▪ Abstract The events leading to transcription of eukaryotic protein-coding genes culminate in the positioning of RNA polymerase II at the correct initiation site. The core promoter, which can extend ∼35 bp upstream and/or downstream of this site, plays a central role in regulating initiation. Specific DNA elements within the core promoter bind the factors that nucleate the assembly of a functional preinitiation complex and integrate stimulatory and repressive signals from factors bound at distal sites. Although core promoter structure was originally thought to be invariant, a remarkable degree of diversity has become apparent. This article reviews the structural and functional diversity of the RNA polymerase II core promoter.

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The RNA polymerase II core promoter.

Ets family transcription factors control the expression of a large number of genes in hematopoietic cells. Here we show strikingly precise differential expression of a subset of these genes marking critical, early stages of mouse lymphocyte cell-type specification. Initially, the Ets family member factor Erg was identified during an arrayed cDNA library screen for genes encoding transcription factors expressed specifically during T cell lineage commitment. Multiparameter fluorescence-activated cell sorting for over a dozen cell surface markers was used to isolate 18 distinct primary-cell populations representing discrete T cell and B cell developmental stages, pluripotent lymphoid precursors, immature NK-like cells and myeloid hematopoietic cells. These populations were monitored for mRNA expression of the Erg, Ets-1, Ets-2, Fli-1, Tel, Elf-1, GABPalpha, PU.1 and Spi-B genes. The earliest stages in T cell differentiation show particularly dynamic Ets family gene regulation, with sharp transitions in expression correlating with specification and commitment events. Ets, Spi-B and PU.1 are expressed in these stages but not by later T-lineage cells. Erg is induced during T-lineage specification and then silenced permanently, after commitment, at the beta-selection checkpoint. Spi-B is transiently upregulated during commitment and then silenced at the same stage as Erg. T-lineage commitment itself is marked by repression of PU.1, a factor that regulates B-cell and myeloid genes. These results show that the set of Ets factors mobilized during T-lineage specification and commitment is different from the set that maintains T cell gene expression during thymocyte repertoire selection and in all classes of mature T cells.

/pdf/precise-developmental-regulation-of-ets-family-transcription-234ll757gx.pdf

Precise developmental regulation of Ets family transcription factors during specification and commitment to the T cell lineage

The interaction of the cyclic AMP response element-binding protein (CREB) with its coactivator, CREB-binding protein (CBP), is one of the best characterized signal-regulated activation domain-coactivator interactions. CREB is a signal-dependent transactivator of the bZIP family which becomes phosphorylated on serine 133 in response to stimuli that result in an increase in intracellular cyclic AMP or Ca2+ (33). CBP, as well as its homologue p300, binds to CREB upon phosphorylation at serine 133 within the kinase-inducible activation domain of CREB (8, 32). CBP recruitment by phosphorylated CREB results in rapid induction of gene expression that is mediated both by direct recruitment of the basal transcription machinery and the intrinsic acetyltransferase activity of CBP (26, 33). A vast number of signal-responsive and developmentally regulated transcription factors interact with different regions of CBP and p300. The CREB-binding (or KIX) domain alone interacts with at least 10 distinct families of transcriptional activators (17). Despite the number of well-established protein interactions involving CBP and p300, the only complex for which structural information is available is the phosphorylated CREB-CBP KIX domain interaction (38, 39).

The solution structure of the phosphorylated CREB-CBP KIX complex revealed that the activation domain of CREB undergoes a dramatic random coil-to-helix transition upon binding to the KIX domain (38). Conversely, the α-3 helix of the KIX domain undergoes a more subtle change upon binding to phosphorylated CREB. Two lines of evidence suggest that this binding event may be regulated at a level beyond serine 133 phosphorylation. First, CREB phosphorylation has been noted in cellular contexts which do not result in the recruitment of CBP or the induction of CREB target genes (4), suggesting that an additional event is required for coactivator recruitment. Second, CREB or CBP KIX domain mutants have been described that exhibit enhanced complex formation compared to the wild type in vitro or in vivo (5, 15). These observations suggest the potential for a third component that regulates CREB-KIX domain interaction by binding to either or both members of the CREB-CBP KIX complex. To isolate proteins that interact with the unique interface formed by the CREB-CBP KIX complex, a genetic screen was devised in which phosphorylated CREB and the CBP KIX domain are coexpressed in yeast to serve as “bait” for such proteins. In contrast to two-component interaction screening, this three-component screen resulted in the isolation of fewer, more specific interacting proteins (63).

