Detection and Visualization of Compositionally Similar <i>cis</i>-Regulatory Element Clusters in Orthologous and Coordinately Controlled Genes

Jegga, Anil G.; Sherwood, Shawn P.; Carman, James W.; Pinski, Andrew T.; Phillips, J. E.; Pestian, John; Aronow, Bruce J.

doi:10.1101/gr.255002

Cited by 89 publications

(81 citation statements)

References 49 publications

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“…One can use computational analysis of genome sequence [5,6,7,8,9], or compare promoter regions of genes that show highly correlated expression patterns [10,11,12,13]. A different, but related, problem is the identification of which DNA sequence binds to known purified transcription factors [14,15,16].…”

Section: Introductionmentioning

confidence: 99%

Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

Mao¹,

Brody²

2007

Biochemical and Biophysical Research Communications

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Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16 bp region of S. Cerevisiae chromosome 5 onto a gold surface. This 16 bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014 s −1 . We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase.

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Section: Introductionmentioning

confidence: 99%

Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

Mao¹,

Brody²

2007

Biochemical and Biophysical Research Communications

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“…The sequences flanking NM_020567 in mouse, XM_214477 in rat, and NM_015895 in human were used for these studies. TraFaC (transcription factor binding site comparison) (19) was used to identify the transcriptional start sites, intron/exon boundaries, and E2F binding sites. Binding sites were determined based on the TRANSFAC (20) data base, version 6.0.…”

Section: Methodsmentioning

confidence: 99%

“…4B). To extend our analysis for E2F-regulatory elements, we used the TraFaC program (trafac.chmcc.org) (19) to search for additional E2F motifs within the murine geminin genomic sequence. This analysis identified four E2F sites that were clustered around the first exon (Fig.…”

Section: Geminin Is Repressedmentioning

confidence: 99%

Geminin Is Targeted for Repression by the Retinoblastoma Tumor Suppressor Pathway through Intragenic E2F Sites

Markey

Siddiqui

Knudsen

2004

Journal of Biological Chemistry

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The geminin protein is a critical regulator of DNA replication. It functions to control replication fidelity by blocking the assembly of prereplication complexes in the S and G 2 phases of the cell cycle. Geminin protein levels, which are low in G 0 /G 1 and increase at the G 1 /S transition, are controlled through coordinate transcriptional and proteolytic regulation. Here we show that geminin is regulated transcriptionally by the retinoblastoma tumor suppressor (RB)/E2F pathway. Initially, we observed that the activation of RB led to the repression of geminin transcription. Conversely, Rb-null mouse embryonic fibroblasts have enhanced the expression of geminin relative to wild type mouse embryonic fibroblasts. Similarly, an acute loss of Rb in mouse adult fibroblasts deregulated geminin RNA and protein levels. To delineate the responsible regulatory motifs, luciferase reporter constructs containing fragments of the geminin promoter were generated. An analysis of the critical regulatory cis-acting elements in the geminin promoter indicated that intragenic E2F sites downstream of the first exon were responsible for RB-mediated repression of geminin. The direct analysis of the endogenous geminin promoter revealed that these intragenic E2F sites are occupied by E2F proteins, and the mutation of these sites eliminates responsiveness to RB. Together, these data link the expression of geminin to the RB/E2F pathway and represent the first promoter analysis of this important regulator of DNA replication.Geminin was identified originally as a protein that is degraded by mitotic Xenopus egg extracts, but not by interphase extracts (1), and concurrently during a screen to identify proteins that affect Xenopus development (2). Geminin is a small (25 kDa) protein expressed during the S and G 2 phases of the cell cycle but degraded in M phase at the metaphase/anaphase transition via the anaphase-promoting complex-mediated ubiquitination (3, 4). Functional analyses demonstrated that geminin acts to prevent the relicensing of replication origins after they have fired once. This is accomplished by binding to CDT1 (5), a requisite factor for loading MCMs 1 into the prereplication complex. Beginning at the G 1 /S transition, geminin protein levels accumulate and become sufficient to inhibit CDT1 activity. As soon as the origins of replication have fired once in the S phase, this inhibition of CDT1 prevents the reloading of MCMs onto chromatin until the completion of mitosis when geminin is degraded. Although the levels of geminin mRNA have been shown to increase 2-3-fold at the G 1 /S transition (6), the mechanism of this transcriptional regulation remains to be elucidated.RB has several emerging roles in the control of diverse processes outside of the G 1 /S transition. These include DNA repair, cell death, and DNA replication (7-10). In the case of DNA replication, the activation of the RB pathway results in the repression of numerous target genes. Classically, this repression is achieved through assembling repressor complexes ...

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“…Specifically, genes from clusters 1-3 were analysed. The murine and human sequences were aligned using the Trafac server (Jegga et al, 2002) and the 500 base pairs upstream and downstream of the transcriptional start site were probed for presence of conserved E2F sites using the CisMols server (Jegga et al, 2005). Initially, we determined the average number of conserved E2F sites present in each gene.…”

Section: Combinatorial Analyses Define Genes Tightly Regulated In Mulmentioning

confidence: 99%

Loss of the retinoblastoma tumor suppressor: differential action on transcriptional programs related to cell cycle control and immune function

et al. 2007

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Functional inactivation of the retinoblastoma tumor suppressor gene product (RB) is a common event in human cancers. Classically, RB functions to constrain cellular proliferation, and loss of RB is proposed to facilitate the hyperplastic proliferation associated with tumorigenesis. To understand the repertoire of regulatory processes governed by RB, two models of RB loss were utilized to perform microarray analysis. In murine embryonic fibroblasts harboring germline loss of RB, there was a striking deregulation of gene expression, wherein distinct biological pathways were altered. Specifically, genes involved in cell cycle control and classically associated with E2F-dependent gene regulation were upregulated via RB loss. In contrast, a program of gene expression associated with immune function and response to pathogens was significantly downregulated with the loss of RB. To determine the specific influence of RB loss during a defined period and without the possibility of developmental compensation as occurs in embryonic fibroblasts, a second system was employed wherein Rb was acutely knocked out in adult fibroblasts. This model confirmed the distinct regulation of cell cycle and immune modulatory genes through RB loss. Analyses of ciselements supported the hypothesis that the majority of those genes upregulated with RB loss are regulated via the E2F family of transcription factors. In contrast, those genes whose expression was reduced with the loss of RB harbored different promoter elements. Consistent with these analyses, we found that disruption of E2F-binding function of RB was associated with the upregulation of gene expression. In contrast, cells harboring an RB mutant protein (RB-750F) that retains E2F-binding activity, but is specifically deficient in the association with LXCXE-containing proteins, failed to upregulate these same target genes. However, downregulation of genes involved in immune function was readily observed with disruption of the LXCXE-binding function of RB.Thus, these studies demonstrate that RB plays a significant role in both the positive and negative regulations of transcriptional programs and indicate that loss of RB has distinct biological effects related to both cell cycle control and immune function.

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Detection and Visualization of Compositionally Similar cis-Regulatory Element Clusters in Orthologous and Coordinately Controlled Genes

Cited by 89 publications

References 49 publications

Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

Geminin Is Targeted for Repression by the Retinoblastoma Tumor Suppressor Pathway through Intragenic E2F Sites

Loss of the retinoblastoma tumor suppressor: differential action on transcriptional programs related to cell cycle control and immune function

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