Foxp1/4 control epithelial cell fate during lung development and regeneration through regulation of anterior gradient 2.

The molecular pathways regulating cell lineage determination and regeneration in epithelial tissues are poorly understood. The secretory epithelium of the lung is required for production of mucus to help protect the lung against environmental insults, including pathogens and pollution, that can lead to debilitating diseases such as asthma and chronic obstructive pulmonary disease. We show that the transcription factors Foxp1 and Foxp4 act cooperatively to regulate lung secretory epithelial cell fate and regeneration by directly restricting the goblet cell lineage program. Loss of Foxp1/4 in the developing lung and in postnatal secretory epithelium leads to ectopic activation of the goblet cell fate program, in part, through de-repression of the protein disulfide isomerase anterior gradient 2 (Agr2). Forced expression of Agr2 is sufficient to promote the goblet cell fate in the developing airway epithelium. Finally, in a model of lung secretory cell injury and regeneration, we show that loss of Foxp1/4 leads to catastrophic loss of airway epithelial regeneration due to default differentiation of secretory cells into the goblet cell lineage. These data demonstrate the importance of Foxp1/4 in restricting cell fate choices during development and regeneration, thereby providing the proper balance of functional epithelial lineages in the lung.

Gene expression profile and functionality of ESC-derived Lin-ckit+Sca-1+ cells are distinct from Lin-ckit+Sca-1+ cells isolated from fetal liver or bone marrow.

In vitro bioreactor-based cultures are being extensively investigated for large-scale production of differentiated cells from embryonic stem cells (ESCs). However, it is unclear whether in vitro ESC-derived progenitors have similar gene expression profiles and functionalities as their in vivo counterparts. This is crucial in establishing the validity of ESC-derived cells as replacements for adult-isolated cells for clinical therapies. In this study, we compared the gene expression profiles of Lin-ckit+Sca-1+ (LKS) cells generated in vitro from mouse ESCs using either static or bioreactor-based cultures, with that of native LKS cells isolated from mouse fetal liver (FL) or bone marrow (BM). We found that in vitro-generated LKS cells were more similar to FL- than to BM LKS cells in gene expression. Further, when compared to cells derived from bioreactor cultures, static culture-derived LKS cells showed fewer differentially expressed genes relative to both in vivo LKS populations. Overall, the expression of hematopoietic genes was lower in ESC-derived LKS cells compared to cells from BM and FL, while the levels of non-hematopoietic genes were up-regulated. In order to determine if these molecular profiles correlated with functionality, we evaluated ESC-derived LKS cells for in vitro hematopoietic-differentiation and colony formation (CFU assay). Although static culture-generated cells failed to form any colonies, they did differentiate into CD11c+ and B220+ cells indicating some hematopoietic potential. In contrast, bioreactor-derived LKS cells, when differentiated under the same conditions failed to produce any B220+ or CD11c+ cells and did not form colonies, indicating that these cells are not hematopoietic progenitors. We conclude that in vitro culture conditions significantly affect the transcriptome and functionality of ESC-derived LKS cells and although in vitro differentiated LKS cells were lineage negative and expressed both ckit and Sca-1, these cells, especially those obtained from dynamic cultures, are significantly different from native cells of the same phenotype.

The ARID family transcription factor bright is required for both hematopoietic stem cell and B lineage development.

Bright/Arid3a has been characterized both as an activator of immunoglobulin heavy-chain transcription and as a proto-oncogene. Although Bright expression is highly B lineage stage restricted in adult mice, its expression in the earliest identifiable hematopoietic stem cell (HSC) population suggests that Bright might have additional functions. We showed that >99% of Bright(-/-) embryos die at midgestation from failed hematopoiesis. Bright(-/-) embryonic day 12.5 (E12.5) fetal livers showed an increase in the expression of immature markers. Colony-forming assays indicated that the hematopoietic potential of Bright(-/-) mice is markedly reduced. Rare survivors of lethality, which were not compensated by the closely related paralogue Bright-derived protein (Bdp)/Arid3b, suffered HSC deficits in their bone marrow as well as B lineage-intrinsic developmental and functional deficiencies in their peripheries. These include a reduction in a natural antibody, B-1 responses to phosphocholine, and selective T-dependent impairment of IgG1 class switching. Our results place Bright/Arid3a on a select list of transcriptional regulators required to program both HSC and lineage-specific differentiation.

Loss of Bright/ARID3a function promotes developmental plasticity.

B-cell regulator of immunoglobulin heavy chain transcription (Bright)/ARID3a, an A+T-rich interaction domain protein, was originally discovered in B lymphocyte lineage cells. However, expression patterns and high lethality levels in knockout mice suggested that it had additional functions. Three independent lines of evidence show that functional inhibition of Bright results in increased developmental plasticity. Bright-deficient cells from two mouse models expressed a number of pluripotency-associated gene products, expanded indefinitely, and spontaneously differentiated into cells of multiple lineages. Furthermore, direct knockdown of human Bright resulted in colonies capable of expressing multiple lineage markers. These data suggest that repression of this single molecule confers adult somatic cells with new developmental options.

Foxp1/2/4-NuRD interactions regulate gene expression and epithelial injury response in the lung via regulation of interleukin-6.

To determine the underlying mechanism of Foxp1/2/4-mediated transcriptional repression, a yeast two-hybrid screen was performed that identified p66beta, a transcriptional repressor and component of the NuRD chromatin-remodeling complex. We show that direct interactions between Foxp1/4 and p66beta are mediated by the CR2 domain within p66beta and the zinc finger/leucine zipper repression domain found in Foxp1/2/4. These direct interactions are functionally relevant as overexpression of p66beta in combination with Foxp factors cooperatively represses Foxp target gene expression, whereas loss of p66 and Foxp factors results in de-repression of endogenous Foxp target genes in lung epithelial cells. Moreover, the NuRD components HDAC1/2 associate in a macromolecular complex with Foxp proteins, and loss of expression or inhibition of HDAC1/2 activity leads to de-repression of Foxp target gene expression. Importantly, we show in vivo that Foxp1 and HDAC2 act cooperatively to regulate expression of the cytoprotective cytokine interleukin-6, which results in increased resistance to hyperoxic lung injury in Foxp1/HDAC2 compound mutant animals. These data reveal an important interaction between the Foxp transcription factors and the NuRD chromatin-remodeling complex that modulates transcriptional repression critical for the lung epithelial injury response.

Aglycosylated IgG variants expressed in bacteria that selectively bind FcgammaRI potentiate tumor cell killing by monocyte-dendritic cells.

The N-linked glycan of immunoglobulin G (IgG) is indispensable for the interaction of the Fc domain with Fcgamma receptors on effector cells and the clearance of target cells via antibody dependent cell-mediated cytotoxicity (ADCC). Escherichia coli expressed, aglycosylated Fc domains bind effector FcgammaRs poorly and cannot elicit ADCC. Using a novel bacterial display/flow cytometric library screening system we isolated Fc variants that bind to FcgammaRI (CD64) with nanomolar affinity. Binding was critically dependent on amino acid substitutions (E382V, and to a lesser extent, M428I) distal to the putative FcgammaRI binding epitope within the CH3 domain. These mutations did not adversely affect its pH-dependent interaction with FcRn in vitro nor its serum persistence in vivo. Remarkably, the anti-Her2 IgG trastuzumab containing the E382V, M428I substitutions and expressed in E. coli exhibited highly selective binding to FcgammaRI but not to the other activating receptors (FcgammaRIIa, FcgammaRIIIa) nor to the inhibitory receptor, FcgammaRIIb. In contrast, the glycosylated version of trastuzumab (E382V, M428I) purified from HEK293T cells bound to all Fcgamma receptors in a manner similar to that of clinical grade trastuzumab. E. coli-purified trastuzumab (E382V, M428I), but not glycosylated trastuzumab (E382V, M428I) or clinical grade trastuzumab, was capable of potentiating the killing of Her2 overexpressing tumor cells with dendritic cells (DCs) as effectors. These results indicate that aglycosylated IgGs can be engineered to display unique FcgammaR selectivity profiles that, in turn, mediate ADCC via mechanisms that are not normally displayed by glycosylated monoclonal antibodies.

Signalling of the BCR is regulated by a lipid rafts-localised transcription factor, Bright.

Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity.

Hsp90--from signal transduction to cell transformation.

The molecular chaperone, Hsp90, facilitates the maturation and/or activation of over 100 'client proteins' involved in signal transduction and transcriptional regulation. Largely an enigma among the families of heat shock proteins, Hsp90 is central to processes broadly ranging from cell cycle regulation to cellular transformation. Here, we review the contemporary body of knowledge regarding the biochemical mechanisms of Hsp90 and update the most current paradigms defining its involvement in both normal and pathological cell physiology.

FOXP1: a potential therapeutic target in cancer.

Forkhead Box P1 (FOXP1) is a member of the FOX family of transcription factors which have a broad range of functions. Foxp1 is widely expressed and has been shown to have a role in cardiac, lung and lymphocyte development. FOXP1 is targeted by recurrent chromosome translocations and its overexpression confers a poor prognosis in a number of types of lymphomas, suggesting it may function as an oncogene. In contrast, FOXP1 localises to a tumour suppressor locus at 3p14.1 and loss of FOXP1 expression in breast cancer is associated with a worse outcome, suggesting FOXP1 may function as a tumour suppressor in other tissue types. These data suggest that FOXP1 may not only be useful in prognosis but also may be used to develop FOXP1-directed therapeutic strategies.

NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma.

The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK(+) ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK(+) cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells.

REKLES is an ARID3-restricted multifunctional domain.

Bright/Dril1/ARID3a is a B cell-specific, matrix association (or attachment) region-binding transcriptional regulator of immunoglobulin heavy chain genes and of E2F1-dependent cell cycle progression. Bright contains a central DNA binding domain termed ARID (AT-rich interacting domain) and a C-terminal region termed REKLES (for a conserved amino acid motif). The ARID domain has been identified in seven highly conserved families of metazoan proteins (ARID1-5 and JARID1-2), whereas REKLES is found only in the ARID3 subfamily (composed of Bright/ARID3a, Bdp/ARID3b, and Bright-like/ARID3c). REKLES consists of two subdomains: a modestly conserved N-terminal REKLESalpha and a highly conserved (among ARID3 orthologous proteins) C-terminal REKLESbeta. Previously we showed that Bright undergoes nucleocytoplasmic shuttling and that REKLESalpha and -beta were required, respectively, for nuclear import and Crm1-dependent nuclear export. Here we show that Bright further requires REKLESbeta for self-association or paralogue association and for nuclear matrix targeting. REK-LES promotes and regulates the extent of Bright multimerization, which occurs in the absence or presence of target DNA and is necessary for specific DNA binding. REKLESbeta-mediated interaction of Bright with Bdp, which localizes strictly to the nucleus, traps Bright within the nucleus via neutralization of its nuclear export activity. These results identify REKLES as a multifunctional domain that has co-evolved with and regulates functional properties of the ARID3 DNA binding domain.

Phosphorylation by SR kinases regulates the binding of PTB-associated splicing factor (PSF) to the pre-mRNA polypyrimidine tract.

PSF (PTB-associated splicing factor) is a multi-functional protein that participates in transcription and RNA processing. While phosphorylation of PSF has been shown to be important for some functions, the sites and the kinases involved are not well understood. Although PSF does not contain a typical RS domain, we report here that PSF is phosphorylated in vivo to generate an epitope(s) that can be recognized by a monoclonal antibody specific for phosphorylated RS motifs within SR proteins. PSF can be phosphorylated by human and yeast SR kinases in vivo and in vitro at an isolated RS motif within its N terminus. A functional consequence of SR phosphorylation of PSF is to inhibit its binding to the 3' polypyrimidine tract of pre-mRNA. These results indicate that PSF is a substrate of SR kinases whose phosphorylation regulates its RNA binding capacity and ultimate biological function.

Open Access and beyond.

Uncensored exchange of scientific results hastens progress. Open Access does not stop at the removal of price and permission barriers; still, censorship and reading disabilities, to name a few, hamper access to information. Here, we invite the scientific community and the public to discuss new methods to distribute, store and manage literature in order to achieve unfettered access to literature.

Identification and characterization of Smyd2: a split SET/MYND domain-containing histone H3 lysine 36-specific methyltransferase that interacts with the Sin3 histone deacetylase complex.

BACKGROUND: Disrupting the balance of histone lysine methylation alters the expression of genes involved in tumorigenesis including proto-oncogenes and cell cycle regulators. Methylation of lysine residues is commonly catalyzed by a family of proteins that contain the SET domain. Here, we report the identification and characterization of the SET domain-containing protein, Smyd2. RESULTS: Smyd2 mRNA is most highly expressed in heart and brain tissue, as demonstrated by northern analysis and in situ hybridization. Over-expressed Smyd2 localizes to the cytoplasm and the nucleus in 293T cells. Although accumulating evidence suggests that methylation of histone 3, lysine 36 (H3K36) is associated with actively transcribed genes, we show that the SET domain of Smyd2 mediates H3K36 dimethylation and that Smyd2 represses transcription from an SV40-luciferase reporter. Smyd2 associates specifically with the Sin3A histone deacetylase complex, which was recently linked to H3K36 methylation within the coding regions of active genes in yeast. Finally, we report that exogenous expression of Smyd2 suppresses cell proliferation. CONCLUSION: We propose that Sin3A-mediated deacetylation within the coding regions of active genes is directly linked to the histone methyltransferase activity of Smyd2. Moreover, Smyd2 appears to restrain cell proliferation, likely through direct modulation of chromatin structure.

Regulation of RNA-polymerase-II-dependent transcription by N-WASP and its nuclear-binding partners.

The presence of actin in the nucleus has been well established, and several studies have implicated nuclear actin in transcriptional regulation. Neuronal Wiskott-Aldrich syndrome protein (N-WASP) is a member of the WASP family of proteins; these proteins function in the cytoplasm as key regulators of cortical actin filament. Interestingly, N-WASP has also been observed in the nucleus. However, a potential nuclear function for N-WASP has not been established. Here, we report the identification of nuclear N-WASP within a large nuclear-protein complex containing PSF-NonO (polypyrimidine-tract-binding-protein-associated splicing factor-non-Pou-domain octamer-binding protein/p54(nrb)), nuclear actin and RNA polymerase II. The PSF-NonO complex is involved in the regulation of many cellular processes, such as transcription, RNA processing, DNA unwinding and repair. We demonstrate that the interaction of N-WASP with the PSF-NonO complex can couple N-WASP with RNA polymerase II to regulate transcription. We also provide evidence that the potential function of N-WASP in promoting polymerization of nuclear actins has an important role in this process. Based on these results, we propose a nuclear function for N-WASP in transcriptional regulation.

Functional studies of BCL11A: characterization of the conserved BCL11A-XL splice variant and its interaction with BCL6 in nuclear paraspeckles of germinal center B cells.

BACKGROUND: Chromosomal aberrations of BCL11A at 2p16.1 have been reported in a variety of B-cell malignancies and its deficiency in mice leads to a profound block in B-cell development. RESULTS: Alternative pre-mRNA splicing of BCL11A produces multiple isoforms sharing a common N-terminus. The most abundant isoform we have identified in human lymphoid samples is BCL11A-XL, the longest transcript produced at this locus, and here we report the conservation of this major isoform and its functional characterization. We show that BCL11A-XL is a DNA-sequence-specific transcriptional repressor that associates with itself and with other BCL11A isoforms, as well as with the BCL6 proto-oncogene. Western blot data for BCL11A-XL expression coupled with data previously published for BCL6 indicates that these genes are expressed abundantly in germinal-center-derived B cells but that expression is extinguished upon terminal differentiation to the plasma cell stage. Although BCL11A-XL/BCL6 interaction can modulate BCL6 DNA binding in vitro, their heteromeric association does not alter the homomeric transcriptional properties of either on model reporter activity. BCL11A-XL partitions into the nuclear matrix and colocalizes with BCL6 in nuclear paraspeckles. CONCLUSION: We propose that the conserved N-terminus of BCL11A defines a superfamily of C2HC zinc-finger transcription factors involved in hematopoietic malignancies.

A regulated nucleocytoplasmic shuttle contributes to Bright's function as a transcriptional activator of immunoglobulin genes.

Bright/ARID3a has been implicated in mitogen- and growth factor-induced up-regulation of immunoglobulin heavy-chain (IgH) genes and in E2F1-dependent G1/S cell cycle progression. For IgH transactivation, Bright binds to nuclear matrix association regions upstream of certain variable region promoters and flanking the IgH intronic enhancer. While Bright protein was previously shown to reside within the nuclear matrix, we show here that a significant amount of Bright resides in the cytoplasm of normal and transformed B cells. Leptomycin B, chromosome region maintenance 1 (CRM1) overexpression, and heterokaryon experiments indicate that Bright actively shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. We mapped the functional nuclear localization signal to the N-terminal region of REKLES, a domain conserved within ARID3 paralogues. Residues within the C terminus of REKLES contain its nuclear export signal, whose regulation is primarily responsible for Bright shuttling. Growth factor depletion and cell synchronization experiments indicated that Bright shuttling during S phase of the cell cycle leads to an increase in its nuclear abundance. Finally, we show that shuttle-incompetent Bright point mutants, even if sequestered within the nucleus, are incapable of transactivating an IgH reporter gene. Therefore, regulation of Bright's cellular localization appears to be required for its function.

Splicing and transcription-associated proteins PSF and p54nrb/nonO bind to the RNA polymerase II CTD.

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II (pol II) plays an important role in promoting steps of pre-mRNA processing. To identify proteins in human cells that bind to the CTD and that could mediate its functions in pre-mRNA processing, we used the mouse CTD expressed in bacterial cells in affinity chromatography experiments. Two proteins present in HeLa cell extract, the splicing and transcription-associated factors, PSF and p54nrb/NonO, bound specifically and could be purified to virtual homogeneity by chromatography on immobilized CTD matrices. Both hypo- and hyperphosphorylated CTD matrices bound these proteins with similar selectivity. PSF and p54nrb/NonO also copurified with a holoenzyme form of pol II containing hypophosphorylated CTD and could be coimmunoprecipitated with antibodies specific for this and the hyperphosphorylated form of pol II. That PSF and p54nrb/NonO promoted the binding of RNA to immobilized CTD matrices suggested these proteins can interact with the CTD and RNA simultaneously. PSF and p54nrb/NonO may therefore provide a direct physical link between the pol II CTD and pre-mRNA processing components, at both the initiation and elongation phases of transcription.

Transcriptional activation of human CYP17 in H295R adrenocortical cells depends on complex formation among p54(nrb)/NonO, protein-associated splicing factor, and SF-1, a complex that also participates in repression of transcription.

The first 57 bp upstream of the transcription initiation site of the human CYP17 (hCYP17) gene are essential for both basal and cAMP-dependent transcription. EMSA carried out by incubating H295R adrenocortical cell nuclear extracts with radiolabeled -57/-38 probe from the hCYP17 promoter showed the formation of three DNA-protein complexes. The fastest complex contained steroidogenic factor (SF-1) and p54(nrb)/NonO, the intermediate complex contained p54(nrb)/NonO and polypyrimidine tract-binding protein-associated splicing factor (PSF), and the slowest complex contained an SF-1/PSF/p54(nrb)/NonO complex. (Bu)(2)cAMP treatment resulted in a cAMP-inducible increase in the binding intensity of only the upper complex and also activated hCYP17 gene transcription. SF-1 coimmunoprecipitated with p54(nrb)/NonO, indicating direct interaction between these proteins. Functional assays revealed that PSF represses basal transcription. Further, the repression of hCYP17 promoter-reporter construct luciferase activity resulted from PSF interacting with the corepressor mSin3A. Trichostatin A attenuated the inhibition of basal transcription, suggesting that a histone deacetylase interacts with the SF-1/PSF/p54(nrb)/NonO/mSin3A complex. Our studies lend support to the idea that the balance between transcriptional activation and repression is essential in the control of adrenocortical steroid hormone biosynthesis.