Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukaemia.

The transforming proteins of acute promyelocytic leukaemias (APL) are fusions of the promyelocytic leukaemia (PML) and the promyelocytic leukaemia zinc-finger (PLZF) proteins with retinoic acid receptor-alpha (RARalpha). These proteins retain the RARalpha DNA- and retinoic acid (RA)-binding domains, and their ability to block haematopoietic differentiation depends on the RARalpha DNA-binding domain. Thus RA-target genes are downstream effectors. However, treatment with RA induces differentiation of leukaemic blast cells and disease remission in PML-RARalpha APLs, whereas PLZF-RARa APLs are resistant to RA. Transcriptional regulation by RARs involves modifications of chromatin by histone deacetylases, which are recruited to RA-target genes by nuclear co-repressors. Here we show that both PML-RARalpha and PLZF-RARalpha fusion proteins recruit the nuclear co-repressor (N-CoR)-histone deacetylase complex through the RARalpha CoR box. PLZF-RARalpha contains a second, RA-resistant binding site in the PLZF amino-terminal region. High doses of RA release histone deacetylase activity from PML-RARalpha, but not from PLZF-RARalpha. Mutation of the N-CoR binding site abolishes the ability of PML-RARalpha to block differentiation, whereas inhibition of histone deacetylase activity switches the transcriptional and biological effects of PLZF-RARalpha from being an inhibitor to an activator of the RA signalling pathway. Therefore, recruitment of histone deacetylase is crucial to the transforming potential of APL fusion proteins, and the different effects of RA on the stability of the PML-RARalpha and PLZF-RARalpha co-repressor complexes determines the differential response of APLs to RA.

Differential recognition of liganded and unliganded thyroid hormone receptor by retinoid X receptor regulates transcriptional repression.

Thyroid hormone receptor (TR) functions as part of multiprotein complexes that also include retinoid X receptor (RXR) and transcriptional coregulators. We have found that both the TR CoR box and ninth heptad are required for RXR interaction and in turn for interaction with corepressor proteins N-CoR and SMRT. Remarkably, the recruitment of RXR to repression-defective CoR box and ninth-heptad mutants via a heterologous dimerization interface restores both corepressor interaction and repression. The addition of thyroid hormone obviates the CoR box requirement for RXR interaction, provided that the AF2 activation helix at the C terminus of TR is intact. These results indicate that RXR differentially recognizes the unliganded and liganded conformations of TR and that these differences appear to play a major role in the recruitment of corepressors to TR-RXR heterodimers.

Stoichiometric and steric principles governing repression by nuclear hormone receptors.

We have defined two principles of corepressor function that account for differences in transcriptional repression by nuclear hormone receptors (NHRs). First, we have determined that receptor stoichiometry is a crucial determinant of transcriptional repression mediated by the corepressors N-CoR and SMRT. This provides a molecular explanation for the observation that NHRs repress transcription as dimers but not monomers. Second, corepressor function is restricted by steric effects related to DNA binding in a receptor-specific manner. Thus, although N-CoR and SMRT are capable of binding to several NHRs in solution, they are highly selective about receptor binding on DNA, a context that reflects their in vivo function more accurately. These stoichiometric and steric principles govern specific interactions between corepressors and NHRs, thus providing evidence that N-CoR and SMRT do not serve redundant functions but rather contribute to receptor-specific transcriptional repression.

The human transcriptional adaptor genes TADA2L and GCN5L2 colocalize to chromosome 17q12-q21 and display a similar tissue expression pattern.

The chromosomal locations and the tissue expression patterns of the human transcriptional adaptors TADA2L and GCN5L2 have been determined. Northern blot analysis across a range of human tissues revealed that both the TADA2L and the GCN5L2 mRNAs are expressed to varying degrees in all tissue types. Furthermore, in most tissue types, the genes are expressed at relatively similar levels, suggesting coordinated regulation of TADA2L and GCN5L2 transcription. Chromosomal mapping by fluorescence in situ hybridization indicated that these genes reside near each other on chromosome 17; TADA2L mapped within bands 17q12-q21, while GCN5L2 mapped distally within band 17q21.

Cloning and characterization of a corepressor and potential component of the nuclear hormone receptor repression complex.

Nuclear hormone receptors are potent repressors of transcription in the unliganded state. We describe here the cloning of a nuclear receptor corepressor that we call SUN-CoR (Small Unique Nuclear receptor CoRepressor), which shows no homology to previously described nuclear hormone receptor corepressors, N-CoR, or SMRT. SUN-CoR is a highly basic, 16-kDa nuclear protein that is expressed at high levels in adult tissues and is induced during adipocyte and myogenic differentiation. SUN-CoR potentiates transcriptional repression by thyroid hormone receptor and RevErb in vivo, represses transcription when fused to a heterologous DNA binding domain, and interacts with RevErb as well as with thyroid hormone receptor in vitro. SUN-CoR also interacts with N-CoR and SMRT in vitro and with endogenous N-CoR in cells. We conclude that SUN-CoR is a corepressor and may function as an additional component of the complex involved in transcriptional repression by unliganded and orphan nuclear hormone receptors.

A nuclear hormone receptor corepressor mediates transcriptional silencing by receptors with distinct repression domains.

Ligand-independent transcriptional repression is an important function of nuclear hormone receptors. An interaction screen with the repression domain of the orphan receptor RevErb identified N-CoR, the corepressor for thyroid hormone receptor (TR) and retinoic acid receptor (RAR). N-CoR is likely to be a bona fide transcriptional corepressor for RevErb because (i) RevErb interacts with endogenous N-CoR, (ii) ectopic N-CoR potentiates RevErb-mediated repression, and (iii) transcriptional repression by RevErb correlates with its ability to bind N-CoR. Remarkably, a region homologous to the CoR box which is necessary for TR and RAR to interact with N-CoR is not required for RevErb. Rather, two short regions of RevErb separated by approximately 200 amino acids are required for interaction with N-CoR. The primary amino acid sequence of the N-terminal region of RevErb essential for N-CoR interaction is not homologous to that of TR or RAR, whereas similarities exist among the C-terminal domains of the receptors. N-CoR contains two adjacent but distinct interaction domains, one of which binds tightly to both RevErb and TR whereas the other binds more weakly and differentially interacts with the nuclear receptors. These results indicate that multiple nuclear receptors, utilizing different primary amino acid sequences, repress transcription by interacting with N-CoR.

GTPase activity in mouse hippocampus membranes following prenatal exposure to heroin and phenobarbital.

Low Km high affinity GTPase activity, with and without muscarinic receptor stimulation with 1 mM carbachol, was measured in membrane preparations of mouse hippocampus prenatally exposed to phenobarbital or heroin. Basal and carbachol-stimulated low Km GTPase activities after prenatal exposure to phenobarbital exhibited a statistically significant (P < 0.05) decrease both in Km and Vmax values. Basal Vmax values were reduced from 152 +/- 10 in controls to 112 +/- 13 (pmol/mg protein/min, mean +/- SEM) in exposed mice. The Km values in the offspring of mice treated with phenobarbital were reduced from 1.55 +/- 0.21 to 0.96 +/- 0.11 (microM, mean +/- SEM); Vmax and Km values after carbachol stimulation were similarly affected. Prenatal exposure to heroin did not change the GTPase activities, basal or carbachol-stimulated, with only a non-significant increase in both Vmax and Km values. It is postulated that these changes in G alpha protein activity may be related to the teratogenic effect of these drugs.

Desmin/vimentin intermediate filaments are dispensable for many aspects of myogenesis.

