p107 in the public eye: an Rb understudy and more

p107 and its related family members Rb and p130 are critical regulators of cellular proliferation and tumorigenesis. Due to the extent of functional overlap within the Rb family, it has been difficult to assess which functions are exclusive to individual members and which are shared. Like its family members, p107 can bind a variety of cellular proteins to affect the expression of many target genes during cell cycle progression. Unlike Rb and p130, p107 is most highly expressed during the G1 to S phase transition of the cell cycle in actively dividing cells and accumulating evidence suggests a role for p107 during DNA replication. The specific roles for p107 during differentiation and development are less clear, although emerging studies suggest that it can cooperate with other Rb family members to control differentiation in multiple cell lineages. As a tumor suppressor, p107 is not as potent as Rb, yet studies in knockout mice have revealed some tumor suppressor functions in mice, depending on the context. In this review, we identify the unique and overlapping functions of p107 during the cell cycle, differentiation, and tumorigenesis

G1 arrest and differentiation can occur independently of Rb family function

Repression of E2F target genes is required for cell cycle arrest in Rb family (Rb, p107, and p130)-deficient cells

Functional Interactions between Retinoblastoma and c-MYC in a Mouse Model of Hepatocellular Carcinoma

Inactivation of the RB tumor suppressor and activation of the MYC family of oncogenes are frequent events in a large spectrum of human cancers. Loss of RB function and MYC activation are thought to control both overlapping and distinct cellular processes during cell cycle progression. However, how these two major cancer genes functionally interact during tumorigenesis is still unclear. Here, we sought to test whether loss of RB function would affect cancer development in a mouse model of c-MYC-induced hepatocellular carcinoma (HCC), a deadly cancer type in which RB is frequently inactivated and c-MYC often activated. We found that RB inactivation has minimal effects on the cell cycle, cell death, and differentiation features of liver tumors driven by increased levels of c-MYC. However, combined loss of RB and activation of c-MYC led to an increase in polyploidy in mature hepatocytes before the development of tumors. There was a trend for decreased survival in double mutant animals compared to mice developing c-MYC-induced tumors. Thus, loss of RB function does not provide a proliferative advantage to c-MYC-expressing HCC cells but the RB and c-MYC pathways may cooperate to control the polyploidy of mature hepatocytes

Altered <i>p107</i> expression affects cellular proliferation.

<p>(A,B) Immortalized wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs were synchronized in G0 through at least three days of serum starvation. DMEM supplemented with 20% BGS was used to stimulate cell-cycle entry. Extracts were collected at the number of hours indicated post-serum stimulation. (A) RT-qPCR analysis of <i>Cdc6</i> mRNA in wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs. (n = 3) (B) Percentage of cells in S-phase, as determined by BrdU/PI staining, at the indicated time points. (n≥4) (C) Cell-cycle profiles of asynchronous primary wild-type, <i>p107^{E2F-1*2*/1*2*}</i>, and <i>p107^−/−</i> MEFs. Percentages of cells in each phase were determined by BrdU/PI staining. (n≥2) (D) Cellular proliferation of primary wild-type, <i>p107^{E2F-1*2*/1*2*}</i>, and <i>p107^−/−</i> MEFs. Equal numbers of cells were plated at day 0. Cells were then counted every other day from day 1 to day 9 post-plating. For statistical analysis, <i>p107^{E2F-1*2*/1*2*}</i> cells were compared to wild-type cells at each time point. (n≥13) (E) Model for the context-dependent regulation of <i>p107</i> transcription by E2F family members. In cycling mESCs, activating members of the E2F family such as E2F3 bind to the <i>p107</i> promoter mostly through the distal consensus E2F binding site (site 1). In quiescent MEFs, binding of the E2F4 repressor is also largely dependent on the presence of the distal consensus site. However, E2F4 may also be recruited to the <i>p107</i> promoter through interactions with other transcription factors and/or by binding to other DNA sequences. The size of the E2F boxes indicates the relative binding activity.</p

Genome Editing in Mouse Spermatogonial Stem/Progenitor Cells Using Engineered Nucleases

<div><p>Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse “GS” (germline stem) cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated <i>in vitro</i>, contribute to the germline <i>in vivo</i> following testicular transplantation or become reprogrammed to pluripotency <i>in vitro</i>.</p></div

ZFN-mediated genome editing in GS cells.

<p>(A) Neon transfection (1200/30/1) was used to transfect 3×10e5 cells with 1.0 µg of em-GFP plasmid DNA (pCDNA6.2/emGFP) or 1.0 µg of capped and poly-adenylated mRNA coding for pmaxGFP and transfection efficiency was quantified by flow cytometry three days after transfection. Lipofectamine-2000 was used to transfect the same ratio of cells:DNA or cells:mRNA as in the Neon experiment. The mean and standard deviation of percentage of GFP+ cells from three experiments are shown. (*p<0.05, **p<0.01,***p<0.001, Student T test). (B) Schematic depicting the two plasmids used in genome editing experiments. The donor DNA (“³⁷GFP”; plasmid BE356) contains a fragment of the GFP coding sequence lacking the first 37 nucleotides and serves as a donor template. “Ubc-ZFN1-T2A-ZFN2” (plasmid M500) contains a bicistronic expression cassette with a human Ubiquitin C promoter driving expression of two ZFNs directed to a recognition site in the GFP gene and separated by a T2A skip sequence. GS cell lines were derived from mice carrying a mutated GFP gene, with a 85 nucleotide stop codon and frame shift insertion (labeled “STOP”), introduced into the <i>ROSA26</i> locus by standard knockin technology <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112652#pone.0112652-Connelly1" target="_blank">[25]</a>. (C) 0.8 µg of Ubc-ZFN1-T2A-ZFN2 (M500) plasmid or 0.8 µg each of synthesized mRNA ZFN1 and ZFN2, together with 2.4 µg donor plasmid (BE356), were transfected (1400/20/1) into MPG6 cells on day 1 and genome editing events were quantified on day 5 or 7 (data pooled). Histogram shows mean +/- standard error mean. The dot plot shows sample results of a single transfection of donor DNA and ZFN mRNAs. (D) GFP fluorescence (left) or corresponding transmitted light image in GT59 cells following two sorts to enrich for GFP+ cells. Bar represents 50 microns. (E) Chromatogram showing corrected GFP gene sequence of PCR amplified genomic DNA from GT59 cells. The ZFN recognition sites are indicated by boxes and the site in which the mutation was replaced by donor DNA is indicated by a line.</p