Shadow Enhancers Foster Robustness of Drosophila Gastrulation

SummaryCritical developmental control genes sometimes contain “shadow” enhancers that can be located in remote positions, including the introns of neighboring genes [1]. They nonetheless produce patterns of gene expression that are the same as or similar to those produced by more proximal primary enhancers. It was suggested that shadow enhancers help foster robustness in gene expression in response to environmental or genetic perturbations [2, 3]. We critically tested this hypothesis by employing a combination of bacterial artificial chromosome (BAC) recombineering and quantitative confocal imaging methods [2, 4]. Evidence is presented that the snail gene is regulated by a distal shadow enhancer located within a neighboring locus. Removal of the proximal primary enhancer does not significantly perturb snail function, including the repression of neurogenic genes and formation of the ventral furrow during gastrulation at normal temperatures. However, at elevated temperatures, there is sporadic loss of snail expression and coincident disruptions in gastrulation. Similar defects are observed at normal temperatures upon reductions in the levels of Dorsal, a key activator of snail expression (reviewed in [5]). These results suggest that shadow enhancers represent a novel mechanism of canalization whereby complex developmental processes “bring about one definite end-result regardless of minor variations in conditions” [6]

Inferring ecological and behavioral drivers of African elephant movement using a linear filtering approach

Understanding the environmental factors influencing animal movements is fundamental to theoretical and applied research in the field of movement ecology. Studies relating fine-scale movement paths to spatiotemporally structured landscape data, such as vegetation productivity or human activity, are particularly lacking despite the obvious importance of such information to understanding drivers of animal movement. In part, this may be because few approaches provide the sophistication to characterize the complexity of movement behavior and relate it to diverse, varying environmental stimuli. We overcame this hurdle by applying, for the first time to an ecological question, a finite impulse–response signal-filtering approach to identify human and natural environmental drivers of movements of 13 free-ranging African elephants (Loxodonta africana) from distinct social groups collected over seven years. A minimum mean-square error (MMSE) estimation criterion allowed comparison of the predictive power of landscape and ecological model inputs. We showed that a filter combining vegetation dynamics, human and physical landscape features, and previous movement outperformed simpler filter structures, indicating the importance of both dynamic and static landscape features, as well as habit, on movement decisions taken by elephants. Elephant responses to vegetation productivity indices were not uniform in time or space, indicating that elephant foraging strategies are more complex than simply gravitation toward areas of high productivity. Predictions were most frequently inaccurate outside protected area boundaries near human settlements, suggesting that human activity disrupts typical elephant movement behavior. Successful management strategies at the human–elephant interface, therefore, are likely to be context specific and dynamic. Signal processing provides a promising approach for elucidating environmental factors that drive animal movements over large time and spatial scales.This research was supported by NSF GRFP (A. N. Boettiger) and NIH grant GM083863-01 and USDI FWS Grant 98210-8-G745 to W. M. Getz.http://www.esajournals.org/loi/ecol

A leukemia-protective germline variant mediates chromatin module formation via transcription factor nucleation

Non-coding variants coordinate transcription factor (TF) binding and chromatin mark enrichment changes over regions spanning >100 kb. These molecularly coordinated regions are named "variable chromatin modules" (VCMs), providing a conceptual framework of how regulatory variation might shape complex traits. To better understand the molecular mechanisms underlying VCM formation, here, we mechanistically dissect a VCM-modulating noncoding variant that is associated with reduced chronic lymphocytic leukemia (CLL) predisposition and disease progression. This common, germline variant constitutes a 5-bp indel that controls the activity of an AXIN2 gene-linked VCM by creating a MEF2 binding site, which, upon binding, activates a super-enhancer-like regulatory element. This triggers a large change in TF binding activity and chromatin state at an enhancer cluster spanning >150 kb, coinciding with subtle, long-range chromatin compaction and robust AXIN2 up-regulation. Our results support a model in which the indel acts as an AXIN2 VCM-activating TF nucleation event, which modulates CLL pathology

Transcriptional Regulation: Effects of Promoter Proximal Pausing on Speed, Synchrony and Reliability

