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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Deletion of Lid's JmjN, ARID, and PHD1 domains abrogate its demethylase activity.Clones of cells expressing Lid or Lid mutant transgenes in fat body were generated by crossing hs-FLP; UAS-Lid females to act>CD2>Gal4, UAS-GFP males. No heat shock was carried out since leaky FLP expression during embryogenesis leads to a small number of fat body clones. Levels of Lid (A', C', E', G', I', K', M') and trimethylated H3K4 (B', D', F', H', J', L', N') were examined. Cells expressing each transgene (as labeled on figure) are marked by co-expression of GFP and are outlined in the other panels.
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pgen-1001221-g001: Deletion of Lid's JmjN, ARID, and PHD1 domains abrogate its demethylase activity.Clones of cells expressing Lid or Lid mutant transgenes in fat body were generated by crossing hs-FLP; UAS-Lid females to act>CD2>Gal4, UAS-GFP males. No heat shock was carried out since leaky FLP expression during embryogenesis leads to a small number of fat body clones. Levels of Lid (A', C', E', G', I', K', M') and trimethylated H3K4 (B', D', F', H', J', L', N') were examined. Cells expressing each transgene (as labeled on figure) are marked by co-expression of GFP and are outlined in the other panels.

Mentions: To assess the ability of our Lid mutants to demethylate, we generated clones of cells overexpressing each protein in larval fat body and examined the levels of Lid and H3K4me3 (Figure 1). Based on the intensity of the immunofluorescence signal and Western analysis, all of our transgenes expressed Lid at similar levels (data not shown; Figure 1), with the exception of LidΔPHD2, for which we were unable to detect Lid overexpression even after combining multiple transgenes (data not shown). To examine the role of Lid's second PHD finger, we created a point mutant in the first cysteine of this C4HC3 zinc finger (UAS-LidC1296A) and found that overexpression could be detected after combining two transgenes (Figure 1K). Mutating the second or deleting the third PHD domain of Lid did not affect its ability to demethylate H3K4me3 (Figure 1K–1N). In contrast, Lid's JmjN, PHD1 or C5HC2 domains were essential for enzymatic activity as overexpression of these deletion mutants resulted in no change in global levels of H3K4me3 (Figure 1D, 1H, 1J). While the role of Lid's PHD1 and C5HC2 domains in demethylation remains to be investigated, our finding that Lid's JmjN domain is required for demethylase activity is not surprising based on structural analysis of the demethylase KDM4a which shows its JmjN domain making extensive contacts within the catalytic core of its immediately adjacent JmjC domain [21]. Unlike other deletions that prevented Lid's enzymatic function, expression of LidΔARID resulted in a variable increase in H3K4me3 levels, indicating that this mutant protein can behave as a dominant negative in fat body cells (Figure 1E, 1F). We do not yet understand the mechanism by which LidΔARID increases H3K4me3 levels, but have observed a similar effect upon overexpression of Lid-JmjC* [7]. The ARID of KDM5a, b and Lid are required for demethylase activity in transient transfection assays, however a dominant interfering effect has not been reported [17], [22]. Our finding that deletion of Lid's ARID can increase H3K4 trimethylation raises the possibility that in addition or concomitant with its ability to bind DNA, this domain may cooperate with Lid's JmjC domain.


Essential functions of the histone demethylase lid.

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

Deletion of Lid's JmjN, ARID, and PHD1 domains abrogate its demethylase activity.Clones of cells expressing Lid or Lid mutant transgenes in fat body were generated by crossing hs-FLP; UAS-Lid females to act>CD2>Gal4, UAS-GFP males. No heat shock was carried out since leaky FLP expression during embryogenesis leads to a small number of fat body clones. Levels of Lid (A', C', E', G', I', K', M') and trimethylated H3K4 (B', D', F', H', J', L', N') were examined. Cells expressing each transgene (as labeled on figure) are marked by co-expression of GFP and are outlined in the other panels.
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Related In: Results  -  Collection

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pgen-1001221-g001: Deletion of Lid's JmjN, ARID, and PHD1 domains abrogate its demethylase activity.Clones of cells expressing Lid or Lid mutant transgenes in fat body were generated by crossing hs-FLP; UAS-Lid females to act>CD2>Gal4, UAS-GFP males. No heat shock was carried out since leaky FLP expression during embryogenesis leads to a small number of fat body clones. Levels of Lid (A', C', E', G', I', K', M') and trimethylated H3K4 (B', D', F', H', J', L', N') were examined. Cells expressing each transgene (as labeled on figure) are marked by co-expression of GFP and are outlined in the other panels.
Mentions: To assess the ability of our Lid mutants to demethylate, we generated clones of cells overexpressing each protein in larval fat body and examined the levels of Lid and H3K4me3 (Figure 1). Based on the intensity of the immunofluorescence signal and Western analysis, all of our transgenes expressed Lid at similar levels (data not shown; Figure 1), with the exception of LidΔPHD2, for which we were unable to detect Lid overexpression even after combining multiple transgenes (data not shown). To examine the role of Lid's second PHD finger, we created a point mutant in the first cysteine of this C4HC3 zinc finger (UAS-LidC1296A) and found that overexpression could be detected after combining two transgenes (Figure 1K). Mutating the second or deleting the third PHD domain of Lid did not affect its ability to demethylate H3K4me3 (Figure 1K–1N). In contrast, Lid's JmjN, PHD1 or C5HC2 domains were essential for enzymatic activity as overexpression of these deletion mutants resulted in no change in global levels of H3K4me3 (Figure 1D, 1H, 1J). While the role of Lid's PHD1 and C5HC2 domains in demethylation remains to be investigated, our finding that Lid's JmjN domain is required for demethylase activity is not surprising based on structural analysis of the demethylase KDM4a which shows its JmjN domain making extensive contacts within the catalytic core of its immediately adjacent JmjC domain [21]. Unlike other deletions that prevented Lid's enzymatic function, expression of LidΔARID resulted in a variable increase in H3K4me3 levels, indicating that this mutant protein can behave as a dominant negative in fat body cells (Figure 1E, 1F). We do not yet understand the mechanism by which LidΔARID increases H3K4me3 levels, but have observed a similar effect upon overexpression of Lid-JmjC* [7]. The ARID of KDM5a, b and Lid are required for demethylase activity in transient transfection assays, however a dominant interfering effect has not been reported [17], [22]. Our finding that deletion of Lid's ARID can increase H3K4 trimethylation raises the possibility that in addition or concomitant with its ability to bind DNA, this domain may cooperate with Lid's JmjC domain.

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