Guidantonio Malagoli Tagliazucchi

Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock

Circadian rhythms control metabolism and energy homeostasis, but the role of the skeletal muscle clock has never been explored. We generated conditional and inducible mouse lines with muscle-specific ablation of the core clock gene Bmal1. Skeletal muscles from these mice showed impaired insulin-stimulated glucose uptake with reduced protein levels of GLUT4, the insulin-dependent glucose transporter, and TBC1D1, a Rab-GTPase involved in GLUT4 translocation. Pyruvate dehydrogenase (PDH) activity was also reduced due to altered expression of circadian genes Pdk4 and Pdp1, coding for PDH kinase and phosphatase, respectively. PDH inhibition leads to reduced glucose oxidation and diversion of glycolytic intermediates to alternative metabolic pathways, as revealed by metabolome analysis. The impaired glucose metabolism induced by muscle-specific Bmal1 knockout suggests that a major physiological role of the muscle clock is to prepare for the transition from the rest/fasting phase to the active/feeding phase, when glucose becomes the predominant fuel for skeletal muscle.

Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation

Summary Human skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations.

The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle

Objective Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle. Methods We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression. Results We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes. Conclusions We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca2+-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression.

Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters

Topoisomerase I-DNA-cleavage complexes (Top1cc) stabilized by camptothecin (CPT) have specific effects at transcriptional levels. We recently reported that Top1cc increase antisense transcript (aRNAs) levels at divergent CpG-island promoters and, transiently, DNA/RNA hybrids (R-loop) in nuclear and mitochondrial genomes of colon cancer HCT116 cells. However, the relationship between R-loops and aRNAs was not established. Here, we show that aRNAs can form R-loops in N-TERA-2 cells under physiological conditions, and that promoter-associated R-loops are somewhat increased and extended in length immediately upon cell exposure to CPT. In contrast, persistent Top1ccs reduce the majority of R-loops suggesting that CPT-accumulated aRNAs are not commonly involved in R-loops. The enhancement of aRNAs by Top1ccs is present both in human colon cancer HCT116 cells and WI38 fibroblasts suggesting a common response of cancer and normal cells. Although Top1ccs lead to DSB and DDR kinases activation, we do not detect a dependence of aRNA accumulation on ATM or DNA-PK activation. However, we showed that the cell response to persistent Top1ccs can involve an impairment of aRNA turnover rather than a higher synthesis rate. Finally, a genome-wide analysis shows that persistent Top1ccs also determine an accumulation of sense transcripts at 5’-end gene regions suggesting an increased occurrence of truncated transcripts. Taken together, the results indicate that Top1 may regulate transcription initiation by modulating RNA polymerase-generated negative supercoils, which can in turn favor R-loop formation at promoters, and that transcript accumulation at TSS is a response to persistent transcriptional stress by Top1 poisoning.

Erratum to “Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock” [Mol Metab 3 (2014) 29–41]

[This corrects the article DOI: 10.1016/j.molmet.2013.10.005.].

Comparative genomics revealed key molecular targets to rapidly convert a reference rifamycin-producing bacterial strain into an overproducer by genetic engineering.

Rifamycins are mainstay agents in treatment of many widespread diseases, but how an improved rifamycin producer can be created is still incompletely understood. Here, we describe a comparative genomic approach to investigate the mutational patterns introduced by the classical mutate-and-screen method in the genome of an improved rifamycin producer. Comparing the genome of the rifamycin B overproducer Amycolatopsis mediterranei HP-130 with those of the reference strains A. mediterranei S699 and U32, we identified 250 variations, affecting 227 coding sequences (CDS), 109 of which were HP-130-specific since they were absent in both S699 and U32. Mutational and transcriptional patterns indicated a series of genomic manipulations that not only proved the causative effect of mutB2 (coding for methylmalonyl-CoA mutase large subunit) and argS2 (coding for arginyl tRNA synthetase) mutations on the overproduction of rifamycin, but also constituted a rational strategy to genetically engineer a reference strain into an overproducer.

