Retrospective analysis of enhancer activity and transcriptome history

Cell state changes in development and disease are controlled by gene regulatory networks, the dynamics of which are difficult to track in real time. Here, we utilize an inducible DCM-RNA-polymerase-subunit-b fusion protein, to label active genes and enhancers with a bacterial methylation mark that does not affect gene transcription and is propagated in S-phase. We applied this DCM-time-machine (DCM-TM) technology to study intestinal homeostasis, following enterocyte differentiation back in time, revealing rapid and simultaneous activation of enhancers and nearby genes during intestinal stem cell (ISC) differentiation. We provide new insights in the absorptive-secretory lineage decision in ISC differentiation, and show that ISCs retain a unique chromatin landscape required to maintain ISC identity and delineate future expression of differentiation associated genes. DCM-TM has wide applicability in tracking cell states, providing new insights in the regulatory networks underlying cell state changes in development and differentiation.

A defined glycosylation regulatory network modulates total glycome dynamics during pluripotency state transition

Embryonic stem cells (ESCs) and epiblast-like cells (EpiLCs) recapitulate in vitro the epiblast first cell lineage decision, allowing characterization of the molecular mechanisms underlying pluripotent state transition. Here, we performed a comprehensive and comparative analysis of total glycomes of mouse ESCs and EpiLCs, revealing that overall glycosylation undergoes dramatic changes from early stages of development. Remarkably, we showed for the first time the presence of a developmentally regulated network orchestrating glycosylation changes and identified polycomb repressive complex 2 (PRC2) as a key component involved in this process. Collectively, our findings provide novel insights into the naïve-to-primed pluripotent state transition and advance the understanding of glycosylation complex regulation during early mouse embryonic development.

Early lineage segregation of the retinal basal glia in the Drosophila eye disc

The retinal basal glia (RBG) is a group of glia that migrates from the optic stalk into the third instar larval eye disc while the photoreceptor cells (PR) are differentiating. The RBGs are grouped into three major classes based on molecular and morphological characteristics: surface glia (SG), wrapping glia (WG) and carpet glia (CG). The SGs migrate and divide. The WGs are postmitotic and wraps PR axons. The CGs have giant nucleus and extensive membrane extension that each covers half of the eye disc. In this study, we used lineage tracing methods to determine the lineage relationships among these glia subtypes and the temporal profile of the lineage decisions for RBG development. We found that the CG lineage segregated from the other RBG very early in the embryonic stage. It has been proposed that the SGs migrate under the CG membrane, which prevented SGs from contacting with the PR axons lying above the CG membrane. Upon passing the front of the CG membrane, which is slightly behind the morphogenetic furrow that marks the front of PR differentiation, the migrating SG contact the nascent PR axon, which in turn release FGF to induce SGs’ differentiation into WG. Interestingly, we found that SGs are equally distributed apical and basal to the CG membrane, so that the apical SGs are not prevented from contacting PR axons by CG membrane. Clonal analysis reveals that the apical and basal RBG are derived from distinct lineages determined before they enter the eye disc. Moreover, the basal SG lack the competence to respond to FGFR signaling, preventing its differentiation into WG. Our findings suggest that this novel glia-to-glia differentiation is both dependent on early lineage decision and on a yet unidentified regulatory mechanism, which can provide spatiotemporal coordination of WG differentiation with the progressive differentiation of photoreceptor neurons.

Lineage Decision-Making within Normal Haematopoietic and Leukemic Stem Cells

To produce the wide range of blood and immune cell types, haematopoietic stem cells can “choose” directly from the entire spectrum of blood cell fate-options. Affiliation to a single cell lineage can occur at the level of the haematopoietic stem cell and these cells are therefore a mixture of some pluripotent cells and many cells with lineage signatures. Even so, haematopoietic stem cells and their progeny that have chosen a particular fate can still “change their mind” and adopt a different developmental pathway. Many of the leukaemias arise in haematopoietic stem cells with the bulk of the often partially differentiated leukaemia cells belonging to just one cell type. We argue that the reason for this is that an oncogenic insult to the genome “hard wires” leukaemia stem cells, either through development or at some stage, to one cell lineage. Unlike normal haematopoietic stem cells, oncogene-transformed leukaemia stem cells and their progeny are unable to adopt an alternative pathway.

