Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development.

Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes-Fgf3, Fgf4, and Fgf15-that regulate several developmental processes. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes-for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, we start to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries. Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.

Enhancer-promoter interactions can bypass CTCF-mediated boundaries and contribute to phenotypic robustness.

How enhancers activate their distal target promoters remains incompletely understood. Here we dissect how CTCF-mediated loops facilitate and restrict such regulatory interactions. Using an allelic series of mouse mutants, we show that CTCF is neither required for the interaction of the Sox2 gene with distal enhancers, nor for its expression. Insertion of various combinations of CTCF motifs, between Sox2 and its distal enhancers, generated boundaries with varying degrees of insulation that directly correlated with reduced transcriptional output. However, in both epiblast and neural tissues, enhancer contacts and transcriptional induction could not be fully abolished, and insertions failed to disrupt implantation and neurogenesis. In contrast, Sox2 expression was undetectable in the anterior foregut of mutants carrying the strongest boundaries, and these animals fully phenocopied loss of SOX2 in this tissue. We propose that enhancer clusters with a high density of regulatory activity can better overcome physical barriers to maintain faithful gene expression and phenotypic robustness.

A year in the salt marsh: Seasonal changes in gill protein expression in the temperate intertidal mussel Geukensia demissa.

Organisms living in temperate and polar regions experience extensive seasonal changes in the physical and biotic environment, including temperature, insolation, and food availability, among other factors. Sessile intertidal organisms respond to such seasonal fluctuations largely through physiological and biochemical means, because their behavioral responses are severely limited. In this study, we used a proteomic approach to examine changes in seasonal protein expression of gill from the intertidal mussel Geukensia demissa, a keystone species of the western Atlantic salt marsh, over the course of one year. Gill tissue of mussels collected in summer had the greatest number of proteins significantly increased in abundance (37 of 592 spots detected on two-dimensional polyacrylamide gels), although autumn mussels revealed a comparable proportion of up-regulated proteins (31 spots). In contrast, the number of proteins changing in abundance in winter and spring mussels were substantially smaller (15 and 9, respectively). Identification of these proteins revealed both expected and unanticipated changes to the proteome. Maintenance of gill cilia dominates in the summer when filter-feeding is most active, as evidenced by cytoskeletal proteins such as tektin-4 and tubulin isoforms; a signal of protection from heat stress is also present in summer (e.g., heat shock cognate 70). In autumn oxidative stress protection (peroxiredoxin-5 and manganese-containing superoxide dismutase) and aerobic ATP synthetic capacity (ATP synthase subunits a and delta) appear to increase. In winter a signal of cold-induced oxidative stress is apparent (Mn-SOD and NADP-dependent isocitrate dehydrogenase), perhaps in association with heavy metal toxicity and exposure to pathogens. Gill tissue from spring shows relatively little environmental acclimatization, other than a possible increase in protein synthesis capacity.