There is considerable interest in the developmental, temporal and tissue-specific patterns of DNA replication in metazoans. Site-specific DNA replication at the chorion loci in Drosophila follicle cells leads to extensive gene amplification, and the organization of the cis-acting DNA elements that regulate this process may provide a model for how such regulation is achieved. Two elements important for amplification of the third chromosome chorion gene cluster, ACE3 and Ori-beta, are directly bound by Orc (origin recognition complex), and two-dimensional gel analysis has revealed that the primary origin used is Ori-beta (refs 7-9). Here we show that the Drosophila homologue of the Myb (Myeloblastosis) oncoprotein family is tightly associated with four additional proteins, and that the complex binds site-specifically to these regulatory DNA elements. Drosophila Myb is required in trans for gene amplification, showing that a Myb protein is directly involved in DNA replication. A Drosophila Myb binding site, as well as the binding site for another Myb complex member (p120), is necessary in cis for replication of reporter transgenes. Chromatin immunoprecipitation experiments localize both proteins to the chorion loci in vivo. These data provide evidence that specific protein complexes bound to replication enhancer elements work together with the general replication machinery for site-specific origin utilization during replication.

We have analyzed the control of developmental expression of the CD4 gene, which encodes an important recognition molecule and differentiation antigen on T cells. We have determined that the CD4 promoter alone functions at high levels in the CD4+ CD8-mature T cell but not at the early CD4+ CD8+ stage of T-cell development. In addition, the CD4 promoter functions only in T lymphocytes; thus, the stage and tissue specificity of the CD4 gene is mediated in part by its promoter. We have determined that a Myb transcription factor binds to the CD4 promoter and is critical for full promoter function. Thus, Myb plays an important role in the expression of T-cell-specific developmentally regulated genes.The CD4 glycoprotein is expressed on specific subsets of mature T cells and thymocytes and plays an important role both in T-cell antigen-specific activation and in T-cell development (37,47). T cells are capable of recognizing antigen only in the form of an oligopeptide bound to a membrane protein encoded within the major histocompatibility complex (MHC) (for a review, see reference 22). The antigenspecific and MHC allele-specific interaction is mediated primarily by the T-cell antigen receptor, whereas CD4 and CD8, another glycoprotein similar in function to CD4, recognize nonpolymorphic regions of the MHC molecule (10, 55). This latter interaction serves both to increase the affinity of the T cell for the antigen-presenting cell (APC) and to provide additional stimulatory signals via the tyrosine kinase pS6Ick (49). CD4 binds to MHC class II molecules and is expressed only on T cells bearing MHC class II-restricted T-cell antigen receptors (TCRs), primarily helper T (TH) cells (66). Thus, CD4 plays an important role in specifying T-cell antigen/MHC recognition and may also influence T-cell functional subclass.CD4 also plays a critical role in T-cell development. Immature thymocytes initially do not express TCR, CD4, or CD8. These CD4-CD8-(double-negative) cells differentiate to express high levels of all three molecules, forming TCR+ double-positive (CD4+ CD8+) cells that compose the largest thymocyte subpopulation (12,70). CD4+ CD8+ thymocytes that bind MHC class II molecules via the TCR and CD4 downregulate the expression of CD8 and maintain the expression of CD4; conversely, thymocytes that ligate MHC class I molecules via the TCR and CD8 molecules downregulate CD4 and maintain CD8 expression (2,29,31,58,59). This process of selection results in the mature TCR+ singlepositive (CD4+ CD8-and CD4-CD8+) populations that seed the peripheral lymphoid organs.

The Drosophila Myb oncoprotein, the E2F2 transcriptional repressor, and the RBF and Mip130/LIN-9 tumor suppressor proteins reside in a conserved Myb-MuvB (MMB)/dREAM complex. We now show that Myb is required in vivo for the expression of Polo kinase and components of the spindle assembly checkpoint (SAC). Surprisingly, the highly conserved DNA-binding domain was not essential for assembly of Myb into MMB/dREAM, for transcriptional regulation in vivo, or for rescue of Myb-null mutants to adult viability. E2F2, RBF, and Mip130/LIN-9 acted in opposition to Myb by repressing the expression of Polo and SAC genes in vivo. Remarkably, the absence of both Myb and Mip130, or of both Myb and E2F2, caused variegated expression in which high or low levels of Polo were stably inherited through successive cell divisions in imaginal wing discs. Restoration of Myb resulted in a uniformly high level of Polo expression similar to that seen in wild-type tissue, whereas restoration of Mip130 or E2F2 extinguished Polo expression. Our results demonstrate epigenetic regulation of gene expression by Myb, Mip130/LIN-9, and E2F2-RBF in vivo, and also provide an explanation for the ability of Mip130-null mutants to rescue the lethality of Myb-null mutants in vivo.[Keywords: Epigenetic; Myb; retinoblastoma; E2F; oncogene; tumor suppressor] Supplemental material is available at http://www.genesdev.org. Received October 16, 2007; revised version accepted December 19, 2007. A central question in developmental biology is the mechanism by which genetically identical cells stably maintain different phenotypic states during successive rounds of mitotic division. Conversely, the disruption of such epigenetic regulation is widely believed to play a key role in cancer (Lund and van Lohuizen 2004). A powerful model for understanding the mechanisms of epigenetic control was provided by Muller's eversporting displacements in Drosophila, which display variegated expression of eye pigment due to the spread of constitutive heterochromatin into adjacent regions of the genome (Muller 1930;Schulze and Wallrath 2007). Parallel systems of epigenetic regulation by Trithorax and Polycomb group proteins were discovered via genetic studies of the Drosophila body plan (Lewis 1978;Schuettengruber et al. 2007).The retinoblastoma (RB) family of tumor suppressor proteins regulates gene expression by binding to E2F-DP heterodimers, which themselves bind directly to DNA (Classon and Harlow 2002). RB family proteins have been proposed to inhibit gene expression by direct interaction with components of the classical position effect variegation (PEV) system and/or the Polycomb system (Nielsen et al. 2001;Vandel et al. 2001;Gonzalo et al. 2005;Kotake et al. 2007). However, evidence for epigenetic regulation of gene expression by RB family proteins in vivo has thus far been lacking.RB is encoded by the retinoblastoma susceptibility gene (RB1), loss of which causes both inherited and sporadic retinoblastoma in humans (Classon and Harlow 2002). Two RB-related proteins, p107 ...

The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myb, differs from its normal cellular counterpart, c-Myb, by (i) expression under the control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl termini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of 11 amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transformation by v-Myb. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have tested them for the transformation of chicken bone marrow cells. We conclude that truncation of either the amino or carboxyl terminus of c-Myb is sufficient for transformation. In contrast, the overexpression of full-length c-Myb does not result in transformation. We have also shown that the amino acid substitutions of v-Myb by themselves are not sufficient for the activation of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to be granulated promyelocytes that express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim-1 protein, in contrast to monoblasts transformed by v-Myb that neither contain granules nor express Mim-1. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells.

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