A Translation Initiation Element Specific to mRNAs with Very Short 5′UTR that Also Regulates Transcription

Transcription is controlled by cis regulatory elements, which if localized downstream to the transcriptional start site (TSS), in the 5′UTR, could influence translation as well. However presently there is little evidence for such composite regulatory elements. We have identified by computational analysis an abundant element located downstream to the TSS up to position +30, which controls both transcription and translation. This element has an invariable ATG sequence, which serves as the translation initiation codon in 64% of the genes bearing it. In these genes the initiating AUG is preceded by an extremely short 5′UTR. We show that translation in vitro and in vivo is initiated exclusively from the AUG of this motif, and that the AUG flanking sequences create a strong translation initiation context. This motif is distinguished from the well-known Kozak in its unique ability to direct efficient and accurate translation initiation from mRNAs with a very short 5′UTR. We therefore named it TISU for Translation Initiator of Short 5′UTR. Interestingly, this translation initiation element is also an essential transcription regulatory element of Yin Yang 1. Our characterization of a common transcription and translation element points to a link between mammalian transcription and translation initiation

Action of transcription factors in the control of transferrin receptor expression in human brain endothelium

Brain endothelium has a distinctive phenotype, including high expression of transferrin receptor, p-glycoprotein, claudin-5 and occludin. Dermal endothelium expresses lower levels of the transferrin receptor and it is absent from lung endothelium. All three endothelia were screened for transcription factors that bind the transferrin receptor promoter and show different patterns of binding between the endothelia. The transcription factor YY1 has distinct DNA-binding activities in brain endothelium and non-brain endothelium. The target-sites on the transferrin receptor promotor for YY1 lie in close proximity to those of the transcription initiation complex containing TFIID, so the two transcription factors potentially compete or interfere. Notably, the DNA-binding activity of TFIID was the converse of YY1, in different endothelia. YY1 knockdown reduced transferrin receptor expression in brain endothelium, but not in dermal endothelium implying that YY1 is involved in tissue-specific regulation of the transferrin receptor. Moreover a distinct YY1 variant is present in brain endothelium and it associates with Sp3. A model is presented, in which expression from the transferrin receptor gene in endothelium requires the activity of both TFIID and Sp3, but whether the gene is transcribed in different endothelia, is related to the balance between activating and suppressive forms of YY1

Characterization of cis- and trans-acting elements in the imprinted human SNURF-SNRPN locus

The imprinted SNRPN locus is a complex transcriptional unit that encodes the SNURF and SmN polypeptides as well as multiple non-coding RNAs. SNRPN is located within the Prader-Willi and Angelman syndrome (PWS/AS) region that contains multiple imprinted genes, which are coordinately regulated by a bipartite imprinting center (IC). The SNRPN 5′ region co-localizes with the PWS-IC and contains two DNase I hypersensitive sites, DHS1 at the SNRPN promoter, and DHS2 within intron 1, exclusively on the paternally inherited chromosome. We have examined DHS1 and DHS2 to identify cis- and trans-acting regulatory elements within the endogenous SNRPN 5′ region. Analysis of DHS1 by in vivo footprinting and chromatin immunoprecipitation identified allele-specific interaction with multiple regulatory proteins, including NRF-1, which regulates genes involved in mitochondrial and metabolic functions. DHS2 acted as an enhancer of the SNRPN promoter and contained a highly conserved region that showed allele-specific interaction with unphosphorylated RNA polymerase II, YY1, Sp1 and NRF-1, further suggesting a key role for NRF-1 in regulation of the SNRPN locus. We propose that one or more of the regulatory elements identified in this study may also contribute to PWS-IC function

The Transcription Factor YY1 Is a Substrate for Polo-Like Kinase 1 at the G2/M Transition of the Cell Cycle

Yin-Yang 1 (YY1) is an essential multifunctional zinc-finger protein. It has been shown over the past two decades to be a critical regulator of a vast array of biological processes, including development, cell proliferation and differentiation, DNA repair, and apoptosis. YY1 exerts its functions primarily as a transcription factor that can activate or repress gene expression, dependent on its spatial and temporal context. YY1 regulates a large number of genes involved in cell cycle transitions, many of which are oncogenes and tumor-suppressor genes. YY1 itself has been classified as an oncogene and was found to be upregulated in many cancer types. Unfortunately, our knowledge of what regulates YY1 is very minimal. Although YY1 has been shown to be a phosphoprotein, no kinase has ever been identified for the phosphorylation of YY1. Polo-like kinase 1 (Plk1) has emerged in the past few years as a major cell cycle regulator, particularly for cell division. Plk1 has been shown to play important roles in the G/M transition into mitosis and for the proper execution of cytokinesis, processes that YY1 has been shown to regulate also. Here, we present evidence that Plk1 directly phosphorylates YY1 in vitro and in vivo at threonine 39 in the activation domain. We show that this phosphorylation is cell cycle regulated and peaks at G2/M. This is the first report identifying a kinase for which YY1 is a substrate

Characterization of functional domains within the multifunctional transcription factor, YY1

