Human mediator subunit MED26 functions as a docking site for transcription elongation factors.

Promoter-proximal pausing by initiated RNA polymerase II (Pol II) and regulated release of paused polymerase into productive elongation has emerged as a major mechanism of transcription activation. Reactivation of paused Pol II correlates with recruitment of super-elongation complexes (SECs) containing ELL/EAF family members, P-TEFb, and other proteins, but the mechanism of their recruitment is an unanswered question. Here, we present evidence for a role of human Mediator subunit MED26 in this process. We identify in the conserved N-terminal domain of MED26 overlapping docking sites for SEC and a second ELL/EAF-containing complex, as well as general initiation factor TFIID. In addition, we present evidence consistent with the model that MED26 can function as a molecular switch that interacts first with TFIID in the Pol II initiation complex and then exchanges TFIID for complexes containing ELL/EAF and P-TEFb to facilitate transition of Pol II into the elongation stage of transcription.

A Structured Workflow for Mapping Human Sin3 Histone Deacetylase Complex Interactions Using Halo-MudPIT Affinity-Purification Mass Spectrometry.

Although a variety of affinity purification mass spectrometry (AP-MS) strategies have been used to investigate complex interactions, many of these are susceptible to artifacts because of substantial overexpression of the exogenously expressed bait protein. Here we present a logical and systematic workflow that uses the multifunctional Halo tag to assess the correct localization and behavior of tagged subunits of the Sin3 histone deacetylase complex prior to further AP-MS analysis. Using this workflow, we modified our tagging/expression strategy with 21.7% of the tagged bait proteins that we constructed, allowing us to quickly develop validated reagents. Specifically, we apply the workflow to map interactions between stably expressed versions of the Sin3 subunits SUDS3, SAP30, or SAP30L and other cellular proteins. Here we show that the SAP30 and SAP30L paralogues strongly associate with the core Sin3 complex, but SAP30L has unique associations with the proteasome and the myelin sheath. Next, we demonstrate an advancement of the complex NSAF (cNSAF) approach, in which normalization to the scaffold protein SIN3A accounts for variations in the proportion of each bait capturing Sin3 complexes and allows a comparison among different baits capturing the same protein complex. This analysis reveals that although the Sin3 subunit SUDS3 appears to be used in both SIN3A and SIN3B based complexes, the SAP30 subunit is not used in SIN3B based complexes. Intriguingly, we do not detect the Sin3 subunits SAP18 and SAP25 among the 128 high-confidence interactions identified, suggesting that these subunits may not be common to all versions of the Sin3 complex in human cells. This workflow provides the framework for building validated reagents to assemble quantitative interaction networks for chromatin remodeling complexes and provides novel insights into focused protein interaction networks.

A High-throughput Automated ELISA Assay for Detection of IgG Antibodies to the SARS-CoV-2 Spike Protein.

The SARS-CoV-2 pandemic and vaccination campaign has illustrated the need for high throughput serological assays to quantitatively measure antibody levels. Here, we present a protocol for a high-throughput colorimetric ELISA assay to detect IgG antibodies against the SARS-CoV-2 spike protein. The assay robustly distinguishes positive from negative samples, while controlling for potential non-specific binding from serum samples. To further eliminate background contributions, we demonstrate a computational pipeline for fitting ELISA titration curves, that produces an extremely sensitive antibody signal metric for quantitative comparisons across samples and time.

The mammalian Mediator complex.

The multiprotein Mediator (Med) complex is an evolutionarily conserved transcriptional regulator that plays important roles in activation and repression of RNA polymerase II transcription. Prior studies identified a set of more than twenty distinct polypeptides that compose the Saccharomyces cerevisiae Mediator. Here we discuss efforts to characterize the subunit composition and associated activities of the mammalian Med complex.

Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors.

Components of multiprotein complexes are routinely determined by using proteomic approaches. However, this information lacks functional content except when new complex members are identified. To analyze quantitatively the abundance of proteins in human Mediator we used normalized spectral abundance factors generated from shotgun proteomics data sets. With this approach we define a common core of mammalian Mediator subunits shared by alternative forms that variably associate with the kinase module and RNA polymerase (pol) II. Although each version of affinity-purified Mediator contained some kinase module and RNA pol II, Mediator purified through F-Med26 contained the most RNA pol II and the least kinase module as demonstrated by the normalized spectral abundance factor approach. The distinct forms of Mediator were functionally characterized by using a transcriptional activity assay, where F-Med26 Mediator/RNA pol II was the most active. This method of protein complex visualization has important implications for the analysis of multiprotein complexes and assembly of protein interaction networks.

Identification of new subunits of the multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase complex.

