The loopometer: a quantitative in vivo assay for DNA-looping proteins.

Proteins that can bring together separate DNA sites, either on the same or on different DNA molecules, are critical for a variety of DNA-based processes. However, there are no general and technically simple assays to detect proteins capable of DNA looping in vivo nor to quantitate their in vivo looping efficiency. Here, we develop a quantitative in vivo assay for DNA-looping proteins in Escherichia coli that requires only basic DNA cloning techniques and a LacZ assay. The assay is based on loop assistance, where two binding sites for the candidate looping protein are inserted internally to a pair of operators for the E. coli LacI repressor. DNA looping between the sites shortens the effective distance between the lac operators, increasing LacI looping and strengthening its repression of a lacZ reporter gene. Analysis based on a general model for loop assistance enables quantitation of the strength of looping conferred by the protein and its binding sites. We use this 'loopometer' assay to measure DNA looping for a variety of bacterial and phage proteins.

Characterization of human variants in obesity-related SIM1 protein identifies a hot-spot for dimerization with the partner protein ARNT2.

The bHLH (basic helix-loop-helix) PAS (Per/Arnt/Sim) transcription factor SIM1 (single-minded 1) is important for development and function of regions of the hypothalamus that regulate energy homoeostasis and the feeding response. Low-activity SIM1 variants have been identified in individuals with severe early-onset obesity, but the underlying molecular causes of impaired function are unknown. In the present study we assess a number of human SIM1 variants with reduced activity and determine that impaired function is frequently due to defects in dimerization with the essential partner protein ARNT2 (aryl hydrocarbon nuclear translocator 2). Equivalent variants generated in the highly related protein SIM2 (single-minded 2) produce near-identical impaired function and dimerization defects, indicating that these effects are not unique to the structure of SIM1. On the basis of these data, we predict that other select SIM1 and SIM2 variants reported in human genomic databases will also be deficient in activity, and identify two new low-activity SIM1 variants (V290E and V326F) present in the population. The cumulative data is used in homology modelling to make novel observations about the dimerization interface between the PAS domains of SIM1 and ARNT2, and to define a mutational 'hot-spot' in SIM1 that is critical for protein function.

Epigenetic Control of Cell Potency and Fate Determination during Mammalian Gastrulation.

Pluripotent embryonic stem cells have a unique and characteristic epigenetic profile, which is critical for differentiation to all embryonic germ lineages. When stem cells exit the pluripotent state and commit to lineage-specific identities during the process of gastrulation in early embryogenesis, extensive epigenetic remodelling mediates both the switch in cellular programme and the loss of potential to adopt alternative lineage programmes. However, it remains to be understood how the stem cell epigenetic profile encodes pluripotency, or how dynamic epigenetic regulation helps to direct cell fate specification. Recent advances in stem cell culture techniques, cellular reprogramming, and single-cell technologies that can quantitatively profile epigenetic marks have led to significant insights into these questions, which are important for understanding both embryonic development and cell fate engineering. This review provides an overview of key concepts and highlights exciting new advances in the field.

The ever-growing world of gastruloids: autogenous models of mammalian embryogenesis.

During early development, extrinsic cues prompt a collection of pluripotent cells to begin the extensive process of cellular differentiation that gives rise to all tissues in the mammalian embryo, a process known as gastrulation. Advances in stem cell biology have resulted in the generation of stem cell-based in vitro models of mammalian gastrulation called gastruloids. Gastruloids and subsequent gastruloid-based models are tractable, scalable and more accessible than mammalian embryos. As such, they have opened an unprecedented avenue for modelling in vitro self-organisation, patterning and fate specification. This review focuses on discussing the recent advances of this rapidly moving research area, clarifying what structures they model and the underlying signal hierarchy. We highlight the exciting potential of these models and where the field might be heading.

An Optimized Protocol for ChIP-Seq from Human Embryonic Stem Cell Cultures.

Chromatin immunoprecipitation (ChIP) followed by next-generation sequencing is a powerful technique that characterizes the genome-wide DNA-binding profile of a protein of interest. The general ChIP-seq workflow has been applied widely to many sample types and target proteins, but sample-specific optimization of various steps is necessary to achieve high-quality data. This protocol is specifically optimized for cultured human embryonic stem cells (hESCs), including steps to check sample quality and non-specific enrichment of "hyper-ChIPable" regions prior to sequencing. For complete details on the use and execution of this protocol, please refer to Gunne-Braden et al. (2020).

Functional Analysis of the SIM1 Variant p.G715V in 2 Patients With Obesity.

