West Coast Worm Meeting,
To ensure equal expression of X-linked genes between XO males and XX hermaphrodites, a complex of proteins named the dosage compensation complex (DCC) binds along the entire length of both hermaphrodite X chromosomes to downregulate gene expression by half. To understand the mechanism of DCC recruitment and binding to X, both the cis -acting DNA elements that distinguish the X from autosomes and the trans -acting factors that bind these elements must be defined. To identify cis -acting X recognition elements we performed a systematic chromosome-wide search for X regions that recruit the DCC when detached from X, using a combination of fluorescent in situ hybridization (to mark X chromosomal territories) and immunoflourescence (to determine sites of DCC binding). We studied strains carrying X chromosome duplications to determine whether the duplicated portion could bind the DCC. We identified multiple X regions that recruited the DCC independently. However, not all detached regions of X were able to recruit the DCC. Yet, the DCC does bind along the entire length of the wild-type X chromosome. Thus, DCC binding to X is first nucleated at recruitment sites followed by spreading into neighboring X chromatin (Csankovszki et al., Science 303:1182, 2004). The dosage compensation complex is similar to the evolutionary conserved 13S condesin complex required for mitotic and meiotic chromosome condensation. DPY-27 and MIX-1 are the SMC-4 and SMC-2 components, DPY-26 is homologous to CAP-H, and DPY-28 to CAP-D2. The homolog of the fifth condensin subunit (CAP-G) had not been identified. The DCC is targeted to hermaphrodite X chromosomes by the trans -acting proteins SDC-2 and SDC-3. Using immunoprecipitation and tandem mass spectrometry (in collaboration with Mcleod, I., Yates, J.), we searched for new factors interacting with the DCC and identified the missing CAP-G homolog. We are currently analyzing the contribution of this protein to the dosage compensation process. To study binding and spreading of individual dosage compensation proteins in more detail, we are analyzing high resolution, 3D reconstructed images of dosage compensation components binding to X. In some regions, particularly those with limited recruitment ability, SDC-3 binding appears more restricted than DPY-27 binding. These results suggest that binding of SDC-3 may be more limited to regions of initial recruitment, while DPY-27 may spread longer distances. Analyzing binding of the dosage compensation complex to X in animals depleted of various dosage compensation proteins may shed new light on the mechanism of recruitment and spreading of the complex.