The CTCF protein is a highly conserved zinc finger protein that is implicated in many aspects of gene regulation and nuclear organization. Its functions include the ability to act as a repressor of genes, including the c-myc oncogene. In this paper, we show that the CTCF protein can be posttranslationally modified by the small ubiquitin-like protein SUMO. CTCF is SUMOylated both in vivo and in vitro, and we identify two major sites of SUMOylation in the protein. The posttranslational modification of CTCF by the SUMO proteins does not affect its ability to bind to DNA in vitro. SUMOylation of CTCF contributes to the repressive function of CTCF on the c-myc P2 promoter. We also found that CTCF and the repressive Polycomb protein, Pc2, are colocalized to nuclear Polycomb bodies. The Pc2 protein may act as a SUMO E3 ligase for CTCF, strongly enhancing its modification by SUMO 2 and 3. These studies expand the repertoire of posttranslational modifications of CTCF and suggest roles for such modifications in its regulation of epigenetic states.

The imprinting of the genes on human chromosome 11p15.5 is thought to be controlled by two imprinting control regions located in two differentially methylated CpG islands upstream of the H19 gene (H19 DMR) and in intron 10 of the KCNQ1 gene (KvDMR). We have examined sequences in the human 11p15.5 genomic imprinted region for the presence of insulators and silencers using a position- and enhancer-dependent stable transfection assay. We have confirmed the existence of insulators in H19 DMR and discovered two novel insulators in the IGF2 gene. We have also found two novel silencer sequences; one is located in KvDMR, a region that is thought to contain the promoter for the KCNQ1OT1 transcript, and another is in the CDKN1C gene. We have demonstrated binding of CTCF protein in vitro to all the insulator and silencer sequences that we have detected. We discuss the differences in the regulation of imprinting controlled by the two imprinting control regions in chromosome 11p.

Calcineurin is highly conserved and regulates growth, conidiation, stress response, and pathogenicity in fungi. However, the functions of calcineurin and its regulatory network in entomopathogenic fungi are not clear. In this study, calcineurin was functionally analyzed by deleting the catalytic subunit MaCnA from the entomopathogenic fungus Metarhizium acridum. The ΔMaCnA mutant had aberrant, compact colonies and blunt, shortened hyphae. Conidia production was reduced, and phialide differentiation into conidiogenous cells was impaired in the ΔMaCnA mutant. ΔMaCnA had thinner cell walls and greatly reduced chitin and β-1,3-glucan content compared to the wild type. The ΔMaCnA mutant was more tolerant to cell wall-perturbing agents and elevated or decreased exogenous calcium but less tolerant to heat, ultraviolet irradiation, and caspofungin than the wild type. Bioassays showed that ΔMaCnA had decreased virulence. Digital gene expression profiling revealed that genes involved in cell wall construction, conidiation, stress tolerance, cell cycle control, and calcium transport were downregulated in ΔMaCnA. Calcineurin affected some components of small G proteins, mitogen-activated protein kinase, and cyclic AMP (cAMP)-protein kinase A signaling pathways in M. acridum. In conclusion, our results gave a global survey of the genes downstream of calcineurin in M. acridum, providing molecular explanations for the changes in phenotypes observed when calcineurin was deleted.

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