Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3′ end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs.

Aims Atrial fibrillation (AF) is a progressive cardiac arrhythmia that increases the risk of hospitalization and adverse cardiovascular events. There is a clear demand for more inclusive and large-scale approaches to understand the molecular drivers responsible for AF, as well as the fundamental mechanisms governing the transition from paroxysmal to persistent and permanent forms. In this study, we aimed to create a molecular map of AF and find the distinct molecular programs underlying cell type-specific atrial remodelling and AF progression. Methods and Results We used a sheep model of long-standing, tachypacing-induced AF, sampled right and left atrial tissue and isolated cardiomyocytes from control, intermediate (transition) and late time points during AF progression, and performed transcriptomic and proteome profiling. We have merged all these layers of information into a meaningful 3-component space in which we explored the genes and proteins detected and their common patterns of expression. Our data-driven analysis points at extracellular matrix remodelling, inflammation, ion channel, myofibril structure, mitochondrial complexes, chromatin remodelling, and genes related to neural function, as well as critical regulators of cell proliferation as hallmarks of AF progression. Most important, we prove that these changes occur at early transitional stages of the disease, but not at later stages, and that the left atrium undergoes significantly more profound changes than the right atrium in its expression program. The pattern of dynamic changes in gene and protein expression replicate the electrical and structural remodelling demonstrated previously in the sheep and in humans, and uncover novel mechanisms potentially relevant for disease treatment. Conclusions Transcriptomic and proteomic analysis of AF progression in a large animal model shows that significant changes occur at early stages, and that among others involve previously undescribed increase in mitochondria, changes to the chromatin of atrial cardiomyocytes, and genes related to neural function and cell proliferation. Translational Perspective We have generated a detailed molecular map of AF progression in a clinically relevant large-animal model. Such data would be very difficult if not impossible to obtain from patients. Our results provide a framework for a comprehensive molecular analysis of the disease, pointing to novel avenues of research toward identifying early events that can lead to therapeutically targets to prevent AF-induced atrial remodelling.

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). By combining single cell transcriptomics and an in-embryo cell tracing approach, we observe an increased fraction of Pitx1 non/low-expressing cells and a decreased fraction of Pitx1 high-expressing cells. We find that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. This locus mis-activation induces a localized heterochrony and a concurrent loss of irregular connective tissue, eventually leading to a clubfoot phenotype. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes.

Pluripotent cells are a transient population present in the early mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while simultaneously suppressing lineage specification. Interestingly, these factors are not exclusive to uncommitted cells, but are also expressed during early phases of differentiation. However, their role in the transition from pluripotency to lineage specification is largely unknown. Using genetic models for controlled Oct4 or Nanog expression during postimplantation stages, we found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that the HoxB cluster is coordinately regulated in this way by OCT4 binding sites located at the 3' end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the pre-committed epiblast, but are also necessary for the proper deployment of subsequent developmental programs. Dual regulation of Hox genes by pluripotency factors Lopez-Jimenez et al. 3

Most developmental genes rely on multiple transcriptional enhancers for their accurate expression during embryogenesis. Because enhancers may have partially redundant activities, the loss of one of them often leads to a partial loss of gene expression and concurrent moderate phenotypic outcome, if any. While such a phenomenon has been observed in many instances, the nature of the underlying mechanisms remains elusive. We used the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion in vivo of one of its enhancers (Pen), which normally accounts for 30 percent of Pitx1 expression in hindlimb buds. By combining single cell transcriptomics and a novel in embryo cell tracing approach, we observed that this global decrease in Pitx1 expression results from both an increase in the number of non- or low-expressing cells, and a decrease in the number of high-expressing cells. We found that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. The resulting increase in Pitx1 non/low-expressing cells eventually affects the proximal limb more severely than the distal limb, leading to a clubfoot phenotype likely produced through a localized heterochrony and concurrent loss of irregular connective tissue. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes.