The 3' Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies' association with histone locus bodies.

Non-polyadenylated mRNAs of replication-dependent histones (RDHs) are synthesized by RNA polymerase II (Pol II) at histone locus bodies (HLBs). HLBs frequently associate with Cajal bodies (CBs), in which 3'-end processing factors for RDH genes are enriched; however, this association's role in transcription termination of RDH genes remains unclear. Here, we show that Pol II pauses immediately upstream of transcript end sites of RDH genes and Mediator plays a role in this Pol II pausing through CBs' association with HLBs. Disruption of the Mediator docking site for Little elongation complex (LEC)-Cap binding complex (CBC)-Negative elongation factor (NELF), components of CBs, interferes with CBs' association with HLBs and 3' Pol II pausing, resulting in increased aberrant unprocessed RDH gene transcripts. Our findings suggest Mediator's involvement in CBs' association with HLBs to facilitate 3' Pol II pausing and subsequent 3'-end processing of RDH genes by supplying 3'-end processing factors.

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.

Elongin functions as a loading factor for Mediator at ATF6α-regulated ER stress response genes.

The bZIP transcription factor ATF6α is a master regulator of endoplasmic reticulum (ER) stress response genes. In this report, we identify the multifunctional RNA polymerase II transcription factor Elongin as a cofactor for ATF6α-dependent transcription activation. Biochemical studies reveal that Elongin functions at least in part by facilitating ATF6α-dependent loading of Mediator at the promoters and enhancers of ER stress response genes. Depletion of Elongin from cells leads to impaired transcription of ER stress response genes and to defects in the recruitment of Mediator and its CDK8 kinase subunit. Taken together, these findings bring to light a role for Elongin as a loading factor for Mediator during the ER stress response.

Multiple roles for PARP1 in ALC1-dependent nucleosome remodeling.

The SNF2 family ATPase Amplified in Liver Cancer 1 (ALC1) is the only chromatin remodeling enzyme with a poly(ADP-ribose) (PAR) binding macrodomain. ALC1 functions together with poly(ADP-ribose) polymerase PARP1 to remodel nucleosomes. Activation of ALC1 cryptic ATPase activity and the subsequent nucleosome remodeling requires binding of its macrodomain to PAR chains synthesized by PARP1 and NAD+ A key question is whether PARP1 has a role(s) in ALC1-dependent nucleosome remodeling beyond simply synthesizing the PAR chains needed to activate the ALC1 ATPase. Here, we identify PARP1 separation-of-function mutants that activate ALC1 ATPase but do not support nucleosome remodeling by ALC1. Investigation of these mutants has revealed multiple functions for PARP1 in ALC1-dependent nucleosome remodeling and provides insights into its multifaceted role in chromatin remodeling.

NRBP1-Containing CRL2/CRL4A Regulates Amyloid ß Production by Targeting BRI2 and BRI3 for Degradation.

Alzheimer's disease (AD) is a progressive neurodegenerative disease caused by accumulations of Aß peptides. Production and fibrillation of Aß are downregulated by BRI2 and BRI3, which are physiological inhibitors of amyloid precursor protein (APP) processing and Aß oligomerization. Here, we identify nuclear receptor binding protein 1 (NRBP1) as a substrate receptor of a Cullin-RING ubiquitin ligase (CRL) that targets BRI2 and BRI3 for degradation. Moreover, we demonstrate that (1) dimerized NRBP1 assembles into a functional Cul2- and Cul4A-containing heterodimeric CRL through its BC-box and an overlapping cryptic H-box, (2) both Cul2 and Cul4A contribute to NRBP1 CRL function, and (3) formation of the NRBP1 heterodimeric CRL is strongly enhanced by chaperone-like function of TSC22D3 and TSC22D4. NRBP1 knockdown in neuronal cells results in an increase in the abundance of BRI2 and BRI3 and significantly reduces Aß production. Thus, disrupting interactions between NRBP1 and its substrates BRI2 and BRI3 may provide a useful therapeutic strategy for AD.

The role of Mediator and Little Elongation Complex in transcription termination.

Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3' end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.

CTD-dependent and -independent mechanisms govern co-transcriptional capping of Pol II transcripts.

