During mouse embryogenesis, totipotency is gradually lost, and, at the 16-cell stage, blastomeres begin to bifurcate into trophectoderm (future placenta) and inner cell mass (future fetus). Although this process is well studied, when and how blastomeres acquire the competence for lineage specification remains unclear. Here, we describe that CEBPa becomes up-regulated at the transition from the two- to the four-cell stage by NR5A2 and is also selectively expressed in the trophectoderm at the blastocyst stage. Its knockout decreases the proportion of trophectoderm cells and delays the morula to blastocyst transition. Conversely, CEBPa overexpression in mouse embryonic stem cells, used as a proxy, drives their differentiation into trophectoderm-like cells, enabling the identification of CEBPa-regulated trophectoderm-specific enhancers. A subset of these enhancers, associated with key trophectoderm-related transcription factor genes, is primed or activated in four- and eight-cell embryos. Together, our data suggest that CEBPa plays a role in the installment of trophectoderm competence before the first lineage bifurcation and in trophectoderm specification.

Here, we describe how the speed of C/EBPα-induced B cell to macrophage transdifferentiation (BMT) can be regulated, using both mouse and human models. The identification of a mutant of C/EBPα (C/EBPαR35A) that greatly accelerates BMT helped to illuminate the mechanism. Thus, incoming C/EBPα binds to PU.1, an obligate partner expressed in B cells, leading to the release of PU.1 from B cell enhancers, chromatin closing and silencing of the B cell program. Released PU.1 redistributes to macrophage enhancers newly occupied by C/EBPα, causing chromatin opening and activation of macrophage genes. All these steps are accelerated by C/EBPαR35A, initiated by its increased affinity for PU.1. Wild-type C/EBPα is methylated by Carm1 at arginine 35 and the enzyme’s perturbations modulate BMT velocity as predicted from the observations with the mutant. Increasing the proportion of unmethylated C/EBPα in granulocyte/macrophage progenitors by inhibiting Carm1 biases the cell’s differentiation toward macrophages, suggesting that cell fate decision velocity and lineage directionality are closely linked processes.

Cell identity is orchestrated through an interplay between transcription factor (TF) action and genome architecture. The mechanisms used by TFs to shape three-dimensional (3D) genome organization remain incompletely understood. Here we present evidence that the lineage-instructive TF CEBPA drives extensive chromatin compartment switching and promotes the formation of long-range chromatin hubs during induced B cell-to-macrophage transdifferentiation. Mechanistically, we find that the intrinsically disordered region (IDR) of CEBPA undergoes in vitro phase separation (PS) dependent on aromatic residues. Both overexpressing B cells and native CEBPA-expressing cell types such as primary granulocyte-macrophage progenitors, liver cells, and trophectoderm cells reveal nuclear CEBPA foci and long-range 3D chromatin hubs at CEBPA-bound regions. In short, we show that CEBPA can undergo PS through its IDR, which may underlie in vivo foci formation and suggest a potential role of PS in regulating CEBPA function.

ABSTRACT Cell fate decisions are driven by lineage-restricted transcription factors but how they are regulated is incompletely understood. The C/EBPα-induced B cell to macrophage transdifferentiation (BMT) is a powerful system to address this question. Here we describe that C/EBPα with a single arginine mutation (C/EBPα R35A ) induces a dramatically accelerated BMT in mouse and human cells. Changes in the expression of lineage-restricted genes occur as early as within 1 hour compared to 18 hours with the wild type. Mechanistically C/EBPα R35A exhibits an increased affinity for PU.1, a bi-lineage transcription factor required for C/EBPα-induced BMT. The complex induces more rapid chromatin accessibility changes and an enhanced relocation (stealing) of PU.1 from B cell to myeloid gene regulatory elements. Arginine 35 is methylated by Carm1 and inhibition of the enzyme accelerates BMT, similar to the mutant. Our data suggest that the relative proportions of methylated and unmethylated C/EBPα in a bipotent progenitor can determine the velocity of cell fate choice and lineage directionality.

