HDAC4 mediates transcriptional repression by the acute promyelocytic leukaemia-associated protein PLZF

International audiencePLZF, the promyelocytic leukaemia zinc-finger protein, is a transcriptional repressor essential to development. In some acute leukaemias, a chromosomal translocation fusing the PLZF gene to that encoding the retinoic acid receptor RARalpha gives rise to a fusion protein, PLZF-RARalpha, thought to be responsible for constitutive repression of differentiation-associated genes in these cells. Repression by both PLZF and PLZF-RARalpha is sensitive to the histone deacetylase inhibitor TSA, and PLZF was previously shown to interact physically with HDAC1, a class I histone deacetylase. We here asked whether class II histone deacetylases, known to be generally involved in differentiation processes, participate in the repression mediated by PLZF and PLZF-RARalpha, and found that PLZF interacts with HDAC4 in both GST-pull-down and co-immunoprecipitation assays. Furthermore, HDAC4 is indeed involved in PLZF and PLZF-RARalpha-mediated repression, since an enzymatically dead mutant of HDAC4 released the repression, as did an siRNA that blocks HDAC4 expression. Taken together, our data indicate that recruitment of HDAC4 is necessary for PLZF-mediated repression in both normal and leukaemic cells

Characterization of Brucella abortus lipopolysaccharide macrodomains as mega rafts

The lipopolysaccharides (LPS) of intracellular Proteobacteria such as Brucella, Chlamydia, Legionella and Rickettsia, have properties distinct from enterobacterial LPSs. These properties include deficient LPS induction of host cell activation, low endotoxicity and resistance to macrophage degradation. Together these constitute key virulence mechanisms for intracellular survival and replication. We previously demonstrated that B. abortus LPS captured by macrophages was recycled back to the plasma membrane where it was found associated with macrodomains. Furthermore, this LPS interferes with the MHC class II (MHC-II) presentation of peptides to specific T cell hybridomas. Here, we characterized the Brucella LPS macrodomains by microscopy and biochemistry approaches. We show for the first time that LPS macrodomains act as detergent resistant membranes (DRMs), segregating several lipid-raft components, LPS-binding proteins and MHC-II molecules. Brucella LPS macrodomains remain intact for several months in macrophages and are resistant to the disruptive effects of methyl β-cyclodextrin. Fluorescent anisotropy measurements show that B. abortus LPS is responsible for the formation of rigid surface membrane complexes. In addition, relocalization of MHC-II molecules is observed in these structures. The effects of B. abortus LPS on membrane properties could be responsible for pathogenic effects such as the inhibition of MHC-II-dependent antigen presentation. © 2005 The Authors; Journal compilation © 2005 Blackwell Publishing Ltd.Los lipopolisacáridos (LPS) de Proteobacterias intracelulares como Brucella, Chlamydia, Legionella y Rickettsia, tienen propiedades distintas a los LPS de las enterobacterias. Estas propiedades incluyen una inducción deficiente de los LPS a la activación de las células del huésped, una baja endotoxicidad y una resistencia a la degradación de los macrófagos. En conjunto, constituyen mecanismos de virulencia clave para la supervivencia y replicación intracelular. Anteriormente demostramos que el LPS de B. abortus capturado por los macrófagos se reciclaba de nuevo a la membrana plasmática, donde se encontraba asociado a macrodominios. Además, este LPS interfiere con la presentación de péptidos del MHC clase II (MHC-II) a hibridomas de células T específicos. Aquí, caracterizamos los macrodominios de LPS de Brucella mediante enfoques de microscopía y bioquímica. Demostramos por primera vez que los macrodominios de LPS actúan como membranas resistentes a los detergentes (DRM), segregando varios componentes de los lípidos, proteínas de unión a LPS y moléculas MHC-II. Los macrodominios de LPS de Brucella permanecen intactos durante varios meses en los macrófagos y son resistentes a los efectos disruptivos de la metil β-ciclodextrina. Las mediciones de anisotropía fluorescente muestran que el LPS de B. abortus es responsable de la formación de complejos rígidos de membrana superficial. Además, se observa la relocalización de las moléculas MHC-II en estas estructuras. Los efectos del LPS de B. abortus sobre las propiedades de la membrana podrían ser responsables de efectos patógenos como la inhibición de la presentación del antígeno dependiente del MHC-IIUniversidad Nacional, Costa RicaEscuela de Medicina Veterinari