A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival.

Approximately a quarter of the human genome consists of gene deserts, large regions devoid of genes often located adjacent to developmental genes and thought to contribute to their regulation. However, defining the regulatory functions embedded within these deserts is challenging due to their large size. Here, we explore the cis-regulatory architecture of a gene desert flanking the Shox2 gene, which encodes a transcription factor indispensable for proximal limb, craniofacial, and cardiac pacemaker development.

Pre-hypertrophic chondrogenic enhancer landscape of limb and axial skeleton development.

Chondrocyte differentiation controls skeleton development and stature. Here we provide a comprehensive map of chondrocyte-specific enhancers and show that they provide a mechanistic framework through which non-coding genetic variants can influence skeletal development and human stature. Working with fetal chondrocytes isolated from mice bearing a Col2a1 fluorescent regulatory sensor, we identify 780 genes and 2'704 putative enhancers specifically active in chondrocytes using a combination of RNA-seq, ATAC-seq and H3K27ac ChIP-seq.

Investigating the etiology of non-carious cervical lesions: Novel µCT analysis.

Objectives: The etiology of non-carious cervical lesions (NCCLs) is not fully understood, limiting treatment and prevention. Our aim was to evaluate the effect of mechanical loading and acid exposure on the cervical tooth region using a random spectrum loading model that simulates the nature of oral mastication.

Methods: Thirty extracted human premolars were divided into three experimental groups: 1) unloaded teeth immersed in acid (erosion group: Er), 2) loaded teeth immersed in acid (erosion with spectrum loading group: Er-SL), and 3) loaded teeth immersed in distilled water (spectrum loading group: SL).

Inhibitory SMAD6 interferes with BMP-dependent generation of muscle progenitor cells and perturbs proximodistal pattern of murine limb muscles.

The mechanism of pattern formation during limb muscle development remains poorly understood. The canonical view holds that naïve limb muscle progenitor cells (MPCs) invade a pre-established pattern of muscle connective tissue, thereby forming individual muscles. Here, we show that early murine embryonic limb MPCs highly accumulate pSMAD1/5/9, demonstrating active signaling of bone morphogenetic proteins (BMP) in these cells.

Genetic cold cases: lessons from solving complex congenital limb disorders.

Congenital genetic disorders affecting limb morphology in humans and other mammals are particularly well described, due to both their rather high frequencies of occurrence and the ease of their detection when expressed as severe forms. In most cases, their molecular and cellular etiology remained unknown long after their initial description, often for several decades, and sometimes close to a century. Over the past 20 yr, however, experimental and conceptual advances in our understanding of gene regulation, in particular over large genomic distances, have allowed these cold cases to be reopened and, eventually, for some of them to be solved.

Context-dependent enhancer function revealed by targeted inter-TAD relocation.

The expression of some genes depends on large, adjacent regions of the genome that contain multiple enhancers. These regulatory landscapes frequently align with Topologically Associating Domains (TADs), where they integrate the function of multiple similar enhancers to produce a global, TAD-specific regulation. We asked if an individual enhancer could overcome the influence of one of these landscapes, to drive gene transcription.

Cell-specific alterations in Pitx1 regulatory landscape activation caused by the loss of a single enhancer.

Developmental genes are frequently controlled by multiple enhancers sharing similar specificities. As a result, deletions of such regulatory elements have often failed to reveal their full function. Here, we use the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion of one of its enhancers (Pen).

Modeling Single-Molecule Conformations of the HoxD Region in Mouse Embryonic Stem and Cortical Neuronal Cells.

Complex architectural rearrangements are associated to the control of the HoxD genes in different cell types; yet, how they are implemented in single cells remains unknown. By use of polymer models, we dissect the locus 3D structure at the single DNA molecule level in mouse embryonic stem and cortical neuronal cells, as the HoxD cluster changes from a poised to a silent state. Our model describes published Hi-C, 3-way 4C, and FISH data with high accuracy and is validated against independent 4C data on the Nsi-SB 0.

Preformed chromatin topology assists transcriptional robustness of during limb development.

Long-range gene regulation involves physical proximity between enhancers and promoters to generate precise patterns of gene expression in space and time. However, in some cases, proximity coincides with gene activation, whereas, in others, preformed topologies already exist before activation. In this study, we investigate the preformed configuration underlying the regulation of the gene by its unique limb enhancer, the , in vivo during mouse development.

Chromatin topology in development and disease.

The discovery of domains of preferential interaction or Topologically Associating Domains (TADs) has provided a framework to understand the relation between enhancers and promoters within intricate regulatory landscapes. It has also enabled the conceptualization of the effect of non-coding structural variants on TADs structure and insulation and reveal new patho-mechanisms leading to disease. Here, we will review current knowledge on enhancer-promoter communication in relation to TAD structure.

Serial genomic inversions induce tissue-specific architectural stripes, gene misexpression and congenital malformations.

