Mechanisms underlying the control of dynamic regulatory element activity and chromatin accessibility during metamorphosis

Cis-regulatory modules of metazoan genomes determine the when and where of gene expression during development. Here we discuss insights into the genetic and molecular mechanisms behind cis-regulatory module usage that have come from recent application of genomics assays to insect metamorphosis. Assays including FAIRE-seq, ATAC-seq, and CUT&RUN indicate that sequential changes in chromatin accessibility play a key role in mediating stage-specific cis-regulatory module activity and gene expression. We review the current understanding of what controls precisely coordinated changes in chromatin accessibility during metamorphosis and describe evidence that points to systemic hormone signaling as a primary signal to trigger genome-wide shifts in accessibility patterns and cis-regulatory module usage

Expression of E93 provides an instructive cue to control dynamic enhancer activity and chromatin accessibility during development

How temporal cues combine with spatial inputs to control gene expression during development is poorly understood. Here, we test the hypothesis that the Drosophila transcription factor E93 controls temporal gene expression by regulating chromatin accessibility. Precocious expression of E93 early in wing development reveals that it can simultaneously activate and deactivate different target enhancers. Notably, the precocious patterns of enhancer activity resemble the wild-type patterns that occur later in development, suggesting that expression of E93 alters the competence of enhancers to respond to spatial cues. Genomic profiling reveals that precocious E93 expression is sufficient to regulate chromatin accessibility at a subset of its targets. These accessibility changes mimic those that normally occur later in development, indicating that precocious E93 accelerates the wild-type developmental program. Further, we find that target enhancers that do not respond to precocious E93 in early wings become responsive after a developmental transition, suggesting that parallel temporal pathways work alongside E93. These findings support a model wherein E93 expression functions as an instructive cue that defines a broad window of developmental time through control of chromatin accessibility

The SWI/SNF nucleosome remodeler constrains enhancer activity during Drosophila wing development

Chromatin remodeling is central to the dynamic changes in gene expression that drive cell fate determination. During development, the sets of enhancers that are accessible for use change globally as cells transition between stages. While transcription factors and nucleosome remodelers are known to work together to control enhancer accessibility, it is unclear how the short stretches of DNA that they individually unmask yield the kilobase-sized accessible regions characteristic of active enhancers. Here, we performed a genetic screen to investigate the role of nucleosome remodelers in control of dynamic enhancer activity. We find that the Drosophila Switch/Sucrose Non-Fermenting complex, BAP, is required for repression of a temporally dynamic enhancer, brdisc. Contrary to expectations, we find that the BAP-specific subunit Osa is dispensable for mediating changes in chromatin accessibility between the early and late stages of wing development. Instead, we find that Osa is required to constrain the levels of brdisc activity when the enhancer is normally active. Genome-wide profiling reveals that Osa directly binds brdisc as well as thousands of other developmentally dynamic regulatory sites, including multiple genes encoding components and targets of the Notch signaling pathway. Transgenic reporter analyses demonstrate that Osa is required for activation and for constraint of different sets of target enhancers in the same cells. Moreover, Osa loss results in hyperactivation of the Notch ligand Delta and development of ectopic sensory structures patterned by Notch signaling early in development. Together, these findings indicate that proper constraint of enhancer activity is necessary for regulation of dose-dependent developmental events

Ecdysone signaling induces two phases of cell cycle exit in Drosophila cells

During development, cell proliferation and differentiation must be tightly coordinated to ensure proper tissue morphogenesis. Because steroid hormones are central regulators of developmental timing, understanding the links between steroid hormone signaling and cell proliferation is crucial to understanding the molecular basis of morphogenesis. Here we examined the mechanism by which the steroid hormone ecdysone regulates the cell cycle in Drosophila. We find that a cell cycle arrest induced by ecdysone in Drosophila cell culture is analogous to a G2 cell cycle arrest observed in the early pupa wing. We show that in the wing, ecdysone signaling at the larva-to-puparium transition induces Broad which in turn represses the cdc25c phosphatase String. The repression of String generates a temporary G2 arrest that synchronizes the cell cycle in the wing epithelium during early pupa wing elongation and flattening. As ecdysone levels decline after the larva-to-puparium pulse during early metamorphosis, Broad expression plummets, allowing String to become re-activated, which promotes rapid G2/M progression and a subsequent synchronized final cell cycle in the wing. In this manner, pulses of ecdysone can both synchronize the final cell cycle and promote the coordinated acquisition of terminal differentiation characteristics in the wing

