Biomedical subjects

Tokameh Mahmoudi

Publications and source records attributed to Tokameh Mahmoudi.

5 recordsLinked to original sources

PubMed2006

The SWI/SNF chromatin-remodeling complex is a cofactor for Tat transactivation of the HIV promoter.

Tat is a critical viral transactivator essential for human immunodeficiency virus (HIV) gene expression. Activation involves binding to an RNA stem-loop structure and recruitment of the positive transcription elongation factor b. Tat also induces the remodeling of a single nucleosome in the HIV promoter. However, the mechanism of this remodeling has remained unclear. Knockdown of INI-1 and BRG-1, two components of the SWI/SNF chromatin-remodeling complex, suppressed Tat-mediated transactivation. Cells lacking INI-1 (G401 and MON) or BRG-1 (C33A) exhibited defective transactivation by Tat that was restored upon INI-1 and BRG-1 expression, respectively. Tat was co-immunoprecipitated with several SWI/SNF subunits, including INI-1, BRG-1, and beta-actin. The SWI/SNF complex interacted with the integrated HIV promoter in a Tat-dependent manner. We also found that INI-1 and BRG-1 synergized with the p300 acetyltransferase to activate the HIV promoter. This synergism depended on the acetyltransferase activity of p300 and on Tat Lys(50) and Lys(51). In conclusion, Tat-mediated activation of the HIV promoter requires the SWI/SNF complex in synergy with the coactivator p300.

Cell Line↗

PubMed2005

DNA looping induced by a transcriptional enhancer in vivo.

Enhancers are DNA sequences that can activate gene transcription from remote positions. In yeast, regulatory sequences that are functionally equivalent to the metazoan enhancers are called upstream activating sequences (UASs). UASs show a lower degree of flexibility than their metazoan counterparts, but can nevertheless activate transcription from a distance of >1000 bp from the promoter. One of several models for the mechanism of action of transcriptional enhancers proposes that enhancer-bound activating proteins contact promoter-bound transcription factors and thereby get in close proximity to the promoter region with concomitant looping of the intervening DNA. We tested the mode of enhancer activity in yeast. A polymerase II-transcribed gene was paired with a remote, inducible enhancer. An independent reporter system was inserted next to the promoter to monitor the potential modes of enhancer activity. Our results show that the enhancer activated the reporter system only in the presence of a functional promoter. We also demonstrate that the heterologous expression of GAGA, a factor known to facilitate DNA loop formation, allows enhancer action in yeast over a distance of 3000 bp.

Animals↗

PubMed2003

GAGA facilitates binding of Pleiohomeotic to a chromatinized Polycomb response element.

Polycomb response elements (PREs) are chromosomal elements, typically comprising thousands of base pairs of poorly defined sequences that confer the maintenance of gene expression patterns by Polycomb group (PcG) repressors and trithorax group (trxG) activators. Genetic studies have indicated a synergistic requirement for the trxG protein GAGA and the PcG protein Pleiohomeotic (PHO) in silencing at several PREs. However, the molecular basis of this cooperation remains unknown. Here, using DNaseI footprinting analysis, we provide a high-resolution map of sites for the sequence- specific DNA-binding PcG protein PHO, trxG proteins GAGA and Zeste and the gap protein Hunchback (HB) on the 1.6 kb Ultrabithorax (Ubx) PRE. Although these binding elements are present throughout the PRE, they display clear patterns of clustering, suggestive of functional collaboration at the level of PRE binding. We found that while GAGA could efficiently bind to a chromatinized PRE, PHO alone was incapable of binding to chromatin. However, PHO binding to chromatin, but not naked DNA, was strongly facilitated by GAGA, indicating interdependence between GAGA and PHO already at the level of PRE binding. These results provide a biochemical explanation for the in vivo cooperation between GAGA and PHO and suggest that PRE function involves the integrated activities of genetically antagonistic trxG and PcG proteins.

Base Sequence↗

PubMed2003

Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics.

A crucial issue in comparative proteomics is the accurate quantification of differences in protein expression levels. To achieve this, several methods have been developed in which proteins are labeled with stable isotopes either in vivo via metabolic labeling or in vitro by protein derivatization. Although metabolic labeling is the only way to obtain labeling of all proteins, it has thus far only been applied to single- celled organisms and cells in culture. Here we describe quantitative 15N metabolic labeling of the multicellular organisms Caenorhabditis elegans, a nematode, and Drosophila melanogaster, the common fruit fly, achieved by feeding them on 15N-labeled Escherichia coli and yeast, respectively. The relative abundance of individual proteins obtained from different samples can then be determined by mass spectrometry (MS). The applicability of the method is exemplified by the comparison of protein expression levels in two C. elegans strains, one with and one without a germ line. The methodology described provides tools for accurate quantitative proteomic studies in these model organisms.

Animals↗

PubMed2002

GAGA can mediate enhancer function in trans by linking two separate DNA molecules.

Enhancers have been defined as cis-acting DNA sequences that stimulate transcription from a linked promoter in a distance- and orientation-independent manner. How enhancers activate gene transcription over vast chromosomal distances within metazoan genomes remains poorly understood. Here, we show that the transcription factor GAGA can stimulate transcription by linking an enhancer to its cognate promoter. Strikingly, in addition to facilitating activation by a remote enhancer in cis, GAGA can direct activation of a promoter by an enhancer located on a separate DNA molecule. Enhancer function in trans is critically dependent on POZ domain-mediated GAGA oligomerization, enabling GAGA to bind two DNA molecules simultaneously. Transcriptional activation by an enhancer functioning in trans was observed both in transfected cells and in reconstituted transcription reactions. We propose that GAGA facilitates long-range activation by providing a protein bridge that mediates enhancer-promoter communication.

Animals↗