Myosin II protein molecules, which consist of a pair of heavy chains (approximately 200 kDa) and two pairs of light chains (15-28 kDa), exist in all eukaryotic cells. Together with actin filaments, they produce contractile force mediated by ATP hydrolysis. While this contractile activity is prominent in differentiated muscle tissues, it is also involved in diverse cellular motile processes such as cytokinesis, cell migration and cell adhesion in nonmuscle cells, as well as in undifferentiated muscle cells. In vertebrates, there are over 10 different myosin II isoforms, each of which contains different myosin II heavy chains (MHCs). MHC isoform diversity is generated by multiple genes as well as by alternative splicing of pre-mRNA. Previous studies have demonstrated cell type-specific expression of MHC isoforms as well as changes in MHC isoforms during the course of muscle and nervous tissue development. This research program has investigated the regulatory mechanisms responsible for the expression of two nonmuscle MHC genes, NMHC-A and NMHC-B. We have focused on NMHC-A gene regulation as it relates to cell type-dependent transcriptional mechanisms and NMHC-B gene regulation as it relates to neural cell-specific alternative pre-mRNA splicing mechanisms. With respect to NMHC-A gene regulation, the region extending 20 kb upstream and 40 kb downstream (which includes the 39 kb intron 1) from the transcriptional start sites were examined by reporter gene analysis, in an attempt to identify cis-regulatory elements. A number of regions located in intron 1 were found to modulate transcription in a cell type-dependent manner. We focused on a 0.5 kb fragment located 33 kb downstream from the transcriptional start sites. Sequence comparisons between the human and mouse genes revealed high levels of conservation in a region of 150 bp within the 0.5 kb fragment. Reporter gene analysis and electrophoretic mobility shift assays defined an interferon-stimulated response element as a major regulatory element. Among a large family of interferon regulatory factors, IRF-2 was found to bind this element in vitro and in intact cells. Furthermore, the results of chromatin immunoprecipitation assays and reporter gene analysis have suggested that IRF-2 is involved in activation of NMHC-A gene transcription during differentiation of promyelocytic leukemia HL60 cells into the macrophage lineage. With respect to NMHC-B, previous studies in this laboratory have demonstrated the existence of a neural cell-specific NMHC-B isoform, in addition to the ubiquitously expressed form of NMHC-B. This neural cell-specific isoform is generated by cassette type alternative splicing of exon N30 which is located between constitutive exons E5 and E6. To characterize regulatory elements required for alternative splicing of N30, neural retinoblastoma Y79 cells, which are capable of including N30 to a large extent during postmitotic and differentiated states, were used as a model system. A cis-acting intronic enhancer (IDDE), located approximately 1.5 kb downstream of N30, and an exonic enhancer located within N30, were found to be indispensable for neural cell-specific inclusion of N30. The IDDE activated the upstream E5-N30 splicing, but not the N30-E6 splicing, in vitro by facilitating early prespliceosome complex formation. The IDDE includes two copies of the hexanucleotide motif UGCAUG. Sequence-specific RNA-binding proteins Fxh and A2BP1, which share an identical RNA recognition motif, have been demonstrated to bind this hexanucleotide motif. Interestingly, both mouse Fxh and A2BP1 transcripts were found to undergo tissue-specific alternative splicing, generating protein isoforms specific to brain and muscle. Co-transfection of the expression constructs for each of the isoforms with minigene reporter constructs showed that the brain isoforms of both Fxh and A2BP1 promoted N30 splicing much more eefficiently than the muscle-specific isoforms. The hexanucleotide UGCAUG in the IDDE was indispensable for splicing activation by these proteins. Moreover, exogenous expression of the brain isoform of Fxh or A2BP1 resulted in an increase in N30 inclusion in the endogenous NMHC-B mRNA. These results support the hypothesis that tissue-dependent isoforms of Fxh and A2BP1 play an important role in regulating neural cell-specific alternative splicing of NMHC-B via the IDDE.
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