The human body is composed of diverse cell types with distinct functions. While it is known that lineage specification depends on cell specific gene expression, which in turn is driven by promoters, enhancers, insulators and other cis-regulatory DNA sequences for each gene1–3, the relative roles of these regulatory elements in this process is not clear. We have previously developed a chromatin immunoprecipitation-based microarray method (ChIP-chip) to locate promoters, enhancers, and insulators in the human genome4–6. Here, we use the same approach to identify these elements in multiple cell types and investigated their roles in cell type-specific gene expression. We observed that chromatin state at promoters and CTCF-binding at insulators are largely invariant across diverse cell types. By contrast, enhancers are marked with highly cell type-specific histone modification patterns, strongly correlate to cell type-specific gene expression programs on a global scale, and are functionally active in a cell type-specific manner. Our results defined over 55,000 potential transcriptional enhancers in the human genome, significantly expanding the current catalog of human enhancers and highlighting the role of these elements in cell type-specific gene expression.

SummaryAs the premier model organism in biomedical research, the laboratory mouse shares the majority of protein-coding genes with humans, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications, and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of other sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

Recent studies of genome-wide chromatin interactions have revealed that the human genome is partitioned into many selfassociating topological domains. The boundary sequences between domains are enriched for binding sites of CTCC-binding factor (CTCF) and the cohesin complex, implicating these two factors in the establishment or maintenance of topological domains. To determine the role of cohesin and CTCF in higher-order chromatin architecture in human cells, we depleted the cohesin complex or CTCF and examined the consequences of loss of these factors on higher-order chromatin organization, as well as the transcriptome. We observed a general loss of local chromatin interactions upon disruption of cohesin, but the topological domains remain intact. However, we found that depletion of CTCF not only reduced intradomain interactions but also increased interdomain interactions. Furthermore, distinct groups of genes become misregulated upon depletion of cohesin and CTCF. Taken together, these observations suggest that CTCF and cohesin contribute differentially to chromatin organization and gene regulation.Hi-C | transcriptional regulation | 4C | HOX cluster R ecent studies of the topological organization of the genome suggest that CTCC-binding factor (CTCF) and cohesin might be involved in establishment or maintenance of topological domains in the mammalian genome, as their binding sites are enriched at the boundaries of these domains (1). It was proposed that CTCF and cohesin might work together to facilitate longrange interactions in the genome (2). First, CTCF and the cohesin complex, consisting of the core subunits SMC3, SMC1, RAD21, and STAG1/SA1 or STAG2/SA2, were found to colocalize extensively throughout mammalian genomes (3-5). Second, both factors are involved in mediating long-range interactions (6-11). Finally, cohesin was shown to be important for CTCF's chromatin insulation function (3-5), whereas CTCF is necessary to recruit cohesin to the shared binding sites but not to chromatin (3). CTCF and cohesin have also been recently correlated with both interaction frequency and gene expression during differentiation (12), indicating that they may play major roles in mediating the impacts of chromatin structure on gene regulation. However, the exact mechanisms these factors use to contribute to chromatin structure and gene regulation are unclear, as depletion of these factors has not yet been systematically tested on a genome-wide basis. Whether the two factors work in concert or independently, through mechanisms, such as long-range enhancer looping (13) or chromatin insulation (2) to control chromatin structure and gene expression, is unknown. To determine the role of cohesin and CTCF in higher-order chromatin architecture in human cells, we depleted the cohesin complex or CTCF and examined the consequences of loss of these factors on domain structure and gene expression. Results Proteolytic Cleavage of RAD21 Leads to Loss of Long-Range ChromatinInteractions. To understand the contribution of cohesin to genom...

Excessive cerebral accumulation of the 42-residue amyloid ␤-protein (A␤) is an early and invariant step in the pathogenesis of Alzheimer's disease. Many studies have examined the cellular production of A␤ from its membrane-bound precursor, including the role of the presenilin proteins therein, but almost nothing is known about how A␤ is degraded and cleared following its secretion. We previously screened neuronal and nonneuronal cell lines for the production of proteases capable of degrading naturally secreted A␤ under biologically relevant conditions and concentrations. The major such protease identified was a metalloprotease released particularly by a microglial cell line, BV-2. We have now purified and characterized the protease and find that it is indistinguishable from insulin-degrading enzyme (IDE), a thiol metalloendopeptidase that degrades small peptides such as insulin, glucagon, and atrial natriuretic peptide. Degradation of both endogenous and synthetic A␤ at picomolar to nanomolar concentrations was completely inhibited by the competitive IDE substrate, insulin, and by two other IDE inhibitors. Immunodepletion of conditioned medium with an IDE antibody removed its A␤-degrading activity. IDE was present in BV-2 cytosol, as expected, but was also released into the medium by intact, healthy cells. To confirm the extracellular occurrence of IDE in vivo, we identified intact IDE in human cerebrospinal fluid of both normal and Alzheimer subjects. In addition to its ability to degrade A␤, IDE activity was unexpectedly found be associated with a time-dependent oligomerization of synthetic A␤ at physiological levels in the conditioned media of cultured cells; this process, which may be initiated by IDE-generated proteolytic fragments of A␤, was prevented by three different IDE inhibitors. We conclude that a principal protease capable of down-regulating the levels of secreted A␤ extracellularly is IDE.Converging lines of evidence support the hypothesis that progressive cerebral accumulation of the 40 -42-residue amyloid ␤-proteins (A␤s) 1 is an early, invariant, and necessary step in the pathogenesis of Alzheimer's disease (AD). As a result, there is growing interest in decreasing cerebral A␤ levels as a therapeutic and preventative approach to the disease. A␤ is generated by endoproteolysis of the ␤-amyloid precursor protein (APP) and secreted constitutively by most mammalian cells throughout life. Whereas many studies have examined the proteolytic processing of APP and the mechanisms of A␤ production, almost nothing is known about how A␤ peptides are normally degraded and cleared following their secretion. We recently screened the conditioned media of several different cell lines for A␤-degrading activity and found that the principal such activity was conferred by a nonmatrix metalloprotease that was released by microglial cells and other cells and efficiently degraded both endogenous and synthetic A␤ (1). The release of the protease from microglial cells was augmented by activating the cells with lipopolysa...