CONTEXT: Single-minded homologue 1 (SIM1) is a transcription factor with several physiological and developmental functions. Haploinsufficiency of SIM1 is associated with early-onset obesity with or without Prader-Willi-like (PWL) features and may exhibit incomplete penetrance. CASE DESCRIPTION: Next-generation sequencing was performed for 2 male patients with obesity, including 1 man presenting with intellectual disability (ID), body mass index (BMI) of 47.4, and impulse-control disorder, and the other man with early obesity (BMI of 36); sequencing revealed a missense variant in SIM1 (c.2144G>T; p.G715V) in both individuals. Previous studies have identified several disease-associated variants that fall near the p.G715V variant within the C-terminal domain of SIM1. We examined p.G715V variant stability and activity in a doxycycline-inducible stable cell line transfected with an artificial reporter construct and either ARNT or ARNT2 as a partner protein. CONCLUSIONS: Functional testing of the p.G715V variant revealed a significant reduction in SIM1-mediated transcriptional activity. We also generated the first ab initio hybrid protein model for full-length SIM1 to show the predicted spatial relationship between p.G715V and other previously described variants in this region and identified a putative mutation hotspot within the C-terminus. Significant clinical heterogeneity has been observed in patients with SIM1 variants, particularly with regards to the PWL phenotype. In the patient with ID, a second variant of uncertain significance in CHD2 was identified that may contribute to his ID and behavioral disturbances, emphasizing the role of additional genetic modifiers.

Disruption of the homeodomain transcription factor orthopedia homeobox (Otp) is associated with obesity and anxiety.

OBJECTIVE: Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis. METHODS: In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity. RESULTS: We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. OtpR108W/+ mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder. CONCLUSIONS: OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis.

MAGED1 is a novel regulator of a select subset of bHLH PAS transcription factors.

Transcription factors of the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) family generally have critical and nonredundant biological roles, but some bHLH PAS proteins compete for common cofactors or recognise similar DNA elements. Identifying factors that regulate function of bHLH PAS proteins, particularly in cells where multiple family members are coexpressed, is important for understanding bHLH PAS factor biology. This study identifies and characterises a novel interaction between melanoma-associated antigen D1 (MAGED1) and select members of the bHLH PAS transcription factor family. MAGED1 binds and positively regulates the transcriptional activity of family members SIM1, SIM2, NPAS4 and ARNT2, but does not interact with AhR, HIF1α and ARNT. This interaction is mediated by PAS repeat regions which also form the interface for bHLH PAS dimerisation, and accordingly MAGED1 is not found in complex with bHLH PAS dimers. We show that MAGED1 does not affect bHLH PAS protein levels and cannot be acting as a coactivator of transcriptionally active heterodimers, but rather appears to interact with nascent bHLH PAS proteins in the cytoplasm to enhance their function prior to nuclear import. As a selective regulator, MAGED1 may play an important role in the biology of these specific factors and in general bHLH PAS protein dynamics.

Inducible and reversible lentiviral and Recombination Mediated Cassette Exchange (RMCE) systems for controlling gene expression.

Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.

bHLH-PAS proteins in cancer.

Mammalian basic HLH (helix-loop-helix)-PER-ARNT-SIM (bHLH-PAS) proteins are heterodimeric transcription factors that sense and respond to environmental signals (such as pollutants) or to physiological signals (for example, hypoxia and circadian rhythms) through their two PAS domains. PAS domains form a generic three-dimensional fold, which commonly contains an internal cavity capable of small-molecule binding and outer surfaces adept at protein-protein interactions. These proteins are important in several pro-tumour and antitumour pathways and their activities can be modulated by both natural metabolites and oncometabolites. Recently determined structures and successful small-molecule screening programmes are now providing new opportunities to discover selective agonists and antagonists directed against this multitasking family of transcription factors.

Rare variants in single-minded 1 (SIM1) are associated with severe obesity.

Single-minded 1 (SIM1) is a basic helix-loop-helix transcription factor involved in the development and function of the paraventricular nucleus of the hypothalamus. Obesity has been reported in Sim1 haploinsufficient mice and in a patient with a balanced translocation disrupting SIM1. We sequenced the coding region of SIM1 in 2,100 patients with severe, early onset obesity and in 1,680 controls. Thirteen different heterozygous variants in SIM1 were identified in 28 unrelated severely obese patients. Nine of the 13 variants significantly reduced the ability of SIM1 to activate a SIM1-responsive reporter gene when studied in stably transfected cells coexpressing the heterodimeric partners of SIM1 (ARNT or ARNT2). SIM1 variants with reduced activity cosegregated with obesity in extended family studies with variable penetrance. We studied the phenotype of patients carrying variants that exhibited reduced activity in vitro. Variant carriers exhibited increased ad libitum food intake at a test meal, normal basal metabolic rate, and evidence of autonomic dysfunction. Eleven of the 13 probands had evidence of a neurobehavioral phenotype. The phenotypic similarities between patients with SIM1 deficiency and melanocortin 4 receptor (MC4R) deficiency suggest that some of the effects of SIM1 deficiency on energy homeostasis are mediated by altered melanocortin signaling.