic single-bp substitutions that break the symmetry of the palindrome and generate activator or repressor motifs critical for restricting expression to photoreceptor subsets. Sensory neuron subtypes can therefore evolve through single-bp changes in short regulatory motifs, allowing the discrimination of a wide spectrum of stimuli.
View details for DOI 10.1126/science.aab3417
View details for Web of Science ID 000365700500079
View details for PubMedID 26785491
Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition.
Proceedings of the National Academy of Sciences of the United States of America
2015; 112 (10): E1086-95
During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development.
View details for DOI 10.1073/pnas.1413990112
View details for PubMedID 25713373
View details for PubMedCentralID PMC4364222
Opposite feedbacks in the Hippo pathway for growth control and neural fate.
2013; 342 (6155): 1238016-?
Signaling pathways are reused for multiple purposes in plant and animal development. The Hippo pathway in mammals and Drosophila coordinates proliferation and apoptosis via the coactivator and oncoprotein YAP/Yorkie (Yki), which is homeostatically regulated through negative feedback. In the Drosophila eye, cross-repression between the Hippo pathway kinase LATS/Warts (Wts) and growth regulator Melted generates mutually exclusive photoreceptor subtypes. Here, we show that this all-or-nothing neuronal differentiation results from Hippo pathway positive feedback: Yki both represses its negative regulator, warts, and promotes its positive regulator, melted. This postmitotic Hippo network behavior relies on a tissue-restricted transcription factor network-including a conserved Otx/Orthodenticle-Nrl/Traffic Jam feedforward module-that allows Warts-Yki-Melted to operate as a bistable switch. Altering feedback architecture provides an efficient mechanism to co-opt conserved signaling networks for diverse purposes in development and evolution.
View details for DOI 10.1126/science.1238016
View details for PubMedID 23989952
The neuronal transcription factor erect wing regulates specification and maintenance of Drosophila R8 photoreceptor subtypes
2013; 381 (2): 482-490
Signaling pathways are often re-used during development in surprisingly different ways. The Hippo tumor suppressor pathway is best understood for its role in the control of growth. The pathway is also used in a very different context, in the Drosophila eye for the robust specification of R8 photoreceptor neuron subtypes, which complete their terminal differentiation by expressing light-sensing Rhodopsin (Rh) proteins. A double negative feedback loop between the Warts kinase of the Hippo pathway and the PH-domain growth regulator Melted regulates the choice between 'pale' R8 (pR8) fate defined by Rh5 expression and 'yellow' R8 (yR8) fate characterized by Rh6 expression. Here, we show that the gene encoding the homolog of human Nuclear respiratory factor 1, erect wing (ewg), is autonomously required to inhibit warts expression and to promote melted expression to specify pR8 subtype fate and induce Rh5. ewg mutants express Rh6 in most R8s due to ectopic warts expression. Further, ewg is continuously required to maintain repression of Rh6 in pR8s in aging flies. Our work shows that Ewg is a critical factor for the stable down-regulation of Hippo pathway activity to determine neuronal subtype fates. Neural-enriched factors, such as Ewg, may generally contribute to the contextual re-use of signaling pathways in post-mitotic neurons.
View details for DOI 10.1016/j.ydbio.2013.07.001
View details for Web of Science ID 000323992800016
View details for PubMedID 23850772
Regional Modulation of a Stochastically Expressed Factor Determines Photoreceptor Subtypes in the Drosophila Retina
2013; 25 (1): 93-105
Stochastic mechanisms are sometimes utilized to diversify cell fates, especially in nervous systems. In the Drosophila retina, stochastic expression of the PAS-bHLH transcription factor Spineless (Ss) controls photoreceptor subtype choice. In one randomly distributed subset of R7 photoreceptors, Ss activates Rhodopsin4 (Rh4) and represses Rhodopsin3 (Rh3); counterparts lacking Ss express Rh3 and repress Rh4. In the dorsal third region of the retina, the Iroquois Complex transcription factors induce Rh3 in Rh4-expressing R7s. Here, we show that Ss levels are controlled in a binary on/off manner throughout the retina yet are attenuated in the dorsal third region to allow Rh3 coexpression with Rh4. Whereas the sensitivity of rh3 repression to differences in Ss levels generates stochastic and regionalized patterns, the robustness of rh4 activation ensures its stochastic expression throughout the retina. Our findings show how stochastic and regional inputs are integrated to control photoreceptor subtype specification in the Drosophila retina.
