Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.

MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.

New therapeutic targets for noncognitive reductions in energy intake, absorption, or storage are crucial given the worldwide epidemic of obesity. The gut microbial community (microbiota) is essential for processing dietary polysaccharides. We found that conventionalization of adult germ-free (GF) C57BL/6 mice with a normal microbiota harvested from the distal intestine (cecum) of conventionally raised animals produces a 60% increase in body fat content and insulin resistance within 14 days despite reduced food intake. Studies of GF and conventionalized mice revealed that the microbiota promotes absorption of monosaccharides from the gut lumen, with resulting induction of de novo hepatic lipogenesis. Fasting-induced adipocyte factor (Fiaf), a member of the angiopoietin-like family of proteins, is selectively suppressed in the intestinal epithelium of normal mice by conventionalization. Analysis of GF and conventionalized, normal and Fiaf knockout mice established that Fiaf is a circulating lipoprotein lipase inhibitor and that its suppression is essential for the microbiota-induced deposition of triglycerides in adipocytes. Studies of Rag1-/- animals indicate that these host responses do not require mature lymphocytes. Our findings suggest that the gut microbiota is an important environmental factor that affects energy harvest from the diet and energy storage in the host. Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AY 667702--AY 668946).

https://www.pnas.org/content/pnas/101/44/15718.full.pdf

The gut microbiota as an environmental factor that regulates fat storage

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

/pdf/integrative-analysis-of-111-reference-human-epigenomes-4bavnjaon6.pdf

Integrative analysis of 111 reference human epigenomes

System-wide profiling of genes and proteins in mammalian cells produce lists of differentially expressed genes/proteins that need to be further analyzed for their collective functions in order to extract new knowledge. Once unbiased lists of genes or proteins are generated from such experiments, these lists are used as input for computing enrichment with existing lists created from prior knowledge organized into gene-set libraries. While many enrichment analysis tools and gene-set libraries databases have been developed, there is still room for improvement. Here, we present Enrichr, an integrative web-based and mobile software application that includes new gene-set libraries, an alternative approach to rank enriched terms, and various interactive visualization approaches to display enrichment results using the JavaScript library, Data Driven Documents (D3). The software can also be embedded into any tool that performs gene list analysis. We applied Enrichr to analyze nine cancer cell lines by comparing their enrichment signatures to the enrichment signatures of matched normal tissues. We observed a common pattern of up regulation of the polycomb group PRC2 and enrichment for the histone mark H3K27me3 in many cancer cell lines, as well as alterations in Toll-like receptor and interlukin signaling in K562 cells when compared with normal myeloid CD33+ cells. Such analyses provide global visualization of critical differences between normal tissues and cancer cell lines but can be applied to many other scenarios. Enrichr is an easy to use intuitive enrichment analysis web-based tool providing various types of visualization summaries of collective functions of gene lists. Enrichr is open source and freely available online at: http://amp.pharm.mssm.edu/Enrichr
 .

/pdf/enrichr-interactive-and-collaborative-html5-gene-list-1dn7g68rn1.pdf

Enrichr: Interactive and collaborative HTML5 gene list enrichment analysis tool

The TRANSFAC database on transcription factors, their binding sites, nucleotide distribution matrices and regulated genes as well as the complementing database TRANSCompel on composite elements have been further enhanced on various levels. A new web interface with different search options and integrated versions of Match and Patch provides increased functionality for TRANSFAC. The list of databases which are linked to the common GENE table of TRANSFAC and TRANSCompel has been extended by: Ensembl, UniGene, EntrezGene, HumanPSD and TRANSPRO. Standard gene names from HGNC, MGI and RGD, are included for human, mouse and rat genes, respectively. With the help of InterProScan, Pfam, SMART and PROSITE domains are assigned automatically to the protein sequences of the transcription factors. TRANSCompel contains now, in addition to the COMPEL table, a separate table for detailed information on the experimental EVIDENCE on which the composite elements are based. Finally, for TRANSFAC, in respect of data growth, in particular the gain of Drosophila transcription factor binding sites (by courtesy of the Drosophila DNase I footprint database) and of Arabidopsis factors (by courtesy of DATF, Database of Arabidopsis Transcription Factors) has to be stressed. The here described public releases, TRANSFAC 7.0 and TRANSCompel 7.0, are accessible under http://www.gene-regulation.com/pub/databases.html.

/pdf/transfac-and-its-module-transcompel-transcriptional-gene-53277i5esk.pdf

TRANSFAC® and its module TRANSCompel®: transcriptional gene regulation in eukaryotes

The TRANSFAC database on eukaryotic transcriptional regulation, comprising data on transcription factors, their target genes and regulatory binding sites, has been extended and further developed, both in number of entries and in the scope and structure of the collected data. Structured fields for expression patterns have been introduced for transcription factors from human and mouse, using the CYTOMER database on anatomical structures and developmental stages. The functionality of Match, a tool for matrix-based search of transcription factor binding sites, has been enhanced. For instance, the program now comes along with a number of tissue-(or state-)specific profiles and new profiles can be created and modified with Match Profiler. The GENE table was extended and gained in importance, containing amongst others links to LocusLink, RefSeq and OMIM now. Further, (direct) links between factor and target gene on one hand and between gene and encoded factor on the other hand were introduced. The TRANSFAC public release is available at http://www.gene-regulation.com. For yeast an additional release including the latest data was made available separately as TRANSFAC Saccharomyces Module (TSM) at http://transfac.gbf.de. For CYTOMER free download versions are available at http://www.biobase.de:8080/index.html.