In this study, we analyzed a fragment of the mixed-lineage leukemia (MLL) gene (Mll) isolated in the CREB-CBP three-hybrid screen. The MLL gene was originally identified as a gene frequently disrupted by chromosomal translocations in childhood leukemias as well as in therapy-induced acute myeloid leukemias (14, 18, 57). Translocations into the MLL locus generally result in fusion of the N terminus of the MLL protein with one of many diverse fusion partners. Two MLL fusion proteins, MLL-AF9 and MLL-ENL, have been demonstrated to be leukemogenic in murine knock-in or retroviral transduction systems, respectively (10, 52). From these studies, regions within the N-terminal portion of MLL as well as a specific motif within the fusion partner were shown to be important for oncogenesis (52). However, haploinsufficiency at the MLL locus also appears to contribute to leukemia in the knock-in system (13).

The mechanism by which the MLL gene maintains target gene expression patterns is of great interest as a starting point for understanding the altered activity of the leukemogenic fusion proteins. Gene-disruption experiments in the mouse illustrated the functional similarity between MLL and its Drosophila homologue, trithorax (60), most dramatically demonstrated by a posterior shift in Hox gene expression and consequent homeotic transformations of the axial skeleton in Mll heterozygotes (19, 59, 60). Mll, like trithorax, positively regulates Hox genes and counters the repressive effect of chromatin-regulating Polycomb-group proteins such as Bmi-1 (19, 24, 36). Analysis of Mll-null embryos indicates that MLL is not involved in the initial specification of Hox gene expression patterns but is essential for maintaining expression patterns once initiated by other sequence-specific transcription factors (59, 60). The mechanism by which MLL acts on its target genes is incompletely understood, in part due to the limited information available regarding protein complexes in which MLL participates (1, 2, 11, 31, 46).

To gain insight into the mechanism by which MLL regulates its target genes, we evaluated the significance of the isolation of MLL in the CREB-CBP three-hybrid screen. We show that MLL interacts directly with CBP as well as the CREB-CBP complex. The MLL-CBP interaction was found to be essential for transcriptional activation by the MLL minimal activation domain. In addition, we demonstrated that interaction of MLL with the CBP KIX domain facilitates binding of phospho-CREB to CBP. This surprising finding leads us to propose that a unique class of target genes may respond synergistically to both CREB and MLL.

MLL and CREB bind cooperatively to the nuclear coactivator CREB-binding protein.

Characterization of the Human Endothelial Nitric-oxide Synthase Promoter

The generation of lymphoid cells in mice depends on the function of the Ikaros protein. Ikaros has been characterized as a lymphoid-restricted, zinc-finger transcription factor that is derived from an alternatively spliced message. Ikaros knockout mice have defects in multiple cell lineages, raising the question of whether the protein regulates multiple committed progenitors and/or multipotent stem cells. To address this issue, we examined Ikaros expression in purified populations of multipotent cells and more committed progenitors. We found that the DNA-binding isoforms of Ikaros were localized in the nucleus of the most primitive hematopoietic stem cell subset. Changes in the RNA splicing pattern of Ikaros occurred at two stages: (i) as long-term self-renewing stem cells differentiated into short-term self-renewing stem cells and (ii) as non-self-renewing multipotent progenitors differentiated into lymphoid-committed progenitors. Unexpectedly, we found Ikaros localized to heterochromatin in Abelson-transformed pre-B lymphocytes by using immunogold electron microscopy. These observations suggest a complex role for Ikaros in lymphoid development.

/pdf/hematopoietic-stem-cells-and-lymphoid-progenitors-express-45lq5vthzu.pdf

Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin in immature lymphocytes

The terminal deoxynucleotidyltransferase (TdT) gene represents an attractive model for the analysis of gene regulation during an early phase of lymphocyte development. In previous studies, we identified a DNA element, termed D', which is essential for TdT promoter activity in immature lymphocytes, and two classes of D'-binding factors, Ikaros proteins and Ets proteins. Here, we report a detailed mutant analysis of the D' element which suggests that an Ets protein, rather than an Ikaros protein, activates TdT transcription. Since multiple Ets proteins are expressed in developing lymphocytes and are capable of binding to the D' element, DNA affinity chromatography was used to determine if one of the Ets proteins might bind to the D' element with a uniquely high affinity, thereby implicating that protein as a potential TdT activator. Indeed, one binding activity was greatly enriched in the high-salt eluates from a D' affinity column. Peptide microsequencing revealed that the enriched protein was Elf-1. Immunoblot analyses confirmed that in nuclear extracts, Elf-1 has a significantly higher affinity for the D' sequence than does another Ets protein, Ets-1. Transactivation and expression studies support the hypothesis that Elf-1 activates TdT transcription in immature T and B cells. Finally, a D' mutation which selectively reduces Elf-1 binding, but not the binding of other Ets proteins, was found to greatly reduce TdT promoter activity. Although Elf-1 previously had been implicated in the inducible activation of genes in mature T and B cells, our results suggest that it also plays an important role in regulating genes during an early phase of lymphocyte development.

A potential role for Elf-1 in terminal transferase gene regulation.

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A potential role for Elf-1 in terminal transferase gene regulation.