YY1 is a multifunctional transcription factor capable of either activation or repression of transcription. The mechanisms underlying its bifunctionality are not well understood. Using a series of mutant YY1 proteins, expressed as GALA fusions, we have characterized domains responsible for transcriptional activation, activation suppression or masking, and repression. The YY1 activation domain resides in the first 100 amino acids of the amino terminus and requires amino acids 16-29 and 80-99 for maximal activity. Since the transcriptional silencing or repressive function of full length YY1 (414 amino acids) appears to be its default state, we were able to identify a minimum region in the carboxyl terminus of YY1, between amino acids 370 and 397, which upon deletion permits YY1 to function as a constitutive activator. This region is also required for transcriptional repression, as the carboxyl terminal 81 amino acids of YY1, amino acids 333-414 are sufficient to repress basal transcription. Deletion of the carboxy terminal 44 amino acids, which include zinc fingers 3 and 4, abolishes repression. However, detailed mutational analyses indicate that the normal structures of zinc fingers 3 and 4, although required for DNA binding, are not required for transcriptional repression. We also provide evidence that YY1 can be sequestered within the nucleus by virtue of its interaction with a large molecular weight complex. The region responsible for this interaction is bounded by amino acids 256 and 341, which we show to be the same region required for the compartmentalization of YY1 within the nuclear matrix. YY1 was also shown to potently repress activated transcription independent of it binding to DNA, by a mechanism which probably involves squelching of a limiting transcription factor. The region responsible for this effect was shown to reside between amino acids 333 and 370. This detailed functional mapping of YY1 should serve as an important basis for future studies aimed at understanding mechanistically its diverse transcriptional effects

Characterization of functional domains within the multifunctional transcription factor, YY1

YY1 is a multifunctional transcription factor capable of either activation or repression of transcription. The mechanisms underlying its bifunctionality are not well understood. Using a series of mutant YY1 proteins, expressed as GALA fusions, we have characterized domains responsible for transcriptional activation, activation suppression or masking, and repression. The YY1 activation domain resides in the first 100 amino acids of the amino terminus and requires amino acids 16-29 and 80-99 for maximal activity. Since the transcriptional silencing or repressive function of full length YY1 (414 amino acids) appears to be its default state, we were able to identify a minimum region in the carboxyl terminus of YY1, between amino acids 370 and 397, which upon deletion permits YY1 to function as a constitutive activator. This region is also required for transcriptional repression, as the carboxyl terminal 81 amino acids of YY1, amino acids 333-414 are sufficient to repress basal transcription. Deletion of the carboxy terminal 44 amino acids, which include zinc fingers 3 and 4, abolishes repression. However, detailed mutational analyses indicate that the normal structures of zinc fingers 3 and 4, although required for DNA binding, are not required for transcriptional repression. We also provide evidence that YY1 can be sequestered within the nucleus by virtue of its interaction with a large molecular weight complex. The region responsible for this interaction is bounded by amino acids 256 and 341, which we show to be the same region required for the compartmentalization of YY1 within the nuclear matrix. YY1 was also shown to potently repress activated transcription independent of it binding to DNA, by a mechanism which probably involves squelching of a limiting transcription factor. The region responsible for this effect was shown to reside between amino acids 333 and 370. This detailed functional mapping of YY1 should serve as an important basis for future studies aimed at understanding mechanistically its diverse transcriptional effects

Variation in EGF-induced EGF receptor downregulation in human hepatoma-derived cell lines expressing different amounts of EGF receptor.

The effect of epidermal growth factor (EGF) receptor overexpression on ligand-induced EGF receptor downregulation was examined using a hepatoma-derived cell line, PLC/PRF/5, which expresses normal amounts of the EGF receptor, and a subline, NPLC/PRF/5, which expresses 10-fold more receptors at its cell surface. PLC/PRF/5 cells efficiently downregulated surface receptor levels upon exposure to saturating and subsaturating concentrations of EGF; the rate of receptor downregulation corresponded to that of ligand-receptor internalization. Upon internalization, EGF receptors were degraded and receptor biosynthesis remained at basal levels. EGF surface receptor remained downregulated for as long as cells were exposed to EGF. By contrast, surface EGF receptor abundance in NPLC/PRF/5 cells decreased by only 5-15% after 1-4 h incubation with subsaturating doses of EGF and actually increased by 67% within 20 h. Exposure of these cells to saturating concentrations of EGF induced modest decreases in surface receptor abundance during the initial 12 h incubation, followed by a progressive decline to 30% of initial values by 24 h. Relative ligand-receptor internalization rates in NPLC/PRF/5 cells were lower than those in PLC/PRF/5, although their surface receptor population was even higher than that predicted by the decreased internalization rates. EGF receptor degradation in NPLC/PRF/5 cells was also inhibited; exposure to saturating levels of EGF for more than 16 h was necessary before significant degradation occurred. Receptor protein and mRNA biosynthesis in NPLC/PRF/5 were stimulated by 8 h exposure to EGF but when saturating concentrations of EGF were present for 16 h, receptor biosynthesis was inhibited. EGF receptor overexpression circumvents the downregulatory effect of EGF by decreasing the rate of receptor internalization, inhibiting degradation of the internalized receptor pool, and stimulating EGF receptor biosynthesis. Conversely, receptor downregulation becomes pronounced at late times when receptor degradation is high and biosynthesis is inhibited