Biomedical subjects

Michael J Pazin

Publications and source records attributed to Michael J Pazin.

5 recordsLinked to original sources

PubMed2006

Crucial roles of Sp1 and epigenetic modifications in the regulation of the CLDN4 promoter in ovarian cancer cells.

Claudins form a large family of tight junction proteins that have essential roles in the control of paracellular ion flux and the maintenance of cell polarity. Many studies have shown that several claudin family members are abnormally expressed in various cancers. In particular, CLDN4 (encoding claudin-4) is overexpressed in ovarian cancer. However, although CLDN4 overexpression is well established, the mechanisms responsible for this abnormal regulation remain unknown. In the present study, we delineate a small region of the CLDN4 promoter critical for its expression. This region contains two Sp1 sites, both of which are required for promoter activity. However, because of the ubiquitous expression of Sp1, these sites, although necessary, are not sufficient to explain the patterns of gene expression of CLDN4 in various ovarian tissues. We show that the CLDN4 promoter is further controlled by epigenetic modifications of the Sp1-containing critical promoter region. Cells that overexpress CLDN4 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN4 promoter region, and the reverse is observed in cells that do not express CLDN4. Moreover, the CLDN4-negative cells can be induced to express CLDN4 through treatment with demethylating and/or acetylating agents. Because CLDN4 is elevated in a large fraction of ovarian cancer, the mechanism leading to deregulation may represent a general pathway in ovarian tumorigenesis and may lead to novel strategies for therapy and an overall better understanding of the biology of this disease.

Base Sequence↗

PubMed2006

Histone H4-K16 acetylation controls chromatin structure and protein interactions.

Acetylation of histone H4 on lysine 16 (H4-K16Ac) is a prevalent and reversible posttranslational chromatin modification in eukaryotes. To characterize the structural and functional role of this mark, we used a native chemical ligation strategy to generate histone H4 that was homogeneously acetylated at K16. The incorporation of this modified histone into nucleosomal arrays inhibits the formation of compact 30-nanometer-like fibers and impedes the ability of chromatin to form cross-fiber interactions. H4-K16Ac also inhibits the ability of the adenosine triphosphate-utilizing chromatin assembly and remodeling enzyme ACF to mobilize a mononucleosome, indicating that this single histone modification modulates both higher order chromatin structure and functional interactions between a nonhistone protein and the chromatin fiber.

Acetylation↗

PubMed2005

hnRNP K binds a core polypyrimidine element in the eukaryotic translation initiation factor 4E (eIF4E) promoter, and its regulation of eIF4E contributes to neoplastic transformation.

Translation initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a key role in regulation of cellular proliferation. Its effects on the m7GpppN mRNA cap are critical because overexpression of eIF4E transforms cells, and eIF4E function is rate-limiting for G1 passage. Although we identified eIF4E as a c-Myc target, little else is known about its transcriptional regulation. Previously, we described an element at position -25 (TTACCCCCCCTT) that was critical for eIF4E promoter function. Here we report that this sequence (named 4EBE, for eIF4E basal element) functions as a basal promoter element that binds hnRNP K. The 4EBE is sufficient to replace TATA sequences in a heterologous reporter construct. Interactions between 4EBE and upstream activator sites are position, distance, and sequence dependent. Using DNA affinity chromatography, we identified hnRNP K as a 4EBE-binding protein. Chromatin immunoprecipitation, siRNA interference, and hnRNP K overexpression demonstrate that hnRNP K can regulate eIF4E mRNA. Moreover, hnRNP K increased translation initiation, increased cell division, and promoted neoplastic transformation in an eIF4E-dependent manner. hnRNP K binds the TATA-binding protein, explaining how the 4EBE might replace TATA in the eIF4E promoter. hnRNP K is an unusually diverse regulator of multiple steps in growth regulation because it also directly regulates c-myc transcription, mRNA export, splicing, and translation initiation.

Animals↗

PubMed2004

Properties of ets-1 binding to chromatin and its effect on platelet factor 4 gene expression.

Ets-1 is important for transcriptional regulation in several hematopoietic lineages, including megakaryocytes. Some transcription factors bind to naked DNA and chromatin with different affinities, while others do not. In the present study we used the megakaryocyte-specific promoters platelet factor 4 (PF4), and glycoprotein IIb (GPIIb) as model systems to explore the properties of Ets-1 binding to chromatin. Chromatin immunoprecipitation assays indicated that Ets-1 binds to proximal regions in the PF4 and GPIIb promoters in vivo. In vitro and in vivo experiments showed that Ets-1 binding to chromatin on lineage-specific promoters does not require lineage-specific factors. Moreover, this binding shows the same order of affinity as the binding to naked DNA and does not require ATP-dependent or Sarkosyl-sensitive factors. The effect of Ets-1 binding on promoter activity was examined using the PF4 promoter as a model. We identified a novel Ets-1 site (at -50), and a novel Sarkosyl-sensitive DNase I-hypersensitive site generated by Ets-1 binding to chromatin, which significantly affect PF4 promoter activity. Taken together, our results suggest a model by which Ets-1 binds to chromatin without the need for lineage-specific accessory factors, and Ets-1 binding induces changes in chromatin and affects transactivation, which are essential for PF4 promoter activation.

Adenosine Triphosphate↗

PubMed2003

Combinatorial control of DNase I-hypersensitive site formation and erasure by immunoglobulin heavy chain enhancer-binding proteins.

DNase I-hypersensitive sites in cellular chromatin are usually believed to be nucleosome-free regions generated by transcription factor binding. Using a cell-free system we show that hypersensitivity does not simply correlate with the number of DNA-bound proteins. Specifically, the leucine zipper containing basic helix-loop-helix protein TFE3 was sufficient to induce a DNase I-hypersensitive site at the immunoglobulin heavy chain micro enhancer in vitro. TFE3 enhanced binding of an ETS protein PU.1 to the enhancer. However, PU.1 binding erased the DNase I-hypersensitive site without abolishing TFE3 binding. Furthermore, TFE3 binding enhanced transcription in the presence and absence of a hypersensitive site, whereas endonuclease accessibility correlated strictly with DNase I hypersensitivity. We infer that chromatin constraints for transcription and nuclease sensitivity can differ.

Animals↗