No abstract

Histone acetyltransferase (HAT) activities of proteins such as p300, CBP, and P/CAF play important roles in activation of gene expression. We now show that the HAT activity of p300 can also be required for down-regulation of transcription by a DNA binding repressor protein. Promyelocytic leukemia zinc finger (PLZF), originally identified as a fusion with retinoic acid receptor alpha in rare cases of all-trans-retinoic acid-resistant acute promyelocytic leukemia, is a transcriptional repressor that recruits histone deacetylasecontaining corepressor complexes to specific DNA binding sites. PLZF associates with p300 in vivo, and its ability to repress transcription is specifically dependent on HAT activity of p300 and acetylation of lysines in its C-terminal C 2 -H 2 zinc finger motif. An acetylation site mutant of PLZF does not repress transcription and is functionally deficient in a colony suppression assay despite retaining its abilities to interact with corepressor/ histone deacetylase complexes. This is due to the fact that acetylation of PLZF activates its ability to bind specific DNA sequences both in vitro and in vivo. Taken together, our results indicate that a histone deacetylase-dependent transcriptional repressor can be positively regulated through acetylation and point to an unexpected role of a coactivator protein in transcriptional repression.Alterations of chromatin structure by covalent modification of nucleosomal histones at specific lysine residues in their amino-terminal tails play a major role in regulation of gene expression (26). Over the past few years, a large number of chromatin-modifying factors and complexes have been identified and characterized (see references 1, 15, and 27) and references therein for reviews). Intrinsic histone acetyltransferase (HAT) activities have been found to be associated with a number of transcriptional coactivator proteins, such as p300 (62) and P/CAF (79) (see also references 12 and 44 for reviews).The results of these studies have provided an explanation for the direct relationship between histone acetylation and gene transcription. However, in addition to histones, acetylation of gene-specific (see below) and basal transcription factors (36) by specific HATs has also been shown to play a role in regulating gene expression. For example, p53 is acetylated at specific lysine residues by p300 in vitro and in vivo (32). Acetylation of p53 contributes to its activation by facilitating a transition to a conformation with a higher affinity to its target DNA (32). Key hematopoietic transcription factors such as GATA1 (9) and ELKF (83) were also shown to be acetylated in their DNA binding domains leading to enhanced DNA binding and transcriptional activation. Acetylation has also been shown to antagonize the activities of transcriptional repressors by inhibiting their association with corepressor proteins (8,35,82). In the case of hypoxia-inducible factor 1␣ (HIF-1␣), acetylation represented a prerequisite for recruitment of the ubiquitin mediated degradation system ...

TrwB is an integral membrane protein linking the relaxosome to the DNA transport apparatus in plasmid R388 conjugation. Native TrwB has been purified in monomeric and hexameric forms, in the presence of dodecylmaltoside from overexpressing bacterial cells. A truncated protein (TrwB⌬N70) that lacked the transmembrane domain could be purified only in the monomeric form. Electron microscopy images revealed the hexameric structure and were in fact superimposable to the previously published atomic structure for TrwB⌬N70. In addition, the electron micrographs showed an appendix, ϳ25 Å wide, corresponding to the transmembrane region of TrwB. TrwB was located in the bacterial inner membrane in agreement with its proposed coupling role. Purified TrwB hexamers and monomers bound tightly the fluorescent ATP analogue TNP-ATP. A mutant in the Walker A motif, TrwB-K136T, was equally purified and found to bind TNP-ATP with a similar affinity to that of the wild type. However, the TNP-ATP affinity of TrwB⌬N70 was significantly reduced in comparison with the TrwB hexamers. Competition experiments in which ATP was used to displace TNP-ATP gave an estimate of ATP binding by TrwB (K d(ATP) ‫؍‬ 0.48 mM for hexamers). The transmembrane domain appears to be involved in TrwB protein hexamerization and also influences its nucleotide binding properties.

No abstract

Bronchoalveolar lavage represents a promising noninvasive source of lung cancer specific protein biomarkers with high diagnostic accuracy. Measurement of APOA1, CO4A, CRP, GSTP1, SAMP, and STMN1 in this fluid may be a useful tool for lung cancer diagnosis, although a further validation in a larger clinical set is required for early stages.

Oncogenic transcription factors such as PML-RARalpha, RUNX1-MTG8, and others work in large part by the recruitment of inhibitors of gene transcription to target promoters leading to aberrant repression of gene expression. PML-RARalpha, an archetypal chimeric oncoprotein, was previously shown to bring complexes of histone deacetylases (HDACs), histone methyltransferases (HMTases), and DNA methyl transferases (DNMTs) to target genes. In this issue of Cancer Cell, Villa et al. show that the full complement of chromatin machinery can be commandeered by these transcription factors with the polycomb group of proteins representing the newest identified recruit.

TrwB is an integral membrane protein encoded by the conjugative plasmid R388. TrwB binds ATP and is essential for R388-directed bacterial conjugation. The protein consists of a cytosolic domain, which contains an ATPbinding site, and a transmembrane domain. The complete protein has been purified in the presence of detergents, and in addition, the cytosolic domain has also been isolated in the form of a soluble truncated protein, TrwB⌬N70. The availability of intact and truncated forms of the protein provides a convenient system to study the role of the transmembrane domain in the stability of TrwB. Protein denaturation was achieved by heat, in the presence of guanidinium HCl, or under low salt conditions. In all three cases TrwB was significantly more stable than TrwB⌬N70 with other conditions being the same. IR spectroscopy of the native and truncated forms revealed significant differences between them. In addition, it was found that TrwB⌬N70 was stabilized in dispersions of non-ionic detergent, suggesting the presence of hydrophobic patches on the surface of the truncated protein. IR spectroscopy also confirmed the conformational stability provided by the detergent. These results suggest that in integral membrane proteins consisting of a transmembrane and a cytosolic domain, the transmembrane portion may have a role beyond the mere anchoring of the protein to the cell membrane. In addition, this study indicates that the truncated soluble parts of two-domain membrane proteins may not reflect the physiological conformation of their native counterparts.

In order to understand the functional significance of the transmembrane domain of TrwB, an integral membrane protein involved in bacterial conjugation, the protein was purified in the native, and also as a truncated soluble form (TrwBDN70). The intact protein (TrwB) binds preferentially purine over pyrimidine nucleotides, NTPs over NDPs, and ribo-over deoxyribonucleotides. In contrast, TrwBDN70 binds uniformly all tested nucleotides. The transmembrane domain has the general effect of making the nucleotide binding site(s) less accessible, but more selective. This is in contrast to other membrane proteins in which most of the protein mass, including the catalytic domain, is outside the membrane, but whose activity is not modified by the presence or absence of the transmembrane segment.