Analysis of a transcriptional network involving PU.1, Notch, and Gata3 in the lymphomyeloid lineage decision during early T-cell development

Hematopoiesis is a well-established system used to study developmental choices amongst cells with multiple lineage potentials, as well as the transcription factor network interactions that drive these developmental paths. Multipotent progenitors travel from the bone marrow to the thymus where T-cell development is initiated and these early T-cell precursors retain lineage plasticity even after initiating a T-cell program. The development of these early cells is driven by Notch signaling and the combinatorial expression of many transcription factors, several of which are also involved in the development of other cell lineages. The ETS family transcription factor PU.1 is involved in the development of progenitor, myeloid, and lymphoid cells, and can divert progenitor T-cells from the T-lineage to a myeloid lineage. This diversion of early T-cells by PU.1 can be blocked by Notch signaling. The PU.1 and Notch interaction creates a switch wherein PU.1 in the presence of Notch promotes T-cell identity and PU.1 in the absence of Notch signaling promotes a myeloid identity. Here we characterized an early T-cell cell line, Scid.adh.2c2, as a good model system for studying the myeloid vs. lymphoid developmental choice dependent on PU.1 and Notch signaling. We then used the Scid.adh.2c2 system to identify mechanisms mediating PU.1 and Notch signaling interactions during early T-cell development. We show that the mechanism by which Notch signaling is protecting pro-T cells is neither degradation nor modification of the PU.1 protein. Instead we give evidence that Notch signaling is blocking the PU.1-driven inhibition of a key set of T-regulatory genes including Myb, Tcf7, and Gata3. We show that the protection of Gata3 from PU.1-mediated inhibition, by Notch signaling and Myb, is important for retaining a T-lineage identity. We also discuss a PU.1-driven mechanism involving E-protein inhibition that leads to the inhibition of Notch target genes. This is mechanism may be used as a lockdown mechanism in pro-T-cells that have made the decision to divert to the myeloid pathway.

Pattern formation during Caenorhabditis Elegans vulval development

Pattern formation during animal development involves at least three processes: establishment of the competence of precursor cells to respond to intercellular signals, formation of a pattern of different cell fates adopted by precursor cells, and execution of the cell fate by generating a pattern of distinct descendants from precursor cells. I have analyzed the fundamental mechanisms of pattern formation by studying the development of Caenorhabditis elegans vulva.

In C. elegans, six multipotential vulval precursor cells (VPCs) are competent to respond to an inductive signal LIN-3 (EGF) mediated by LET- 23 (RTK) and a lateral signal via LIN-12 (Notch) to form a fixed pattern of 3°-3°-2°-1°-2°-3°. Results from expressing LIN-3 as a function of time in animals lacking endogenous LIN-3 indicate that both VPCs and VPC daughters are competent to respond to LIN-3. Although the daughters of VPCs specified to be 2° or 3° can be redirected to adopt the 1°fate, the decision to adopt the 1° fate is irreversible. Coupling of VPC competence to cell cycle progression reveals that VPC competence may be periodic during each cell cycle and involve LIN-39 (HOM-C). These mechanisms are essential to ensure a bias towards the 1° fate, while preventing an excessive response.

After adopting the 1° fate, the VPC executes its fate by dividing three rounds to form a fixed pattern of four inner vulF and four outer vulE descendants. These two types of descendants can be distinguished by a molecular marker zmp-1::GFP. A short-range signal from the anchor cell (AC), along with signaling between the inner and outer 1° VPC descendants and intrinsic polarity of 1° VPC daughters, patterns the 1° lineage. The Ras and the Wnt signaling pathways may be involved in these mechanisms.

The temporal expression pattern of egl-17::GFP, another marker ofthe 1° fate, correlates with three different steps of 1° fate execution: the commitment to the 1° fate, as well as later steps before and after establishment of the uterine-vulval connection. Six transcription factors, including LIN-1(ETS), LIN-39 (HOM-C), LIN-11(LIM), LIN-29 (zinc finger), COG-1 (homeobox) and EGL-38 (PAX2/5/8), are involved in different steps during 1° fate execution.

Investigations of Pyrrole-Imidazole Polyamide Effects on DNA Replication

Pyrrole–Imidazole polyamides are programmable, cell-permeable small molecules that bind in the minor groove of double-stranded DNA sequence-specifically. Polyamide binding has been shown to alter the local helical structure of DNA, disrupt protein-DNA interactions, and modulate endogenous gene expression. Py–Im polyamides targeted to the androgen receptor-DNA interface have been observed to decrease expression of androgen-regulated genes, upregulate p53, and induce apoptosis in a hormone-sensitive prostate cancer cell line. Here we report that androgen response element (ARE)-targeted polyamides induced DNA replication stress in a hormone-insensitive prostate cancer cell line. The ATR checkpoint kinase was activated in response to this stress, causing phosphorylation of MCM2, and FANCD2 was monoubiquitinated. Surprisingly, little single-stranded DNA was exhibited, and the ATR targets RPA2 and Chk1 were not phosphorylated. We conclude that polyamide induces relatively low level replication stress, and suggest inhibition of the replicative helicase as a putative mechanism based on in vitro assays. We also demonstrate polyamide-induced inhibition of DNA replication in cell free extracts from X. laevis oocytes. In this system, inhibition of chromatin decondensation is observed, preventing DNA replication initiation. Finally, we show that Py-Im polyamides targeted to the ARE and ETS binding sequence downregulate AR- and ERG-driven signaling in a prostate cancer cell line harboring the TMPRSS2-ERG fusion. In a mouse xenograft model, ARE-targeted polyamide treatment reduced growth of the tumor.