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. 2015 Jun 15;5(1):10-24.
eCollection 2015.

AF4 and AF4N protein complexes: recruitment of P-TEFb kinase, their interactome and potential functions

Bastian Scholz  1 , Eric Kowarz  1 , Tanja Rössler  1 , Khalil Ahmad  2 , Dieter Steinhilber  2 , Rolf Marschalek  1
Affiliations

AF4 and AF4N protein complexes: recruitment of P-TEFb kinase, their interactome and potential functions

Bastian Scholz et al. Am J Blood Res. .

Abstract

AF4/AFF1 and AF5/AFF4 are the molecular backbone to assemble "super-elongation complexes" (SECs) that have two main functions: (1) control of transcriptional elongation by recruiting the positive transcription elongation factor b (P-TEFb = CyclinT1/CDK9) that is usually stored in inhibitory 7SK RNPs; (2) binding of different histone methyltransferases, like DOT1L, NSD1 and CARM1. This way, transcribed genes obtain specific histone signatures (e.g. H3K79me2/3, H3K36me2) to generate a transcriptional memory system. Here we addressed several questions: how is P-TEFb recruited into SEC, how is the AF4 interactome composed, and what is the function of the naturally occuring AF4N protein variant which exhibits only the first 360 amino acids of the AF4 full-length protein. Noteworthy, shorter protein variants are a specific feature of all AFF protein family members. Here, we demonstrate that full-length AF4 and AF4N are both catalyzing the transition of P-TEFb from 7SK RNP to their N-terminal domain. We have also mapped the protein-protein interaction network within both complexes. In addition, we have first evidence that the AF4N protein also recruits TFIIH and the tumor suppressor MEN1. This indicate that AF4N may have additional functions in transcriptional initiation and in MEN1-dependend transcriptional processes.

Keywords: 7SK RNP; AF4/AF4N; P-TEFb; RNA polymerase II; elongation control.

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Figures

Figure 1
Figure 1
Characterization of the TCZP-AF4ST stable cell line. A. Stable integrated vectors used in this study. B. Quantitative analysis of relative amounts of AF4 mRNA by qPCR from prepared cDNA with (TCZP+) or without (TCZP-) application of doxycyclin compared to regular 293T cells. Amounts are normalized to Actin [n = 3, +S.E.M.]. C. Top: Representative Western-Blot experiments from TCZP-AF4ST whole cell lysates after 2 passages with or without doxycyclin compared to constitutively expressing constructs pEZP and pEZP-BGH. Faster migrating bands are degradation products of AF4. Bottom: Western-Blot experiments from TCZP-AF4ST whole cell lysates 0-72 h after induction of expression. Lanes 481 and 483 differ in the amount of applied doxycyclin (481 = 1 µg/ml, 483 = 3 µg/ml). D. CCK-8 proliferation assays of induced TCZP-AF4ST cells over a time course of 10 days, compared to non-treated 293T cells [n = 3, ±S.E.M.]. Decline of untransfected cells is due to reaching confluency. E. Representative in-vitro GFP fluorescence microscopy images of TCZP-AF4ST cells during the indicated passages (magnification 100x, 0.5 s. exposure).
Figure 2
Figure 2
Characterization of the AF4kd V100 stable cell line. A. Results of the AF4-specific ELISA after transduction of HeLa cells with viral particles and establishment of resistance to puromycin. Relative amounts of AF4 protein in regular HeLa cells (100% = 1) and knock-down lines V10, V50 and V100 are depicted [n = 4, +S.E.M., *p < 0.05]. B. Quantitative analysis of relative amounts of AF4 mRNA by qPCR from prepared cDNA of the V10, V50 and V100 infected HeLa cells (100% = 1). Amounts are normalized to Actin [n = 3, +S.E.M., *p < 0.05]. C. CCK-8 proliferation assays of AF4kd V100 cells over a time course of 7 days, compared to non-treated HeLa cells [n = 3, ±S.E.M.]. D. Representative in-vitro microscopy images of AF4kd V100 cells during day 6 of the proliferation assays (magnification 100x, 10 ms. exposure) showing cowered morphology and non-commenced confluence.
Figure 3
Figure 3
Interaction and dependency between AF4 and transcription-associated factors. A. Western-Blot experiment for CDK9, CDK9-T186A, CYCT1, NFkB, AF4, CDK2, CDK7 and MEN1 from whole cell lysates of non-transfected HeLa (left) compared to AF4kd V100 cells (right). Relative quantification was carried out by using the Image Lab 3.0 Software (Bio-Rad). Molecular weight of all proteins are indicated. B. Western-Blot experiments with affinity purified (AP) strep-tagged AF4 (left) and AF4N (right) probed with the same antibodies. C. Immunoprecipitation of BFP-CYCT1 from nuclear fractions of transiently transfected 293T cells. (mock, pETP-BFPCYCT1, pEZP-AF4ST or pEZP-AF4NST; input 0.2%, eluate 33%). BFP-CYCT1 (112 kDa) as well as endogenous CYCT1 (87 kDa) were precipitated. Lower panel: Western blot with antiserum against the AF4 N-terminus.
Figure 4
Figure 4
AF4/AF4N interactome mapping. A. The Yeast-2-hybrid Matchmaker III system was used to analyze the interactome of the core components of the protein assembly that occur within the AF4 N-terminal portion. B. More detailed interaction map displaying which portion of each protein which is binding to other proteins of the AF4/AF4N interactome. The experimentally observed strength of protein interactions is indicated by different sizes or dotted lines. Mapping of protein-protein interactions by others is indicated by colored lines.
Figure 5
Figure 5
Recruitment of P-TEFb from storage complexes into AF4/AF4N. A. Schematic representation of the 7SK RNP. B. Western blots after CDK9 immunoprecipitation from whole cell lysates of transiently transfected 293T cells (mock, Strep-tagged AF4, HCMV IE1 or 5-LO). blots were probed with antibodies against CDK9 and LARP7. C. 7SK RNA binding assay. Results of specific quantitative PCR of 7SK RNA purified from elution fractions. Samples were subjected to quantitative PCR and quantified against a 7SK RNA dilution series. Shown is the x-fold binding of 7SK RNA compared to the GST negative control (set as 1) [n = 3, +S.E.M., *p < 0.05]. D. Results of quantitative PCR of 7SK RNA after incubation of whole cell lysates with RNase, recombinant GST-AF4N or a distinct whole cell lysate with overexpressed AF4ST. A non-treated lysate was used as negative control. Shown is the relative amount of 7SK RNA after incubation as the quotient of 7SK RNA/β-Actin copy number.
Figure 6
Figure 6
AF4N/AF4 transfers P-TEFb from 7SK RNPs to AF4N/AF4. 293T cells were transiently transfected with empty vector (mock), pEZP-AF4ST or pEZP-AF4NST. Whole cell lysates were prepared and separated on a 0-40% glycerol gradient by ultracentrifugation. Gradient were fractionated (n = 10) and analyzed for the presence of CDK9, LARP7 and AF4/AF4N. Dissociation of LARP7 from 7SK RNPs is equivalent to the disruption of existing 7SK RNPs. Bottom: AF4 immunoprecipitation from glycerol gradient fractions 2 to 7 was probed for the presence of CDK9 to demonstrate the P-TEFb transition from 7SK RNP to AF4N/AF4.
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