Chromatin profiling of Drosophila CNS subpopulations identifies active transcriptional enhancers

One of the key issues in studying transcriptional regulation during development is how to employ genome-wide assays that reveals sites of open chromatin and transcription factor binding to efficiently identify biologically relevant genes and enhancers. Analysis of Drosophila CNS midline cell development provides a useful system for studying transcriptional regulation at the genomic level due to a large, well-characterized set of midline-expressed genes and in vivo validated enhancers. In this study, FAIRE-seq on FACS-purified midline cells was performed and the midline FAIRE data were compared with whole-embryo FAIRE data. We find that regions of the genome with a strong midline FAIRE peak and weak whole-embryo FAIRE peak overlap with known midline enhancers and provide a useful predictive tool for enhancer identification. In a complementary analysis, we compared a large dataset of fragments that drive midline expression in vivo with the FAIRE data. Midline enhancer fragments with a midline FAIRE peak tend to be near midline-expressed genes, whereas midline enhancers without a midline FAIRE peak were often distant from midline-expressed genes and unlikely to drive midline transcription in vivo

A Common Set of DNA Regulatory Elements Shapes Drosophila Appendages

Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different "master regulator" transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. Here, we use genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. We propose that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules

A Common Set of DNA Regulatory Elements Shapes Drosophila Appendages

Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different "master regulator" transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. Here, we use genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. We propose that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules

Characterization of AbiR, a novel multicomponent abortive infection mechanism encoded by plasmid pKR223 of Lactococcus lactis subsp. lactis KR2

The native lactococcal plasmid pKR223 encodes two distinct phage resistance mechanisms, a restriction and modification (R/M) system designated LlaKR2I and an abortive infection mechanism (Abi) which affects prolate- headed-phage proliferation. The nucleotide sequence of a 16,174-bp segment of pKR223 encompassing both the R/M and Abi determinants has been determined, and sequence analysis has validated the novelty of the Abi system, which has now been designated AbiR. Analysis of deletion and insertion clones demonstrated that AbiR was encoded by two genetic loci, separated by the LlaKR2I R/M genes. Mechanistic studies on the AbiR phenotype indicated that it was heat sensitive and that it impeded phage DNA replication. These data indicated that AbiR is a novel multicomponent, heat-sensitive, 'early'- functioning Abi system and is the first lactococcal Abi system described which is encoded by two separated genetic loci.Facultad de Ciencias ExactasCentro de Investigación y Desarrollo en Criotecnología de Alimento

Alternative Transcript Initiation and Splicing as a Response to DNA Damage

Humans are exposed to the DNA damaging agent, ionizing radiation (IR), from background radiation, medical treatments, occupational and accidental exposures. IR causes changes in transcription, but little is known about alternative transcription in response to IR on a genome-wide basis. These investigations examine the response to IR at the exon level in human cells, using exon arrays to comprehensively characterize radiation-induced transcriptional expression products. Previously uncharacterized alternative transcripts that preferentially occur following IR exposure have been discovered. A large number of genes showed alternative transcription initiation as a response to IR. Dose-response and time course kinetics have also been characterized. Interestingly, most genes showing alternative transcript induction maintained these isoforms over the dose range and times tested. Finally, clusters of co-ordinately up- and down-regulated radiation response genes were identified at specific chromosomal loci. These data provide the first genome-wide view of the transcriptional response to ionizing radiation at the exon level. This study provides novel insights into alternative transcripts as a mechanism for response to DNA damage and cell stress responses in general