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 PHD fingers of Lid bind specific forms of methylated histone tails.GST fusion proteins of Lid's PHD1 (top panel), PHD2 (2nd panel) and PHD3 (3rd panel) were tested for binding to biotinylated histone peptides of histone H3 amino acids 1–21, 21–44 and histone H4 1–21. In addition, peptides mono, di or tri methylated at histone H3 K4, K9 and K27 were also tested for their ability to bind these PHD fingers.
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pgen-1001221-g005: The PHD fingers of Lid bind specific forms of methylated histone tails.GST fusion proteins of Lid's PHD1 (top panel), PHD2 (2nd panel) and PHD3 (3rd panel) were tested for binding to biotinylated histone peptides of histone H3 amino acids 1–21, 21–44 and histone H4 1–21. In addition, peptides mono, di or tri methylated at histone H3 K4, K9 and K27 were also tested for their ability to bind these PHD fingers.

Mentions: The primary characterized function of Lid is its histone H3 lysine 4 demethylase activity. However, since we have demonstrated that this activity is not Lid's essential function, we chose to further characterize Lid's third PHD finger as this domain is required for it to function with dMyc and is essential for development. Moreover, PHD domains have recently emerged as important interpreters the histone code that act by binding to histone tails that are unmodified, mono-, di- or tri- methylated at specific lysine residues [20]. To address whether Lid's third PHD finger is able to bind methylated histones, we incubated bacterially expressed and purified GST-PHD finger proteins with biotinylated histone peptides mono-, di- or trimethylated at K4, K9 or K27 in vitro and compared this to the binding of Lid's other two PHD fingers and the known H3K4me2/3 binding protein hING2 [27], [28](Figure 5; data not shown).


Essential functions of the histone demethylase lid.

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

The PHD fingers of Lid bind specific forms of methylated histone tails.GST fusion proteins of Lid's PHD1 (top panel), PHD2 (2nd panel) and PHD3 (3rd panel) were tested for binding to biotinylated histone peptides of histone H3 amino acids 1–21, 21–44 and histone H4 1–21. In addition, peptides mono, di or tri methylated at histone H3 K4, K9 and K27 were also tested for their ability to bind these PHD fingers.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991268&req=5

pgen-1001221-g005: The PHD fingers of Lid bind specific forms of methylated histone tails.GST fusion proteins of Lid's PHD1 (top panel), PHD2 (2nd panel) and PHD3 (3rd panel) were tested for binding to biotinylated histone peptides of histone H3 amino acids 1–21, 21–44 and histone H4 1–21. In addition, peptides mono, di or tri methylated at histone H3 K4, K9 and K27 were also tested for their ability to bind these PHD fingers.
Mentions: The primary characterized function of Lid is its histone H3 lysine 4 demethylase activity. However, since we have demonstrated that this activity is not Lid's essential function, we chose to further characterize Lid's third PHD finger as this domain is required for it to function with dMyc and is essential for development. Moreover, PHD domains have recently emerged as important interpreters the histone code that act by binding to histone tails that are unmodified, mono-, di- or tri- methylated at specific lysine residues [20]. To address whether Lid's third PHD finger is able to bind methylated histones, we incubated bacterially expressed and purified GST-PHD finger proteins with biotinylated histone peptides mono-, di- or trimethylated at K4, K9 or K27 in vitro and compared this to the binding of Lid's other two PHD fingers and the known H3K4me2/3 binding protein hING2 [27], [28](Figure 5; data not shown).

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