IL-6 has been shown to be required for somatic cell reprogramming into induced pluripotent stem cells (iPSCs). However, how Il6 expression is regulated and whether it plays a role during embryo development remains unknown. Here, we describe that IL-6 is necessary for C/EBPα-enhanced reprogramming of B cells into iPSCs but not for B cell to macrophage transdifferentiation. C/EBPα overexpression activates both Il6 and Il6ra genes in B cells and in PSCs. In embryo development, Cebpa is enriched in the trophectoderm of blastocysts together with Il6, while Il6ra is mostly expressed in the inner cell mass (ICM). In addition, Il6 expression in blastocysts requires Cebpa. Blastocysts secrete IL-6 and neutralization of the cytokine delays the morula to blastocyst transition. The observed requirement of C/EBPα-regulated IL-6 signaling for pluripotency during somatic cell reprogramming thus recapitulates a physiologic mechanism in which the trophectoderm acts as niche for the ICM through the secretion of IL-6.

A key problem in understanding chordate evolution has been the origin of sessility of ascidians, and whether the appendicularian free-living style represents a primitive or derived condition of tunicates. To address this problem, we performed comprehensive developmental and genomic comparative analyses of the cardiopharyngeal gene regulatory network (GRN) between appendicularians and ascidians. Our results reveal that the cardiopharyngeal GRN has suffered a process of evolutionary deconstruction with massive ancestral losses of genes ( Mesp , Ets1/2 , Gata4/5/6 , Mek1/2 , Tbx1/10 , and RA- and FGF-signaling related genes) and subfunctions (e.g. FoxF , Islet , Ebf , Mrf , Dach and Bmp signaling). These losses have led to the deconstruction of two modules of the cardiopharyngeal GRN that in ascidians are related to early and late multipotent state cells involved in lineage fate determination towards first and secondary heart fields, and siphon muscle. Our results allow us to propose an evolutionary scenario, in which the evolutionary deconstruction of the cardiopharyngeal GRN has had an adaptive impact on the acceleration of the developmental cardiac program, the redesign of the cardiac architecture into an open-wide laminar structure, and the loss of pharyngeal muscle. Our findings, therefore, provide evidence supporting that the ancestral tunicate had a sessile ascidian-like lifestyle, and points to the deconstruction of the cardiopharyngeal GRN in appendicularians as a key event that facilitated the evolution of their pelagic free-living style connected to the innovation of the house.

The COVID-19 pandemic has posed and is continuously posing enormous societal and health challenges worldwide. The research community has mobilized to develop novel projects to find a cure or a vaccine, as well as to contribute to mass testing, which has been a critical measure to contain the infection in several countries. Through this article, we share our experiences and learnings as a group of volunteers at the Centre for Genomic Regulation (CRG) in Barcelona, Spain. As members of the ORFEU project, an initiative by the Government of Catalonia to achieve mass testing of people at risk and contain the epidemic in Spain, we share our motivations, challenges and the key lessons learnt, which we feel will help better prepare the global society to address similar situations in the future.

The COVID-19 pandemic has posed and is continuously posing enormous societal and health challenges worldwide. The research community has mobilized to develop novel projects to find a cure or a vaccine, as well as to contribute to mass testing, which has been a critical measure to contain the infection in several countries. Through this article, we share our experiences and learnings as a group of volunteers at the Centre for Genomic Regulation (CRG) in Barcelona, Spain. As members of the ORFEU project, an initiative by the Government of Catalonia to achieve mass testing of people at risk and contain the epidemic in Spain, we share our motivations, challenges and the key lessons learnt, which we feel will help better prepare the global society to address similar situations in the future.

Forced transcription factor expression can transdifferentiate somatic cells into other specialised cell types or reprogram them into induced pluripotent stem cells (iPSCs) with variable efficiency. To better understand the heterogeneity of these processes, we used single-cell RNA sequencing to follow the transdifferentation of murine pre-B cells into macrophages as well as their reprogramming into iPSCs. Even in these highly efficient systems, there was substantial variation in the speed and path of fate conversion. We predicted and validated that these differences are inversely coupled and arise in the starting cell population, with Mychigh large pre-BII cells transdifferentiating slowly but reprogramming efficiently and Myclow small pre-BII cells transdifferentiating rapidly but failing to reprogram. Strikingly, differences in Myc activity predict the efficiency of reprogramming across a wide range of somatic cell types. These results illustrate how single cell expression and computational analyses can identify the origins of heterogeneity in cell fate conversion processes.