/pdf/transfac-transcriptional-regulation-from-patterns-to-1dumqi58b4.pdf

TRANSFAC®: transcriptional regulation, from patterns to profiles

In T-cell acute lymphoblastic leukemia, alternative t(5;14)(q35;q32.2) forms effect dysregulation of either TLX3 or NKX2-5 homeobox genes at 5q35 by juxtaposition with 14q32.2 breakpoints dispersed across the BCL11B downstream genomic desert. Leukemic gene dysregulation by t(5;14) was investigated by DNA inhibitory treatments with 26-mer double-stranded DNA oligonucleotides directed against candidate enhancers at, or near, orphan T-cell DNase I hypersensitive sites located between 3′- BCL11B and VRK1. NKX2-5 down-regulation in t(5;14) PEER cells was almost entirely restricted to DNA inhibitory treatment targeting enhancers within the distal breakpoint cluster region and was dose and sequence dependent, whereas enhancers near 3′- BCL11B regulated that gene only. Chromatin immunoprecipitation assays showed that the four most effectual NKX2-5 ectopic enhancers were hyperacetylated. These enhancers clustered ∼1 Mbp downstream of BCL11B , within a region displaying multiple regulatory stigmata, including a TCRA enhancer motif, deep sequence conservation, and tight nuclear matrix attachment relaxed by trichostatin A treatment. Intriguingly, although TLX3 / NKX2-5 promoter/exon 1 regions were hypoacetylated, their expression was trichostatin A sensitive, implying extrinsic regulation by factor(s) under acetylation control. Knockdown of PU.1 , known to be trichostatin A responsive and which potentially binds TLX3 / NKX2-5 promoters, effected down-regulation of both homeobox genes. Moreover, genomic analysis showed preferential enrichment near ectopic enhancers of binding sites for the PU.1 cofactor HMGA1, the knockdown of which also inhibited NKX2-5 . We suggest that HMGA1 and PU.1 coregulate ectopic homeobox gene expression in t(5;14) T-cell acute lymphoblastic leukemia by interactions mediated at the nuclear matrix. Our data document homeobox gene dysregulation by a novel regulatory region at 3′- BCL11B responsive to histone deacetylase inhibition and highlight a novel class of potential therapeutic target amid noncoding DNA. [Cancer Res 2007;67(4):1461–71]

https://cancerres.aacrjournals.org/content/canres/67/4/1461.full.pdf

Activation of TLX3 and NKX2-5 in t(5;14)(q35;q32) T-Cell Acute Lymphoblastic Leukemia by Remote 3′-BCL11B Enhancers and Coregulation by PU.1 and HMGA1

TiProD is a database of human promoter sequences for which some functional features are known. It allows a user to query individual promoters and the expression pattern they mediate, gene expression signatures of individual tissues, and to retrieve sets of promoters according to their tissue-specific activity or according to individual Gene Ontology terms the corresponding genes are assigned to. We have defined a measure for tissue-specificity that allows the user to discriminate between ubiquitously and specifically expressed genes. The database is accessible at http://tiprod.cbi.pku.edu.cn:8080/index.html.

TiProD: the Tissue-specific Promoter Database

Known transcription regulatory signals which generally act as transcription factor binding sites (TFs) differ significantly in their base composition. Therefore, their occurrence in a genome largely depends on the local base composition. In an attempt to initiate an all human genome analysis for the occurrence of potential TFs, we systematically analyzed the GC-content of distinct functional regions (e.g., upstream and downstream gene regions, exons, long and short introns, repetitive elements) and correlated the frequencies of potential binding sites of a rep- resentative set of TFs in these regions. For these analyses, we used the pattern collection of the TRANSFAC ® data- base on transcriptional regulation, the information about functionally relevant combinations of them from the data- base TRANSCompel ® , and our new resource, TRANSGenome TM , which provides an overall annotation of the hu- man genome with emphasis on its regulatory characteristics. We show that the occurrence of sequence patterns with regulatory potential may be supported by, but cannot be fully explained by either the GC content of a whole chro- mosome or its putative promoter regions, nor by the information content of the patterns. Several patterns, HNF-3, NFAT, and GC box, show a clear overrepresentation in all promoter groups as well as in all chromosomes. Other patterns, like E2F and CRE-BP1, are underrepresented in all promoter groups as well as in all chromosomes in com- parison with random sequences. Simultaneously, both patterns are over-represented in promoters in comparison with repetitive elements. We define several structural characteristics of the proximal promoters that differentiate them from other functional genomic regions. Two well-known promoter elements, GC- and TATA-boxes, are statistically enriched in promoters in comparison with random sequences, repetitive elements and exons. Altogether, our findings provide insights into the macroheterogeneity amongst the individual chromosomes, into the microheterogeneity among different functional regions of individual chromosomes, contribute to further understand- ing of structural organization of gene regulatory regions, and give first hints on the development of regulatory fea- tures during evolution.

Klaus Hornischer

Papers

TRANSFAC® and its module TRANSCompel®: transcriptional gene regulation in eukaryotes

TRANSFAC®: transcriptional regulation, from patterns to profiles

Activation of TLX3 and NKX2-5 in t(5;14)(q35;q32) T-Cell Acute Lymphoblastic Leukemia by Remote 3′-BCL11B Enhancers and Coregulation by PU.1 and HMGA1

TiProD: the Tissue-specific Promoter Database

Composition-sensitive analysis of the human genome for regulatory signals.