Novel critical role of a human Mediator complex for basal RNA polymerase II transcription

Abstract

Human Mediator complexes have been described as important bridging factors that enhance the effect of activators in purified systems and in chromatin. Here we report a novel basal function of a human Mediator complex. A monoclonal antibody was generated that depleted the majority of Mediator components from crude cell extracts. The removal of human Mediator abolished transcription by RNA polymerase II. This was observed on all genes tested, on TATA-containing and TATA-less promoters, both in the presence and absence of activators. To identify the relevant complex a combined biochemical and immunopurification protocol was applied. Two variants termed Mediator and basal Mediator were functionally and structurally distinguished. Basal Mediator function relies on additional constraints, which is reflected in the observation that it is essential in crude but not in purified systems. We conclude that basal Mediator is a novel general transcription factor of RNA polymerase II.

Fig. 1. PAQ-associated complex is a general factor in physiological transcription systems. (A) PAQ depletion (ΔPAQ) abolishes RNA polymerase II transcription in HeLa nuclear extracts (IN), whereas depletion with control antibody (ΔControl) does not affect formation of transcripts (pol II). (B) PAQ depletion is specific for RNA polymerase II. Formation of RNA polymerase III transcripts (pol III) is not influenced. (C) Capacity of PAQ-associated complex to restore transcription in depleted nuclear extracts is resistant to high salt washes, arguing for specificity of the procedure. Titration of the respective complexes was performed in a four-fold range. (D) and (E) PAQ-associated complex is essential for TATA-less and TATA-containing promoters driven by Gal4-VP16. (D) PAQ depletion (ΔPAQ) abolishes RNA polymerase II transcription in Jurkat nuclear extracts (IN). Transcription reactions were performed with pCD4GAL (TATA-less reporter) and pMLGAL (TATA-containing reporter) as templates in the absence (–) and presence (+) of 20 ng of activators Gal-VP16:H1 (H1) and Gal-VP16:H1F442P (H1mt), respectively. (E) PAQ-associated complex restores basal and activated transcription in depleted Jurkat nuclear extracts (ΔPAQ). Titration of the complex was achieved in a three-fold range in the absence (–) and presence (+) of Gal147-VP16.

Fig. 2. PAQ-monoclonal antibody depletes the majority of Mediator components from nuclear extracts. Four per cent of input (IN-JNE), 4.4% of PAQ depleted (ΔPAQ) and mock depleted (Δcontrol) Jurkat nuclear extract together with 20% of the PAQ (E-PAQ)- or the mock precipitated (E-control) material were analyzed by western blotting. Cross-reactions of secondary antibodies with IgG-HC and IgG-LC are marked (*).

Fig. 3. PAQ-associated complex purified from the phosphocellulose (P11) 0.85 M KCl fraction restores transcription in PAQ-depleted nuclear extracts. (A) Analysis of P11 fractions for capacity to reconstitute transcription by PAQ1H7-IPs. Transcription reactions were conducted with titration of immunopurified complexes in a five-fold range. Untreated nuclear extracts (IN) and IPs (NE IP) from the latter were employed as positive controls. (B) PAQ1H7-IPs from DE52 fractions restore transcription in PAQ-depleted nuclear extracts. PAQ-IPs from input (IN) and various fractions (#) of the second DE52 column [IP(DE52)] were processed and used for add-back experiments similar to those in (A). Activity peaks at 100 mM salt (#18) on the gradient. (C) Western blot analysis of input fractions for IPs described in (B). The functional complex coelutes with the majority of Mediator components tested, except TRAP230, which exhibits a sharper elution profile.

Fig. 4. Two forms of PAQ associated Mediators. (A) Purified B-Med (IP-DE52; see text) exhibits basal activity only in crude but not in purified transcription systems. Immunopurified Mediator from P11 0.5 M KCl fraction (IP-0.5), P11 0.85 M KCl (IP-0.85) and DE52 0.1 M KCl fraction (IP-DE52), respectively, was tested either in a depleted (NEΔPAQ) nuclear extract or in a purified system (SYSTEM) containing TBP (in the absence of TAFs). (B) B-Med purified from the DE52 0.1 M KCl fraction (IP-DE52) contains neither RNA polymerase II (CTD) nor TRAP95. The presence of CRSP70 and the absence of TRAP95 distinguish B-Med from the P11 0.5 M KCl derived complex (IP-0.5). The amount of cdk8 is substantially reduced.