Gene regulatory mechanisms underlying the intestinal innate immune response

In the mammalian gastrointestinal tract, distinct types of cells, including epithelial cells and macrophages, collaborate to eliminate ingested pathogens while striving to preserve the commensal microbiota. The underlying innate immune response is driven by significant gene expression changes in each cell, and recent work has provided novel insights into the gene regulatory mechanisms that mediate such transcriptional changes. These mechanisms differ from those underlying the canonical cellular differentiation model in which a sequential deposition of DNA methylation and histone modification marks progressively restricts the chromatin landscape. Instead, inflammatory macrophages and intestinal epithelial cells appear to largely rely on transcription factors that explore an accessible chromatin landscape to generate dynamic stimulus-specific and spatial-specific physiological responses

Genome-Wide Ultrabithorax Binding Analysis Reveals Highly Targeted Genomic Loci at Developmental Regulators and a Potential Connection to Polycomb-Mediated Regulation

Hox homeodomain transcription factors are key regulators of animal development. They specify the identity of segments along the anterior-posterior body axis in metazoans by controlling the expression of diverse downstream targets, including transcription factors and signaling pathway components. The Drosophila melanogaster Hox factor Ultrabithorax (Ubx) directs the development of thoracic and abdominal segments and appendages, and loss of Ubx function can lead for example to the transformation of third thoracic segment appendages (e.g. halters) into second thoracic segment appendages (e.g. wings), resulting in a characteristic four-wing phenotype. Here we present a Drosophila melanogaster strain with a V5-epitope tagged Ubx allele, which we employed to obtain a high quality genome-wide map of Ubx binding sites using ChIP-seq. We confirm the sensitivity of the V5 ChIP-seq by recovering 7/8 of well-studied Ubx-dependent cis-regulatory regions. Moreover, we show that Ubx binding is predictive of enhancer activity as suggested by comparison with a genome-scale resource of in vivo tested enhancer candidates. We observed densely clustered Ubx binding sites at 12 extended genomic loci that included ANTP-C, BX-C, Polycomb complex genes, and other regulators and the clustered binding sites were frequently active enhancers. Furthermore, Ubx binding was detected at known Polycomb response elements (PREs) and was associated with significant enrichments of Pc and Pho ChIP signals in contrast to binding sites of other developmental TFs. Together, our results show that Ubx targets developmental regulators via strongly clustered binding sites and allow us to hypothesize that regulation by Ubx might involve Polycomb group proteins to maintain specific regulatory states in cooperative or mutually exclusive fashion, an attractive model that combines two groups of proteins with prominent gene regulatory roles during animal development

The environment is everything that isn't me: molecular mechanisms and evolutionary dynamics of insect clocks in variable surroundings

Circadian rhythms are oscillations in behavior, metabolism and physiology that have a period close to 24h. These rhythms are controlled by an internal pacemaker that evolved under strong selective pressures imposed by environmental cyclical changes, mainly of light and temperature. The molecular nature of the circadian pacemaker was extensively studied in a number of organisms under controlled laboratory conditions. But although these studies were fundamental to our understanding of the circadian clock, most of the conditions used resembled in nature only the relatively constant situation at lower latitudes. At higher latitudes light-dark and temperature cycles vary considerably across different seasons, with summers having long and hot days and winters short and cold ones. Considering these differences and other external cues, such as moonlight, recent studies in more natural and semi-natural situations revealed unexpected features at both molecular and behavioral levels, highlighting the dramatic influence of multiple environmental variables in the molecular clockwork. This emphasizes the importance of studying the circadian clock in the wild, where seasonal environmental changes fine-tune the underlying circadian mechanism, affecting population dynamics and impacting the geographical variation in clock genes. Indeed, latitudinal clines in clock gene frequencies suggest that natural selection and demography shape the circadian clock over wide geographical ranges. In this review we will discuss the recent advances in understanding the molecular underpinnings of the circadian clock, how it resonates with the surrounding variables (both in the laboratory and in semi-natural conditions) and its impact on the population dynamics and evolution. In addition, we will elaborate on how next-generation sequencing technologies will complement classical reductionist approaches by identifying causal variants in natural populations that will link genetic variation to circadian phenotypes, illuminating how the circadian clock functions in the real world

Creation of an epitope-tagged Ubx allele by homologous recombination.

