Biomedical subjects

Marjorie Brand

Publications and source records attributed to Marjorie Brand.

4 recordsLinked to original sources

PubMed2004

Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network.

Small Maf proteins serve as dual-function transcription factors through an exchange of their heterodimerization partners. For example, as heterodimers with hematopoietic cell-specific p45 NF-E2 or NF-E2-related factors (Nrf), they activate the beta-globin or antioxidative stress enzyme heme oxygenase 1 (HO-1) genes, respectively. In contrast, together with Bach1, they repress these same genes. However, the signals leading to this partner exchange are not known. Using chromatin immunoprecipitation assays in NIH 3T3 cells, we show that heme, an inducer of ho-1, promotes displacement of Bach1 from the MafK-occupied ho-1 enhancers, which is followed by Nrf2 binding to these elements. Whereas histone H3 at the ho-1 enhancers and promoter is hyperacetylated irrespective of gene activity, exposure of cells to heme results in de novo hyperacetylation and hypermethylation of histone H3 in the transcribed region. These data indicate that, under normal conditions, the chromatin structure of ho-1 is in a preactivation state, but transcription is repressed by Bach1. Heme induces switching of Maf dimers, resulting in ho-1 expression. Heme also promotes displacement of Bach1 from the beta-globin locus control region without affecting MafK binding in murine erythroleukemia cells. Thus, heme functions as a signaling molecule for gene expression in higher eukaryotes.

Acetylation↗

PubMed2003

Dynamic changes in transcription factor complexes during erythroid differentiation revealed by quantitative proteomics.

During erythroid differentiation, beta-globin gene expression is regulated by the locus control region (LCR). The transcription factor NF-E2p18/MafK binds within this region and is essential for beta-globin expression in murine erythroleukemia (MEL) cells. Here we use the isotope-coded affinity tag (ICAT) technique of quantitative mass spectrometry to compare proteins interacting with NF-E2p18/MafK during differentiation. Our results define MafK as a 'dual-function' molecule that shifts from a repressive to an activating mode during erythroid differentiation. The exchange of MafK dimerization partner from Bach1 to NF-E2p45 is a key step in the switch from the repressed to the active state. This shift is associated with changes in the interaction of MafK with co-repressors and co-activators. Thus, our results suggest that in addition to its role as a cis-acting activator of beta-globin gene expression in differentiated erythroid cells, the LCR also promotes an active repression of beta-globin transcription in committed cells before terminal differentiation.

Animals↗

PubMed2003

Novel subunits of the TATA binding protein free TAFII-containing transcription complex identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry following one-dimensional gel electrophoresis.

Initiation of transcription of protein-encoding genes by RNA polymerase II was thought to require the transcription factor II D (TF(II)D), a complex comprising the TATA binding protein (TBP) and TBP-associated factors. However, another multiprotein complex isolated more recently and called TFTC (TBP-free TAF(II )containing complex), was shown to mediate initiation of RNA polymerase II (Pol II) transcription in the absence of TF(II)D as well as specific acetylation of histone H3 in a nucleosomal context. Several subunits of the TFTC complex were already identified using classical methods such as Edman based microsequencing and Western blot analysis. In this article we present a mass spectrometry based proteomic approach to confirm previous results and to identify other possible subunits of the TFTC complex. The TFTC complex was separated on one-dimensional sodium dodecyl sulfate polyacrylamide electrophoresis and analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting. Identifications were realized after databank searches. This new characterization of TFTC complex confirmed the presence of already described subunits (TRRAP, GCN5, SAP130/KIA0017, TAF(II)150, TAF(II)135, TAF(II)100, TAF(II)80, TAF(II)20, SPT3 and PAF65beta). Moreover, a good coverage of these sequences was obtained. Interestingly, TAF(II)32 and PAF6alpha were also determined as potential novel subunits of TFTC. These results together show the suitability and the great potential of this method and offer new perspectives in fundamental studies of transcription factor complexes.

Blotting, Western↗

PubMed2002

TATA-binding protein-free TAF-containing complex (TFTC) and p300 are both required for efficient transcriptional activation.

Initiation of transcription of protein-encoding genes by RNA polymerase II was thought to require transcription factor TFIID, a complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs). In the presence of TBP-free TAF complex (TFTC), initiation of polymerase II transcription can occur in the absence of TFIID. TFTC contains several subunits that have been shown to play the role of transcriptional coactivators, including the GCN5 histone acetyltransferase (HAT), which acetylates histone H3 in a nucleosomal context. Here we analyze the coactivator function of TFTC. We show direct physical interactions between TFTC and the two distinct activation regions (H1 and H2) of the VP16 activation domain, whereas the HAT-containing coactivators, p300/CBP (CREB-binding protein), interact only with the H2 subdomain of VP16. Accordingly, cell transfection experiments demonstrate the requirement of both p300 and TFTC for maximal transcriptional activation by GAL-VP16. In agreement with this finding, we show that in vitro on a chromatinized template human TFTC mediates the transcriptional activity of the VP16 activation domain in concert with p300 and in an acetyl-CoA-dependent manner. Thus, our results suggest that these two HAT-containing co-activators, p300 and TFTC, have complementary rather than redundant roles during the transcriptional activation process.

Adaptor Proteins, Signal Transducing↗