A multiprotein occupancy map of the mRNP on the 3' end of histone mRNAs.

The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stem-loop (SL) structure. This SL structure interacts with the stem-loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3'hExo. We also performed a secondary analysis of an iCLIP data set to map UPF1 occupancy across the RD-histone mRNAs and found that UPF1 is bound adjacent to the SLBP-binding site. Multiple proteins likely bind the 3' end of RD-histone mRNAs together with SLBP.

Microdissection of shoot meristem functional domains.

The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection-microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize.

Specificity in PDZ-peptide interaction networks: Computational analysis and review.

Globular PDZ domains typically serve as protein-protein interaction modules that regulate a wide variety of cellular functions via recognition of short linear motifs (SLiMs). Often, PDZ mediated-interactions are essential components of macromolecular complexes, and disruption affects the entire scaffold. Due to their roles as linchpins in trafficking and signaling pathways, PDZ domains are attractive targets: both for controlling viral pathogens, which bind PDZ domains and hijack cellular machinery, as well as for developing therapies to combat human disease. However, successful therapeutic interventions that avoid off-target effects are a challenge, because each PDZ domain interacts with a number of cellular targets, and specific binding preferences can be difficult to decipher. Over twenty-five years of research has produced a wealth of data on the stereochemical preferences of individual PDZ proteins and their binding partners. Currently the field lacks a central repository for this information. Here, we provide this important resource and provide a manually curated, comprehensive list of the 271 human PDZ domains. We use individual domain, as well as recent genomic and proteomic, data in order to gain a holistic view of PDZ domains and interaction networks, arguing this knowledge is critical to optimize targeting selectivity and to benefit human health.

Structural characterization and computational analysis of PDZ domains in Monosiga brevicollis.

Identification of the molecular networks that facilitated the evolution of multicellular animals from their unicellular ancestors is a fundamental problem in evolutionary cellular biology. Choanoflagellates are recognized as the closest extant nonmetazoan ancestors to animals. These unicellular eukaryotes can adopt a multicellular-like "rosette" state. Therefore, they are compelling models for the study of early multicellularity. Comparative studies revealed that a number of putative human orthologs are present in choanoflagellate genomes, suggesting that a subset of these genes were necessary for the emergence of multicellularity. However, previous work is largely based on sequence alignments alone, which does not confirm structural nor functional similarity. Here, we focus on the PDZ domain, a peptide-binding domain which plays critical roles in myriad cellular signaling networks and which underwent a gene family expansion in metazoan lineages. Using a customized sequence similarity search algorithm, we identified 178 PDZ domains in the Monosiga brevicollis proteome. This includes 11 previously unidentified sequences, which we analyzed using Rosetta and homology modeling. To assess conservation of protein structure, we solved high-resolution crystal structures of representative M. brevicollis PDZ domains that are homologous to human Dlg1 PDZ2, Dlg1 PDZ3, GIPC, and SHANK1 PDZ domains. To assess functional conservation, we calculated binding affinities for mbGIPC, mbSHANK1, mbSNX27, and mbDLG-3 PDZ domains from M. brevicollis. Overall, we find that peptide selectivity is generally conserved between these two disparate organisms, with one possible exception, mbDLG-3. Overall, our results provide novel insight into signaling pathways in a choanoflagellate model of primitive multicellularity.

Genomic analysis of atypical fibroxanthoma.

Atypical fibroxanthoma (AFX), is a rare type of skin cancer affecting older individuals with sun damaged skin. Since there is limited genomic information about AFX, our study seeks to improve the understanding of AFX through whole-exome and RNA sequencing of 8 matched tumor-normal samples. AFX is a highly mutated malignancy with recurrent mutations in a number of genes, including COL11A1, ERBB4, CSMD3, and FAT1. The majority of mutations identified were UV signature (C>T in dipyrimidines). We observed deletion of chromosomal segments on chr9p and chr13q, including tumor suppressor genes such as KANK1 and CDKN2A, but no gene fusions were found. Gene expression profiling revealed several biological pathways that are upregulated in AFX, including tumor associated macrophage response, GPCR signaling, and epithelial to mesenchymal transition (EMT). To further investigate the presence of EMT in AFX, we conducted a gene expression meta-analysis that incorporated RNA-seq data from dermal fibroblasts and keratinocytes. Ours is the first study to employ high throughput sequencing for molecular profiling of AFX. These data provide valuable insights to inform models of carcinogenesis and additional research towards tumor-directed therapy.

