The Carboxy-Terminal Domain of Dictyostelium C-Module-Binding Factor Is an Independent Gene Regulatory Entity

Abstract

The C-module-binding factor (CbfA) is a multidomain protein that belongs to the family of jumonji-type (JmjC) transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF) motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD). An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo.

Conserved Gene Regulatory Function of the Carboxy-Terminal Domain of Dictyostelid C-Module-Binding Factor

“…Consistent with the previous DNA microarray data (18), we found with RNA-seq that the CbfA CTD core exerts the majority of its gene regulatory activity without requiring the other domains of CbfA. Thus, CbfA can be divided into two independently functioning regions: the JmjC/ZF region and the CTD.…”

“…In fact, we found that approximately 87% of the CbfA CTD-responsive genes were restored to at least 80% of the wild-type level by both the D. discoideum and P. pallidum CbfA CTDs. This result is notable because D. discoideum and P. pallidum have different percentages of GC content in their genomes and promoter alignments of the two species are nearly impossible (4); however, the gene regulatory function of the D. discoideum CbfA CTD requires direct binding to the AT-rich DNA sequences within the target promoters, as suggested by in vitro and in vivo data (14,18).…”

“…However, there is evidence that the JmjC/ZF portion of CbfA is required for gene regulatory functions that cannot be carried out by the CbfA CTD alone. For example, the CbfA CTD was not able to rescue the developmental defect in CbfA-deficient JH.D cells (18). The fact that CbfA-deficient cells have a strong developmental phenotype indicates that CbfB, which is expressed in the CbfA mutant at normal levels, is not able to complement the loss of CbfA.…”

“…In this study, we performed Illumina RNA-seq to extend our knowledge concerning the function of the CbfA protein. A previous DNA microarray experiment performed with CbfA-deficient mutant JH.D cells (18) had limited sensitivity and included only approximately half of the D. discoideum genes. Comparing the two transcriptomic approaches, we observed that only 35% of the 1,030 CbfA-dependent genes identified by RNA-seq were present on the DNA microarray.…”

“…However, multicellular development of JH.D cells can be restored either by the application of exogenous cAMP pulses or by mixing mutant and wild-type cells, suggesting that the developmental phenotype of JH.D cells is not cell autonomous (17). The aggregation block in JH.D cells can be overcome by the ectopic expression of the full-length CbfA protein (17), but not by the expression of the isolated CbfA CTD (18).…”