Methylation of Xilf3 by Xprmt1b alters its DNA, but not RNA, binding activity.

Modification of proteins by methylation has emerged as a key regulatory mechanism in many cellular processes, including gene control. Eighty to ninety percent of the arginine methylation in the cell is performed by the protein arginine methyl transferase PRMT1. ILF3, a protein involved in gene regulation at several levels, has been shown to be a substrate and regulator of PRMT1 in mammals. Here we show that the Xenopus orthologue of ILF3 (Xilf3) is methylated in vivo, and, at least in vitro, this methylation is carried out by Xprmt1b. The in vitro methylation of Xilf3 inhibits its ability to bind to DNA while leaving RNA binding activity unaltered. Consistent with these activities having a role in vivo, the DNA binding activity of the Xilf3-containing CBTF complex and the transcription of its target gene, Xgata2, are both decreased by overexpression of Xprmt1b in embryos. However, in contrast to other RNA binding proteins, a changing degree of methylation does not alter the subcellular localization of Xilf3. Several other proteins involved in gene regulation can bind both RNA and DNA; these data demonstrate a mechanism by which such binding activities may be controlled independently.

mef2 activity levels differentially affect gene expression during Drosophila muscle development.

Cell differentiation is controlled by key transcription factors, and a major question is how they orchestrate cell-type-specific genetic programs. Muscle differentiation is a well studied paradigm in which the conserved Mef2 transcription factor plays a pivotal role. Recent genomic studies have identified a large number of mef2-regulated target genes with distinct temporal expression profiles during Drosophila myogenesis. However, the question remains as to how a single transcription factor can control such diverse patterns of gene expression. In this study we used a strategy combining genomics and developmental genetics to address this issue in vivo during Drosophila muscle development. We found that groups of mef2-regulated genes respond differently to changes in mef2 activity levels: some require higher levels for their expression than others. Furthermore, this differential requirement correlates with when the gene is first expressed during the muscle differentiation program. Genes that require higher levels are activated later. These results implicate mef2 in the temporal regulation of muscle gene expression, and, consistent with this, we show that changes in mef2 activity levels can alter the start of gene expression in a predictable manner. Together these results indicate that Mef2 is not an all-or-none regulator; rather, its action is more subtle, and levels of its activity are important in the differential expression of muscle genes. This suggests a route by which mef2 can orchestrate the muscle differentiation program and contribute to the stringent regulation of gene expression during myogenesis.

The Him gene reveals a balance of inputs controlling muscle differentiation in Drosophila.

Tissue development requires the controlled regulation of cell-differentiation programs. In muscle, the Mef2 transcription factor binds to and activates the expression of many genes and has a major positive role in the orchestration of differentiation. However, little is known about how Mef2 activity is regulated in vivo during development. Here, we characterize a gene, Holes in muscle (Him), which our results indicate is part of this control in Drosophila. Him expression rapidly declines as embryonic muscle differentiates, and consistent with this, Him overexpression inhibits muscle differentiation. This inhibitory effect is suppressed by mef2, implicating Him in the mef2 pathway. We then found that Him downregulates the transcriptional activity of Mef2 in both cell culture and in vivo. Furthermore, Him protein binds Groucho, a conserved, transcriptional corepressor, through a WRPW motif and requires this motif and groucho function to inhibit both muscle differentiation and Mef2 activity during development. Together, our results identify a mechanism that can inhibit muscle differentiation in vivo. We conclude that a balance of positive and negative inputs, including Mef2, Him, and Groucho, controls muscle differentiation during Drosophila development and suggest that one outcome is to hold developing muscle cells in a state with differentiation genes poised to be expressed.

Intact RNA-binding domains are necessary for structure-specific DNA binding and transcription control by CBTF122 during Xenopus development.

CBTF122 is a subunit of the Xenopus CCAAT box transcription factor complex and a member of a family of double-stranded RNA-binding proteins that function in both transcriptional and post-transcriptional control. Here we identify a region of CBTF122 containing the double-stranded RNA-binding domains that is capable of binding either RNA or DNA. We show that these domains bind A-form DNA in preference to B-form DNA and that the -59 to -31 region of the GATA-2 promoter (an in vivo target of CCAAT box transcription factor) adopts a partial A-form structure. Mutations in the RNA-binding domains that inhibit RNA binding also affect DNA binding in vitro. In addition, these mutations alter the ability of CBTF122 fusions with engrailed transcription repressor and VP16 transcription activator domains to regulate transcription of the GATA-2 gene in vivo. These data support the hypothesis that the double-stranded RNA-binding domains of this family of proteins are important for their DNA binding both in vitro and in vivo.