Rebecca A. Keough

Molecular Cloning Reveals that the p160 Myb-Binding Protein Is a Novel, Predominantly Nucleolar Protein Which May Play a Role in Transactivation by Myb

ABSTRACT We have previously detected two related murine nuclear proteins, p160 and p67, that can bind to the leucine zipper motif within the negative regulatory domain of the Myb transcription factor. We now describe the molecular cloning of cDNA corresponding to murine p160. The P160 gene is located on mouse chromosome 11, and related sequences are found on chromosomes 1 and 12. The predicted p160 protein is novel, and in agreement with previous studies, we find that the corresponding 4.5-kb mRNA is ubiquitously expressed. We showed that p67 is an N-terminal fragment of p160 which is generated by proteolytic cleavage in certain cell types. The protein encoded by the cloned p160 cDNA and an engineered protein (p67*) comprising the amino-terminal region of p160 exhibit binding specificities for the Myb and Jun leucine zipper regions identical to those of endogenous p160 and p67, respectively. This implies that the Myb-binding site of p160 lies within the N-terminal 580 residues and that the Jun-binding site is C-terminal to this position. Moreover, we show that p67* but not p160 can inhibit transactivation by Myb. Unexpectedly, immunofluorescence studies show that p160 is localized predominantly in the nucleolus. The implications of these results for possible functions of p160 are discussed.

L-Proline induces differentiation of ES cells: a novel role for an amino acid in the regulation of pluripotent cells in culture

The development of cell therapeutics from embryonic stem (ES) cells will require technologies that direct cell differentiation to specific somatic cell lineages in response to defined factors. The initial step in formation of the somatic lineages from ES cells, differentiation to an intermediate, pluripotent primitive ectoderm-like cell, can be achieved in vitro by formation of early primitive ectoderm-like (EPL) cells in response to a biological activity contained within the conditioned medium MEDII. Fractionation of MEDII has identified two activities required for EPL cell formation, an activity with a molecular mass of <3 kDa and a second, much larger species. Here, we have identified the low-molecular-weight activity as l-proline. An inhibitor of l-proline uptake, glycine, prevented the differentiation of ES cells in response to MEDII. Supplementation of the culture medium of ES cells with >100 M l-proline and some l-proline-containing peptides resulted in changes in colony morphology, cell proliferation, gene expression, and differentiation kinetics consistent with differentiation toward a primitive ectoderm-like cell. This activity appeared to be associated with l-proline since other amino acids and analogs of proline did not exhibit an equivalent activity. Activation of the mammalian target of rapamycin (mTOR) signaling pathway was found to be necessary but not sufficient for l-proline activity; addition of other activators of the mTOR signaling pathway failed to alter the ES cell phenotype. This is the first report describing a role for amino acids in the regulation of pluripotency and cell differentiation and identifies a novel role for the imino acid l-proline.

p160 Myb-binding protein interacts with Prep1 and inhibits its transcriptional activity

ABSTRACT Prep1 is known to interact in vivo with Pbx1 to regulate development and organogenesis. We have identified a novel Prep1-interacting protein, p160 c-Myb binding protein (p160). p160 and Pbx1 compete for Prep1 in vitro, and p160 inhibits Prep1-dependent HoxB2 expression in retinoic acid-treated NT2-D1 cells. The N-terminal physiologically truncated form of p160, p67, binds the sequence 63LFPLL67 in the HR1 domain of Prep1. Mutation of both L63 and L66 impairs the binding of Prep1 to both p160/p67 and Pbx1. The sequences required to bind Prep1 are mainly located in residues 51 to 151. Immunofluorescence colocalization and coimmunoprecipitation of endogenous p160 and Prep1 are induced by ActD, which translocates p160 from the nucleolus to the nucleoplasm. These data therefore show that p160 is a novel regulator of Prep1-Pbx1 transcriptional activity.

Ribosomal stress induces processing of Mybbp1a and its translocation from the nucleolus to the nucleoplasm

Myb‐binding protein 1a (Mybbp1a) was originally identified as a c‐myb proto‐oncogene product (c‐Myb)‐interacting protein, and also binds to various other transcription factors. The 160‐kDa Mybbp1a protein (p160MBP) is ubiquitously expressed and is post‐translationally processed in some types of cells to generate an amino‐terminal 67 kDa fragment (p67MBP). Despite its interaction with various transcription factors, Mybbp1a is localized predominantly, but not exclusively, in nucleoli. Here, we have purified the two Mybbp1a‐containing complexes. The smaller complex contained p67MBP and p140MBP, which lacked the C‐terminal region of p160MBP containing the nucleolar localization sequences. The larger complex contained the intact p160MBP and various ribosomal subunits. Treatment of cells with actinomycin D (ActD), cisplatin or UV, all of which inhibit ribosome biogenesis, induced processing of p160MBP into p140MBP and p67MBP. ActD, cisplatin and UV also induced a translocation of Mybbp1a from the nucleolus to the nucleoplasm. Both small and large Mybbp1a complexes contained nucleophosmin and nucleolin. In contrast, nucleostemin was detected only in the large complex, while the cell cycle‐regulated protein EBP1 was only in the small complex. These results suggest that Mybbp1a may connect the ribosome biogenesis and the Myb‐dependent transcription, which controls cell cycle progression and proliferation.