Co-transcriptional capping of RNA polymerase II (Pol II) transcripts by capping enzyme proceeds orders of magnitude more efficiently than capping of free RNA. Previous studies brought to light a role for the phosphorylated Pol II carboxyl-terminal domain (CTD) in activation of co-transcriptional capping; however, CTD phosphorylation alone could not account for the observed magnitude of activation. Here, we exploit a defined Pol II transcription system that supports both CTD phosphorylation and robust activation of capping to dissect the mechanism of co-transcriptional capping. Taken together, our findings identify a CTD-independent, but Pol II-mediated, mechanism that functions in parallel with CTD-dependent processes to ensure optimal capping, and they support a "tethering" model for the mechanism of activation.

Role for the MED21-MED7 Hinge in Assembly of the Mediator-RNA Polymerase II Holoenzyme.

Mediator plays an integral role in activation of RNA polymerase II (Pol II) transcription. A key step in activation is binding of Mediator to Pol II to form the Mediator-Pol II holoenzyme. Here, we exploit a combination of biochemistry and macromolecular EM to investigate holoenzyme assembly. We identify a subset of human Mediator head module subunits that bind Pol II independent of other subunits and thus probably contribute to a major Pol II binding site. In addition, we show that binding of human Mediator to Pol II depends on the integrity of a conserved "hinge" in the middle module MED21-MED7 heterodimer. Point mutations in the hinge region leave core Mediator intact but lead to increased disorder of the middle module and markedly reduced affinity for Pol II. These findings highlight the importance of Mediator conformation for holoenzyme assembly.

TRIM29 regulates the assembly of DNA repair proteins into damaged chromatin.

Although DNA double-strand break (DSB) repair is mediated by numerous proteins accumulated at DSB sites, how DNA repair proteins are assembled into damaged chromatin has not been fully elucidated. Here we show that a member of the tripartite motif protein family, TRIM29, is a histone-binding protein responsible for DNA damage response (DDR). We found that TRIM29 interacts with BRCA1-associated surveillance complex, cohesion, DNA-PKcs and components of TIP60 complex. The dynamics of the TRIM29-containing complex on H2AX nucleosomes is coordinated by a cross-talk between histone modifications. TRIM29 binds to modified histone H3 and H4 tails in the context of nucleosomes. Furthermore, chromatin binding of TRIM29 is required for the phosphorylation of H2AX and cell viability in response to ionizing radiation. Our results suggest that TRIM29 functions as a scaffold protein to assemble DNA repair proteins into chromatin followed by efficient activation of DDR.

MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex.

Regulation of transcription elongation by RNA polymerase II (Pol II) is a key regulatory step in gene transcription. Recently, the little elongation complex (LEC)-which contains the transcription elongation factor ELL/EAF-was found to be required for the transcription of Pol II-dependent small nuclear RNA (snRNA) genes. Here we show that the human Mediator subunit MED26 plays a role in the recruitment of LEC to a subset of snRNA genes through direct interaction of EAF and the N-terminal domain (NTD) of MED26. Loss of MED26 in cells decreases the occupancy of LEC at a subset of snRNA genes and results in a reduction in their transcription. Our results suggest that the MED26-NTD functions as a molecular switch in the exchange of TBP-associated factor 7 (TAF7) for LEC to facilitate the transition from initiation to elongation during transcription of a subset of snRNA genes.

Subunit architecture and functional modular rearrangements of the transcriptional mediator complex.

The multisubunit Mediator, comprising ∼30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA-binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism.

A conserved Mediator-CDK8 kinase module association regulates Mediator-RNA polymerase II interaction.

The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) C-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a pre-eminent complex in eukaryotic transcription regulation. We used macromolecular EM and biochemistry to investigate the subunit organization, structure and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator's middle module interferes with CTD-dependent RNAPII binding to a previously unknown middle-module CTD-binding site and with the holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the mechanism of transcription regulation by Mediator.

Immunoaffinity purification of protein complexes from Mammalian cells.

In this chapter, we describe a purification scheme designed to isolate multisubunit protein complexes gently and quickly from crude extracts of mammalian cells using immunoaffinity purification of epitope tagged proteins and the multisubunit complexes with which they associate. As an example we describe isolation of the mammalian Mediator complex from HeLa S3 cells.

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.

DNA-PKcs-PIDDosome: a nuclear caspase-2-activating complex with role in G2/M checkpoint maintenance.

