DIMM transactivates the PHM via the subsets of E boxes in its first intron and binds the E1 box directly. (A) A schematic of the first PHM intron indicating the positions of seven E boxes, E1 to E7 (see Table 1 for sequences of wild-type and mutated E boxes). Arrows indicate the original orientation of the DNA fragments. (B) Activity of the two constructs and the control vector diagrammed in panel A. In response to DIMM cotransfection, the first intron of PHM displays strong enhancer activity when fused to a heterologous simian virus 40 (SV40) promoter. (C to F) Effects on PHM transactivation by DIMM following site-directed mutagenesis of different E-box sequences within PHM's first intron. A gray box indicates a particular E-box mutation site. Ratios were calculated by dividing values resulting from dimm cotransfection by those resulting from no cotransfection. The histogram represents the means and standard errors of the means of the results. *, P < 0.05; **, P < 0.01. P values in comparison to the wild type were obtained by Student's t test.

High coincidence between the expression pattern of the ∼1.0-kb PHM transgenic reporter and that of DIMM in the third instar larval CNS. The diagram at the top describes the PHM-gal4 construct. Arrows indicate the orientation of gene transcription, and the X indicates mutation of the PHM AUG sequence. (A to C) Double-staining with anti-DIMM and anti-GFP in the third instar larval brain of PHM-WT line. (D) Quantification of the coincidence of patterns in PHM-WT flies. About 41% of GFP⁺ [GFP(+)] cells in the brain were also DIMM⁺ (n = 112 total cells); more than 90% of those that were DIMM⁺ in the VNC are GFP⁺ (n = 156 total cells). The number of GFP⁻/DIMM⁺ cells was typically less than 1% and, for clarity, was not reported in the graphs.

Misexpressed DIMM drives ectopic expression of the PHM-GFP reporter in vivo. (A) Top diagram indicates the positions of two of the three normal ap cell groups present in three thoracic segments of the larval VNC: the four-cell Tv cluster (red circles) and the pair of vAP neurons (blue circles). Two of the Tv cluster neurons in the larval stage (called Tv and Tvb) are peptidergic, and both express DIMM and PHM (3, 44). The bottom diagram schematizes the expression of DIMM and PHM in the Tv cluster in the wild type (left) and following DIMM misexpression driven by ap-GAL4 (right). (B) ap-gal4 reporter (UAS-2X EGFP line) is expressed in all four cells of the Tv cluster. (C) Without ap-GAL4 activity, PHM-GFP displays normal restriction in the DIMM⁺ Tv and Tvb cells and not in the Tv3 or Tv4 cells and is not found in vAPs (not shown). (D) When DIMM is misexpressed in all ap neurons, PHM-GFP is seen in all four Tv cluster neurons and in the two vAP neurons (data not shown). Anti-MYC recognizes the epitope-tagged DIMM (UAS-dimm-myc). (E and F) Misexpressed MIST1, the mammalian homologue of DIMM, also drives PHM-GFP throughout the four-cell Tv cluster. (E) An example is shown without the UAS-Mist1 gene. (F) When the UAS-Mist1 gene was included, GFP was found to be elevated in all four Tv cells. The results shown are representative for at least five specimens studied per experiment. α, anti. In designations of genotypes, + indicates the wild type.

A proposed mechanism and model for DIMM activity. (A) DIMM homodimers activate PHM directly via three specific E boxes (E1, E2, and E4). Presumed coactivators present in HEK 293 cells and in vivo are not shown. In vitro analysis showed that the three E boxes contribute differentially to the total amount of transactivation: the E1 box is absolutely required for in vitro activity, while E2 and E4 have only partial activity. To display full activity, the DIMM-bound E1 box must interact with E2-bound DIMM and E4-bound DIMM independently and cooperatively. However, in vivo, all three E boxes contribute equally to the activity. (B) DIMM plays two distinct roles in the regulation of neuroendocrine cell fate. (i) The left panel shows a “core program” of neuroendocrine differentiation that defines that set of genes that is utilized by all cells displaying an amidated peptide secretory cell fate. It is directly and completely controlled by DIMM. The core program consists of PHM and other as-yet-unidentified gene products. (ii) The right panel shows a “shell program” of neuroendocrine differentiation that defines that set of genes which varies among neuroendocrine cell types and which are controlled in combinatorial fashion. DIMM contributes partial control in concert with diverse other transcription factors which are symbolized as different shapes. Genes in the shell program include neuropeptide-encoding genes, as well as other as-yet-unidentified ones.

Transactivation of PHM fragments by DIMM in mammalian HEK 293 cells. (A) Schematic diagram of the PHM gene and PHM-luciferase constructs used for these experiments. When present, the PHM ATG site (shown by X) was mutated. (B) The activities of PHM promoter constructs with and without cotransfected effectors (DIMM or DIMM and DA). (C) Evaluating the specificity of PHM activation by DIMM. NAU, NAUTILUS; ATO, ATONAL; AP, APTEROUS; Mist1, murine Mist1. (D) PHM transactivation depends on the presence of a wild-type basic (DNA binding) domain in DIMM. Histograms represent means and standard errors of the means of the results. *, P < 0.05; **, P < 0.01. P values in comparison to the control values were obtained by using Student's t test.

Direct binding of DIMM to the E1 E box. (A) E1, the sequence of the probe representing the wild-type E1-box region; E1^M and E1to7, the sequences of two E1-box variants that were used as competitors for EMSA. The box demarcates the E box and 2 bp on either side. (B) HA-tagged DIMM binds to the radiolabeled E1-box probe directly and specifically. (C) The E1-DIMM complex was supershifted by its specific antibody, and not by control antibodies. Arrows indicate the shifted bands; asterisks indicate supershifted bands. α, anti. (D) The E1 box competes differently with the other E boxes of the first intron of the PHM gene. Arrowheads in panels C and D indicate nonspecific bands observed when lysate (control or experimental) was added. These assays were performed twice, with similar results. +, present; −, absent.

The activity of the ∼1.0-kb transgenic PHM reporter largely depends on DIMM. PHM-GAL4 expression in the second instar larval CNS is much stronger in dimm heterozygotes (Rev8/+) (A to C) than in dimm transheterozygotes (Rev8/Rev4) (D to F). At least 10 samples were tested per genotype. Note that the PHM-GAL4 activity is not as prevalent in these second instar tissues as in the older tissues displayed in Fig. 4.

The three E boxes E1, E2, and E4 are essential for PHM-GFP activity in vivo. The in vivo activities of three transgenic PHM-gal4 lines were evaluated with UAS-2X EGFP in the CNS of third instar larvae. Xs in diagrams on the left indicate positions of site-directed alterations in the ∼1.0-kb PHM genomic fragment; all constructs altered the PHM AUG to prevent initiation at that site. PHM-WT was otherwise wild type in sequence. PHM-E1^M, PHM-E2^M, PHM-E4^M, PHM-E24^M, and PHM-E124^M harbor alterations of specific E-box sequences. Each altered PHM transgene displayed significant reductions of promoter activity, as indicated by the results shown in the histogram on the right for the average number of GFP⁺ neurons (n = 5) observed in a representative line for each construct. Error bars show the standard errors of the means. **, P < 0.01. For each construct, there was good consensus in the results for independent transgenic lines: PHM-WT (5 lines), PHM-E1^M (8 lines) PHM-E2^M (3 lines), PHM-E4^M (3 lines), PHM-E24^M (3 lines), and PHM-E124^M (10 lines).