The mammalian ATM/PI 3-kinase-related TRRAP protein was previously found to be a component of a multi-protein histone acetyltransferase (HAT) complex containing the HAT TIP60. In this report, we identify a previously uncharacterized protein encoded by the FLJ10914 ORF, which we designate MRGBP, as a new component of the TRRAP/TIP60 HAT complex. In addition, through purification of MRGBP and its associated proteins from HeLa cell nuclear extracts, we identify the thyroid receptor coactivating protein (TRCp120), DMAP1, and the related MRG15 and MRGX proteins as MRGBP-associating proteins, and we present biochemical evidence that they are previously unrecognized components of the TRRAP/TIP60 HAT complex. Taken together, our findings shed new light on the structure and function of the mammalian TRRAP/TIP60 histone acetyltransferase complex.

A mammalian homolog of Drosophila melanogaster transcriptional coactivator intersex is a subunit of the mammalian Mediator complex.

The multiprotein Mediator complex is a coactivator required for transcriptional activation of RNA polymerase II transcribed genes by DNA binding transcription factors. We previously partially purified a Med8-containing Mediator complex from rat liver nuclei (Brower, C. S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R. D., Malik, S., Lane, W. S., Sorokina, I., Roeder, R. G., Conaway, J. W., and Conaway, R. C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353-10358). Analysis of proteins present in the most highly enriched Mediator fractions by tandem mass spectrometry led to the identification of several new mammalian Mediator subunits, as well as several potential Mediator subunits. Here we identify one of these proteins, encoded by the previously uncharacterized AK000411 open reading frame, as a new subunit of the mammalian Mediator complex. The AK000411 protein, which we designate hIntersex (human Intersex), shares significant sequence similarity with the Drosophila melanogaster intersex protein, which has functional properties expected of a transcriptional coactivator specific for the Drosophila doublesex transactivator. In addition, we show that hIntersex assembles into a subcomplex with Mediator subunits p28b and TRFP. Taken together, our findings identify a new subunit of the mammalian Mediator and shed new light on the architecture of the mammalian Mediator complex.

Identification of mammalian Mediator subunits with similarities to yeast Mediator subunits Srb5, Srb6, Med11, and Rox3.

The Mediator is a multiprotein coactivator required for activation of RNA polymerase II transcription by DNA binding transactivators. We recently identified a mammalian homologue of yeast Mediator subunit Med8 and partially purified a Med8-containing Mediator complex from rat liver nuclei (Brower, C. S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R. D., Malik, S., Lane, W. S., Sorokina, I., Roeder, R. G., Conaway, J. W., and Conaway, R. C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353-10358). Analysis of proteins present in the most highly purified Med8-containing fractions by tandem mass spectrometry led to the identification of many known mammalian Mediator subunits, as well as four potential Mediator subunits exhibiting sequence similarity to yeast Mediator subunits Srb5, Srb6, Med11, and Rox3. Here we present direct biochemical evidence that these four proteins are bona fide mammalian Mediator subunits. In addition, we identify direct pairwise binding partners of these proteins among the known mammalian Mediator subunits. Taken together, our findings identify a collection of novel mammalian Mediator subunits and shed new light on the underlying architecture of the mammalian Mediator complex.

A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology.

The Mediator is a multiprotein transcriptional coactivator that is expressed ubiquitously in eukaryotes from yeast to mammals and is required for induction of RNA polymerase II (pol II) transcription by DNA binding transcription factors. In the work described here, we exploit multidimensional protein identification technology (MudPIT) to carry out a proteomic analysis of the subunit composition of the mammalian Mediator complex. By comparing MudPIT data sets obtained from six independent Mediator preparations immunoaffinity purified through their Nut2 (MED10), Med25 (MED9), Intersex (MED29), LCMR1 (MED19), AK007855 (MED28), or CRSP70 (MED26) subunits, we identify a set of consensus mammalian Mediator subunits. In addition, we identify as Mediator-associated proteins the CDK8-like cyclin-dependent kinase CDK11 and the TRAP240-like KIAA1025 protein (MED13L), which is mutated in patients with the congenital heart defect transposition of the great arteries (TGA).

A mammalian mediator subunit that shares properties with Saccharomyces cerevisiae mediator subunit Cse2.

The multiprotein Mediator complex is a coactivator required for activation of RNA polymerase II transcription by DNA bound transcription factors. We previously identified and partially purified a mammalian Mediator complex from rat liver nuclei (Brower, C.S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R.D., Malik, S., Lane, W.S., Sorokina, I., Roeder, R.G., Conaway, J.W., and Conaway, R.C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353-10358). Analysis by tandem mass spectrometry of proteins present in the most highly purified rat Mediator fractions led to the identification of a collection of new mammalian Mediator subunits, as well as several potential Mediator subunits including a previously uncharacterized protein encoded by the FLJ10193 open reading frame. In this study, we present direct biochemical evidence that the FLJ10193 protein, which we designate Med25, is a bona fide subunit of the mammalian Mediator complex. In addition, we present evidence that Med25 shares structural and functional properties with Saccharomyces cerevisiae Mediator subunit Cse2 and may be a mammalian Cse2 ortholog. Taken together, our findings identify a novel mammalian Mediator subunit and shed new light on the architecture of the mammalian Mediator complex.