Search

The cnidarian model organism Hydra has long been studied for its remarkable ability to regenerate its head, which is controlled by a head organizer located near the hypostome. The canonical Wnt pathway plays a central role in head organizer function during regeneration and during bud formation, which is the asexual mode of reproduction in Hydra. However, it is unclear how shared the developmental programs of head organizer genesis are in budding and regeneration. Time-series analysis of gene expression changes during head regeneration and budding revealed a set of 298 differentially expressed genes during the 48-h head regeneration and 72-h budding time courses. In order to understand the regulatory elements controlling Hydra head regeneration, we first identified 27,137 open-chromatin elements that are open in one or more sections of the organism body or regenerating tissue. We used histone modification ChIP-seq to identify 9,998 candidate proximal promoter and 3,018 candidate enhancer-like regions respectively. We show that a subset of these regulatory elements is dynamically remodeled during head regeneration and identify a set of transcription factor motifs that are enriched in the enhancer regions activated during head regeneration. Our results show that Hydra displays complex gene regulatory structures of developmentally dynamic enhancers, which suggests that the evolution of complex developmental enhancers predates the split of cnidarians and bilaterians.

We have compared the thermodynamics of substrate and redox partner binding of P450cam to its close homologue, CYP101D1, using isothermal titration calorimetry (ITC). CYP101D1 binds camphor about 10-fold more weakly than P450cam which is consistent with the inability of camphor to cause a complete low- to high-spin shift in CYP101D1. Even so molecular dynamics simulations show that camphor is very stable in the CYP101D1 active site similar to P450cam. ITC data on the binding of the CYP101D1 ferredoxin redox partner (abbreviated Arx) shows that the substrate-bound closed state of CYP101D1 binds Arx more tightly than the substrate-free open form. This is just the opposite to P450cam where Pdx (ferredoxin redox partner of P450cam) favors binding to the P450cam open state. In addition, CYP101D1-Arx binding has a large negative ΔS while the P450cam-Pdx has a much smaller ΔS indicating that interactions at the docking interface are different. The most obvious difference is that PDXD38 which forms an important ion pair with P450camR112 at the center of the interface is ArxL39 in Arx. This suggests that Arx may adopt a different orientation than Pdx in order to optimize nonpolar interactions with ArxL39.

Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting.

The α and β cells act in concert to maintain blood glucose. The α cells release glucagon in response to low levels of glucose to stimulate glycogenolysis in the liver. In contrast, β cells release insulin in response to elevated levels of glucose to stimulate peripheral glucose disposal. Despite these opposing roles in glucose homeostasis, α and β cells are derived from a common progenitor and share many proteins important for glucose sensing and hormone secretion. Results from recent work have underlined these similarities between the two cell types by revealing that β-to-α as well as α-to-β transdifferentiation can take place under certain experimental circumstances. These exciting findings highlight unexpected plasticity of adult islets and offer hope of novel therapeutic paths to replenish β cells in diabetes. In this review, we focus on the transcription factor networks that establish and maintain pancreatic endocrine cell identity and how they may be perturbed to facilitate transdifferentiation.

Mutations in the Aristaless related homeodomain transcription factor (ARX) are associated with a diverse set of X-linked mental retardation and epilepsy syndromes in humans. Although most studies have been focused on its function in the forebrain, ARX is also expressed in other regions of the developing nervous system including the floor plate (FP) of the spinal cord where its function is incompletely understood. To investigate the role of Arx in the FP, we performed gain-of-function studies in the chick using in ovo electroporation, and loss-of-function studies in Arx-deficient mice. We have found that Arx, in conjunction with FoxA2, directly induces Sonic hedgehog (Shh) expression through binding to a Shh floor plate enhancer (SFPE2). We also observed that FoxA2 induces Arx through its transcriptional activation domain whereas Nkx2.2, induced by Shh, abolishes this induction. Our data support a feedback loop model for Arx function; through interactions with FoxA2, Arx positively regulates Shh expression in the FP, and Shh signaling in turn activates Nkx2.2, which suppresses Arx expression. Furthermore, our data are evidence that Arx plays a role as a context dependent transcriptional activator, rather than a primary inducer of Shh expression, potentially explaining how mutations in ARX are associated with diverse, and often subtle, defects.