Revisiting the erythroid-myeloid lineage decision – a data-driven dynamical model analysis

It is widely conjectured that the mutually antagonizing pair of transcription factors GATA1 and PU.1, deter-mines the choice between the erythroid and myeloid lineages in hematopoiesis. In theoretical approaches, this appears natural with a bistable switch driving the decision. Recent extensive binding and gene expression experiments with some focus on the triad GATA1, GATA2 and PU.1 indicate that GATA2 may be more involved in this lineage decision than previously anticipated. Here, we analyze these experimental data by modeling regulatory sub-networks with deterministic rate equations. Using network dynamical parameters determined by the data, we deduce from increasing the self-interaction bindings in silico among the triad genes that GATA2 and PU.1 exhibit non-linear behavior with one unstable and one stable state. This is in contrast to GATA1, which shows smoother behavior. We extend the network to include the downstream regulators FOG1 and CEBPA, and extract the nature of the corresponding regulatory interactions, excitatory or suppressing, between this pair and the triad by fitting to experimental gene expression time series. Based on this extended network, we simulate and explore different knockout scenarios, providing insight into the role of these regulators in the process of lineage specification, as well as predictions for future experimental validation. We address the mechanism of GATA switching as a mechanism of lineage differentiation by investigating the dynamics of FOG1 regulation by GATA2 and GATA1. Overall, this analysis strongly suggests that within this network, GATA2 is the key driver of erythroid lineage specification through its repression of PU.1, whereas GATA1 appears to be more relevant for the downstream differentiation events.

Discovering sparse transcription factor codes for cell states and state transitions during development

Computational analysis of gene expression to determine both the sequence of lineage choices made by multipotent cells and to identify the genes influencing these decisions is challenging. Here we discover a pattern in the expression levels of a sparse subset of genes among cell types in B- and T-cell developmental lineages that correlates with developmental topologies. We develop a statistical framework using this pattern to simultaneously infer lineage transitions and the genes that determine these relationships. We use this technique to reconstruct the early hematopoietic and intestinal developmental trees. We extend this framework to analyze single-cell RNA-seq data from early human cortical development, inferring a neocortical-hindbrain split in early progenitor cells and the key genes that could control this lineage decision. Our work allows us to simultaneously infer both the identity and lineage of cell types as well as a small set of key genes whose expression patterns reflect these relationships.

Abstract 139: Role of Histone Lysine Demethylase PHF8 in Epigenetic Regulation of Embryonic Stem Cell Cardiac Differentiation

Epigenetic control mechanisms play a key role in the regulation of lineage commitment of stem/progenitor cells, while the epigenetic regulators involved in the determination of cardiomyogenic lineage are incompletely defined. Using in vitro cardiac differentiation system of mESCs with Brachyury and Nkx2.5 selection, we analyzed expression profiles of epigenetic regulators at critical stages of cardiomyogenesis by RT 2 profiler PCR arrays. To identify the potential epigenetic regulators in the cardiac lineage decision, we compared their expression levels in Brachyury + mesodermal cells with Brachyury - cells, and in Nkx2.5 + cardiac progenitor cells with the Nkx2.5 - cells, respectively. To further understand the role of these epigenetic regulators in cardiac lineage commitment, we knockout these genes and investigate their function. For example, deletion of the H3K9me2 demethylase PHF8 in mESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis, without significant differences between differentiating wide-type (ph8 +/Y ) and ph8 -/Y ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. These results reveal the new epigenetic control mechanism in mesodemal and cardiac differentiation and establish a link between the apoptosis and cell lineage decision as well as cardiogenesis.