Davide Cacchiarelli

A Long Noncoding RNA Controls Muscle Differentiation by Functioning as a Competing Endogenous RNA

Summary Recently, a new regulatory circuitry has been identified in which RNAs can crosstalk with each other by competing for shared microRNAs. Such competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules on their targets and thereby impose an additional level of post-transcriptional regulation. Here we identify a muscle-specific long noncoding RNA, linc-MD1, which governs the time of muscle differentiation by acting as a ceRNA in mouse and human myoblasts. Downregulation or overexpression of linc-MD1 correlate with retardation or anticipation of the muscle differentiation program, respectively. We show that linc-MD1 “sponges” miR-133 and miR-135 to regulate the expression of MAML1 and MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we demonstrate that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells. We conclude that the ceRNA network plays an important role in muscle differentiation.

The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells

Defining the transcriptional dynamics of a temporal process such as cell differentiation is challenging owing to the high variability in gene expression between individual cells. Time-series gene expression analyses of bulk cells have difficulty distinguishing early and late phases of a transcriptional cascade or identifying rare subpopulations of cells, and single-cell proteomic methods rely on a priori knowledge of key distinguishing markers. Here we describe Monocle, an unsupervised algorithm that increases the temporal resolution of transcriptome dynamics using single-cell RNA-Seq data collected at multiple time points. Applied to the differentiation of primary human myoblasts, Monocle revealed switch-like changes in expression of key regulatory factors, sequential waves of gene regulation, and expression of regulators that were not known to act in differentiation. We validated some of these predicted regulators in a loss-of function screen. Monocle can in principle be used to recover single-cell gene expression kinetics from a wide array of cellular processes, including differentiation, proliferation and oncogenic transformation.Single-cell expression profiling by RNA-Seq promises to exploit cell-to-cell variation in gene expression to reveal regulatory circuitry governing cell differentiation and other biological processes. Here, we describe Monocle, a novel unsupervised algorithm for ordering cells by progress through differentiation that dramatically increases temporal resolution of expression measurements in a model of skeletal muscle differentiation. This reordering unmasks switch-like changes in expression of key regulatory factors, reveals sequentially organized waves of gene regulation, and exposes novel regulators of cell differentiation. A loss-of function screen revealed that many of these inhibitors act through regulatory elements also used by pro-myogenic factors to activate downstream genes. This study demonstrates that single-cell expression analysis by Monocle can uncover novel regulatory interactions governing differentiation.

miRNAs as serum biomarkers for Duchenne muscular dystrophy

Dystrophin absence in Duchenne muscular dystrophy (DMD) causes severe muscle degeneration. We describe that, as consequence of fibre damage, specific muscle‐miRNAs are released in to the bloodstream of DMD patients and their levels correlate with the severity of the disease. The same miRNAs are abundant also in the blood of mdx mice and recover to wild‐type levels in animals ‘cured’ through exon skipping. Even though creatine kinase (CK) blood levels have been utilized as diagnostic markers of several neuromuscular diseases, including DMD, we demonstrate that they correlate less well with the disease severity. Although the analysis of a larger number of patients should allow to obtain more refined correlations with the different stages of disease progression, we propose that miR‐1, miR‐133, and miR‐206 are new and valuable biomarkers for the diagnosis of DMD and possibly also for monitoring the outcomes of therapeutic interventions in humans. Despite many different DMD therapeutic approaches are now entering clinical trials, a unifying method for assessing the benefit of different treatments is still lacking.

miR-31 modulates dystrophin expression: new implications for Duchenne muscular dystrophy therapy

Duchenne muscular dystrophy (DMD)—which is caused by mutations in the dystrophin gene—is one of the most severe myopathies. Among therapeutic strategies, exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional messenger RNA. Here, we report the identification of a microRNA—miR‐31—that represses dystrophin expression by targeting its 3′ untranslated region. In human DMD myoblasts treated with exon skipping, we demonstrate that miR‐31 inhibition increases dystrophin rescue. These results indicate that interfering with miR‐31 activity can provide an ameliorating strategy for those DMD therapies that are aimed at efficiently recovering dystrophin synthesis.

Dissecting neural differentiation regulatory networks through epigenetic footprinting

Models derived from human pluripotent stem cells that accurately recapitulate neural development in vitro and allow for the generation of specific neuronal subtypes are of major interest to the stem cell and biomedical community. Notch signalling, particularly through the Notch effector HES5, is a major pathway critical for the onset and maintenance of neural progenitor cells in the embryonic and adult nervous system. Here we report the transcriptional and epigenomic analysis of six consecutive neural progenitor cell stages derived from a HES5::eGFP reporter human embryonic stem cell line. Using this system, we aimed to model cell-fate decisions including specification, expansion and patterning during the ontogeny of cortical neural stem and progenitor cells. In order to dissect regulatory mechanisms that orchestrate the stage-specific differentiation process, we developed a computational framework to infer key regulators of each cell-state transition based on the progressive remodelling of the epigenetic landscape and then validated these through a pooled short hairpin RNA screen. We were also able to refine our previous observations on epigenetic priming at transcription factor binding sites and suggest here that they are mediated by combinations of core and stage-specific factors. Taken together, we demonstrate the utility of our system and outline a general framework, not limited to the context of the neural lineage, to dissect regulatory circuits of differentiation.

