Along the Bos taurus genome, uncover candidate imprinting control regions.

BACKGROUND: In mammals, Imprinting Control Regions (ICRs) regulate a subset of genes in a parent-of-origin-specific manner. In both human and mouse, previous studies identified a set of CpG-rich motifs occurring as clusters in ICRs and germline Differentially Methylated Regions (gDMRs). These motifs consist of the ZFP57 binding site (ZFBS) overlapping a subset of MLL binding units known as MLL morphemes. MLL or MLL1 (Mixed Lineage Leukemia 1) is a relatively large multidomain protein that plays a central role in the regulation of transcription. The structures of both MLL1 and MLL2 include a domain (MT) that binds CpG-rich DNA and a conserved domain (SET) that methylates lysine 4 in histone H3 producing H3K4me3 marks in chromatin. RESULTS: Since genomic imprinting impacts many developmental and key physiological processes, we followed a previous bioinformatics strategy to pinpoint ICR positions in the Bos taurus genome. Initial genome-wide analyses involved finding the positions of ZFP57 binding sites, and the CpG-rich motifs (ZFBS-morph overlaps) along cattle chromosomal DNA. By creating plots displaying the density of ZFBS-morph overlaps, we removed background noise and thus improved signal detection. With the density-plots, we could view the positions of peaks locating known and candidate ICRs in cattle DNA. Our evaluations revealed the correspondence of peaks in plots to reported known and inferred ICRs/DMRs in cattle. Beside peaks pinpointing such ICRs, the density-plots also revealed additional peaks. Since evaluations validated the robustness of our approach, we inferred that the additional peaks may correspond to candidate ICRs for imprinted gene expression. CONCLUSION: Our bioinformatics strategy offers the first genome-wide approach for systematically localizing candidate ICRs. Furthermore, we have tailored our datasets for upload onto the UCSC genome browser so that researchers could find known and candidate ICRs with respect to a wide variety of annotations at all scales: from the positions of Single Nucleotide Polymorphisms (SNPs), to positions of genes, transcripts, and repeated DNA elements. Furthermore, the UCSC genome browser offers tools to produce enlarged views: to uncover the genes in the vicinity of candidate ICRs and thus discover potential imprinted genes for experimental validations.

Datasets on the genomic positions of the MLL1 morphemes, the ZFP57 binding site, and ZFBS-Morph overlaps in the build mm9 of the mouse genome.

While MLL1 activates gene expression in most tissues, ZFP57 represses transcription. MLL1 selectively interacts with a group of nonmethylated DNA sequences known as the MLL1 morphemes. ZFP57 associates with a methylated hexamer (ZFBS), dispersed in the genomic DNA segments known as Imprinted Control Regions (ICRs) and germline Differentially Methylated Regions (gDMRs), to maintain allele-specific gene repression. We have identified a set of composite DNA elements (ZFBS-Morph overlaps) that provides the sequence context of ZFBS in the canonical ICRs/gDMRs. This report provides tables listing the nucleotide sequences of the MLL1 morphemes and ZFBS-Morph overlaps. The report also offers links to the data repository at Purdue University, for downloading the positions of the MLL1 morphemes, the ZFP57 binding site, and the ZFBS-Morph overlaps in the mouse genome.

Impact of the MLL1 morphemes on codon utilization and preservation in CpG islands.

Previous studies have shown that Mixed Lineage Leukemia 1 (MLL1 or MLL) binds a group of CpG-rich motifs known as morphemes. To examine whether occurrences of MLL1 morphemes in genomic DNA may influence codon utilization, we analyzed the frequency of various 9-mers in human cDNAs and in total human genomic DNA. We uncovered preferential utilization of GGC for Gly, GCG for Ala, CCG for Pro, and TCG for Ser, in coding sequences (CDSs) that included MLL1 morphemes. We also examined weighted occurrences of CDS 9-mers in a 30-base window that moved along each human chromosome. In plots, we observed peaks with fluctuating intensities. High intensity peaks appeared within promoter and exons localized in CpG islands, encompassing sequences that included MLL1 morphemes. High intensity peaks included CCG/GGC repeats, whose expansion may cause neurological disorders and congenital malformations. Such repeats are generated from overlap of a morpheme (CGCCG/CGGCG), which depending on reading frame and orientation would produce runs of Ala, Gly, or Pro in proteins. Overall, our results point to a role for morpheme occurrences on synonymous codon utilization in human genomic DNA and indicate that regulatory instructions are dispersed not only in promoters but also in exons of human genes.

Discovery of MLL1 binding units, their localization to CpG Islands, and their potential function in mitotic chromatin.

BACKGROUND: Mixed Lineage Leukemia 1 (MLL1) is a mammalian ortholog of the Drosophila Trithorax. In Drosophila, Trithorax complexes transmit the memory of active genes to daughter cells through interactions with Trithorax Response Elements (TREs). However, despite their functional importance, nothing is known about sequence features that may act as TREs in mammalian genomic DNA. RESULTS: By analyzing results of reported DNA binding assays, we identified several CpG rich motifs as potential MLL1 binding units (defined as morphemes). We find that these morphemes are dispersed within a relatively large collection of human promoter sequences and appear densely packed near transcription start sites of protein-coding genes. Genome wide analyses localized frequent morpheme occurrences to CpG islands. In the human HOX loci, the morphemes are spread across CpG islands and in some cases tail into the surrounding shores and shelves of the islands. By analyzing results of chromatin immunoprecipitation assays, we found a connection between morpheme occurrences, CpG islands, and chromatin segments reported to be associated with MLL1. Furthermore, we found a correspondence of reported MLL1-driven "bookmarked" regions in chromatin to frequent occurrences of MLL1 morphemes in CpG islands. CONCLUSION: Our results implicate the MLL1 morphemes in sequence-features that define the mammalian TREs and provide a novel function for CpG islands. Apparently, our findings offer the first evidence for existence of potential TREs in mammalian genomic DNA and the first evidence for a connection between CpG islands and gene-bookmarking by MLL1 to transmit the memory of highly active genes during mitosis. Our results further suggest a role for overlapping morphemes in producing closely packed and multiple MLL1 binding events in genomic DNA so that MLL1 molecules could interact and reside simultaneously on extended potential transcriptional maintenance elements in human chromosomes to transmit the memory of highly active genes during mitosis.

