Rivka Dikstein

miR-22 Forms a Regulatory Loop in PTEN/AKT Pathway and Modulates Signaling Kinetics

Background The tumor suppressor PTEN (phosphatase and tensin homolog) is a lipid phosphatase that converts PIP3 into PIP2 and downregulates the kinase AKT and its proliferative and anti-apoptotic activities. The FoxO transcription factors are PTEN downstream effectors whose activity is negatively regulated by AKT-mediated phosphorylation. PTEN activity is frequently lost in many types of cancer, leading to increased cell survival and cell cycle progression. Principal Findings Here we characterize the widely expressed miR-22 and report that miR-22 is a novel regulatory molecule in the PTEN/AKT pathway. miR-22 downregulates PTEN levels acting directly through a specific site on PTEN 3′UTR. Interestingly, miR-22 itself is upregulated by AKT, suggesting that miR-22 forms a feed-forward circuit in this pathway. Time-resolved live imaging of AKT-dependent FoxO1 phosphorylation revealed that miR-22 accelerated AKT activity upon growth factor stimulation, and attenuated its down regulation by serum withdrawal. Conclusions Our results suggest that miR-22 acts to fine-tune the dynamics of PTEN/AKT/FoxO1 pathway.

Mechanism of Rapid Transcriptional Induction of Tumor Necrosis Factor Alpha-Responsive Genes by NF-κB

ABSTRACT NF-κB induces the expression of genes involved in immune response, apoptosis, inflammation, and the cell cycle. Certain NF-κB-responsive genes are activated rapidly after the cell is stimulated by cytokines and other extracellular signals. However, the mechanism by which these genes are activated is not entirely understood. Here we report that even though NF-κB interacts directly with TAFIIs, induction of NF-κB by tumor necrosis factor alpha (TNF-α) does not enhance TFIID recruitment and preinitiation complex formation on some NF-κB-responsive promoters. These promoters are bound by the transcription apparatus prior to TNF-α stimulus. Using the immediate-early TNF-α-responsive gene A20 as a prototype promoter, we found that the constitutive association of the general transcription apparatus is mediated by Sp1 and that this is crucial for rapid transcriptional induction by NF-κB. In vitro transcription assays confirmed that NF-κB plays a postinitiation role since it enhances the transcription reinitiation rate whereas Sp1 is required for the initiation step. Thus, the consecutive effects of Sp1 and NF-κB on the transcription process underlie the mechanism of their synergy and allow rapid transcriptional induction in response to cytokines.

TAFII105 mediates activation of anti‐apoptotic genes by NF‐κB

The transcription factor NF‐κB is important for expression of genes involved in immune responses, viral infections, cytokine signaling and stress. In addition NF‐κB plays a crucial role in protecting cells from TNF‐α‐induced apoptotic stimuli, presumably by activating anti‐apoptotic genes. Here we report that the sub‐stoichiometric TFIID subunit TAFII105 is essential for activation of anti‐apoptotic genes in response to TNF‐α, serving as a transcriptional coactivator for NF‐κB. The putative coactivator domain of TAFII105 interacts with the activation domain of the p65/RelA member of the NF‐κB family, and further stimulates p65‐induced transcription in human 293 cells. Moreover, inhibition of TAFII105 activity by overexpression of a dominant negative mutant of TAFII105 decreased NF‐κB transcriptional activity and severely reduced cell survival in response to TNF‐α. Similarly, expression of anti‐sense TAFII105 RNA sensitized the cells to TNF‐α cytotoxicity. These results suggest that TAFII105 is involved in activation of anti‐apoptotic genes by NF‐κB.

Transcriptional Control by NF-κB: Elongation in Focus

The NF-κB family of transcription factors governs the cellular reaction to a variety of extracellular signals. Following stimulation, NF-κB activates genes involved in inflammation, cell survival, cell cycle, immune cell homeostasis and more. This review focuses on studies of the past decade that uncover the transcription elongation process as a key regulatory stage in the activation pathway of NF-κB. Of interest are studies that point to the elongation phase as central to the selectivity of target gene activation by NF-κB. Particularly, the cascade leading to phosphorylation and acetylation of the NF-κB subunit p65 on serine 276 and lysine 310, respectively, was shown to mediate the recruitment of Brd4 and P-TEFb to many pro-inflammatory target genes, which in turn facilitate elongation and mRNA processing. On the other hand, some anti-inflammatory genes are refractory to this pathway and are dependent on the elongation factor DSIF for efficient elongation and mRNA processing. While these studies have advanced our knowledge of NF-κB transcriptional activity, they have also raised unresolved issues regarding the specific genomic and physiological contexts by which NF-κB utilizes different mechanisms for activation.

Functional organization of the hepatitis B virus enhancer.

We have studied the functional constituents of the hepatitis B virus enhancer in a number of cell lines. The sequence of this enhancer, being embedded within an open reading frame of the virus, is in part evolutionarily frozen and therefore serves as a good model to investigate the fundamental enhancer elements. The hepatitis B virus enhancer contains three functionally important DNA sequence elements, EP, E, and NF-1a, each of which is bound by a distinct protein(s). The synergistic action of these elements accounts for all of the enhancer activity in a nonliver cell line and for most, but not all, of the activity in liver-derived cell lines. Multimers of the E but not of the EP element act as an autonomous enhancer. Conversely, a single element of either the E or the NF-1a element can act only when linked to the EP element. These results suggest that EP is a crucial enhancer element that acts only in interaction with a second enhancer element with intrinsic enhancer activity. Interestingly, a highly similar enhancer structure is found in a number of distinct viruses.

Core Promoter Binding by Histone-Like TAF Complexes

ABSTRACT A major function of TFIID is core promoter recognition. TFIID consists of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Most of them contain a histone fold domain (HFD) that lacks the DNA-contacting residues of histones. Whether and how TAF HFDs contribute to core promoter DNA binding are yet unresolved. Here we examined the DNA binding activity of TAF9, TAF6, TAF4b, and TAF12, which are related to histones H3, H4, H2A, and H2B, respectively. Each of these TAFs has intrinsic DNA binding activity adjacent to or within the HFD. The DNA binding domains were mapped to evolutionarily conserved and essential regions. Remarkably, HFD-mediated interaction enhanced the DNA binding activity of each of the TAF6-TAF9 and TAF4b-TAF12 pairs and of a histone-like octamer complex composed of the four TAFs. Furthermore, HFD-mediated interaction stimulated sequence-specific binding by TAF6 and TAF9. These results suggest that TAF HFDs merge with other conserved domains for efficient and specific core promoter binding.

Differential regulation of NF-κB by elongation factors is determined by core promoter type

ABSTRACT NF-κB transcription factors activate genes important for immune response, inflammation, and cell survival. P-TEFb and DSIF, which are positive and negative transcription elongation factors, respectively, both regulate NF-κB-induced transcription, but the mechanism underlying their recruitment to NF-κB target genes is unknown. We show here that upon induction of NF-κB, a subset of target genes is regulated differentially by either P-TEFb or DSIF. The regulation of these genes and their occupancy by these elongation factors are dependent on the NF-κB enhancer and the core promoter type. Converting a TATA-less promoter to a TATA promoter switches the regulation of NF-κB from DSIF to P-TEFb. Accumulation or displacement of DSIF and P-TEFb is dictated by the formation of distinct initiation complexes (TFIID dependent or independent) on the two types of core promoter. The underlying mechanism for the dissociation of DSIF from TATA promoters upon NF-κB activation involves the phosphorylation of RNA polymerase II by P-TEFb. The results highlight a regulatory link between the initiation and the elongation phases of the transcription reaction and broaden our comprehension of the NF-κB pathway.

c-abl has a sequence-specific enhancer binding activity

The enhancers of several distinct viruses contain a common functional element, termed EP. This element binds ubiquitous cellular proteins and generates specific complexes in gel retardation analysis. Ultraviolet cross-linking and Southwestern analysis showed that a 140 kd polypeptide is the major EP DNA-binding protein. Using a combination of DNA binding and immunological techniques, we have identified the c-abl protein in a nuclear complex that binds to the EP element. abl was found to have both a specific and high affinity DNA binding activity. The ability to bind DNA is abolished in the mutant abl protein, p210bcr-abl, consistent with its cytoplasmic localization in chronic myelogenous leukemia.

Unique translation initiation of mRNAs-containing TISU element

Translation Initiator of Short 5′ UTR (TISU) is a unique regulatory element of both transcription and translation initiation. It is present in a sizable number of genes with basic cellular functions and a very short untranslated region (5′ UTR). Here, we investigated translation initiation from short 5′ UTR mRNAs with AUG in various contexts. Reducing 5′ UTR length to the minimal functional size increases leaky scanning from weak and strong initiators but hardly affects translation initiation and ribosomal binding directed by TISU. Ribosome interaction with TISU mRNA is cap dependent and involves AUG downstream nucleotides that compensate for the absent 5′ UTR contacts. Interestingly, eIF1 inhibits cap-proximal AUG selection within weak or strong contexts but not within TISU. Furthermore, TISU-directed translation is unaffected by inhibition of the RNA helicase eIF4A. Thus, TISU directs efficient cap-dependent translation initiation without scanning, a mechanism that would be advantageous when intracellular levels of eIF1 and eIF4A fluctuate.

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.