Joseph M. Lee

RBP1 Recruits Both Histone Deacetylase-Dependent and -Independent Repression Activities to Retinoblastoma Family Proteins

ABSTRACT Retinoblastoma (RB) tumor suppressor family proteins block cell proliferation in part by repressing certain E2F-specific promoters. Both histone deacetylase (HDAC)-dependent and -independent repression activities are associated with the RB “pocket.” The mechanism by which these two repression functions occupy the pocket is unknown. A known RB-binding protein, RBP1, was previously found by our group to be an active corepressor which, if overexpressed, represses E2F-mediated transcription via its association with the pocket. We show here that RBP1 contains two repression domains, one of which binds all three known HDACs and represses them in an HDAC-dependent manner while the other domain functions independently of the HDACs. Thus, RB family members repress transcription by recruiting RBP1 to the pocket. RBP1, in turn, serves as a bridging molecule to recruit HDACs and, in addition, provides a second HDAC-independent repression function.

Toxicity of human adenovirus E4orf4 protein in Saccharomyces cerevisiae results from interactions with the Cdc55 regulatory B subunit of PP2A.

The E4orf4 protein of human adenovirus induces p53-independent apoptosis, a process that may promote cell death and viral spread. When expressed alone, E4orf4 kills transformed cells but not normal human cells. The only clear target of E4orf4 in mammalian cells is the Bα (B55) subunit of protein phosphatase 2A (PP2A), a member of one of three classes of regulatory B subunits. Here we report the effects of E4orf4 in Saccharomyces cerevisiae, which encodes two PP2A regulatory B subunits, CDC55 and RTS1, that share homology with mammalian B and B′ subunits, respectively. E4orf4 expression was found to be toxic in yeast, resulting in the accumulation of cells in G2/M phase that failed to grow upon removal of E4orf4. E4orf4-expressing yeast also displayed an elongated cell morphology similar to cdc55 deletion strains. E4orf4 required CDC55 to elicit its effect, whereas RTS1 was dispensable. The recruitment of the PP2A holoenzyme by E4orf4 was entirely dependent on Cdc55. These studies indicate that E4orf4-induced apoptosis in mammalian cells and cell death in yeast require functional interactions with B-type subunits of PP2A. However, some inhibition of growth by E4orf4 was observed in the cdc55 strain and with an E4orf4 mutant that fails to interact with Cdc55, indicating that E4orf4 may possess a second Cdc55-independent function affecting cell growth.

Use of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells

Significance Pancreatic islets are complex structures composed of four cell types whose primary function is to maintain glucose homeostasis. Owing to the scarcity and heterogeneity of the islet cell types, little is known about their individual gene expression profiles. Here we used the Fluidigm C1 platform to obtain high-quality gene expression profiles of each islet cell type from mice. We identified cell-type–specific transcription factors and pathways providing previously unrecognized insights into genes characterizing islet cells. Unexpectedly, our data uncover technical limitations with the C1 Fluidigm cell capture process, which should be considered when analyzing single-cell transcriptomics data. This study provides an assessment of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells. The system combines microfluidic technology and nanoliter-scale reactions. We sequenced 622 cells, allowing identification of 341 islet cells with high-quality gene expression profiles. The cells clustered into populations of α-cells (5%), β-cells (92%), δ-cells (1%), and pancreatic polypeptide cells (2%). We identified cell-type–specific transcription factors and pathways primarily involved in nutrient sensing and oxidation and cell signaling. Unexpectedly, 281 cells had to be removed from the analysis due to low viability, low sequencing quality, or contamination resulting in the detection of more than one islet hormone. Collectively, we provide a resource for identification of high-quality gene expression datasets to help expand insights into genes and pathways characterizing islet cell types. We reveal limitations in the C1 Fluidigm cell capture process resulting in contaminated cells with altered gene expression patterns. This calls for caution when interpreting single-cell transcriptomics data using the C1 Fluidigm system.

The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site.

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.

Internal Translation Initiation Mediated by the Angiogenic Factor Tie2

Tie2 is an endothelium-specific receptor tyrosine kinase required for normal blood vessel maturation. We report that Tie2 mRNA translation is maintained under hypoxic conditions. To identify the mechanism responsible for this, we undertook structure/function analysis of the Tie2 5′-untranslated region (UTR). Transcription start site mapping indicates the existence of a several mRNA isoforms containing unusually long 5′-UTRs (>350 nucleotides) with five upstream open reading frames. We find internal ribosome binding activity that allows the Tie2 mRNA to initiate in a cap-independent fashion. Our data provide a framework for understanding how Tie2 mRNA is translated despite a cumbersome structured 5′-UTR and how its production is secured under unfavorable environmental conditions.

Modulation of EWS/WT1 activity by the v‐Src protein tyrosine kinase

Desmoplastic small round cell tumor (DSRCT) is a malignant human cancer that is associated with a specific t(11;22) chromosome translocation, where 265 amino acids from the EWS amino‐terminus are fused to the DNA binding domain of the WT1 tumor suppressor gene. We have noticed the presence of several SH3 interacting domains within the amino‐terminus of EWS and have assessed the potential of EWS/WT1 to interact with such motifs. We find that EWS/WT1 can associate with the SH3 domain of several proteins, including v‐Src. Ectopic expression of v‐Src phosphorylates EWS/WT1 in vivo, as well as enhances the transactivation ability of the EWS amino‐terminal domain. Structural alteration of the v‐Src SH2 or SH3 domains produced mutants that could not interact with EWS/WT1 nor augment the transcriptional properties of EWS. Taken together, our results suggest the possibility that some transcriptional properties of EWS/WT1 may be regulated by a cytoplasmic signaling pathway.

The Tie2 5' untranslated region is inhibitory to 5' end-mediated translation initiation.

Tie2 is an endothelium‐specific receptor tyrosine kinase required for normal blood vessel maturation, remodeling, and stability. Tie2 expression is also upregulated in various cancers implicating a role in tumor angiogenesis. Its mRNA transcript contains an unusually long (372 nucleotides) 5′ untranslated region (UTR) with five upstream open reading frames (uORFs) and an internal ribosome entry site (IRES) that allows this mRNA to be translated under hypoxic conditions. This sets up an alternative initiation pathway with the potential to clash with 5′ end‐mediated initiation from the same template. Herein, we define experimental conditions under which the Tie2 IRES is not active, allowing us to assess the contribution of the 5′ UTR to cap‐dependent translation on the Tie2 transcript. We find that the Tie2 5′ UTR is inhibitory to translation initiation with ribosome flow decreasing following encounters with each uORF. No single uORF was found to harbor significant cis‐acting inhibitory activity. Our results suggest that the uORFs within the Tie2 5′ UTR serve to decrease the percent of ribosomes competent for reinitiation as these traverse the mRNA 5′ UTR, thus minimizing interference with the IRES.