Limits...
Essential functions of the histone demethylase lid.

Li L, Greer C, Eisenman RN, Secombe J - PLoS Genet. (2010)

Bottom Line: Strikingly, we find that lid mutants are rescued to adulthood by either wildtype or enzymatically inactive Lid expressed under the control of its endogenous promoter, demonstrating that Lid's demethylase activity is not essential for development.In contrast, ubiquitous expression of UAS-Lid transgenes lacking its JmjN, C-terminal PHD domain, and C(5)HC(2) zinc finger were unable to rescue lid homozygous mutants, indicating that these domains carry out Lid's essential developmental functions.We also show that Lid's essential C-terminal PHD finger binds specifically to di- and trimethylated H3K4 and that this activity is required for Lid to function in dMyc-induced cell growth.

View Article: PubMed Central - PubMed

Affiliation: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

ABSTRACT
Drosophila Little imaginal discs (Lid) is a recently described member of the JmjC domain class of histone demethylases that specifically targets trimethylated histone H3 lysine 4 (H3K4me3). To understand its biological function, we have utilized a series of Lid deletions and point mutations to assess the role that each domain plays in histone demethylation, in animal viability, and in cell growth mediated by the transcription factor dMyc. Strikingly, we find that lid mutants are rescued to adulthood by either wildtype or enzymatically inactive Lid expressed under the control of its endogenous promoter, demonstrating that Lid's demethylase activity is not essential for development. In contrast, ubiquitous expression of UAS-Lid transgenes lacking its JmjN, C-terminal PHD domain, and C(5)HC(2) zinc finger were unable to rescue lid homozygous mutants, indicating that these domains carry out Lid's essential developmental functions. Although Lid-dependent demethylase activity is not essential, dynamic removal of H3K4me3 may still be an important component of development, as we have observed a genetic interaction between lid and another H3K4me3 demethylase, dKDM2. We also show that Lid's essential C-terminal PHD finger binds specifically to di- and trimethylated H3K4 and that this activity is required for Lid to function in dMyc-induced cell growth. Taken together, our findings highlight the importance of Lid function in the regulated removal and recognition of H3K4me3 during development.

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The third PHD finger of Lid is required for it to function with dMyc.(A) Schematic representation of UAS Lid deletion and point mutant transgenes (domains are not shown to scale) summarizing their histone demethylation activity, rescue of lid mutants (lid10424), and their ability to enhance the dMyc overexpression eye phenotype. (B–E) Scanning electron micrographs of GMR-Gal4 alone (B), GMR-Gal4, UAS-dMyc (3 copies of transgene; GMM) (C), GMM, UAS-Lid (D) and GMM, UAS-LidΔPHD3 (E). B through E all contain 3 copies of the UAS-dMyc transgene. (F) Western analysis of GMR-Gal4 alone (lane 1), GMM (lane 2), GMM, UAS-Lid (lane 3) and GMM, UAS-LidΔPHD3 (lane 4). (G) In vitro binding assays demonstrating that GST-dMyc binds full-length Lid, Lid-JmjC*, LidΔJmjN, LidΔARID, LidΔPHD1, LidΔC5HC2, LidC1296A and LidΔPHD3 (lanes 1–8, respectively).
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pgen-1001221-g004: The third PHD finger of Lid is required for it to function with dMyc.(A) Schematic representation of UAS Lid deletion and point mutant transgenes (domains are not shown to scale) summarizing their histone demethylation activity, rescue of lid mutants (lid10424), and their ability to enhance the dMyc overexpression eye phenotype. (B–E) Scanning electron micrographs of GMR-Gal4 alone (B), GMR-Gal4, UAS-dMyc (3 copies of transgene; GMM) (C), GMM, UAS-Lid (D) and GMM, UAS-LidΔPHD3 (E). B through E all contain 3 copies of the UAS-dMyc transgene. (F) Western analysis of GMR-Gal4 alone (lane 1), GMM (lane 2), GMM, UAS-Lid (lane 3) and GMM, UAS-LidΔPHD3 (lane 4). (G) In vitro binding assays demonstrating that GST-dMyc binds full-length Lid, Lid-JmjC*, LidΔJmjN, LidΔARID, LidΔPHD1, LidΔC5HC2, LidC1296A and LidΔPHD3 (lanes 1–8, respectively).

Mentions: We originally isolated lid in a genetic screen for regulators and mediators of dMyc-dependent cell growth based on an adult eye phenotype generated by dMyc expression in post-mitotic cells of the developing eye using GMR-Gal4 [7]. Furthermore, we showed that Lid's demethylase activity was not required for its dMyc-dependent functions. To pursue the mechanism by which Lid functions in cell growth induced by dMyc, we crossed our UAS-Lid mutant transgenes to the dMyc overexpressing fly strain and compared their ability to enhance this eye phenotype to that observed in response to expression of wildtype Lid (Figure 4A). Expression of Lid lacking its JmjN, C5HC2 or PHD3 domains failed to enhance the dMyc overexpression eye phenotype while not altering the levels of overexpressed dMyc (Figure 4B–4F; data not shown). As controls, we expressed the Lid deletion transgenes in a wildtype background and found that they resulted in no adult eye phenotype and, unlike fat body cells, Lid-JmjC* and LidΔARID do not have a dominant negative effects in post mitotic cells of the developing eye. We have previously shown that dMyc binds to two regions of Lid: its JmjC domain and its C5HC2 zinc finger [7]. To verify that all Lid deletion proteins retain their ability to bind dMyc, we carried out in vitro binding assays and found that they all bind equivalently (Figure 4G), suggesting that the JmjN, C5HC2 and PHD3 domains of Lid are likely to be required for its Myc-dependent functions in cell growth.