Balanced chromosomal rearrangements such as inversions and translocations can cause congenital disease or cancer by inappropriately rewiring promoter-enhancer contacts. To study the potentially pathogenic consequences of balanced chromosomal rearrangements, we generated a series of genomic inversions by placing an active limb enhancer cluster from the Epha4 regulatory domain at different positions within a neighbouring gene-dense region and investigated their effects on gene regulation in vivo in mice. Expression studies and high-throughput chromosome conformation capture from embryonic limb buds showed that the enhancer cluster activated several genes downstream that are located within asymmetric regions of contact, the so-called architectural stripes.

Similarities and differences in the regulation of HoxD genes during chick and mouse limb development.

In all tetrapods examined thus far, the development and patterning of limbs require the activation of gene members of the HoxD cluster. In mammals, they are regulated by a complex bimodal process that controls first the proximal patterning and then the distal structure. During the shift from the former to the latter regulation, this bimodal regulatory mechanism allows the production of a domain with low Hoxd gene expression, at which both telomeric (T-DOM) and centromeric regulatory domains (C-DOM) are silent.

Dynamic 3D chromatin architecture contributes to enhancer specificity and limb morphogenesis.

The regulatory specificity of enhancers and their interaction with gene promoters is thought to be controlled by their sequence and the binding of transcription factors. By studying Pitx1, a regulator of hindlimb development, we show that dynamic changes in chromatin conformation can restrict the activity of enhancers. Inconsistent with its hindlimb-restricted expression, Pitx1 is controlled by an enhancer (Pen) that shows activity in forelimbs and hindlimbs.

An SSP-PCR method for the rapid detection of disease-associated alleles HLA-A*29 and HLA-B*51.

HLA-A*29 and HLA-B*51 are associated with birdshot uveitis and Behçet's disease, respectively, and are used as a diagnostic criterion in patients with suspected disease, requiring their detection in diagnostic laboratories. While commercial tests for individual HLA alleles are available for other disease-associated HLA variants, no similar allele-specific assays are available for HLA-A*29 and HLA-B*51. Here, we report sequence-specific priming-polymerase chain reaction (SSP-PCR) methods for the detection of HLA-A*29 and HLA-B*51 using a single PCR reaction per allele.

Polymer physics predicts the effects of structural variants on chromatin architecture.

Structural variants (SVs) can result in changes in gene expression due to abnormal chromatin folding and cause disease. However, the prediction of such effects remains a challenge. Here we present a polymer-physics-based approach (PRISMR) to model 3D chromatin folding and to predict enhancer-promoter contacts.

The three-dimensional genome: regulating gene expression during pluripotency and development.

The precise expression of genes in time and space during embryogenesis is largely influenced by communication between enhancers and promoters, which is propagated and governed by the physical proximity of these elements in the nucleus. Here, we review how chromatin domains organize the genome by guiding enhancers to their target genes thereby preventing non-specific interactions with other neighboring regions. We also discuss the dynamics of chromatin interactions between enhancers and promoters, as well as the consequent changes in gene expression, that occur in pluripotent cells and during development.

Integration of Shh and Fgf signaling in controlling gene expression in cultured limb cells.

During embryonic development, fields of progenitor cells form complex structures through dynamic interactions with external signaling molecules. How complex signaling inputs are integrated to yield appropriate gene expression responses is poorly understood. In the early limb bud, for instance, Sonic hedgehog () is expressed in the distal posterior mesenchyme, where it acts as a mediator of anterior to posterior (AP) patterning, whereas fibroblast growth factor 8 () is produced by the apical ectodermal ridge (AER) at the distal tip of the limb bud to direct outgrowth along the proximal to distal (PD) axis.

Genome-Wide Binding of Posterior HOXA/D Transcription Factors Reveals Subgrouping and Association with CTCF.

Homeotic genes code for key transcription factors (HOX-TFs) that pattern the animal body plan. During embryonic development, Hox genes are expressed in overlapping patterns and function in a partially redundant manner. In vitro biochemical screens probing the HOX-TF sequence specificity revealed largely overlapping sequence preferences, indicating that co-factors might modulate the biological function of HOX-TFs.

Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding.

Complex regulatory landscapes control the pleiotropic transcriptional activities of developmental genes. For most genes, the number, location, and dynamics of their associated regulatory elements are unknown. In this work, we characterized the three-dimensional chromatin microarchitecture and regulatory landscape of 446 limb-associated gene loci in mouse using Capture-C, ChIP-seq, and RNA-seq in forelimb, hindlimb at three developmental stages, and midbrain.

Control of Hoxd gene transcription in the mammary bud by hijacking a preexisting regulatory landscape.

Vertebrate Hox genes encode transcription factors operating during the development of multiple organs and structures. However, the evolutionary mechanism underlying this remarkable pleiotropy remains to be fully understood. Here, we show that Hoxd8 and Hoxd9, two genes of the HoxD complex, are transcribed during mammary bud (MB) development.

Formation of new chromatin domains determines pathogenicity of genomic duplications.

Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood.