Estrogen receptor-α recruits P-TEFb to overcome transcriptional pausing in intron 1 of the MYB gene.

The MYB proto-oncogene is expressed in most estrogen receptor-positive (ERα(+)) breast tumors and cell lines. Expression of MYB is controlled, in breast cancer and other cell types, by a transcriptional pausing mechanism involving an attenuation site located ∼1.7 kb downstream from the transcription start site. In breast cancer cells, ligand-bound ERα binds close to, and drives transcription beyond this attenuation site, allowing synthesis of complete transcripts. However, little is known, in general, about the factors involved in relieving transcriptional attenuation, or specifically how ERα coordinates such factors to promote transcriptional elongation. Using cyclin dependent kinase 9 (CDK9) inhibitors, reporter gene assays and measurements of total and intronic MYB transcription, we show that functionally active CDK9 is required for estrogen-dependent transcriptional elongation. We further show by ChIP and co-immunoprecipitation studies that the P-TEFb complex (CDK9/CyclinT1) is recruited to the attenuation region by ligand-bound ERα, resulting in increased RNA polymerase II Ser-2 phosphorylation. These data provide new insights into MYB regulation, and given the critical roles of MYB in tumorigenesis, suggest targeting MYB elongation as potential therapeutic strategy.

The Mix family of homeobox genes--key regulators of mesendoderm formation during vertebrate development.

The Mix/Bix family of paired-like homeobox genes encode evolutionarily conserved, sequence specific, DNA-binding transcription factors that have been implicated in the co-ordination of gene expression, axis formation and cell fate determination during gastrulation in vertebrates. When mutated, these genes give rise to dramatic phenotypes in amphibians, zebrafish and mice, that can be traced back to defects in the formation and specification of mesoderm and endoderm. We review here the biochemical properties of the Mix/Bix proteins and summarise genetic, molecular and embryological studies of Mix/Bix function in mesendoderm development. We emphasise recent data generated using embryonic stem cell differentiation systems that have provided important new insights into Mix/Bix function and the biological roles of these proteins in regulating the earliest phases of vertebrate development.

NC2alpha interacts with BTAF1 and stimulates its ATP-dependent association with TATA-binding protein.

Transcriptional activity of the TATA-binding protein (TBP) is controlled by a variety of proteins. The BTAF1 protein (formerly known as TAF(II)170/TAF-172 and the human ortholog of Saccharomyces cerevisiae Mot1p) and the NC2 complex composed of NC2alpha (DRAP1) and NC2beta (Dr1) are able to bind to TBP directly and regulate RNA polymerase II transcription both positively and negatively. Here, we present evidence that the NC2alpha subunit interacts with BTAF1. In contrast, the NC2beta subunit is not able to associate with BTAF1 and seems to interfere with the BTAF1-TBP interaction. Addition of NC2alpha or the NC2 complex can stimulate the ability of BTAF1 to interact with TBP. This function is dependent on the presence of ATP in cell extracts but does not involve the ATPase activity of BTAF1 nor phosphorylation of NC2alpha. Together, our results constitute the first evidence of the physical cooperation between BTAF1 and NC2alpha in TBP regulation and provide a framework to understand transcription functions of NC2alpha and NC2beta in vivo.

MYB elongation is regulated by the nucleic acid binding of NFκB p50 to the intronic stem-loop region.

MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFκBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFκBp65. We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation. We observed that the TAR RNA-binding region of Tat is homologous to the NFκBp50 RHD lysine-rich motif, a finding consistent with HIV Tat acting as an effector of MYB transcriptional elongation in an SLR dependent manner. Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB. Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.

Roles for BTAF1 and Mot1p in dynamics of TATA-binding protein and regulation of RNA polymerase II transcription.

Regulation of RNA polymerase II (pol II) transcription is a highly dynamic process requiring the coordinated interaction of an array of regulatory proteins. Central to this process is the TATA-binding protein (TBP), the key component of the multiprotein complex TFIID. Interaction of TBP with core promoters nucleates the assembly of the preinitiation complex and subsequent recruitment of pol II. Despite recent advances in our understanding of the dynamic nature of the pol II transcription apparatus, the dynamics of TBP function on pol II promoters has remained largely unexplored. Human BTAF1 (TAF(II)170/TAF-172) and its yeast ortholog, Mot1p, are evolutionarily conserved members of the SNF2-like family of ATPase proteins. Genetic identification of Mot1p as a repressor of pol II transcription was supported by findings that Mot1p and BTAF1 could dissociate TBP from TATA DNA complexes using the energy of ATP hydrolysis. Recent data have revealed new aspects of BTAF1 and Mot1p as positive regulators of TBP function in the pol II system and have described new observations relating to their molecular mechanism of action. We review these data in the context of previous findings with particular attention paid to how human BTAF1 and Mot1p may dynamically regulate TBP function on pol II promoters in cells.

WNT3A promotes hematopoietic or mesenchymal differentiation from hESCs depending on the time of exposure.

We investigated the role of canonical WNT signaling in mesoderm and hematopoietic development from human embryonic stem cells (hESCs) using a recombinant human protein-based differentiation medium (APEL). In contrast to prior studies using less defined culture conditions, we found that WNT3A alone was a poor inducer of mesoderm. However, WNT3A synergized with BMP4 to accelerate mesoderm formation, increase embryoid body size, and increase the number of hematopoietic blast colonies. Interestingly, inclusion of WNT3A or a GSK3 inhibitor in methylcellulose colony-forming assays at 4 days of differentiation abrogated blast colony formation but supported the generation of mesospheres that expressed genes associated with mesenchymal lineages. Mesospheres differentiated into cells with characteristics of bone, fat, and smooth muscle. These studies identify distinct effects for WNT3A, supporting the formation of hematopoietic or mesenchymal lineages from human embryonic stem cells, depending upon differentiation stage at the time of exposure.

Pdgfrα and Flk1 are direct target genes of Mixl1 in differentiating embryonic stem cells.

The Mixl1 homeodomain protein plays a key role in mesendoderm patterning during embryogenesis, but its target genes remain to be identified. We compared gene expression in differentiating heterozygous Mixl1(GFP/w) and homozygous null Mixl1(GFP/Hygro) mouse embryonic stem cells to identify potential downstream transcriptional targets of Mixl1. Candidate Mixl1 regulated genes whose expression was reduced in GFP+ cells isolated from differentiating Mixl1(GFP/Hygro) embryoid bodies included Pdgfrα and Flk1. Mixl1 bound to ATTA sequences located in the Pdgfrα and Flk1 promoters and chromatin immunoprecipitation assays confirmed Mixl1 occupancy of these promoters in vivo. Furthermore, Mixl1 transactivated the Pdgfrα and Flk1 promoters through ATTA sequences in a DNA binding dependent manner. These data support the hypothesis that Mixl1 directly regulates Pdgfrα and Flk1 gene expression and strengthens the position of Mixl1 as a key regulator of mesendoderm development during mammalian gastrulation.

Brachyury and related Tbx proteins interact with the Mixl1 homeodomain protein and negatively regulate Mixl1 transcriptional activity.

Mixl1 is a homeodomain transcription factor required for mesoderm and endoderm patterning during mammalian embryogenesis. Despite its crucial function in development, co-factors that modulate the activity of Mixl1 remain poorly defined. Here we report that Mixl1 interacts physically and functionally with the T-box protein Brachyury and related members of the T-box family of transcription factors. Transcriptional and protein analyses demonstrated overlapping expression of Mixl1 and Brachyury during embryonic stem cell differentiation. In vitro protein interaction studies showed that the Mixl1 with Brachyury associated via their DNA-binding domains and gel shift assays revealed that the Brachyury T-box domain bound to Mixl1-DNA complexes. Furthermore, luciferase reporter experiments indicated that association of Mixl1 with Brachyury and related T-box factors inhibited the transactivating potential of Mixl1 on the Gsc and Pdgfrα promoters. Our results indicate that the activity of Mixl1 can be modulated by protein-protein interactions and that T-box factors can function as negative regulators of Mixl1 activity.

