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Trithorax maintains the functional heterogeneity of neural stem cells through the transcription factor buttonhead.

Komori H, Xiao Q, Janssens DH, Dou Y, Lee CY - Elife (2014)

Bottom Line: Consistently, btd is necessary and sufficient for eliciting a type II neuroblast functional identity.Furthermore, over-expression of btd restores the competence to generate INPs in trx mutant type II neuroblasts.Thus, Trx instructs a type II neuroblast functional identity by epigenetically promoting Btd expression, thereby maintaining neuroblast functional heterogeneity.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell Biology, Life Sciences Institute, University of Michigan Medical School, Ann Arbor, United States.

ABSTRACT
The mechanisms that maintain the functional heterogeneity of stem cells, which generates diverse differentiated cell types required for organogenesis, are not understood. In this study, we report that Trithorax (Trx) actively maintains the heterogeneity of neural stem cells (neuroblasts) in the developing Drosophila larval brain. trx mutant type II neuroblasts gradually adopt a type I neuroblast functional identity, losing the competence to generate intermediate neural progenitors (INPs) and directly generating differentiated cells. Trx regulates a type II neuroblast functional identity in part by maintaining chromatin in the buttonhead (btd) locus in an active state through the histone methyltransferase activity of the SET1/MLL complex. Consistently, btd is necessary and sufficient for eliciting a type II neuroblast functional identity. Furthermore, over-expression of btd restores the competence to generate INPs in trx mutant type II neuroblasts. Thus, Trx instructs a type II neuroblast functional identity by epigenetically promoting Btd expression, thereby maintaining neuroblast functional heterogeneity.

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Global H3K4 mono- or tri-methylation is not required for maintenance of a type II neuroblast functional identity.(A–H) The core component of the SET1/MLL complex is required for the global methylation of H3K4. (A, C, E, G) Knocking down the function of ash2 or trr leads to global loss of the H3K4 mono-methylation while knocking down the function of dSet1 does not. Scale bar, 10 μm. (B, D, F, H) Knocking down the function of ash2 or dSet1 leads to global loss of the H3K4 mono-methylation while knocking down the function of trr does not. (I–J) trr and dSet1 are dispensable for the maintenance of type II neuroblasts. (I–J) The average number of type II neuroblasts or INPs per brain lobe of the indicated genotypes after knocking down the function of trr or dSet1 for 72 hr. (K–N) trx mutant type II neuroblasts do not display appreciable reduction in the global methylation pattern. Scale bar, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.03502.011
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fig4s1: Global H3K4 mono- or tri-methylation is not required for maintenance of a type II neuroblast functional identity.(A–H) The core component of the SET1/MLL complex is required for the global methylation of H3K4. (A, C, E, G) Knocking down the function of ash2 or trr leads to global loss of the H3K4 mono-methylation while knocking down the function of dSet1 does not. Scale bar, 10 μm. (B, D, F, H) Knocking down the function of ash2 or dSet1 leads to global loss of the H3K4 mono-methylation while knocking down the function of trr does not. (I–J) trr and dSet1 are dispensable for the maintenance of type II neuroblasts. (I–J) The average number of type II neuroblasts or INPs per brain lobe of the indicated genotypes after knocking down the function of trr or dSet1 for 72 hr. (K–N) trx mutant type II neuroblasts do not display appreciable reduction in the global methylation pattern. Scale bar, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.03502.011

Mentions: Knocking down the function of trr or dset1 drastically reduced the global H3K4 mono- or tri-methylation in type II neuroblasts but had no effects on the maintenance of their functional identity (Figure 4—figure supplement 1A–J). By contrast, removing trx function had no appreciable effects on the global H3K4 pattern in type II neuroblasts (Figure 4—figure supplement 1K–N). These data led us to hypothesize that Trx maintains the type II neuroblast functional identity by regulating a small number of genes that are specifically expressed in the type II neuroblast. We compared gene transcription profiles by using mRNAs isolated from dissected larval brains enriched with type I or II neuroblasts to identify the candidate Trx target genes (Bowman et al., 2008; Weng et al., 2010; Carney et al., 2012; Haenfler et al., 2012). pnt and btd were among a small number of genes that were dramatically up-regulated in the mRNAs isolated from larval brains enriched with type II neuroblasts as compared to the mRNAs isolated from larval brains enriched with type I neuroblasts. We confirmed that both pntP1 and btd transcripts were indeed highly enriched in the brain lysate enriched with type II neuroblasts by qRT-PCR (Figure 4A). Furthermore, we detected the binding of Trx to the transcription start site for both the pntP1 and btd transcription units (Figure 4B, Figure 4—figure supplement 2A). In addition, the promoter region of both the pntP1 and btd transcription units also displayed a high level of H3K4 di-methylation, consistent with Trx-maintaining chromatin in an active state in these two loci through the H3K4 methylation (Figure 4B, Figure 4—figure supplement 2A). By contrast, we did not detect Trx binding to the negative control region located 7.5 kilobases 3ʹ from the btd transcription unit (Figure 4B; data not presented) (Petruk et al., 2012). Thus, both pnt and btd are the direct target genes of Trx.10.7554/eLife.03502.010Figure 4.Btd likely acts downstream of Trx to maintain a type II neuroblast functional identity.