Caspase-2 is unique among all the mammalian caspases in that it is the only caspase that is present constitutively in the cell nucleus, in addition to other cellular compartments. However, the functional significance of this nuclear localization is unknown. Here we show that DNA damage induced by gamma-radiation triggers the phosphorylation of nuclear caspase-2 at the S122 site within its prodomain, leading to its cleavage and activation. This phosphorylation is carried out by the nuclear serine/threonine protein kinase DNA-PKcs and promoted by the p53-inducible death-domain-containing protein PIDD within a large nuclear protein complex consisting of DNA-PKcs, PIDD, and caspase-2, which we have named the DNA-PKcs-PIDDosome. This phosphorylation and the catalytic activity of caspase-2 are involved in the maintenance of a G2/M DNA damage checkpoint and DNA repair mediated by the nonhomologous end-joining (NHEJ) pathway. The DNA-PKcs-PIDDosome thus represents a protein complex that impacts mammalian G2/M DNA damage checkpoint and NHEJ.

MED19 and MED26 are synergistic functional targets of the RE1 silencing transcription factor in epigenetic silencing of neuronal gene expression.

A key hub for the orchestration of epigenetic modifications necessary to restrict neuronal gene expression to the nervous system is the RE1 silencing transcription factor (REST; also known as neuron restrictive silencer factor, NRSF). REST suppresses the nonspecific and premature expression of neuronal genes in non-neuronal and neural progenitor cells, respectively, via recruitment of enzymatically diverse corepressors, including G9a histone methyltransferase (HMTase) that catalyzes di-methylation of histone 3-lysine 9 (H3K9me2). Recently, we identified the RNA polymerase II transcriptional Mediator to be an essential link between RE1-bound REST and G9a in epigenetic suppression of neuronal genes in non-neuronal cells. However, the means by which REST recruits Mediator to facilitate G9a-dependent extra-neuronal gene silencing remains to be elucidated. Here, we identify the MED19 and MED26 subunits in Mediator as direct physical and synergistic functional targets of REST. We show that although REST independently binds to both MED19 and MED26 in isolation, combined depletion of both subunits is required to disrupt the association of REST with Mediator. Furthermore, combined, but not individual, depletion of MED19/MED26 impairs REST-directed recruitment to RE1 elements of Mediator and G9a, leading to a reversal of G9a-dependent H3K9me2 and de-repression of REST-target gene expression. Together, these findings identify MED19/MED26 as a probable composite REST interface in Mediator and further clarify the mechanistic basis by which Mediator facilitates REST-imposed epigenetic restrictions on neuronal gene expression.

Neuralized-like 1 (Neurl1) targeted to the plasma membrane by N-myristoylation regulates the Notch ligand Jagged1.

Notch signaling constitutes an evolutionarily conserved mechanism that mediates cell-cell interactions in various developmental processes. Numerous regulatory proteins interact with the Notch receptor and its ligands and control signaling at multiple levels. Ubiquitination and endocytosis followed by endosomal sorting of both the receptor and its ligands is essential for Notch-mediated signaling. The E3 ubiquitin ligases, Neuralized (Neur) and Mind Bomb (Mib1), are crucial for regulating the activity and stability of Notch ligands in Drosophila; however, biochemical evidence that the Notch ligands are directly targeted for ubiquitination by Neur and/or Mib1 has been lacking. In this report, we explore the function of Neurl1, a mouse ortholog of Drosophila Neur. We show that Neurl1 can function as an E3 ubiquitin ligase to activate monoubiquitination in vitro of Jagged1, but not other mammalian Notch ligands. Neurl1 expression decreases Jagged1 levels in cells and blocks signaling from Jagged1-expressing cells to neighboring Notch-expressing cells. We demonstrate that Neurl1 is myristoylated at its N terminus, and that myristoylation of Neurl1 targets it to the plasma membrane. Point mutations abolishing either Neurl1 myristoylation and plasma membrane localization or Neurl1 ubiquitin ligase activity impair its ability to down-regulate Jagged1 expression and to block signaling. Taken together, our results argue that Neurl1 at the plasma membrane can affect the signaling activity of Jagged1 by directly enhancing its ubiquitination and subsequent turnover.

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.

The mammalian Mediator complex and its role in transcriptional regulation.

Mediator is an essential component of the RNA polymerase II general transcriptional machinery and plays a crucial part in the activation and repression of eukaryotic mRNA synthesis. The Saccharomyces cerevisiae Mediator was the first to be defined and is a high molecular mass complex composed of >20 distinct subunits that performs multiple activities in transcription. Recent studies have defined the subunit composition and associated activities of mammalian Mediator, and revealed a striking evolutionary conservation of Mediator structure and function from yeast to man.