Our genetic and biochemical analyses have defined the conserved PHD1-PHD2 module of ZFP-1/AF10 as a chromatin-binding domain with a discrete function essential for viability. The conservation of this region between the human AF10 protein and C. elegans
ZFP-1 was noted in the initial study describing the translocation of AF10 in acute myeloid leukemias (2
) and thereafter (10
). However, the biological significance of this conserved module has not been revealed until now.
The AF10 protein function has been studied mostly in the context of its oncogenic variants MLL-AF10 (6
) and CALM-AF10 (43
). The oncogenic potential of the MLL-AF10 fusion protein is linked to the recruitment of the DOT1L H3K79 methyltransferase by the leucine zipper and octapeptide motifs of AF10, present in the C-terminal portion of the protein (8
). However, a normal copy of the AF10 gene is also present in cancer cells. Since endogenous AF10 exists in complex with DOT1L and contributes to the normal levels of global H3K79 methylation (9
), oncogenic fusion forms are likely to compete with wild-type AF10 for interaction with DOT1L, which has been shown for CALM-AF10 (44
). This may cause aberrant expression of genes normally regulated by AF10, further contributing to cancer progression. Our study suggests that the PHD1-PHD2 module within the ZFP-1/AF10 protein possesses an autonomous function, which likely consists of binding chromatin and recruiting other factors necessary for gene expression regulation. Therefore, the PHD1-PHD2 and leucine zipper domains may recruit distinct partners, and the balance of these interactions is likely to be important for normal cell function.
Tissue-specific expression analyses of human AF10 detected mRNA expression in peripheral blood lymphocytes, thymus, ovaries, and testes (2
). The murine ortholog of AF10 is also expressed in the brain and kidneys, as seen by Northern blotting (45
), and expression in the white matter of the cerebellum was determined by in situ
). To date, there have been no studies addressing the developmental role of AF10 by using genetic knockdown techniques in mice. More recently, AF10 was found to coimmunoprecipitate with β-catenin in the HEK293T cell line (9
) and with Tcf4 and β-catenin in mouse intestinal crypt cells (46
). Since these interactors mediate downstream transcriptional effects of the Wnt signaling pathway, small interfering RNA (siRNA) knockdown of AF10 in tissue culture demonstrated that many downstream targets of Wnt signaling have reduced levels of expression in both AF10-depleted colorectal cancer cells and HEK293T cells (46
). Similarly, lower expression of Wingless targets was obtained after reducing expression of the Drosophila
ortholog of AF10 (Alhambra
) by RNAi in fly larvae (9
) and after morpholino depletion of AF10 in zebrafish (46
). Although the potential involvement of ZFP-1/AF10 in regulation of Wnt targets may contribute significantly to the developmental role of this protein, it is notable that the above studies connected both AF10 and DOT1L to Wnt signaling. Our discovery of the essential developmental function of the PHD1-PHD2 module suggests that it is independent of the role of ZFP-1/AF10 in the recruitment of DOT1L to chromatin. Also, ZFP-1 expression is much broader than the expression of β-catenin homologs in C. elegans
), indicating that its targets are not limited to those regulated by Wnt pathways.
Although transcription from the AF10 locus has not been studied extensively in humans or mice, expression of at least two major isoforms was previously detected in flies (48
), similar to our findings in C. elegans
. Consistent with our results, high expression of the PHD-containing long isoform was noted in oocytes and embryos (48
), and this isoform was also found to associate with euchromatin on polytene chromosomes of salivary glands (50
). The existing loss-of-function mutants of Alhambra
(also called Dalf
and dAF10) appear to affect expression of the C-terminal portion of both isoforms of the protein (48
). However, these mutant alleles may still allow expression of the PHD1-PHD2 module. Nonetheless, although Drosophila Alhambra
mutant larvae are very retarded in growth and eventually die (48
), the analogous C. elegans
) mutants, in which only PHD1-PHD2 function is retained, are slow growing but viable (22
). Therefore, it appears that the function of the C-terminal portion of AF10 that interacts with DOT1L is more essential for development in flies than in nematodes. We predict that the conserved PHD1-PHD2 module may have an additional essential function in both flies and mammals.
Since the long isoform of ZFP-1 is exclusively expressed in the adult germ line and predominantly expressed in the embryo, we predict that its localization to H3K4 methylation-enriched promoters contributes to the regulation of genes essential for the early development of nematodes. We have shown that PHD1 contributes to the interaction of PHD1-PHD2 with H3K4me and that the extended PHD2 is likely responsible for the multimerization of the protein. Although the DOT1L methyltransferase is a known interacting partner of AF10 (8
), it interacts with the C-terminal region of the protein. Since PHD1-PHD2 is essential for viability, we predict that additional interacting partners of ZFP-1 regulate ZFP-1 targets during early development.
Our work has highlighted the structural autonomy of the PHD1-PHD2 domain and suggested a novel mechanism of interaction between a PHD zinc finger and an H3K4-methylated histone tail. Future structural studies will determine the precise details of PHD1-PHD2 binding to nucleosomes and of its multimerization. Possibilities for the connection between the structure and function of this domain are intriguing. One prediction is that PHD1-PHD2 may recruit effector proteins that regulate gene expression; another possibility is that the histone binding of PHD1-PHD2, along with its multimerization properties, confers an autonomous role for this domain in configuring higher-order chromatin structure and thus contributing to proper gene regulation and development.