Trithorax maintains the functional heterogeneity of neural stem cells through the transcription factor buttonhead.

Komori H, Xiao Q, Janssens DH, Dou Y, Lee CY - Elife (2014)

Global H3K4 mono- or tri-methylation is not required for maintenance of a type II neuroblast functional identity.(A–H) The core component of the SET1/MLL complex is required for the global methylation of H3K4. (A, C, E, G) Knocking down the function of ash2 or trr leads to global loss of the H3K4 mono-methylation while knocking down the function of dSet1 does not. Scale bar, 10 μm. (B, D, F, H) Knocking down the function of ash2 or dSet1 leads to global loss of the H3K4 mono-methylation while knocking down the function of trr does not. (I–J) trr and dSet1 are dispensable for the maintenance of type II neuroblasts. (I–J) The average number of type II neuroblasts or INPs per brain lobe of the indicated genotypes after knocking down the function of trr or dSet1 for 72 hr. (K–N) trx mutant type II neuroblasts do not display appreciable reduction in the global methylation pattern. Scale bar, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.03502.011
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221733&req=5

fig4s1: Global H3K4 mono- or tri-methylation is not required for maintenance of a type II neuroblast functional identity.(A–H) The core component of the SET1/MLL complex is required for the global methylation of H3K4. (A, C, E, G) Knocking down the function of ash2 or trr leads to global loss of the H3K4 mono-methylation while knocking down the function of dSet1 does not. Scale bar, 10 μm. (B, D, F, H) Knocking down the function of ash2 or dSet1 leads to global loss of the H3K4 mono-methylation while knocking down the function of trr does not. (I–J) trr and dSet1 are dispensable for the maintenance of type II neuroblasts. (I–J) The average number of type II neuroblasts or INPs per brain lobe of the indicated genotypes after knocking down the function of trr or dSet1 for 72 hr. (K–N) trx mutant type II neuroblasts do not display appreciable reduction in the global methylation pattern. Scale bar, 10 μm.DOI:http://dx.doi.org/10.7554/eLife.03502.011
Mentions: Knocking down the function of trr or dset1 drastically reduced the global H3K4 mono- or tri-methylation in type II neuroblasts but had no effects on the maintenance of their functional identity (Figure 4—figure supplement 1A–J). By contrast, removing trx function had no appreciable effects on the global H3K4 pattern in type II neuroblasts (Figure 4—figure supplement 1K–N). These data led us to hypothesize that Trx maintains the type II neuroblast functional identity by regulating a small number of genes that are specifically expressed in the type II neuroblast. We compared gene transcription profiles by using mRNAs isolated from dissected larval brains enriched with type I or II neuroblasts to identify the candidate Trx target genes (Bowman et al., 2008; Weng et al., 2010; Carney et al., 2012; Haenfler et al., 2012). pnt and btd were among a small number of genes that were dramatically up-regulated in the mRNAs isolated from larval brains enriched with type II neuroblasts as compared to the mRNAs isolated from larval brains enriched with type I neuroblasts. We confirmed that both pntP1 and btd transcripts were indeed highly enriched in the brain lysate enriched with type II neuroblasts by qRT-PCR (Figure 4A). Furthermore, we detected the binding of Trx to the transcription start site for both the pntP1 and btd transcription units (Figure 4B, Figure 4—figure supplement 2A). In addition, the promoter region of both the pntP1 and btd transcription units also displayed a high level of H3K4 di-methylation, consistent with Trx-maintaining chromatin in an active state in these two loci through the H3K4 methylation (Figure 4B, Figure 4—figure supplement 2A). By contrast, we did not detect Trx binding to the negative control region located 7.5 kilobases 3ʹ from the btd transcription unit (Figure 4B; data not presented) (Petruk et al., 2012). Thus, both pnt and btd are the direct target genes of Trx.10.7554/eLife.03502.010Figure 4.Btd likely acts downstream of Trx to maintain a type II neuroblast functional identity.

Bottom Line: Consistently, btd is necessary and sufficient for eliciting a type II neuroblast functional identity.Furthermore, over-expression of btd restores the competence to generate INPs in trx mutant type II neuroblasts.Thus, Trx instructs a type II neuroblast functional identity by epigenetically promoting Btd expression, thereby maintaining neuroblast functional heterogeneity.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell Biology, Life Sciences Institute, University of Michigan Medical School, Ann Arbor, United States.

ABSTRACT
The mechanisms that maintain the functional heterogeneity of stem cells, which generates diverse differentiated cell types required for organogenesis, are not understood. In this study, we report that Trithorax (Trx) actively maintains the heterogeneity of neural stem cells (neuroblasts) in the developing Drosophila larval brain. trx mutant type II neuroblasts gradually adopt a type I neuroblast functional identity, losing the competence to generate intermediate neural progenitors (INPs) and directly generating differentiated cells. Trx regulates a type II neuroblast functional identity in part by maintaining chromatin in the buttonhead (btd) locus in an active state through the histone methyltransferase activity of the SET1/MLL complex. Consistently, btd is necessary and sufficient for eliciting a type II neuroblast functional identity. Furthermore, over-expression of btd restores the competence to generate INPs in trx mutant type II neuroblasts. Thus, Trx instructs a type II neuroblast functional identity by epigenetically promoting Btd expression, thereby maintaining neuroblast functional heterogeneity.

Show MeSH