Global genome decompaction leads to stochastic activation of gene expression as a first step toward fate commitment in human hematopoietic cells.

When human cord blood-derived CD34+ cells are induced to differentiate, they undergo rapid and dynamic morphological and molecular transformations that are critical for fate commitment. In particular, the cells pass through a transitory phase known as "multilineage-primed" state. These cells are characterized by a mixed gene expression profile, different in each cell, with the coexpression of many genes characteristic for concurrent cell lineages. The aim of our study is to understand the mechanisms of the establishment and the exit from this transitory state. We investigated this issue using single-cell RNA sequencing and ATAC-seq. Two phases were detected. The first phase is a rapid and global chromatin decompaction that makes most of the gene promoters in the genome accessible for transcription. It results 24 h later in enhanced and pervasive transcription of the genome leading to the concomitant increase in the cell-to-cell variability of transcriptional profiles. The second phase is the exit from the multilineage-primed phase marked by a slow chromatin closure and a subsequent overall down-regulation of gene transcription. This process is selective and results in the emergence of coherent expression profiles corresponding to distinct cell subpopulations. The typical time scale of these events spans 48 to 72 h. These observations suggest that the nonspecificity of genome decompaction is the condition for the generation of a highly variable multilineage expression profile. The nonspecific phase is followed by specific regulatory actions that stabilize and maintain the activity of key genes, while the rest of the genome becomes repressed again by the chromatin recompaction. Thus, the initiation of differentiation is reminiscent of a constrained optimization process that associates the spontaneous generation of gene expression diversity to subsequent regulatory actions that maintain the activity of some genes, while the rest of the genome sinks back to the repressive closed chromatin state.

SINCERITIES: inferring gene regulatory networks from time-stamped single cell transcriptional expression profiles.

MOTIVATION: Single cell transcriptional profiling opens up a new avenue in studying the functional role of cell-to-cell variability in physiological processes. The analysis of single cell expression profiles creates new challenges due to the distributive nature of the data and the stochastic dynamics of gene transcription process. The reconstruction of gene regulatory networks (GRNs) using single cell transcriptional profiles is particularly challenging, especially when directed gene-gene relationships are desired. RESULTS: We developed SINCERITIES (SINgle CEll Regularized Inference using TIme-stamped Expression profileS) for the inference of GRNs from single cell transcriptional profiles. We focused on time-stamped cross-sectional expression data, commonly generated from transcriptional profiling of single cells collected at multiple time points after cell stimulation. SINCERITIES recovers directed regulatory relationships among genes by employing regularized linear regression (ridge regression), using temporal changes in the distributions of gene expressions. Meanwhile, the modes of the gene regulations (activation and repression) come from partial correlation analyses between pairs of genes. We demonstrated the efficacy of SINCERITIES in inferring GRNs using in silico time-stamped single cell expression data and single cell transcriptional profiles of THP-1 monocytic human leukemia cells. The case studies showed that SINCERITIES could provide accurate GRN predictions, significantly better than other GRN inference algorithms such as TSNI, GENIE3 and JUMP3. Moreover, SINCERITIES has a low computational complexity and is amenable to problems of extremely large dimensionality. Finally, an application of SINCERITIES to single cell expression data of T2EC chicken erythrocytes pointed to BATF as a candidate novel regulator of erythroid development. AVAILABILITY AND IMPLEMENTATION: MATLAB and R version of SINCERITIES are freely available from the following websites: http://www.cabsel.ethz.ch/tools/sincerities.html and https://github.com/CABSEL/SINCERITIES. The single cell THP-1 and T2EC transcriptional profiles are available from the original publications (Kouno et al., 2013; Richard et al., 2016). The in silico single cell data are available on SINCERITIES websites. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Single-Cell-Based Analysis Highlights a Surge in Cell-to-Cell Molecular Variability Preceding Irreversible Commitment in a Differentiation Process.

In some recent studies, a view emerged that stochastic dynamics governing the switching of cells from one differentiation state to another could be characterized by a peak in gene expression variability at the point of fate commitment. We have tested this hypothesis at the single-cell level by analyzing primary chicken erythroid progenitors through their differentiation process and measuring the expression of selected genes at six sequential time-points after induction of differentiation. In contrast to population-based expression data, single-cell gene expression data revealed a high cell-to-cell variability, which was masked by averaging. We were able to show that the correlation network was a very dynamical entity and that a subgroup of genes tend to follow the predictions from the dynamical network biomarker (DNB) theory. In addition, we also identified a small group of functionally related genes encoding proteins involved in sterol synthesis that could act as the initial drivers of the differentiation. In order to assess quantitatively the cell-to-cell variability in gene expression and its evolution in time, we used Shannon entropy as a measure of the heterogeneity. Entropy values showed a significant increase in the first 8 h of the differentiation process, reaching a peak between 8 and 24 h, before decreasing to significantly lower values. Moreover, we observed that the previous point of maximum entropy precedes two paramount key points: an irreversible commitment to differentiation between 24 and 48 h followed by a significant increase in cell size variability at 48 h. In conclusion, when analyzed at the single cell level, the differentiation process looks very different from its classical population average view. New observables (like entropy) can be computed, the behavior of which is fully compatible with the idea that differentiation is not a "simple" program that all cells execute identically but results from the dynamical behavior of the underlying molecular network.

CALISTA: Clustering and LINEAGE Inference in Single-Cell Transcriptional Analysis.

We present Clustering and Lineage Inference in Single-Cell Transcriptional Analysis (CALISTA), a numerically efficient and highly scalable toolbox for an end-to-end analysis of single-cell transcriptomic profiles. CALISTA includes four essential single-cell analyses for cell differentiation studies, including single-cell clustering, reconstruction of cell lineage specification, transition gene identification, and cell pseudotime ordering, which can be applied individually or in a pipeline. In these analyses, we employ a likelihood-based approach where single-cell mRNA counts are described by a probabilistic distribution function associated with stochastic gene transcriptional bursts and random technical dropout events. We illustrate the efficacy of CALISTA using single-cell gene expression datasets from different single-cell transcriptional profiling technologies and from a few hundreds to tens of thousands of cells. CALISTA is freely available on https://www.cabselab.com/calista.

Constraints on Human CD34+ Cell Fate due to Lentiviral Vectors Can Be Relieved by Valproic Acid.

The initial stages following the in vitro cytokine stimulation of human cord blood CD34+ cells overlap with the period when lentiviral gene transfer is typically performed. Single-cell transcriptional profiling and time-lapse microscopy were used to investigate how the vector-cell crosstalk impacts on the fate decision process. The single-cell transcription profiles were analyzed using a new algorithm, and it is shown that lentiviral transduction during the early stages of stimulation modifies the dynamics of the fate choice process of the CD34+ cells. The cells transduced with a lentiviral vector are biased toward the common myeloid progenitor lineage. Valproic acid, a histone deacetylase inhibitor known to increase the grafting potential of the CD34+ cells, improves the transduction efficiency to almost 100%. The cells transduced in the presence of valproic acid can subsequently undergo normal fate commitment. The higher gene transfer efficiency did not alter the genomic integration profile of the vector. These observations open the way to substantially improving lentiviral gene transfer protocols.