In vertebrates, there are over 15 different myosin II isoforms, each of which contains different myosin II heavy chains (MHC IIs). MHC II 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 II isoforms as well as changes in MHC II isoforms during the course of muscle and neural tissue development. This research program has investigated the regulatory mechanisms responsible for the expression of three nonmuscle MHC II (NMHC II) genes, NMHC II-A, NMHC II-B, and NMHC II-C. We have been studying the transcriptional regulation of NMHC II-A and II-C genes as well as tissue-dependent regulation of alternative splicing of NMHC II-B and C genes. In this report, we focus on regulation of alternative splicing of NMHC II-B. The gene encoding NMHC II-B generates alternatively spliced isoforms, which include or exclude a cassette of amino acids (aa) near the ATP-binding domain. Inclusion of alternative exon B1 (also called exon N30) encoding 10 aa in NMHC II-B mRNAs is restricted to some types of neural cells. We have previously reported that the intronic region downstream of B1, called the intronic distal downstream enhancer (IDDE), is required for activation of B1 splicing. Two copies of an RNA element UGCAUG within the IDDE are essential for B1 inclusion in neural cells. Recently a vertebrate homolog of C. elegans Fox-1 was reported to bind to UGCAUG in a highly sequence-specific manner. Database search revealed that there are three genes for Fox-1 homologs (Fox family) in mammals, Fox-1 (also called A2BP1), Fox-2 (also called Fxh and Rbm9) and Fox-3. Fox-1 is expressed in brain and striated muscles whereas Fox-2 is expressed in various tissues including brain and muscles. Notably, the Fox-3 expression is restricted to neural tissues. We raised antibodies specific to each of the Fox proteins and histologically analyzed distribution of Fox proteins in mouse brain and spinal cord. Expressions of Fox-1, 2 and 3 overlap in many kinds of neural cells, but we also observed differences in their expression in certain types of neural cells. Comparison of these Fox expression patterns with expression of the B1-included NMHC II-B mRNA detected by in situ hybridization suggested that the Fox-3 expressing cells tend to include the B1 insert. This notion was further supported by the RT-PCR analysis of B1 splicing patterns of brain and spinal cord cells which were dissociated and sorted according to Fox-3 expression. Fox-3 positive cells from cerebellum, brainstem and spinal cord include the B1 insert in a Fox-3 concentration-dependent manner. Fox-3 negative cells almost completely exclude the B1 insert, despite expression of Fox-1 and 2. Therefore there is a better correlation of the extent of B1 inclusion with the level of Fox-3 expression rather than with that of Fox-1 or 2 expression in intact tissues, although Fox-3 and some of the isoforms of Fox-1 and 2 are similarly capable of enhancing B1 splicing when they are over-expressed in cultured cells. To understand a mechanism for Fox-mediated regulation of alternative splicing, we searched for nuclear factor(s) which interact with Fox-3. We identified PTB-associated splicing factor (PSF) as an interacting protein with Fox-3 by affinity-chromatography. The C-terminal region of Fox-3 directly binds to the N-terminal region of PSF. In cultured cells, enhancement of B1 inclusion by Fox-3 depends on the presence of PSF. PSF enhances B1 inclusion in a UGCAUG-dependent manner, although it does not bind directly to this element. Fox-3 is recruited to the UGCAUG element downstream of B1 in the endogenous NMHC II-B transcript in a PSF-dependent manner. Therefore PSF functions as an essential co-activator of Fox-3. Fox-3 and PSF interaction is an integral part of the mechanism by which Fox proteins regulate activation of alternative exons via a downstream intronic enhancer.