Myb-binding protein 1a is a nucleocytoplasmic shuttling protein that utilizes CRM1-dependent and independent nuclear export pathways

Myb-binding protein 1a (Mybbp1a) is a novel nuclear protein localized predominantly, but not exclusively, in nucleoli. Although initially isolated as a c-Myb interacting protein, Mybbp1a is expressed ubiquitously, associates with a number of different transcription factors, and may play a role in both RNA polymerase I- and II-mediated transcriptional regulation. However, its precise function remains unclear. In this study we show that Mybbp1a is a nucleocytoplasmic shuttling protein and investigate the mechanisms responsible for both nuclear import and export. The carboxyl terminus of Mybbp1a, which contains seven short basic amino acid repeat sequences, is responsible for both nuclear and nucleolar localization, and this activity can be transferred to a heterologous protein. Deletion mapping demonstrated that these repeat sequences appear to act incrementally, with successive deletions resulting in a corresponding increase in the proportion of protein localized in the cytoplasm. Glutathione S-transferase pulldown experiments showed that the nuclear receptor importin-alpha/beta mediates Mybbp1a nuclear import. Interspecies heterokaryons were used to demonstrate that Mybbp1a was capable of shuttling between the nucleus and the cytoplasm. Deletion analysis and in vivo export studies using a heterologous assay system identified several nuclear export sequences which facilitate Mybbp1a nuclear export of Mybbp1a by CRM1-dependent and -independent pathways.

Understanding pluripotency—how embryonic stem cells keep their options open

Embryonic stem (ES) cells have the capacity to proliferate indefinitely in culture while maintaining the ability to differentiate to form any of the cells of the body. This unique combination of functions suggests that these cells could provide a potentially unlimited source of differentiated cells for the treatment of disease and aging. Understanding the molecular processes that underpin these functions in ES cells will allow us to harness their potential and develop strategies that control their differentiation. Combination of controlled differentiation with ground-breaking technologies for the reversal of somatic cells to an ES cell-like state promise the generation of patient-derived pluripotent cell lines for the treatment of disease in the future.

Myb-binding Protein 1a (Mybbp1a) Regulates Levels and Processing of Pre-ribosomal RNA

Background: rRNA gene transcription and processing have to be coordinated, as they are intimately linked. Results: Mybbp1a regulates RNA Pol I transcription and pre-rRNA processing. Conclusion: Mybbp1a coordinates both processes. Significance: Mybbp1a was shown to be an important regulator of cellular proliferation, related to RNA Pol II genes. We link its function now as well to regulating RNA Pol I genes and to control ribosome biogenesis. Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes.

Modulation of CP2 family transcriptional activity by CRTR-1 and sumoylation.

CRTR-1 is a member of the CP2 family of transcription factors. Unlike other members of the family which are widely expressed, CRTR-1 expression shows specific spatio-temporal regulation. Gene targeting demonstrates that CRTR-1 plays a central role in the maturation and function of the salivary glands and the kidney. CRTR-1 has also recently been identified as a component of the complex transcriptional network that maintains pluripotency in embryonic stem (ES) cells. CRTR-1 was previously shown to be a repressor of transcription. We examine the activity of CRTR-1 in ES and other cells and show that CRTR-1 is generally an activator of transcription and that it modulates the activity of other family members, CP2, NF2d9 and altNF2d9, in a cell specific manner. We also demonstrate that CRTR-1 activity is regulated by sumoylation at a single major site, residue K30. These findings imply that functional redundancy with other family members may mask important roles for CRTR-1 in other tissues, including the blastocyst stage embryo and embryonic stem cells.

Regulation of the gene encoding glutathione S -transferase M1 (GSTM1) by the Myb oncoprotein

The identification of Myb ‘target’ genes will not only aid in the understanding of how overexpression of Myb, or expression of activated forms of Myb, leads to cellular transformation but will also shed light on its role in normal cells. Using a combination of an estrogen-regulated Myb-transformed cell line (ERMYB) and PCR-based subtractive hybridization, we have identified the gene (GSTM1) encoding the detoxification enzyme glutathione S-transferase M1 as being transcriptionally upregulated by Myb. Functional analysis of the GSTM1 promoter using reporter assays indicated that both the DNA binding and transactivation domains of Myb were required for transcriptional activation. Mutational ana-lysis of consensus Myb-binding sites (MBS) in the promoter and electrophoretic mobility gel shift analysis indicated that one of the three potential MBS can bind Myb protein, and is the primary site involved in the regulation of this promoter by Myb.

Mutation screening of the c-MYB negative regulatory domain in acute and chronic myeloid leukaemia.

Over‐expression of the c‐myb gene and expression of activated forms of myb are known to transform haemopoietic cells, particularly cells of the myeloid lineage. Truncations or mutations that disrupt the negative regulatory domain (NRD) of the Myb protein confer an increased ability to transform cells. Although it has proved difficult to link mutations in c‐MYB to human leukaemia, no studies investigating the presence of mutations within the c‐MYB NRD have been reported. Therefore, we have performed mutational analysis of this region, using polymerase chain reaction–single‐stranded conformation polymorphism and sequence analysis, in 26 patients with acute or chronic myeloid leukaemia. No mutations were detected, indicating that mutation of this region of the Myb protein is not common in the pathogenesis or progression of these diseases.