View details for DOI 10.1016/j.devcel.2013.02.016
View details for Web of Science ID 000318058400010
View details for PubMedID 23597484
Binary Regulation of Hippo Pathway by Merlin/NF2, Kibra, Lgl, and Melted Specifies and Maintains Postmitotic Neuronal Fate
2011; 21 (5): 874-887
Patterning the Drosophila retina for color vision relies on postmitotic specification of photoreceptor subtypes. R8 photoreceptors express one of two light-sensing Rhodopsins, Rh5 or Rh6. This fate decision involves a bistable feedback loop between Melted, a PH-domain protein, and Warts, a kinase in the Hippo growth pathway. Here, we show that a subset of the Hippo pathway-Merlin, Kibra, and Lethal(2)giant larvae (Lgl), but not Expanded or Fat-is required for Warts expression and activity in R8 to specify Rh6 fate. Melted represses warts transcription to disrupt Hippo pathway activity and specify Rh5 fate. Therefore, R8 Hippo signaling exhibits ON-or-OFF regulation, promoting mutually exclusive fates. Furthermore, Merlin and Lgl are continuously required to maintain R8 neuronal subtypes. These results reveal roles for Merlin, Kibra, and Lgl in neuronal specification and maintenance and show that the Hippo pathway is reimplemented for sensory neuron fate by combining canonical and noncanonical regulatory steps.
View details for DOI 10.1016/j.devcel.2011.10.004
View details for Web of Science ID 000297233700010
View details for PubMedID 22055343
Binary fate decisions in differentiating neurons
CURRENT OPINION IN NEUROBIOLOGY
2010; 20 (1): 6-13
Neural cell fate programs must generate an enormous number of neurons with distinct adult functions. The decision to choose one neuronal subtype from two alternatives--a binary fate decision--is one way to diversify neuronal subtypes during nervous system development. Recent progress has been made in describing the genetic programs that define late-stage neuronal identity. Here, we review mechanisms that control how such fate decisions generate two different postmitotic, terminally differentiated neuronal subtypes. We survey examples from Caenorhabditis elegans and Drosophila that demonstrate different modes of binary neuronal fate specification that depend on cell division, lineage, stochastic gene expression, or extracellular signals. Comparison of these strategies reveals that, although organisms use diverse approaches to generate neural diversity, some common themes do exist.
View details for DOI 10.1016/j.conb.2009.11.002
View details for Web of Science ID 000275521900002
View details for PubMedID 20022236
- Rhodopsins in Drosophila Color Vision Visual Transduction and Non-Visual Light Perception edited by Tombran-Tink, J., Barnstable, C. J. Humana Press. 2008: 251–266
Deficient pheromone responses in mice lacking a cluster of vomeronasal receptor genes
2002; 419 (6902): 70-74
The mammalian vomeronasal organ (VNO), a part of the olfactory system, detects pheromones--chemical signals that modulate social and reproductive behaviours. But the molecular receptors in the VNO that detect these chemosensory stimuli remain undefined. Candidate pheromone receptors are encoded by two distinct and complex superfamilies of genes, V1r and V2r (refs 3 and 4), which code for receptors with seven transmembrane domains. These genes are selectively expressed in sensory neurons of the VNO. However, there is at present no functional evidence for a role of these genes in pheromone responses. Here, using chromosome engineering technology, we delete in the germ line of mice an approximately 600-kilobase genomic region that contains a cluster of 16 intact V1r genes. These genes comprise two of the 12 described V1r gene families, and represent approximately 12% of the V1r repertoire. The mutant mice display deficits in a subset of VNO-dependent behaviours: the expression of male sexual behaviour and maternal aggression is substantially altered. Electrophysiologically, the epithelium of the VNO of such mice does not respond detectably to specific pheromonal ligands. The behavioural impairment and chemosensory deficit support a role of V1r receptors as pheromone receptors.
View details for DOI 10.1038/nature00955
View details for Web of Science ID 000177788600038
View details for PubMedID 12214233