ObjectiveTo assess the prevalence of diabetes and its risk factors.DesignPopulation based, cross sectional study.Setting31 provinces in mainland China with nationally representative cross sectional data from 2015 to 2017.Participants75 880 participants aged 18 and older—a nationally representative sample of the mainland Chinese population.Main outcome measuresPrevalence of diabetes among adults living in China, and the prevalence by sex, regions, and ethnic groups, estimated by the 2018 American Diabetes Association (ADA) and the World Health Organization diagnostic criteria. Demographic characteristics, lifestyle, and history of disease were recorded by participants on a questionnaire. Anthropometric and clinical assessments were made of serum concentrations of fasting plasma glucose (one measurement), two hour plasma glucose, and glycated haemoglobin (HbA1c).ResultsThe weighted prevalence of total diabetes (n=9772), self-reported diabetes (n=4464), newly diagnosed diabetes (n=5308), and prediabetes (n=27 230) diagnosed by the ADA criteria were 12.8% (95% confidence interval 12.0% to 13.6%), 6.0% (5.4% to 6.7%), 6.8% (6.1% to 7.4%), and 35.2% (33.5% to 37.0%), respectively, among adults living in China. The weighted prevalence of total diabetes was higher among adults aged 50 and older and among men. The prevalence of total diabetes in 31 provinces ranged from 6.2% in Guizhou to 19.9% in Inner Mongolia. Han ethnicity had the highest prevalence of diabetes (12.8%) and Hui ethnicity had the lowest (6.3%) among five investigated ethnicities. The weighted prevalence of total diabetes (n=8385) using the WHO criteria was 11.2% (95% confidence interval 10.5% to 11.9%).ConclusionThe prevalence of diabetes has increased slightly from 2007 to 2017 among adults living in China. The findings indicate that diabetes is an important public health problem in China.

Progressive cerebral accumulation of amyloid beta-protein (Abeta) is an early and invariant feature of Alzheimer's disease. Little is known about how Abeta, after being secreted, is degraded and cleared from the extracellular space of the brain. Defective Abeta degradation could be a risk factor for the development of Alzheimer's disease in some subjects. We reported previously that microglial cells release substantial amounts of an Abeta-degrading protease that, after purification, is indistinguishable from insulin-degrading enzyme (IDE). Here we searched for and characterized a role for IDE in Abeta degradation by neurons, the principal cell type that produces Abeta. Whole cultures of differentiated pheochromocytoma (PC12) cells and primary rat cortical neurons actively degraded endogenously secreted Abeta via IDE. However, unlike that in microglia, IDE in differentiated neurons was not released but localized to the cell surface, as demonstrated by biotinylation. Undifferentiated PC12 cells released IDE into their medium, whereas after differentiation, IDE was cell associated but still degraded Abeta in the medium. Overexpression of IDE in mammalian cells markedly reduced the steady-state levels of extracellular Abeta(40) and Abeta(42), and the catalytic site mutation (E111Q) abolished this effect. We observed a novel membrane-associated form of IDE that is approximately 5 kDa larger than the known cytosolic form in a variety of cells, including differentiated PC12 cells. Our results support a principal role for membrane-associated and secreted IDE isoforms in the degradation and clearance of naturally secreted Abeta by neurons and microglia.

The transition from juvenile to adult life is accompanied by programmed remodeling in many tissues and organs, which is key for organisms to adapt to the demand of the environment. Here we report a novel regulated alternative splicing program that is crucial for postnatnal heart remodeling in the mouse. We identify the essential splicing factor ASF/SF2 as a key component of the program, regulating a restricted set of tissue-specific alternative splicing events during heart remodeling. Cardiomyocytes deficient in ASF/SF2 display an unexpected hypercontraction phenotype due to a defect in postnatal splicing switch of the Ca(2+)/calmodulin-dependent kinase IIdelta (CaMKIIdelta) transcript. This failure results in mistargeting of the kinase to sarcolemmal membranes, causing severe excitation-contraction coupling defects. Our results validate ASF/SF2 as a fundamental splicing regulator in the reprogramming pathway and reveal the central contribution of ASF/SF2-regulated CaMKIIdelta alternative splicing to functional remodeling in developing heart.

Summary In mammals, cytosine methylation (5mC) is widely distributed throughout the genome, but is notably depleted from active promoters and enhancers. While the role of DNA methylation in promoter silencing has been well documented, the function of this epigenetic mark at enhancers remains unclear. Recent experiments have demonstrated that enhancers are enriched for 5-hydroxymethylcytosine (5hmC), an oxidization product of the Tet family of 5mC dioxygenases and an intermediate of DNA demethylation. These results support the involvement of Tet proteins in regulation of dynamic DNA methylation at enhancers. By mapping DNA methylation and hydroxymethylation at base resolution, we find that deletion of Tet2 causes extensive loss of 5hmC at enhancers, accompanied by enhancer hypermethylation, reduction of enhancer activity, and delayed gene induction in the early steps of differentiation. Our results reveal that DNA demethylation modulates enhancer activity, and its disruption influences the timing of transcriptome reprogramming during cellular differentiation.