The Promoter of the Immunoglobulin J Chain Gene Receives Its Authentic Enhancer Activity through the Abutting MEF2 and PU.1 Sites in a DNA-Looping Interaction

Immunoglobulin J chain (IgJ) promoter had previously been dissected in the context of a heterologous enhancer and/or promoter because its strength was weak and its authentic enhancer was not available at that time. Thus, it has been questioned whether the previous dissection of the IgJ promoter might also be relevant in the context of its authentic enhancer. Now that the authentic IgJ enhancer has been identified, redelineation of the IgJ promoter could be performed in the context of this authentic enhancer. In this redelineation, the previously identified MEF2 and PU.1 sites were shown to be critical for communicating with its authentic enhancer and thereby for receiving enhancer activity. In accordance with this finding, a DNA-looping interaction between the IgJ promoter and its enhancer was demonstrated using chromosome conformation capture assays not only in IgJ-expressing S194 plasma cells but also during interleukin-2-induced BCL1 B-cell terminal differentiation. Furthermore, MEF2 was shown to be reciprocally coimmunoprecipitated with E47, which had been identified to bind to the IgJ enhancer, suggesting that the DNA-looping interaction between the IgJ promoter and its enhancer might be mediated by these proteins. However, the previously identified USF and BSAP sites were shown to be not important for IgJ promoter activity in the context of its authentic enhancer. These findings were further supported by in vivo footprinting and/or chromatin immunoprecipitation assays, which showed the binding of MEF2 and PU.1—but not the binding of USF and BSAP—to the IgJ promoter.     

The Him gene inhibits the development of Drosophila flight muscles during metamorphosis

During Drosophila metamorphosis some larval tissues escape the general histolysis and are remodelled to form adult tissues. One example is the dorso-longitudinal muscles (DLMs) of the indirect flight musculature. They are formed by an intriguing process in which residual larval oblique muscles (LOMs) split and fuse with imaginal myoblasts associated with the wing disc. These myoblasts arise in the embryo, but remain undifferentiated throughout embryogenesis and larval life, and thus share characteristics with mammalian satellite cells. However, the mechanisms that maintain the Drosophila myoblasts in an undifferentiated state until needed for LOM remodelling are not understood. Here we show that the Him gene is expressed in these myoblasts, but is undetectable in developing DLM fibres. Consistent with this, we found that Him could inhibit DLM development: it inhibited LOM splitting and resulted in fibre degeneration. We then uncovered a balance between mef2, a positive factor required for proper DLM development, and the inhibitory action of Him. Mef2 suppressed the inhibitory effect of Him on DLM development, while Him could suppress the premature myosin expression induced by mef2 in myoblasts. Furthermore, either decreased Him function or increased mef2 function disrupted DLM development. These findings, together with the co-expression of Him and Mef2 in myoblasts, indicate that Him may antagonise mef2 function during normal DLM development and that Him participates in a balance of signals that controls adult myoblast differentiation and remodelling of these muscle fibres. Lastly, we provide evidence for a link between Notch function and Him and mef2 in this balance.     

Identification of a new hybrid serum response factor and myocyte enhancer factor 2-binding element in MyoD enhancer required for MyoD expression during myogenesis

MyoD is a critical myogenic factor induced rapidly upon activation of quiescent satellite cells, and required for their differentiation during muscle regeneration. One of the two enhancers of MyoD, the distal regulatory region, is essential for MyoD expression in postnatal muscle. This enhancer contains a functional divergent serum response factor (SRF)-binding CArG element required for MyoD expression during myoblast growth and muscle regeneration in vivo. Electrophoretic mobility shift assay, chromatin immunoprecipitation, and microinjection analyses show this element is a hybrid SRF- and MEF2 Binding (SMB) sequence where myocyte enhancer factor 2 (MEF2) complexes can compete out binding of SRF at the onset of differentiation. As cells differentiate into postmitotic myotubes, MyoD expression no longer requires SRF but instead MEF2 binding to this dual-specificity element. As such, the MyoD enhancer SMB element is the site for a molecular relay where MyoD expression is first initiated in activated satellite cells in an SRF-dependent manner and then increased and maintained by MEF2 binding in differentiated myotubes. Therefore, SMB is a DNA element with dual and stage-specific binding activity, which modulates the effects of regulatory proteins critical in controlling the balance between proliferation and differentiation.