Recruitment of human cyclin T1 to nuclear bodies through direct interaction with the PML protein

Abstract

Human cyclin T1, the cyclin partner of Cdk9 kinase in the positive transcription elongation factor b (P-TEFb), is an essential cellular cofactor that is recruited by the human immunodeficiency virus type 1 (HIV-1) Tat transactivator to promote transcriptional elongation from the HIV-1 long terminal repeat (LTR). Here we exploit fluorescence resonance energy transfer (FRET) to demonstrate that cyclin T1 physically interacts in vivo with the promyelocytic leukaemia (PML) protein within specific subnuclear compartments that are coincident with PML nuclear bodies. Deletion mutants at the C-terminal region of cyclin T1 are negative for FRET with PML and fail to localize to nuclear bodies. Cyclin T1 and PML are also found associated outside of nuclear bodies, and both proteins are present at the chromatinized HIV-1 LTR promoter upon Tat transactivation. Taken together these results suggest that PML proteins regulate Tat- mediated transcriptional activation by modulating the availability of cyclin T1 and other essential cofactors to the transcription machinery.

Subnuclear distribution of the largest subunit of the human origin recognition complex during the cell cycle

“…Pull-down experiments using the recombinant GST-HP1α and GST proteins were performed as described (Marcello et al, 2003;Marzio et al, 1998).…”

“…Cells were fixed in 2% paraformaldehyde after 48 hours and mounted directly in 70% glycerol for FRET analysis. FRET measurements were carried out with an epifluorescence Axioskop 2 Zeiss microscope mounting a 103 W HBO lamp, a 100×/NA=1.3, oilimmersion Plan-Neofluar objective and Nomarsky optics, performed as described in detail elsewhere (Marcello et al, 2001;Marcello et al, 2003). Briefly, FRET experiments consisted of collecting two EGFP emission signals by exciting the EGFP with two different excitation wavelengths, 480 and 350 nm, the former being optimal for EGFP and the latter for BFP.…”

“…FRET experiments were performed by transfection of human HeLa cells with plasmids expressing HP1α and Orc1p fused at their N-terminus with EGFP and BFP respectively. We have already observed that the BFP:EGFP fluorescent protein pair has excitation and emission properties favorable for FRET and we have exploited these properties to study the in vivo interactions of other nuclear proteins in the same experimental conditions (Marcello et al, 2001;Marcello et al, 2003). FRET image analysis of individual transfected cells are shown in Fig.…”

Primitive hematopoietic cells resist HIV-1 infection via p21Waf1/Cip1/Sdi1

“…In this report, we show that p21 plays an indispensable role in maintaining the intrinsic cellular defense against HIV-1 infection in HSCs. Of note, the effect of p21 on HIV susceptibility was not due to an effect on cell cycle entry and was independent of the known mediators of HIV-1 resistance, tripartite motif protein 5α (Trim5α), promyelocytic leukemia protein (PML), copper metabolism domain containing 1 (Murr1), and IFN-α (12)(13)(14). Rather, it was associated with restricting viral DNA integration into the host cell genome.…”

Nuclear positional control of HIV transcription in 4D

“…In the case of constitutive virus expression, chromatin has to be open and permissive, while in the case of a latent infection it is expected to be open and permissive initially to allow integration, but then the provirus has to be silenced remaining inducible by external stimuli. This happens in cell types like resting memory T cells by a combination of various mechanisms: (1) the availability of factors essential for transcription; 23 (2) transcriptional interference from the endogenous gene harboring the provirus; [24][25][26] (3) formation of heterochromatin at the site of provirus integration; 27 (4) CpG methylation of the HIV-1 promoter; 28,29 (5) post-transcriptional mechanisms. [30][31][32] Our studies indicate another level of control determined by the three-dimensional conformation of chromatin at the provirus that we may call (6) silencing in trans as described below.…”