Recent whole genome polymerase binding assays in the Drosophila embryo have shown that a substantial proportion of uninduced genes have pre-assembled RNA polymerase-II transcription initiation complex (PIC) bound to their promoters. These constitute a subset of promoter proximally paused genes for which mRNA elongation instead of promoter access is regulated. This difference can be described as a rearrangement of the regulatory topology to control the downstream transcriptional process of elongation rather than the upstream transcriptional initiation event. It has been shown experimentally that genes with the former mode of regulation tend to induce faster and more synchronously, and that promoter-proximal pausing is observed mainly in metazoans, in accord with a posited impact on synchrony. However, it has not been shown whether or not it is the change in the regulated step per se that is causal. We investigate this question by proposing and analyzing a continuous-time Markov chain model of PIC assembly regulated at one of two steps: initial polymerase association with DNA, or release from a paused, transcribing state. Our analysis demonstrates that, over a wide range of physical parameters, increased speed and synchrony are functional consequences of elongation control. Further, we make new predictions about the effect of elongation regulation on the consistent control of total transcript number between cells. We also identify which elements in the transcription induction pathway are most sensitive to molecular noise and thus possibly the most evolutionarily constrained. Our methods produce symbolic expressions for quantities of interest with reasonable computational effort and they can be used to explore the interplay between interaction topology and molecular noise in a broader class of biochemical networks. We provide general-purpose code implementing these methods

Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function

Spatial and Temporal Organization of the Genome: Current State and Future Aims of the 4D Nucleome Project

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function

Molecular Mechanisms of Precise and Robust Gene Regulation in Drosophila

The ornate arrangement of diverse cells into specialized tissues, organs, and higher structures characteristic of multicellular organisms is all encoded from the same genome sequence. Despite their differences, morphologically distinct cells (e.g. muscle cells and neurons) must transcribe many of the same genes. Morphological indistinguishable cells must often transcribe distinct sets of genes (e.g. different odorant receptor cells). The ensemble of genes expressed in a given cell -- and the relative frequency they are expressed at, give each cell its characteristic identity more so than the presence of individual genes. Therefore understanding the genetic control of development and differentiation is a question not so much of the understanding the gene sequences themselves, but the regulatory structure of the genome which determines how they are deployed.In order for development to unravel in such a manner that each embryo makes it through the process with all the correct parts in the correct positions at the end, this process must be exceedingly precise. Though often taken for granted, this precision becomes particular impressive if one considers the frequency with which mistakes are made in intelligently designed human built assembly processes. The developing animal must position components correctly on scales of microns (e.g. tissue boundaries) and nanometers (e.g. neuron-junctions), has no external direction of assembly, and requires thermal noise to position many of its components (including essentially all transcription factors - proteins which regulate read access to the genome). It is not sufficient for the process to be precise. It must also be robust to changes in the conditions in which it operates, such as different thermal environments, nutrient conditions, and chemical environments. This robustness enables a certain degree of plasticity, such that some components of the system can change and evolve new functions, without causing catastrophic failure of the rest of the system. In my thesis research I have tried to explore some of the molecular mechanisms of gene regulation which support the precise and robust expression of multicellular genomes. Rapid advances in post-genomic technologies have exposed a broad range of fundamental differences in the organization and regulation of multicellular genomes such as Drosophila. I have worked primarily on two phenomena, the use of promoter proximal pausing as a regulatory strategy, and the use of multiple apparently redundant regulatory sequences to drive expression of the same gene. Discovery of both of these phenomena emerged from analysis of whole genome polymerase and transcription factor binding data. Using quantitative high resolution in situ and semi-automated computational image processing I have studied the detailed differences in the transcriptional activation and transcription frequency of genes regulated by these mechanisms. Through this analysis I have shown a strong correlation through more rapid and synchronous gene expression and regulation through release of promoter proximal paused polymerase. Theoretical modeling demonstrates that such an effect can be expected from regulating release of stable downstream state in a general assembly process (such as construction of the RNA Pol II pre-initiation complex). Analysis of gene expression driven by multiple enhancers with overlapping activity compared to constructs with only a single active enhancer revealed that the process by which an enhancer binds its target transcription factors and activates expression is often limiting enough that having a second independent copy can produce detectable changes in the frequency of transcription. This reduction of natural variation in gene activation is especially important under stress conditions, such as thermal stress or reduced levels of some of the activating factors. Robustness to this sort of variation may be important both for adaptation within a species and the flexibility to allow modification of interacting pathways in the course of evolutionary modification. These investigations also revealed a corrective propensity whereby the simultaneous activity of multiple enhancers, responding to repressors as well as activators, can give rise to correctly restricted gene expression even when the elements taken in isolation drive some degree of ectopic expression. So far both of these mechanisms have only been reliably documented in multicellular systems, suggesting that the precision and robustness they confer may be an innovation of metazoans in response to increased levels of coordination required to keep many cells functioning in the tight cooperation of a multicellular organism. Doubtless this is but scratching the surface of the mechanisms which ensure such precision and control. However the rapid improvements to both genomic tools and imaging technology make it like to be a promising field for further exploration for years to come