Laszlo Tora

Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B.

The human progesterone receptor (hPR) cDNA, synthesized from T47D breast cancer cells, and the hPR gene 5′‐flanking region were cloned and sequenced. Comparison of the cDNA‐deduced amino acid sequence with other PR homologues demonstrated the modular structure characteristic of nuclear receptors. As in the case of the chicken homologue, there are two hPR forms, A and B, which originate from translational initiation at AUG2 (codon 165) and AUG1, respectively. Northern blot analysis of T47D mRNA using various cDNA derived probes identified two classes of hPR mRNAs, one of which could code for hPR form B, while the other one lacked the 5′ region upstream of AUG1. S1 nuclease mapping and primer extension analyses confirmed that the second class of hPR transcripts are initiated between +737 and +842 and thus encode hPR form A, but not form B. By using the hPR gene 5′‐flanking sequences as promoter region in chimeric genes, we show that a functional promoter (located between ‐711 and +31) directs initiation of hPR mRNAs from the authentic start sites located at +1 and +15. Most importantly, initiation of transcription from chimeric genes demonstrated the existence of a second promoter located between +464 and +1105. Transient co‐transfection experiments with vectors expressing the human estrogen receptor showed that both promoters were estrogen inducible, although no classical estrogen responsive element was detected in the corresponding sequences. When transiently expressed, the two hPR forms similarly activated transcription from reporter genes containing a single palindromic progestin responsive element (PRE), while form B was more efficient at activating the PRE of the mouse mammary tumor virus long terminal repeat. Transcription from the ovalbumin promoter, however, was induced by hPR form A, but not by form B.

The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18.

Nuclear receptors (NRs) bound to response elements mediate the effects of cognate ligands on gene expression. Their ligand‐dependent activation function, AF‐2, presumably acts on the basal transcription machinery through intermediary proteins/mediators. We have isolated a mouse nuclear protein, TIF1, which enhances RXR and RAR AF‐2 in yeast and interacts in a ligand‐dependent manner with several NRs in yeast and mammalian cells, as well as in vitro. Remarkably, these interactions require the amino acids constituting the AF‐2 activating domain conserved in all active NRs. Moreover, the oestrogen receptor (ER) AF‐2 antagonist hydroxytamoxifen cannot promote ER‐TIF1 interaction. We propose that TIF1, which contains several conserved domains found in transcriptional regulatory proteins, is a mediator of ligand‐dependent AF‐2. Interestingly, the TIF1 N‐terminal moiety is fused to B‐raf in the mouse oncoprotein T18.

Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor.

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxia and dioxin signalling, respectively. Both proteins are conditionally regulated basic helix-loop-helix (bHLH) transcription factors that, in addition to the bHLH motif, share a Per-Arnt-Sim (PAS) region of homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt. Here we demonstrate that HIF-1 alpha required Arnt for DNA binding in vitro and functional activity in vivo. Both the bHLH and PAS motifs of Arnt were critical for dimerization with HIF-1 alpha. Strikingly, HIF-1 alpha exhibited very high affinity for Arnt in coimmunoprecipitation assays in vitro, resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistent with these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alpha inhibited ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor function on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediated signalling pathways were not mutually exclusive, since activation of dioxin receptor function did not impair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAs were expressed constitutively in a large number of human tissues and cell lines, and these steady-state expression levels were not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionally regulated by a mechanism that is distinct from induced expression levels, the prevalent model of activation of HIF-1 alpha function. Interestingly, we observed that HIF-1 alpha was associated with the molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activation of the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism, possibly by binding an as yet unknown class of ligands.

Human TAFII30 is present in a distinct TFIID complex and is required for transcriptional activation by the estrogen receptor

We showed previously that coactivators mediating stimulation by different activators were associated with the TATA-binding protein (TBP) in distinct TFIID complexes. We have characterized a human TBP-associated factor (TAF), hTAFII30, associated with a subset of TFIID complexes. hTAFII30 interacts with the AF-2-containing region E of the human estrogen receptor (ER), but not with ER AF-1 or VP16. An antibody against hTAFII30 inhibited transcriptional stimulation by the ER AF-2 without affecting basal or VP16-activated transcription and allowed the separation of TFIID complex(es) containing hTAFII30 from complexes mediating the activity of VP16. These results directly demonstrate the existence of functionally distinct TFIID populations that share common TAFIIs but differ in specific TAFIIs.

The cloned human oestrogen receptor contains a mutation which alters its hormone binding properties.

We demonstrate here that the human oestrogen receptor (hER) cDNA clone pOR8 obtained from MCF‐7 cells contains an artefactual point mutation which results in the substitution of a valine for a glycine at amino acid position 400 (Gly‐400––Val‐400). This mutation in the hormone binding domain of the cloned hER destabilizes its structure and decreases its apparent affinity for oestradiol at 25 degrees C, but not at 4 degrees C, when compared with the wild‐type hER with a Gly‐400.

hTAF(II)68, a novel RNA/ssDNA-binding protein with homology to the pro-oncoproteins TLS/FUS and EWS is associated with both TFIID and RNA polymerase II.

