An Integrative Genomic and Epigenomic Approach for the Study of Transcriptional Regulation

The molecular heterogeneity of acute leukemias and other tumors constitutes a major obstacle towards understanding disease pathogenesis and developing new targeted-therapies. Aberrant gene regulation is a hallmark of cancer and plays a central role in determining tumor phenotype. We predicted that integration of different genome-wide epigenetic regulatory marks along with gene expression levels would provide greater power in capturing biological differences between leukemia subtypes. Gene expression, cytosine methylation and histone H3 lysine 9 (H3K9) acetylation were measured using high-density oligonucleotide microarrays in primary human acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) specimens. We found that DNA methylation and H3K9 acetylation distinguished these leukemias of distinct cell lineage, as expected, but that an integrative analysis combining the information from each platform revealed hundreds of additional differentially expressed genes that were missed by gene expression arrays alone. This integrated analysis also enhanced the detection and statistical significance of biological pathways dysregulated in AML and ALL. Integrative epigenomic studies are thus feasible using clinical samples and provide superior detection of aberrant transcriptional programming than single-platform microarray studies

Deregulation of DUX4 and ERG in acute lymphoblastic leukemia

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL).1,2 Here, we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG are hallmarks of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases, and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt utilizes a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivating domains of ERG, but inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia, in which DUX4 deregulation results in loss-of-function of ERG, either by deletion or induction of expression of an isoform that is a dominant negative inhibitor of wild type ERG function

Biochemical Genetic Analysis of Blue Light Responses in Neurospora Crassa

136 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1982.Physiological responses to blue light are widespread among prokaryotic and eukaryotic organisms. The photoreceptor for some of these responses, long a matter of controversy, may be a flavin or carotenoid. This dissertation applies a biochemical genetic approach to an analysis of the blue light responses in the fungus Neurospora. A major goal was to determine the nature of the Neurospora blue light photoreceptor. The key findings are as follows: (i) A correlation was found in riboflavin mutants between flavin deficiency and a reduced sensitivity to light. Flavin deficiency resulted in an 80-fold decrease in sensitivity for the photosuppression of circadian conidiation and reduced the photosensitivity for phase shifting and carotenogenesis to about 1/16th and 1/4th of normal, respectively. (ii) Riboflavin analogs were used to demonstrate that flavins can function as photoreceptors. The analogs 1-deazariboflavin and roseoflavin, which have red-shifted absorption, were effective photoreceptors for the photosuppression and phase shifting of circadian conidiation by 540 nm light, but were relatively ineffective for carotenogenesis. These results (i and ii) provide evidence for flavin photoreception in the blue light responses of Neurospora. (iii) The flavoprotein nitrate reductase was found not to be involved in the photoresponses being studied. The responses of photosuppression and phase shifting of circadian conidiation, as assayed in nitrate reductase mutants, showed no significant differences as compared to the responses in a strain which would utilize nitrate. The responses also occur on a medium which represses nitrate reductase activity. (iv) As a start towards the genetic dissection of the photoreception process, three mutants were isolated which did not exhibit photosuppression of circadian conidiation. The three lis ("light insensitive") mutations segregate as single nuclear genes, are nonallelic, and are recessive to wild type. The light insensitive phenotype of the mutants is restricted to the photosuppression response in that phase shifting and carotenogenesis are not altered. (v) A theoretical consideration of the role of photooxidiative processes in the evolution of circadian rhythmicity is also included.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Cloning and Characterization of scon-3(+), a New Member of the Neurospora crassa Sulfur Regulatory System

The sulfur regulatory system of Neurospora crassa consists of a group of sulfur-regulated structural genes (e.g., arylsulfatase) that are under coordinate control of the CYS3 positive regulator and sulfur controller (SCON) negative regulators. Here we report on the cloning of scon-3(+), which encodes a polypeptide of 171 amino acids and is a Skp1 family homolog. Repeat-induced point mutation of scon-3(+) resulted in a phenotype of constitutive expression of arylsulfatase, a phenotype consistent with other sulfur controller mutants. Northern analysis indicated that, unlike other members of the sulfur regulatory system, expression of scon-3(+) is not under the direct control of the CYS3 transcriptional activator. In particular, scon-3(+) mRNA was detectable under sulfur repressing or derepressing conditions in a Δcys-3 mutant. In yeast, Skp1p and an F-box protein binding partner are core constituents of a class of E3 ubiquitin ligases known as SCF complexes. The N. crassa negative regulator SCON2 contains an F-box motif essential for the operation of the sulfur regulatory system and suggests a role for an SCF complex in the N. crassa sulfur regulatory system. A crucial set of experiments, by using a yeast two-hybrid approach with confirming coimmunoprecipitation assays, demonstrated that SCON3 interacts with SCON2 in a manner dependent upon the F-box motif of SCON2. The protein-protein interaction detected between SCON2 and SCON3 represents the initial demonstration in a filamentous fungus of functional interaction between putative core components of a SCF complex