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The skeletal muscle is a highly heterogeneous tissue comprised of different fiber types with varying contractile and metabolic properties. The complexity in the analysis of skeletal muscle fibers associated with their small size (30–50 μm) and mosaic-like distribution across the tissue tnecessitates the use of high-resolution imaging to differentiate between fiber types. Herein, we use a multimodal approach to characterize the chemical composition of skeletal fibers in a limb muscle, the gastrocnemius. Specifically, we combine high-resolution nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging (MSI) with immunofluorescence (IF)-based fiber type identification. Computational image registration and segmentation approaches are used to integrate the information obtained with both techniques. Our results indicate that the transition between oxidative and glycolytic fibers is associated with shallow chemical gradients (<2.5 fold change in signals). Interestingly, we did not find any fiber type-specific molecule. We hypothesize that these findings might be linked to muscle plasticity thereby facilitating a switch in the metabolic properties of fibers in response to different conditions such as exercise and diet, among others. Despite the shallow chemical gradients, cardiolipins (CLs), acylcarnitines (CAR), monoglycerides (MGs), fatty acids, highly polyunsaturated phospholipids, and oxidized phospholipids, were identified as molecular signatures of oxidative metabolism. In contrast, histidine-related compounds were found as molecular signatures of glycolytic fibers. Additionally, the presence of highly polyunsaturated acyl chains in phospholipids was found in oxidative fibers whereas more saturated acyl chains in phospholipids were found in glycolytic fibers which suggests an effect of the membrane fluidity on the metabolic properties of skeletal myofibers. 

Knowledge of locations and activities ofcis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state regulatory potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbor distinctive transcription factor binding motifs that are similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we show that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment. 

Abstract DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability 1,2 . At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs) 3–6 , subTADs 7 and loops 8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.