The transcriptome dynamics of single cells during the cell cycle.

The cell cycle is among the most basic phenomena in biology. Despite advances in single-cell analysis, dynamics and topology of the cell cycle in high-dimensional gene expression space remain largely unknown. We developed a linear analysis of transcriptome data which reveals that cells move along a planar circular trajectory in transcriptome space during the cycle. Non-cycling gene expression adds a third dimension causing helical motion on a cylinder. We find in immortalized cell lines that cell cycle transcriptome dynamics occur largely independently from other cellular processes. We offer a simple method ("Revelio") to order unsynchronized cells in time. Precise removal of cell cycle effects from the data becomes a straightforward operation. The shape of the trajectory implies that each gene is upregulated only once during the cycle, and only two dynamic components represented by groups of genes drive transcriptome dynamics. It indicates that the cell cycle has evolved to minimize changes of transcriptional activity and the related regulatory effort. This design principle of the cell cycle may be of relevance to many other cellular differentiation processes.

Germinal center output is sustained by HELLS-dependent DNA-methylation-maintenance in B cells.

HELLS/LSH (Helicase, Lymphoid Specific) is a SNF2-like chromatin remodelling protein involved in DNA methylation. Its loss-of-function in humans causes humoral immunodeficiency, called ICF4 syndrome (Immunodeficiency, Centromeric Instability, Facial anomalies). Here we show by our newly generated B-cell-specific Hells conditional knockout mouse model that HELLS plays a pivotal role in T-dependent B-cell responses. HELLS deficiency induces accelerated decay of germinal center (GC) B cells and impairs the generation of high affinity memory B cells and circulating antibodies. Mutant GC B cells undergo dramatic DNA hypomethylation and massive de-repression of evolutionary recent retrotransposons, which surprisingly does not directly affect their survival. Instead, they prematurely upregulate either memory B cell markers or the transcription factor ATF4, which is driving an mTORC1-dependent metabolic program typical of plasma cells. Treatment of wild type mice with a DNMT1-specific inhibitor phenocopies the accelerated kinetics, thus pointing towards DNA-methylation maintenance by HELLS being a crucial mechanism to fine-tune the GC transcriptional program and enable long-lasting humoral immunity.

Characterization of Transcription Termination-Associated RNAs: New Insights into their Biogenesis, Tailing, and Expression in Primary Tumors.

Next-generation sequencing has uncovered novel classes of small RNAs (sRNAs) in eukaryotes, in addition to the well-known miRNAs, siRNAs, and piRNAs. In particular, sRNA species arise from transcription start sites (TSSs) and the transcription termination sites (TTSs) of genes. However, a detailed characterization of these new classes of sRNAs is still lacking. Here, we present a comprehensive study of sRNAs derived from TTSs of expressed genes (TTSa-RNAs) in human cell lines and primary tissues. Taking advantage of sRNA-sequencing, we show that TTSa-RNAs are present in the nuclei of human cells, are loaded onto both AGO1 and AGO2, and their biogenesis does not require DICER and AGO2 endonucleolytic activity. TTSa-RNAs display a strong bias against a G residue in the first position at 5' end, a known feature of AGO-bound sRNAs, and a peculiar oligoA tail at 3' end. AGO-bound TTSa-RNAs derive from genes involved in cell cycle progression regulation and DNA integrity checkpoints. Finally, we provide evidence that TTSa-RNAs can be detected by sRNA-Seq in primary human tissue, and their expression increases in tumor samples as compared to nontumor tissues, suggesting that in the future, TTSa-RNAs might be explored as biomarker for diagnosis or prognosis of human malignancies.