Coupled RNA processing and transcription of intergenic primary microRNAs.

ABSTRACT The first step in microRNA (miRNA) biogenesis occurs in the nucleus and is mediated by the Microprocessor complex containing the RNase III-like enzyme Drosha and its cofactor DGCR8. Here we show that the 5′→3′ exonuclease Xrn2 associates with independently transcribed miRNAs and, in combination with Drosha processing, attenuates transcription in downstream regions. We suggest that, after Drosha cleavage, a torpedo-like mechanism acts on nascent long precursor miRNAs, whereby Xrn2 exonuclease degrades the RNA polymerase II-associated transcripts inducing its release from the template. While involved in primary transcript termination, this attenuation effect does not restrict clustered miRNA expression, which, in the majority of cases, is separated by short spacers. We also show that transcripts originating from a miRNA promoter are retained on the chromatin template and are more efficiently processed than those produced from mRNA or snRNA Pol II-dependent promoters. These data imply that coupling between transcription and processing promotes efficient expression of independently transcribed miRNAs.

Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency

Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here, we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PAPERCLIP.

Phenotypic Characterization of a Comprehensive Set of MAPK1/ERK2 Missense Mutants

Tumor-specific genomic information has the potential to guide therapeutic strategies and revolutionize patient treatment. Currently, this approach is limited by an abundance of disease-associated mutants whose biological functions and impacts on therapeutic response are uncharacterized. To begin to address this limitation, we functionally characterized nearly all (99.84%) missense mutants of MAPK1/ERK2, an essential effector of oncogenic RAS and RAF. Using this approach, we discovered rare gain- and loss-of-function ERK2 mutants found in human tumors, revealing that, in the context of this assay, mutational frequency alone cannot identify all functionally impactful mutants. Gain-of-function ERK2 mutants induced variable responses to RAF-, MEK-, and ERK-directed therapies, providing a reference for future treatment decisions. Tumor-associated mutations spatially clustered in two ERK2 effector-recruitment domains yet produced mutants with opposite phenotypes. This approach articulates an allele-characterization framework that can be scaled to meet the goals of genome-guided oncology.

Genetic determinants and epigenetic effects of pioneer-factor occupancy

Transcription factors (TFs) direct developmental transitions by binding to target DNA sequences, influencing gene expression and establishing complex gene-regultory networks. To systematically determine the molecular components that enable or constrain TF activity, we investigated the genomic occupancy of FOXA2, GATA4 and OCT4 in several cell types. Despite their classification as pioneer factors, all three TFs exhibit cell-type-specific binding, even when supraphysiologically and ectopically expressed. However, FOXA2 and GATA4 can be distinguished by low enrichment at loci that are highly occupied by these factors in alternative cell types. We find that expression of additional cofactors increases enrichment at a subset of these sites. Finally, FOXA2 occupancy and changes to DNA accessibility can occur in G1-arrested cells, but subsequent loss of DNA methylation requires DNA replication.Investigation of FOXA2, GATA4 and OCT4 binding across several cell types provides insights into the genetic determinants and epigenetic effects of pioneer-factor occupancy. The data suggest that FOXA2 samples most of its potential binding sites but is stabilized at only a subset of targets.

Epigenetic restriction of extraembryonic lineages mirrors the somatic transition to cancer

In mammals, the canonical somatic DNA methylation landscape is established upon specification of the embryo proper and subsequently disrupted within many cancer types. However, the underlying mechanisms that direct this genome-scale transformation remain elusive, with no clear model for its systematic acquisition or potential developmental utility. Here, we analysed global remethylation from the mouse preimplantation embryo into the early epiblast and extraembryonic ectoderm. We show that these two states acquire highly divergent genomic distributions with substantial disruption of bimodal, CpG density-dependent methylation in the placental progenitor. The extraembryonic epigenome includes specific de novo methylation at hundreds of embryonically protected CpG island promoters, particularly those that are associated with key developmental regulators and are orthologously methylated across most human cancer types. Our data suggest that the evolutionary innovation of extraembryonic tissues may have required co-option of DNA methylation-based suppression as an alternative to regulation by Polycomb-group proteins, which coordinate embryonic germ-layer formation in response to extraembryonic cues. Moreover, we establish that this decision is made deterministically, downstream of promiscuously used—and frequently oncogenic—signalling pathways, via a novel combination of epigenetic cofactors. Methylation of developmental gene promoters during tumorigenesis may therefore reflect the misappropriation of an innate trajectory and the spontaneous reacquisition of a latent, developmentally encoded epigenetic landscape.