Essential functions of the histone demethylase lid.

Li L, Greer C, Eisenman RN, Secombe J - PLoS Genet. (2010)

The third PHD finger of Lid is required for it to function with dMyc.(A) Schematic representation of UAS Lid deletion and point mutant transgenes (domains are not shown to scale) summarizing their histone demethylation activity, rescue of lid mutants (lid10424), and their ability to enhance the dMyc overexpression eye phenotype. (B–E) Scanning electron micrographs of GMR-Gal4 alone (B), GMR-Gal4, UAS-dMyc (3 copies of transgene; GMM) (C), GMM, UAS-Lid (D) and GMM, UAS-LidΔPHD3 (E). B through E all contain 3 copies of the UAS-dMyc transgene. (F) Western analysis of GMR-Gal4 alone (lane 1), GMM (lane 2), GMM, UAS-Lid (lane 3) and GMM, UAS-LidΔPHD3 (lane 4). (G) In vitro binding assays demonstrating that GST-dMyc binds full-length Lid, Lid-JmjC*, LidΔJmjN, LidΔARID, LidΔPHD1, LidΔC5HC2, LidC1296A and LidΔPHD3 (lanes 1–8, respectively).
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Related In: Results  -  Collection

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pgen-1001221-g004: The third PHD finger of Lid is required for it to function with dMyc.(A) Schematic representation of UAS Lid deletion and point mutant transgenes (domains are not shown to scale) summarizing their histone demethylation activity, rescue of lid mutants (lid10424), and their ability to enhance the dMyc overexpression eye phenotype. (B–E) Scanning electron micrographs of GMR-Gal4 alone (B), GMR-Gal4, UAS-dMyc (3 copies of transgene; GMM) (C), GMM, UAS-Lid (D) and GMM, UAS-LidΔPHD3 (E). B through E all contain 3 copies of the UAS-dMyc transgene. (F) Western analysis of GMR-Gal4 alone (lane 1), GMM (lane 2), GMM, UAS-Lid (lane 3) and GMM, UAS-LidΔPHD3 (lane 4). (G) In vitro binding assays demonstrating that GST-dMyc binds full-length Lid, Lid-JmjC*, LidΔJmjN, LidΔARID, LidΔPHD1, LidΔC5HC2, LidC1296A and LidΔPHD3 (lanes 1–8, respectively).
Mentions: We originally isolated lid in a genetic screen for regulators and mediators of dMyc-dependent cell growth based on an adult eye phenotype generated by dMyc expression in post-mitotic cells of the developing eye using GMR-Gal4 [7]. Furthermore, we showed that Lid's demethylase activity was not required for its dMyc-dependent functions. To pursue the mechanism by which Lid functions in cell growth induced by dMyc, we crossed our UAS-Lid mutant transgenes to the dMyc overexpressing fly strain and compared their ability to enhance this eye phenotype to that observed in response to expression of wildtype Lid (Figure 4A). Expression of Lid lacking its JmjN, C5HC2 or PHD3 domains failed to enhance the dMyc overexpression eye phenotype while not altering the levels of overexpressed dMyc (Figure 4B–4F; data not shown). As controls, we expressed the Lid deletion transgenes in a wildtype background and found that they resulted in no adult eye phenotype and, unlike fat body cells, Lid-JmjC* and LidΔARID do not have a dominant negative effects in post mitotic cells of the developing eye. We have previously shown that dMyc binds to two regions of Lid: its JmjC domain and its C5HC2 zinc finger [7]. To verify that all Lid deletion proteins retain their ability to bind dMyc, we carried out in vitro binding assays and found that they all bind equivalently (Figure 4G), suggesting that the JmjN, C5HC2 and PHD3 domains of Lid are likely to be required for its Myc-dependent functions in cell growth.

Bottom Line: Strikingly, we find that lid mutants are rescued to adulthood by either wildtype or enzymatically inactive Lid expressed under the control of its endogenous promoter, demonstrating that Lid's demethylase activity is not essential for development.In contrast, ubiquitous expression of UAS-Lid transgenes lacking its JmjN, C-terminal PHD domain, and C(5)HC(2) zinc finger were unable to rescue lid homozygous mutants, indicating that these domains carry out Lid's essential developmental functions.We also show that Lid's essential C-terminal PHD finger binds specifically to di- and trimethylated H3K4 and that this activity is required for Lid to function in dMyc-induced cell growth.

View Article: PubMed Central - PubMed

Affiliation: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

ABSTRACT
Drosophila Little imaginal discs (Lid) is a recently described member of the JmjC domain class of histone demethylases that specifically targets trimethylated histone H3 lysine 4 (H3K4me3). To understand its biological function, we have utilized a series of Lid deletions and point mutations to assess the role that each domain plays in histone demethylation, in animal viability, and in cell growth mediated by the transcription factor dMyc. Strikingly, we find that lid mutants are rescued to adulthood by either wildtype or enzymatically inactive Lid expressed under the control of its endogenous promoter, demonstrating that Lid's demethylase activity is not essential for development. In contrast, ubiquitous expression of UAS-Lid transgenes lacking its JmjN, C-terminal PHD domain, and C(5)HC(2) zinc finger were unable to rescue lid homozygous mutants, indicating that these domains carry out Lid's essential developmental functions. Although Lid-dependent demethylase activity is not essential, dynamic removal of H3K4me3 may still be an important component of development, as we have observed a genetic interaction between lid and another H3K4me3 demethylase, dKDM2. We also show that Lid's essential C-terminal PHD finger binds specifically to di- and trimethylated H3K4 and that this activity is required for Lid to function in dMyc-induced cell growth. Taken together, our findings highlight the importance of Lid function in the regulated removal and recognition of H3K4me3 during development.

Show MeSH