Stereochemical preferences modulate affinity and selectivity among five PDZ domains that bind CFTR: comparative structural and sequence analyses.

PDZ domain interactions are involved in signaling and trafficking pathways that coordinate crucial cellular processes. Alignment-based PDZ binding motifs identify the few most favorable residues at certain positions along the peptide backbone. However, sequences that bind the CAL (CFTR-associated ligand) PDZ domain reveal only a degenerate motif that overpredicts the true number of high-affinity interactors. Here, we combine extended peptide-array motif analysis with biochemical techniques to show that non-motif "modulator" residues influence CAL binding. The crystallographic structures of 13 CAL:peptide complexes reveal defined, but accommodating stereochemical environments at non-motif positions, which are reflected in modulator preferences uncovered by multisequence substitutional arrays. These preferences facilitate the identification of high-affinity CAL binding sequences and differentially affect CAL and NHERF PDZ binding. As a result, they also help determine the specificity of a PDZ domain network that regulates the trafficking of CFTR at the apical membrane.

Identification of cell cycle-regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors.

We identify the cell cycle-regulated mRNA transcripts genome-wide in the osteosarcoma-derived U2OS cell line. This results in 2140 transcripts mapping to 1871 unique cell cycle-regulated genes that show periodic oscillations across multiple synchronous cell cycles. We identify genomic loci bound by the G2/M transcription factor FOXM1 by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and associate these with cell cycle-regulated genes. FOXM1 is bound to cell cycle-regulated genes with peak expression in both S phase and G2/M phases. We show that ChIP-seq genomic loci are responsive to FOXM1 using a real-time luciferase assay in live cells, showing that FOXM1 strongly activates promoters of G2/M phase genes and weakly activates those induced in S phase. Analysis of ChIP-seq data from a panel of cell cycle transcription factors (E2F1, E2F4, E2F6, and GABPA) from the Encyclopedia of DNA Elements and ChIP-seq data for the DREAM complex finds that a set of core cell cycle genes regulated in both U2OS and HeLa cells are bound by multiple cell cycle transcription factors. These data identify the cell cycle-regulated genes in a second cancer-derived cell line and provide a comprehensive picture of the transcriptional regulatory systems controlling periodic gene expression in the human cell division cycle.

Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.

We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle-dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.

RAGGED SEEDLING2 is required for expression of KANADI2 and REVOLUTA homologues in the maize shoot apex.

The anatomy of ragged seedling2-R (rgd2-R) leaves is disorganized along the adaxial/abaxial axis. Mutant leaves often exhibit radial morphology but unlike similar mutants, rgd2-R radial leaves have no reduction of adaxial or abaxial identity. Previously, we proposed that RGD2 does not establish adaxial or abaxial fate but performs a signaling function during primordial leaf development. Here we describe genetic and molecular tests of our model for RGD2 function. Genetic analyses explored the phenotypic effects of rgd2-R on maize leaf mutations disrupting mediolateral and/or dorsiventral development. In situ hybridization and qRT-PCR analyses reveal that rgd2-R mutants show reduced accumulation of adaxial and abaxial molecular markers in the maize shoot apex. These data contribute to a revised model whereby RGD2 is required for normal accumulation of leaf patterning transcripts in the developing shoot. Molecular and genetic analyses suggest that RGD2 and LEAF BLADELESS1 share overlapping functions affecting shoot development in maize.