Transcriptional, epigenetic and retroviral signatures identify regulatory regions involved in hematopoietic lineage commitment

Genome-wide approaches allow investigating the molecular circuitry wiring the genetic and epigenetic programs of human somatic stem cells. Hematopoietic stem/progenitor cells (HSPC) give rise to the different blood cell types; however, the molecular basis of human hematopoietic lineage commitment is poorly characterized. Here, we define the transcriptional and epigenetic profile of human HSPC and early myeloid and erythroid progenitors by a combination of Cap Analysis of Gene Expression (CAGE), ChIP-seq and Moloney leukemia virus (MLV) integration site mapping. Most promoters and transcripts were shared by HSPC and committed progenitors, while enhancers and super-enhancers consistently changed upon differentiation, indicating that lineage commitment is essentially regulated by enhancer elements. A significant fraction of CAGE promoters differentially expressed upon commitment were novel, harbored a chromatin enhancer signature, and may identify promoters and transcribed enhancers driving cell commitment. MLV-targeted genomic regions co-mapped with cell-specific active enhancers and super-enhancers. Expression analyses, together with an enhancer functional assay, indicate that MLV integration can be used to identify bona fide developmentally regulated enhancers. Overall, this study provides an overview of transcriptional and epigenetic changes associated to HSPC lineage commitment, and a novel signature for regulatory elements involved in cell identity.

Genome-Wide Definition of Promoter and Enhancer Usage during Neural Induction of Human Embryonic Stem Cells

Genome-wide mapping of transcriptional regulatory elements is an essential tool for understanding the molecular events orchestrating self-renewal, commitment and differentiation of stem cells. We combined high-throughput identification of transcription start sites with genome-wide profiling of histones modifications to map active promoters and enhancers in embryonic stem cells (ESCs) induced to neuroepithelial-like stem cells (NESCs). Our analysis showed that most promoters are active in both cell types while approximately half of the enhancers are cell-specific and account for most of the epigenetic changes occurring during neural induction, and most likely for the modulation of the promoters to generate cell-specific gene expression programs. Interestingly, the majority of the promoters activated or up-regulated during neural induction have a “bivalent” histone modification signature in ESCs, suggesting that developmentally-regulated promoters are already poised for transcription in ESCs, which are apparently pre-committed to neuroectodermal differentiation. Overall, our study provides a collection of differentially used enhancers, promoters, transcription starts sites, protein-coding and non-coding RNAs in human ESCs and ESC-derived NESCs, and a broad, genome-wide description of promoter and enhancer usage and of gene expression programs characterizing the transition from a pluripotent to a neural-restricted cell fate.

An Ultraconserved Element Containing lncRNA Preserves Transcriptional Dynamics and Maintains ESC Self-Renewal

Summary Ultraconserved elements (UCEs) show the peculiar feature to retain extended perfect sequence identity among human, mouse, and rat genomes. Most of them are transcribed and represent a new family of long non-coding RNAs (lncRNAs), the transcribed UCEs (T-UCEs). Despite their involvement in human cancer, the physiological role of T-UCEs is still unknown. Here, we identify a lncRNA containing the uc.170+, named T-UCstem1, and provide in vitro and in vivo evidence that it plays essential roles in embryonic stem cells (ESCs) by modulating cytoplasmic miRNA levels and preserving transcriptional dynamics. Specifically, while T-UCstem1::miR-9 cytoplasmic interplay regulates ESC proliferation by reducing miR-9 levels, nuclear T-UCstem1 maintains ESC self-renewal and transcriptional identity by stabilizing polycomb repressive complex 2 on bivalent domains. Altogether, our findings provide unprecedented evidence that T-UCEs regulate physiological cellular functions and point to an essential role of T-UCstem1 in preserving ESC identity.

Genome-wide analysis of promoter and enhancer usage in hematopoietic stem cell differentiation

Background Somatic stem cells are the basic tools of regenerative medicine and gene therapy, providing unique opportunities for the therapy of genetic and acquired disorders. The molecular mechanisms underlying fundamental characteristics of human somatic stem cells, such as selfrenewal, commitment and differentiation, are still poorly understood. A better knowledge of these mechanisms is crucial to the understanding of stem cell biology and to the development of stem cell-based therapies. The rapidly expanding information on the structural and functional characteristics of the human genome allows the development of genome-wide approaches to investigate the molecular circuitry wiring the genetic and epigenetic programs of somatic stem cells. High-throughput approaches are essential to study the transcriptome, the epigenome and the usage of regulatory elements in the genome.