TFIID is the main sequence‐specific DNA‐binding component of the RNA polymerase II (Pol II) transcriptional machinery. It is a multiprotein complex composed of the TATA‐binding protein (TBP) and TBP‐associated factors (TAF(II)s). Here we report the cloning and characterization of a novel human TBP‐associated factor, hTAF(II)68. It contains a consensus RNA‐binding domain (RNP‐CS) and binds not only RNA, but also single stranded (ss) DNA. hTAF(II)68 shares extensive sequence similarity with TLS/FUS and EWS, two human nuclear RNA‐binding pro‐oncoproteins which are products of genes commonly translocated in human sarcomas. Like hTAF(II)68, TLS/FUS is also associated with a sub‐population of TFIID complexes chromatographically separable from those containing hTAF(II)68. Therefore, these RNA and/or ssDNA‐binding proteins may play specific roles during transcription initiation at distinct promoters. Moreover, we demonstrate that hTAF(II)68 co‐purifies also with the human RNA polymerase II and can enter the preinitiation complex together with Pol II.

Distinct GCN5/PCAF-containing complexes function as co-activators and are involved in transcription factor and global histone acetylation.

Transcription in eukaryotes is a tightly regulated, multistep process. Gene-specific transcriptional activators, several different co-activators and general transcription factors are necessary to access specific loci to allow precise initiation of RNA polymerase II transcription. As the dense chromatin folding of the genome does not allow the access of these sites by the huge multiprotein transcription machinery, remodelling is required to loosen up the chromatin structure for successful transcription initiation. In the present review, we summarize the recent evolution of our understanding of the function of two histone acetyl transferases (ATs) from metazoan organisms: GCN5 and PCAF. Their overall structure and the multiprotein complexes in which they are carrying out their activities are discussed. Metazoan GCN5 and PCAF are subunits of at least two types of multiprotein complexes, one having a molecular weight of 2 MDa (SPT3-TAF9-GCN5 acetyl transferase/TATA binding protein (TBP)–free–TAF complex/PCAF complexes) and a second type with about a size of 700 kDa (ATAC complex). These complexes possess global histone acetylation activity and locus-specific co-activator functions together with AT activity on non-histone substrates. Thus, their biological functions cover a wide range of tasks and render them indispensable for the normal function of cells. That deregulation of the global and/or specific AT activities of these complexes leads to the cancerous transformation of the cells highlights their importance in cellular processes. The possible effects of GCN5 and PCAF in tumorigenesis are also discussed.

A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5′-TGACC-3′ motifs acting synergistically

We have identified an estrogen-responsive enhancer element (DH3 ERE) in the estrogen-induced DNAase I-hypersensitive region III of the chicken ovalbumin gene, which is located approximately 3.3 kb upstream from the mRNA start site and does not contain palindromic ERE. Four TGACC half-palindromic motifs, separated from each other by more than 100 bp, are responsible for conferring estrogen inducibility either to the proximal ovalbumin gene promoter or to heterologous promoters. Thus, widely spaced half-palindromic ERE motifs can act synergistically. Each half-palindromic motif was shown to bind the estrogen receptor (ER) with a low efficiency in vitro. However, two widely spaced half-palindromic motifs bound the ER cooperatively, much more efficiently than expected from binding to isolated half-ERE motifs. The ovalbumin promoter half-palindromic ERE motif located close to the TATA box was required for the activity of the distal DH3 ERE, but could be replaced by the binding sites of other transactivators.

Distinct classes of transcriptional activating domains function by different mechanisms

We have previously shown that the two transcriptional activation functions (TAF-1 and TAF-2) of the human estrogen receptor (hER) have synergistic properties different from one another and from those of acidic activating domains (AADs). Here we compare the transcriptional interference/squelching properties of the hER TAFs with those of the AADs of yeast GAL4 and chimeric GAL-VP16 activators. Our results indicate that AADs interact with a factor(s) that, while required for activation by AADs, is not essential for activation by hER TAFs. In contrast, hER TAFs appear to interact with factors indispensable for mediating both their activation function and that of AADs. Thus, different classes of trans-activators may interact with different factors. In addition, the synergistic and transcriptional interference/squelching properties of the two TAFs of the human glucocorticoid receptor (hGR) indicate that both are composed of acidic and nonacidic activation functions.

Collisions between Replication and Transcription Complexes Cause Common Fragile Site Instability at the Longest Human Genes

We show that the time required to transcribe human genes larger than 800 kb spans more than one complete cell cycle, while their transcription speed equals that of smaller genes. Independently of their expression status, we find the long genes to replicate late. Regions of concomitant transcription and replication in late S phase exhibit DNA break hot spots known as common fragile sites (CFSs). This CFS instability depends on the expression of the underlying long genes. We show that RNA:DNA hybrids (R-loops) form at sites of transcription/replication collisions and that RNase H1 functions to suppress CFS instability. In summary, our results show that, on the longest human genes, collisions of the transcription machinery with a replication fork are inevitable, creating R-loops and consequent CFS formation. Functional replication machinery needs to be involved in the resolution of conflicts between transcription and replication machineries to ensure genomic stability.