An expression vector was prepared containing a cDNA coding for a truncated version of the intermediate filament (IF) protein desmin. The encoded truncated desmin protein lacks a portion of the highly conserved alpha-helical rod region as well as the entire nonhelical carboxy-terminal domain. When transiently expressed in primary fibroblasts, or in differentiating postmitotic myoblasts and multinucleated myotubes, the truncated protein induces the complete dismantling of the preexisting vimentin or desmin/vimentin IF networks, respectively. Instead, in both cell types vimentin and desmin are packaged into hybrid spheroid bodies scattered throughout the cytoplasm. Despite the complete lack of intact IFs, myoblasts and myotubes expressing truncated desmin assemble and laterally align normal striated myofibrils and contract spontaneously in a manner indistinguishable from that of control myogenic cells. In older cultures the spheroid bodies shift from a longitudinal to a predominantly transverse orientation and loosely align along the I-Z-I-regions of striated myofibrils (Bennett, G.S., S. Fellini, Y. Toyama, and H. Holtzer. 1979. J. Cell Biol. 82:577-584), analogous to the translocation of intact desmin/vimentin IFs in control muscle. These results suggest the need for a critical reexamination of currently held concepts regarding the functions of desmin IFs during myogenesis.

Separation and determination of saturated very-long-chain free fatty acids in plasma of patients with adrenoleukodystrophy using solid-phase extraction and high-performance liquid chromatographic analysis.

An improved method was developed for the isolation of very-long-chain free fatty acids (VLCFFAs) in plasma and their separation and determination by high-performance liquid chromatography (HPLC). The method includes sample clean-up using solid-phase extraction, fluorophoric labelling of the FFAs and reversed-phase HPLC separation. The solid-phase extraction was carried out with aminopropyl-bonded phase columns. The FFAs were then derivatized with 9-anthryldiazomethane (fluorescent) reagent and separated by HPLC on an RP-18 column with methanol as the mobile phase. Using this method, the concentrations of C20:0, C22:0, C24:0 and C26:0 were determined in the plasma of five adrenoleukodystrophy (ALD) patients, one obligatory heterozygote, four healthy male volunteers and one child with cerebral leukodystrophy but without any other ALD symptoms. Statistically significant differences were found in the levels of C24 and C26 and in the ratios C24/C22 and C26/C22 in ALD patients and in normal controls. The values were higher in patients with X-ALD. This method therefore provides a rapid and accurate procedure for the laboratory confirmation of X-ALD.

High-performance liquid chromatographic analysis of free palmitic and stearic acids in cerebrospinal fluid.

A relatively simple method for extraction of free fatty acids from cerebrospinal fluid with aminopropyl bonded-phase columns, and the estimation of palmitic acid (C16:0) and stearic acid (C18:0) concentrations by high-performance liquid chromatographic analysis is described. The values of C16:0 and C18:0 in patients with non-neurological disorders lie within a narrow range, with a mean (+/- S.D.) of 4.02 +/- 0.33 micrograms/ml for C16:0 and 2.72 +/- 0.39 micrograms/ml for C18:0.

Characteristics of antifriction devices used for ski bindings.

These tests suggest that various antifriction devices do reduce the amount of torsion that can be transmitted to the skier's tibia during a slow twisting fall. It was shown that under various environmental conditions the antifriction devices are effective. Dirt negatively affected the performance of some devices. The importance of protecting bindings and antifriction devices from contamination cannot be overemphasized. It was shown that some antifriction devices perform better than others. In most instances the performance of the plate type of binding compares favorable with that of toe-heel bindings using antifriction devices. For the plate type of binding the dirt particularly had a detrimental effect. It should also be pointed out that these tests were run with one particular binding. These results would not necessarily be the same if the tests were conducted with another binding system. For instance, the limited motion of some of the antifriction devices (Lotork, Skidder) may impede their performance when used with bindings requiring a greater travel distance before release. The performance with other boot soles may be different from that with the particular boot sole used for these tests. Performance might be improved with a harder, smoother boot sole. Similarly, the effectiveness of the antifriction device may be reduced by the use of boot soles with a coarse pattern or relatively soft treads. In this test series a relatively small number of antifriction devices were evaluated. One should not presume that all devices will perform with equal effectiveness. Another factor, equally important, is the correction for the height of antifriction devices. Antifriction devices raise the boot sole from the ski surface, and if this change in height is not accounted for, the boot sole may effectively interfere with the operation of the toe or the heel unit. In summary, it can be stated that when properly applied and selected, antifriction devices make a significant contribution to skiing safety.