Xiaokang Pan

Maize-targeted mutagenesis: A knockout resource for maize

We describe an efficient system for site-selected transposon mutagenesis in maize. A total of 43,776 F1 plants were generated by using Robertsons Mutator (Mu) pollen parents and self-pollinated to establish a library of transposon-mutagenized seed. The frequency of new seed mutants was between 10–4 and 10–5 per F1 plant. As a service to the maize community, maize-targeted mutagenesis selects insertions in genes of interest from this library by using the PCR. Pedigree, knockout, sequence, phenotype, and other information is stored in a powerful interactive database (maize-targeted mutagenesis database) that enables analysis of the entire population and the handling of knockout requests. By inhibiting Mu activity in most F1 plants, we sought to reduce somatic insertions that may cause false positives selected from pooled tissue. By monitoring the remaining Mu activity in the F2, however, we demonstrate that seed phenotypes depend on it, and false positives occur in lines that appear to lack it. We conclude that more than half of all mutations arising in this population are suppressed on losing Mu activity. These results have implications for epigenetic models of inbreeding and for functional genomics.

Gramene: development and integration of trait and gene ontologies for rice

Gramene (http://www.gramene.org/) is a comparative genome database for cereal crops and a community resource for rice. We are populating and curating Gramene with annotated rice (Oryza sativa) genomic sequence data and associated biological information including molecular markers, mutants, phenotypes, polymorphisms and Quantitative Trait Loci (QTL). In order to support queries across various data sets as well as across external databases, Gramene will employ three related controlled vocabularies. The specific goal of Gramene is, first to provide a Trait Ontology (TO) that can be used across the cereal crops to facilitate phenotypic comparisons both within and between the genera. Second, a vocabulary for plant anatomy terms, the Plant Ontology (PO) will facilitate the curation of morphological and anatomical feature information with respect to expression, localization of genes and gene products and the affected plant parts in a phenotype. The TO and PO are both in the early stages of development in collaboration with the International Rice Research Institute, TAIR and MaizeDB as part of the Plant Ontology Consortium. Finally, as part of another consortium comprising macromolecular databases from other model organisms, the Gene Ontology Consortium, we are annotating the confirmed and predicted protein entries from rice using both electronic and manual curation.

SynBrowse: a synteny browser for comparative sequence analysis

MOTIVATION The recent efforts of various sequence projects to sequence deeply into various phylogenies provide great resources for comparative sequence analysis. A generic and portable tool is essential for scientists to visualize and analyze sequence comparisons. RESULTS We have developed SynBrowse, a synteny browser for visualizing and analyzing genome alignments both within and between species. It is intended to help scientists study macrosynteny, microsynteny and homologous genes between sequences. It can also aid with the identification of uncharacterized genes, putative regulatory elements and novel structural features of a species. SynBrowse is a GBrowse (the Generic Genome Browser) family software tool that runs on top of the open source BioPerl modules. It consists of two components: a web-based front end and a set of relational database back ends. Each database stores pre-computed alignments from a focus sequence to reference sequences in addition to the genome annotations of the focus sequence. The user interface lets end users select a key comparative alignment type and search for syntenic blocks between two sequences and zoom in to view the relationships among the corresponding genome annotations in detail. SynBrowse is portable with simple installation, flexible configuration, convenient data input and easy integration with other components of a model organism system. AVAILABILITY The software is available at http://www.gmod.org CONTACT vbrendel@iastate.edu

Site Preferences of Insertional Mutagenesis Agents in Arabidopsis

We have performed a comparative analysis of the insertion sites of engineered Arabidopsis (Arabidopsis thaliana) insertional mutagenesis vectors that are based on the maize (Zea mays) transposable elements and Agrobacterium T-DNA. The transposon-based agents show marked preference for high GC content, whereas the T-DNA-based agents show preference for low GC content regions. The transposon-based agents show a bias toward insertions near the translation start codons of genes, while the T-DNAs show a predilection for the putative transcriptional regulatory regions of genes. The transposon-based agents also have higher insertion site densities in exons than do the T-DNA insertions. These observations show that the transposon-based and T-DNA-based mutagenesis techniques could complement one another well, and neither alone is sufficient to achieve the goal of saturation mutagenesis in Arabidopsis. These results also suggest that transposon-based mutagenesis techniques may prove the most effective for obtaining gene disruptions and for generating gene traps, while T-DNA-based agents may be more effective for activation tagging and enhancer trapping. From the patterns of insertion site distributions, we have identified a set of nucleotide sequence motifs that are overrepresented at the transposon insertion sites. These motifs may play a role in the transposon insertion site preferences. These results could help biologists to study the mechanisms of insertions of the insertional mutagenesis agents and to design better strategies for genome-wide insertional mutagenesis.

