Evaluation of Myc E-Box Phylogenetic Footprints in Glycolytic Genes by Chromatin Immunoprecipitation Assays

Abstract

Prediction of gene regulatory sequences using phylogenetic footprinting has advanced considerably but lacks experimental validation. Here, we report whether transcription factor binding sites predicted by dot plotting or web-based Trafac analysis could be validated by chromatin immunoprecipitation assays. MYC overexpression enhances glycolysis without hypoxia and hence may contribute to altered tumor metabolism. Because the full spectrum of glycolytic genes directly regulated by Myc is not known, we chose Myc as a model transcription factor to determine whether it binds target glycolytic genes that have conserved canonical Myc binding sites or E boxes (5-CACGTG-3). Conserved canonical E boxes in ENO1, HK2, and LDHA occur in 31-to 111-bp islands with high interspecies sequence identity (>65%). Trafac analysis revealed another region in ENO1 that corresponds to a murine region with a noncanonical E box. Myc bound all these conserved regions well in the human P493-6 B lymphocytes. We also determined whether Myc could bind nonconserved canonical E boxes found in the remaining human glycolytic genes. Myc bound PFKM, but it did not significantly bind GPI, PGK1, and PKM2. Binding to BPGM, PGAM2, and PKLR was not detected. Both GAPD and TPI1 do not have conserved E boxes but are induced and bound by Myc through regions with noncanonical E boxes. Our results indicate that Myc binds well to conserved canonical E boxes, but not nonconserved E boxes. However, the binding of Myc to unpredicted genomic regions with noncanonical E boxes reveals a limitation of phylogenetic footprinting. In aggregate, these observations indicate that Myc is an important regulator of glycolytic genes, suggesting that MYC plays a key role in a switch to glycolytic metabolism during cell proliferation or tumorigenesis.Defining transcriptional regulatory networks is essential for our understanding of embryonic development, cell growth, and tumorigenesis. Throughout evolution, biologically important genes and their regulatory elements have been selectively conserved (34). Completing sequencing the genomes of a variety of species provides a unique opportunity for the identification of transcriptional regulatory regions through interspecies sequence comparison, also known as phylogenetic footprinting. In particular, noncoding sequences with interspecies sequence identity approaching that of exonic sequences are enriched with putative transcription factor binding sites.A number of approaches that allow the prediction of transcriptional regulatory regions in any genomic region through comparisons of mouse and human sequences have been reported (4,19,21,24). These approaches identify conserved regions that contain putative transcription factor binding sites. However, a purely computational approach tends to be replete with the uncertainty as to whether a predicted cis-regulatory module is biologically functional.Several algorithms have been developed and experimentally evaluated for the discovery of candidate regulatory regions, such as those in the Dros...

Glycolytic rate and lymphomagenesis depend on PARP14, an ADP ribosyltransferase of the B aggressive lymphoma (BAL) family

“…We found substantially increased glycolysis in premalignant B lineage-committed Eμ-myc bone marrow cells, consistent with the increased size of these cells and analyses of cell lines showing that c-Myc directly binds regulatory elements in the chromatin of genes encoding glycolytic enzymes to increase their expression (35). Of note, we found that PARP14 is crucial for Eμ-myc cells fully to increase their glycolysis and size.…”

Metabolic reprogramming of the tumor

“…MYC activates glycolysis through transcriptional induction of various glycolytic enzymes and LDH-A (Shim et al, 1997;Kim et al, 2004). Recent experiments using double transgenic mice expressing c-MYC in a doxycycline-dependent manner specifically in hepatocytes revealed that overexpression of a multitude of glycolysis genes in tumors was abrogated by switching off c-MYC expression .…”

AMP-activated protein kinase and human cancer: cancer metabolism revisited

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