<p>(A) Design of the targeting construct (top), the integration to the endogenous Ubx locus and the location of the probe for Southern blotting (middle) and the locus after the cassette removal (bottom). (B) The eye color and haltere morphology for candidate flies before (left) and after cassette removal (white-eyed fly) (right). (C) Southern blot confirming the correct integration for two independently recombined <i>Drosophila</i> lines #11 and #12 (heterozygous for the insert). w¹¹¹⁸ flies were used as a control.</p

Genomic location of Ubx binding sites and recovery of known Ubx-dependent enhancers.

<p>(A) Genomic distribution of Ubx peaks (right) in comparison to the genome (left). (B-H) UCSC Genome Browser screenshots [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref083" target="_blank">83</a>] of Ubx (blue), mock (green) ChIP-seq fragment density tracks and the Ubx peak calls at known Ubx-dependent enhancers (red bars) (see the main text for references). Panel (B) also contains the fragment density tracks for the two input samples (grey). (I) UCSC Genome Browser view of the <i>hth</i> locus and examples of Ubx-bound embryonic enhancers and their activity patterns [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref022" target="_blank">22</a>]. (J) UCSC Genome Browser view of the <i>Con</i> locus and Ubx-bound embryonic enhancer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref022" target="_blank">22</a>]. Vienna tiles (VT) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref022" target="_blank">22</a>] are marked by slate blue boxes and their ID numbers are indicated.</p

Clustered Ubx binding sites at the genomic loci of important developmental regulators.

<p>(A, B) UCSC Genome Browser screenshots at BX-C and <i>hth</i> gene loci show ChIP-seq fragment density tracks and Ubx peak calls, revealing many clustered binding sites. (C, D) The number of Ubx peaks per 100 kb on chromosomes 3R and 2L (each 100 kb window starts at a Ubx peak, covering all possible windows that contain at least one Ubx peak). The plots for chromosomes 2R, 3L, X and 4 are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.s001" target="_blank">S1 Fig</a> Representative genes for all windows containing ≥25 Ubx peaks are labeled.</p

Strong overlap of Ubx and Polycomb complex binding sites in entire embryos.

<p>(A-D) UCSC Genome Browser screenshots of Ubx and mock ChIP-seq tracks at known PRE/TRE elements (purple shading) at BX-C, ANTP-C, <i>en</i>, <i>ph-p</i> and <i>ph-d</i>. The coordinates are from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref060" target="_blank">60</a>]. Pho track is from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref068" target="_blank">68</a>]. (E) The box plots show the Pc and Pho ChIP-chip signal (ChIP/input ratio [log2]) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref067" target="_blank">67</a>] at the Ubx peak summits and at control regions. The left panel represents all regions, the middle panel positions that overlap HOT regions and the right panel those that do not overlap HOT regions. NS–not significant; Wicoxon test: **P<10⁻²⁰; equivalent plots for other TFs are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.s003" target="_blank">S3 Fig</a>. (F) The plots show the percentage of TFs binding sites that have Pc or Pho signal [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref067" target="_blank">67</a>] greater than a given threshold value (X-axis; red line: Ubx, black: control regions and grey: other TFs from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref014" target="_blank">14</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161997#pone.0161997.ref024" target="_blank">24</a>]).</p

Ubx binds to active enhancers.

<p>(A) The bar plot shows the percentage of Vienna tiles (VTs) without or with Ubx binding sites (black and red bars, respectively) that is active at any stage of embryogenesis (left) or at the indicated embryonic stages. Hypergeometric p-value: **P<10⁻¹⁰. (B) The left panel shows the fraction of all VTs (top) and the fraction of Ubx-bound VTs (bottom) that overlaps HOT regions (dark shading). The bar plot on the right shows the percentage of active tiles for the four subsets of VTs defined on the left panel. NS–not significant. Hypergeometric p-value: **P<10⁻¹⁹.</p