Targeting casein kinase II restores Ikaros tumor suppressor activity and demonstrates therapeutic efficacy in high-risk leukemia

Ikaros (IKZF1) is a tumor suppressor that binds DNA and regulates expression of its target genes. The mechanism of Ikaros activity as a tumor suppressor and the regulation of Ikaros function in leukemia are unknown. Here, we demonstrate that Ikaros controls cellular proliferation by repressing expression of genes that promote cell cycle progression and the phosphatidylinositol-3 kinase (PI3K) pathway. We show that Ikaros function is impaired by the pro-oncogenic casein kinase II (CK2), and that CK2 is overexpressed in leukemia. CK2 inhibition restores Ikaros function as transcriptional repressor of cell cycle and PI3K pathway genes, resulting in an antileukemia effect. In high-risk leukemia where one IKZF1 allele has been deleted, CK2 inhibition restores the transcriptional repressor function of the remaining wild-type IKZF1 allele. CK2 inhibition demonstrated a potent therapeutic effect in a panel of patient-derived primary high-risk B-cell acute lymphoblastic leukemia xenografts as indicated by prolonged survival and a reduction of leukemia burden. We demonstrate the efficacy of a novel therapeutic approach for high-risk leukemia: restoration of Ikaros tumor suppressor activity via inhibition of CK2. These results provide a rationale for the use of CK2 inhibitors in clinical trials for high-risk leukemia, including cases with deletion of one IKZF1 allele.

Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacetylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B-cell Acute Lymphoblastic Leukemia

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 trimethylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the upstream regulatory element of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed, and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic casein kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of trimethylated histone H3 lysine 27 and decreased histone H3 Lys-9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. The presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL.

Epigenetic regulation of gene expression by Ikaros, HDAC1 and Casein Kinase II in leukemia.

IKZF1 (Ikaros) encodes a DNA-binding protein that acts as a master regulatory of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL).1, 2, 3, 4 The deletion and/or mutation of Ikaros is associated with the development of B-cell acute lymphoblastic leukemia (B-ALL) with poor outcome.5, 6, 7, 8, 9, 10, 11 Ikaros directly associates with components of the histone deacetylase complex (NuRD), HDAC1, HDAC2 and Mi-2.12, 13, 14 Although Ikaros is hypothesized to regulate the transcription of target genes by recruiting the NuRD complex, the mechanism of Ikaros-mediated transcriptional regulation in leukemia is still unknown. Here we use a systems biology approach to determine the mechanism through which Ikaros and HDAC1 regulate gene expression in human B-ALL.

A graphical approach to tracking and reporting target status in structural genomics

Determination of a protein structure requires a series of decisions and processes, starting with target selection, through cloning, expression, purification, and finally structure determination. Structural genomics projects may distribute these steps among several different groups of researchers. Although this division may achieve a lower cost per solved structure, it creates a unique set of challenges for integrating and passing information on the progress of a given target across several functional divisions. Laboratory information management systems (LIMS) are essential for gathering this information, but may not display the progress of a given target in an intuitive way. In addition, structural genomics projects funded by the Protein Structure Initiative (PSI) are obliged to disseminate data regularly to the TargetDB and PepcDB data repositories, and this requires the creation of specialized views of the data. We report here how the flow of a target through a structural genomics pipeline and reports to TargetDB and PepcDB can be abstracted as directed acyclic graphs or trees. To implement this kind of display, we created software that tracks the flow of activity leading toward protein structure determination and prepares XML reports as input to TargetDB and PepcDB. The target tracing software consists of a set of Perl CGI scripts that integrate with the Graphviz visualization system to provide a graphical, user-friendly Web interface. The database reporting software, also coded in Perl, transfers large-scale genomics data from our LIMS into a PepcDB reportable XML file. This software package has facilitated inter-group communication, improved the quality and accuracy of information in our LIMS, and increased the efficiency and accuracy of our reports to PepcDB.

