Characterization of the Role of Drosophila Myb in Regulation as part of the Myb-MuvB/dREAM Complex

Drosophila Myb, a member of the evolutionarily well-conserved myeloblastosis proto-oncogene family, is an important regulator of development and cell growth. Myb binds DNA site-specifically to drive replication and control expression of differentiation and late cell cycle genes. This latter function of Myb in regulating the G2/M transition appears to be its most essential one: loss of Myb causes flies to die late in larval development with an accumulation of mitotically-arrested cells displaying a variety of abnormalities associated with genomic instability. The C-terminal portion of Myb proteins, which is deleted in oncogenic viral Myb forms, is known to provide an autoregulatory function. However, the mechanism of this activity is not yet understood./nMyb resides in a conserved multisubunit complex known as Myb-MuvB/dREAM (MMB). In addition to Myb, the MMB complex also contains the Drosophila homologues of the retinoblastoma protein (RBFs) and its binding partners E2F/DP, which are well-studied transcriptional repressors and regulators of the G1/S transition. By coordinating the opposing activities of Myb and E2F/DP/RBF, the MMB complex can both positively and negatively regulate transcription and replication according to developmental cues. Myb provides an essential function during development by counteracting the otherwise repressive MMB complex. An outstanding question is how the Myb-MuvB/dREAM complex achieves a switch between repressive and activating functions, and how Myb acts to promote such a switch./nMyb is dependent on association with MMB complex members for stability and executes its functions within this context. Yet, little is known about the molecular mechanisms governing the assembly of Myb into the full complex. To understand how Myb incorporates into the MMB complex, I have mapped the region of Myb important for this interaction. I have found that the region of Myb required for interaction with MMB core members in vivo is distinct from that required when expressed recombinantly. In Drosophila S2 tissue culture cells, I have found that the last 78 residues of the protein are critical for association with the core MMB complex members, Mip120 and Mip130. In flies, the C-terminus of Myb is required for efficient localization to the polytene chromosomes of larval salivary glands, and cannot rescue viability of a myb null mutation. Surprisingly, when co-expressed recombinantly, the central region of Myb, and not the C-terminus, is required for Myb association with MMB members. These findings suggest that the Myb C-terminus is indirectly required for stably maintaining Myb in the full Myb-MuvB/dREAM complex, while the central portion is required for direct physical interactions./nGiven that the activities of Myb are restricted to the MMB complex, I have sought to understand the mechanisms that govern Myb levels. I have shown that Myb turnover occurs at at least two levels. At a global level, Myb is constitutively degraded, determining the steady-state levels of the protein. Myb is a short-lived protein with a half-life of between one and two hours. This rapid turnover likely limits Myb activity outside of MMB. Moreover, a C-terminal truncation of Myb accumulates in S2 cells, indicating this region may contain a degron that is masked upon association with MMB complex members. Secondly, Myb undergoes cell cycle-specific ubiquitin-mediated proteolysis as a target of the Anaphase-Promoting Complex/Cyclosome (APC/C). A three-residue motif, termed the KEN Box, is required for APC/C-dependent turnover in an in vitro assay and is essential for viability in vivo./nTogether these data suggest the Myb C-terminus serves to indirectly facilitate its assembly into the MMB complex in vivo and to target Myb for ubiquitin-mediated turnover. Thus, the C-terminus appears to be critical for regulating Myb protein levels, for restricting Myb activity to the context of the larger MMB complex, and for determining the overall Myb-MuvB regulatory functions.

Advisor: Botchan, Michael R