Expression from a betageo gene trap in the Slain1 gene locus is predominantly associated with the developing nervous system.

Slain1 was originally identified as a novel stem cell-associated gene in transcriptional profiling experiments comparing mouse and human embryonic stem cells (ESCs) and their immediate differentiated progeny. In order to obtain further insight into the potential function of Slain1, we examined the expression of beta-galactosidase in a gene-trap mouse line in which a beta-geo reporter gene was inserted into the second intron of Slain1. In early stage embryos (E7.5), the Slain1-betageo fusion protein was expressed within the entire epiblast, but by E9.5 became restricted to the developing nervous system and gastrointestinal tract. In later stage embryos (E11.5 - E13.5), expression was predominantly within the developing nervous system. Lower level expression was also observed in the developing limb buds, in the condensing mesenchyme, along the apical epidermal ridge and, at later stages, within the digital zones. These observations suggest that Slain1 may play a role in the development of the nervous system, as well as in the morphogenesis of several embryonic structures.

Molecular architecture of the basal transcription factor B-TFIID.

BTAF1 (formerly named TAF(II)170/TAF-172) is an essential, evolutionarily conserved member of the SNF2-like family of ATPase proteins and together with TATA-binding protein (TBP) forms the B-TFIID complex. BTAF1 has been proposed to play a key role in the dynamic regulation of TBP function in RNA polymerase II transcription. We have determined the structure of native B-TFIID purified from human cells by electron microscopy and by image analysis of single particles at a resolution of 28 A. B-TFIID is 15 x 9 nm in size and is organized into a large domain of about 170 kDa, which can be subdivided into two domains. Extending from this domain is a long thumb, which in turn is divided into subdomains of about 25, 15, and 35 kDa, the largest of which is located at the end of the thumb. Immunolabeling experiments localize the extreme carboxyl terminus of BTAF1 within the 170-kDa domain, whereas the amino terminus and TBP co-localize to the end of the protruding thumb. The central portion of BTAF1 localizes to the base of the thumb. Comparison of the native B-TFIID with its recombinant form shows that both share a similar domain organization. Collectively, these data provide the first structural model of the B-TFIID complex and map its key functional domains.

Characterization of interactions between transcription factors and a regulatory region spanning nt -320 to -281 of the HIV-1 LTR in T-lymphoid and non-T-lymphoid cells.

HIV-1 gene expression is regulated by the interplay of transcription factors with multiple binding motifs present within the U3, R and U5 regions of the long terminal repeat (LTR). Here we report novel DNA binding complexes (termed 9a, 9b and 9c) between nuclear proteins from T-lymphoid and non-T-lymphoid cells and a region of the U3 LTR between nucleotides (nts) -320 to -281 in the HIV strain HXB2. Complex 9b bound a motif predicted to bind E-box or c-Myb proteins and a partially overlapping dyad symmetrical motif, and included basic helix-loop-helix proteins (E12, E47 or ITF-1) but surprisingly not c-Myb. Complex 9c, which bound to a pair of GATA sites, included GATA-3 and GATA-4 in Jurkat and MT-2 cells, respectively. We also demonstrate that the c-Myb/E-box and GATA sites form a bipartite motif required for the formation of complex 9a. Transient transfection experiments with T cells revealed that in the context of a minichromosome assembled full-length LTR, mutation of region -320 to -281 increased basal and PMA-stimulated LTR activity. These findings suggest that this region is an important component of the HIV-1 LTR required for response to different cellular transcription factors.