Domain view: a web tool for protein domain visualization and analysis

The identification of sequence-based protein domains and their boundaries is often a prelude to structure determination. An accurate prediction of disordered regions, secondary structures and low complexity segments of target protein sequences can improve the efficiency of selection in structural genomics and also aid in design of constructs for directed structural biology studies. At the Center for Eukaryotic Structural Genomics (CESG) we have developed DomainView, a web tool to visualize and analyze predicted protein domains, disordered regions, secondary structures and low complexity segments of target protein sequences for selection of experimental protein structure attempts. DomainView consists of a relational database and a web graphical-user interface. The database was developed based on MySQL, which stores data from target protein sequences and their domains, disordered regions, secondary structures and low complexity segments. The program of the web user interface is a Perl CGI script. When a user searches for a target protein sequence, the script displays the combinational information about the domains and other features of that target sequence graphically on a web page by querying the database. The graphical representation for each feature is linked to a web page showing more detailed annotation information or to a new window directly running the corresponding prediction program to show further information about that feature.

Abstract PR09: CK2 inhibition exerts a therapeutic effect in high-risk ALL by restoring IKZF1-mediated repression of cell cycle progression and the PI3K pathway

The IKZF1 (Ikaros) gene encodes a DNA-binding protein that acts as a tumor suppressor in leukemia. Deletion of one Ikaros allele results in B-cell acute lymphoblastic leukemia (B-ALL) with a high rate of relapse and poor outcome. The mechanisms through which Ikaros suppresses leukemogenesis and that regulate Ikaros activity as a tumor suppressor in leukemia are unknown. Using a systems biology approach, we determined that Ikaros regulates transcription of genes that control two pathways crucial for proliferation of leukemia cells: 1) cell cycle progression and 2) the phosphatidylinositol 3-kinase (PI3K) pathway. Using gain-of-function and loss-of-function experiments we demonstrate that Ikaros transcriptionally represses genes that promote cell cycle progression and the PI3K pathway and activates transcription of a gene that suppresses the PI3K pathway. In high-risk B-ALL with deletion of one Ikaros allele, we show that the function of Ikaros as a transcriptional regulator is impaired due to reduced binding at promoters of its target genes. Previous work shows that Ikaros DNA-binding affinity is regulated via direct phosphorylation by the pro-oncogenic kinase, CK2 (Casein Kinase II). We show that CK2 is overexpressed in high-risk B-ALL as compared to normal B-cell precursors, further reducing Ikaros function. Treatment of primary high-risk B-ALL (with deletion of one IKZF1allele) using the CK2 specific inhibitor, CX-4945, restored Ikaros function as a transcriptional regulator of genes that control cell cycle progression and the PI3K pathway. Treatment with CK2 inhibitor was also associated with cell cycle arrest and reduced phosphorylation of the AKT kinase, a downstream PI3K pathway target. Using serial quantitative chromatin immunoprecipitation (qChIP) analyses spanning the promoters of Ikaros target genes, we demonstrated that Ikaros can repress transcription of its target genes through two distinct mechanisms: 1) via recruitment of histone deacetylase 1 (HDAC1), which is associated with the formation of repressive chromatin characterized by H3K27me3 and the loss of H3K9ac; and 2) by an HDAC1-independent mechanism that is associated with the formation of repressive chromatin characterized by H3K9me3, along with the loss of H3K9ac. The therapeutic efficacy of CK2 inhibition using CX-4945 against high-risk B-ALL was demonstrated in vivo using 4 different xenografts: 3 different high-risk primary pre-B-ALL patient-derived xenografts and Nalm6 xenografts. CX-4945 showed strong therapeutic effects in all 4 xenografts, as evidence by reduced leukemia cell numbers in bone marrow and in spleen, together with prolonged survival of treated xenograft animals. Expression analysis of Ikaros target genes in leukemia cells treated in vivo with CX-4945 revealed an expression pattern cell cycle regulatory and PI3K pathway genes that was highly similar to that observed with Ikaros overexpression. These data suggest that CK2 inhibition in vivo exerts its therapeutic effect on high-risk B-ALL by restoring Ikaros function as a transcriptional regulator of genes that promote cell cycle progression and the PI3K pathway. In summary, our results reveal that: 1) Ikaros functions as a tumor suppressor by suppressing cell cycle progression and the PI3K pathway; 2) Ikaros regulates transcription by inducing two distinct epigenetic alterations at promoters of its target genes and 3) CK2 inhibition with CX-4945 restores Ikaros function as a transcriptional regulator in vivo, and has a strong therapeutic effect in primary xenografts of high-risk B-ALL. These results provide support for the use of CK2 inhibitors in clinical trials for high-risk B-ALL. Supported by the National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment. This abstract is also presented as Poster B10. Citation Format: Chunhua Song, Chandrika Gowda, XiaoKang Pan, Kimberly J. Payne, Sinisa Dovat. CK2 inhibition exerts a therapeutic effect in high-risk ALL by restoring IKZF1-mediated repression of cell cycle progression and the PI3K pathway. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR09.