Absorption mode Fourier transform electrostatic linear ion trap mass spectrometry.

In Fourier transform mass spectrometry, it is well-known that plotting the spectrum in absorption mode rather than magnitude mode has several advantages. However, magnitude spectra remain commonplace due to difficulties associated with determining the phase of each frequency at the onset of data acquisition, which is required for generating absorption spectra. The phasing problem for electrostatic traps is much simpler than for Fourier transform ion cyclotron resonance (FTICR) instruments, which greatly simplifies the generation of absorption spectra. Here, we present a simple method for generating absorption spectra from a Fourier transform electrostatic linear ion trap mass spectrometer. The method involves time shifting the data prior to Fourier transformation in order to synchronize the onset of data acquisition with the moment of ion acceleration into the electrostatic trap. Under these conditions, the initial phase of each frequency at the onset of data acquisition is zero. We demonstrate that absorption mode provides a 1.7-fold increase in resolution (full width at half maximum, fwhm) as well as reduced peak tailing. We also discuss methodology that may be applied to unsynchronized data in order to determine the time shift required to generate an absorption spectrum.

Photoelectron spectroscopy of chloro-substituted phenylnitrene anions.

The 355 nm time-of-flight negative ion photoelectron spectra of (o-, m-, and p-chlorophenyl)nitrene radical anions are reported. Electron affinities are obtained from the photoelectron spectra, and are 1.79 +/- 0.05, 1.82 +/- 0.05, and 1.72 +/- 0.05 eV for the (o-, m-, and p-chlorophenyl)nitrenes, respectively. Singlet-triplet splittings are determined to be 14 +/- 2, 15 +/- 1, and 14 +/- 2 kcal/mol, respectively. The shapes of the photoelectron bands indicate resonance interactions in the singlet states for the ortho- and para-substituted isomers, which is attributed to quinoidal structures of the open-shell singlet states. Reanalysis of the photoelectron spectrum of phenylnitrene anion leads to a revised experimental singlet-triplet splitting of 14.8 kcal/mol in the unsubstituted phenylnitrene.

Discovering sequences with potential regulatory characteristics.

We developed a computational model to explore the hypothesis that regulatory instructions are context dependent and conveyed through specific 'codes' in human genomic DNA. We provide examples of correlation of computational predictions to reported mapped DNase I hypersensitive segments in the HOXA locus in human chromosome 7. The examples show that statistically significant 9-mers from promoter regions may occur in sequences near and upstream of transcription initiation sites, in intronic regions, and within intergenic regions. Additionally, a subset of 9-mers from coding sequences appears frequently, as clusters, in regulatory regions dispersed in noncoding regions in genomic DNA. The results suggest that the computational model has the potential of decoding regulatory instructions to discover candidate transcription factor binding sites and to discover candidate epigenetic signals that appear in both coding and regulatory regions of genes.

A database of 9-mers from promoter regions of human protein-coding genes.

Discovery of lexical characteristics of specific sequence motifs in human genomic DNA can help with predicting and classifying regulatory cis elements according to the genes they control. In lexical models, some "words" may serve as downstream targets of signaling systems, whereas other "words" may specify sequences that selectively control the expression of a subset of genes to produce the various cell types and tissues. To discover lexical features of potential regulatory "words," we have created a database of 9-mers derived from the promoter regions of a subset of human protein-coding genes. This report describes the procedure for extracting information from that database through the web.

A program toolkit for the analysis of regulatory regions of genes.

A major challenge in systems biology is to discover and reconstruct the cis-regulatory networks through which the expression of genes is controlled. Even though a variety of sequences have been shown to interact with the transcription factors that bind DNA, extensive work is needed to discover and classify regulatory "codes" and to elucidate the role played by the sequence context of genomic DNA in the regulation of genes. Databases of sequence elements extracted from regulatory regions may facilitate this process. This report provides a Toolkit and instructions for creating a database for collecting and analyzing 9-base elements (9-mers) from a large collection of DNA sequences. A reference set consisting of all possible 9-mers is included for extracting potential control elements, irrespective of their orientation and order in DNA.

Exploring the characteristics of sequence elements in proximal promoters of human genes.

Central to reconstruction of cis-regulatory networks is identification and classification of naturally occurring transcription factor-binding sites according to the genes that they control. We have examined salient characteristics of 9-mers that occur in various orders and combinations in the proximal promoters of human genes. In evaluations of a dataset derived with respect to experimentally defined transcription initiation sites, in some cases we observed a clear correspondence of highly ranked 9-mers with protein-binding sites in genomic DNA. Evaluations of the larger dataset, derived with respect to the 5' end of human ESTs, revealed that a subset of the highly ranked 9-mers corresponded to sites for several known transcription factor families (including CREB, ETS, EGR-1, SP1, KLF, MAZ, HIF-1, and STATs) that play important roles in the regulation of vertebrate genes. We identified several highly ranked CpG-containing 9-mers, defining sites for interactions with the CREB and ETS families of proteins, and identified potential target genes for these proteins. The results of the studies imply that the CpG-containing transcription factor-binding sites regulate the expression of genes with important roles in pathways leading to cell-type-specific gene expression and pathways controlled by the complex networks of signaling systems.