Limits...
Multifaceted genome control by Set1 Dependent and Independent of H3K4 methylation and the Set1C/COMPASS complex.

Mikheyeva IV, Grady PJ, Tamburini FB, Lorenz DR, Cam HP - PLoS Genet. (2014)

Bottom Line: Here, we show that the fission yeast Schizosaccharomyces pombe Set1 utilizes distinct domain modules to regulate disparate classes of repetitive elements associated with euchromatin and heterochromatin via H3K4me-dependent and -independent pathways.Intriguingly, we uncover a genome organization role for Set1C and H3K4me in mediating the clustering of Tf2s into Tf bodies by antagonizing the acetyltransferase Mst1-mediated H3K4 acetylation.Our study provides unexpected insights into the regulatory intricacies of a highly conserved chromatin-modifying complex with diverse roles in genome control.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Boston College, Chestnut Hill, Massachusetts, United States of America.

ABSTRACT
Histone modifiers are critical regulators of chromatin-based processes in eukaryotes. The histone methyltransferase Set1, a component of the Set1C/COMPASS complex, catalyzes the methylation at lysine 4 of histone H3 (H3K4me), a hallmark of euchromatin. Here, we show that the fission yeast Schizosaccharomyces pombe Set1 utilizes distinct domain modules to regulate disparate classes of repetitive elements associated with euchromatin and heterochromatin via H3K4me-dependent and -independent pathways. Set1 employs its RNA-binding RRM2 and catalytic SET domains to repress Tf2 retrotransposons and pericentromeric repeats while relying on its H3K4me function to maintain transcriptional repression at the silent mating type (mat) locus and subtelomeric regions. These repressive functions of Set1 correlate with the requirement of Set1C components to maintain repression at the mat locus and subtelomeres while dispensing Set1C in repressing Tf2s and pericentromeric repeats. We show that the contributions of several Set1C subunits to the states of H3K4me diverge considerably from those of Saccharomyces cerevisiae orthologs. Moreover, unlike S. cerevisiae, the regulation of Set1 protein level is not coupled to the status of H3K4me or histone H2B ubiquitination by the HULC complex. Intriguingly, we uncover a genome organization role for Set1C and H3K4me in mediating the clustering of Tf2s into Tf bodies by antagonizing the acetyltransferase Mst1-mediated H3K4 acetylation. Our study provides unexpected insights into the regulatory intricacies of a highly conserved chromatin-modifying complex with diverse roles in genome control.

Show MeSH
Set1 represses different classes of repetitive elements via distinct functional domains.(A) Schematic of S. pombe Set1 protein domain architecture. (B) H3K4 methylation (H3K4me) in set1 domain mutants. Mono (H3K4me1), di (H3K4me2), and tri (H3K4me3) methylation of H3K4 was analyzed from histone extracts of indicated set1 mutant strains by western blotting. Full-length FLAG-set1 and the indicated deleted domain mutants of set1 contain an N-terminal FLAG (3×) epitope. set1FH3K4me- denotes an H3K4me  mutant due to the presence of a FLAG (3×) epitope at the C-terminus of set1. (C) Tf2 repression requires intact RRM2 and SET domains. (D–F) Set1 represses heterochromatic loci dependent and independent of H3K4me. Expression of Tf2 ORF and heterochromatic loci was analyzed by qRT-PCR. Fold changes relative to wildtype were normalized by act1 expression. (s.d., error bars; n = 3). Pericentromeric repeat dg (cen), silent mating type cenH (mat), subtelomeric prl70 (subtel).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214589&req=5

pgen-1004740-g001: Set1 represses different classes of repetitive elements via distinct functional domains.(A) Schematic of S. pombe Set1 protein domain architecture. (B) H3K4 methylation (H3K4me) in set1 domain mutants. Mono (H3K4me1), di (H3K4me2), and tri (H3K4me3) methylation of H3K4 was analyzed from histone extracts of indicated set1 mutant strains by western blotting. Full-length FLAG-set1 and the indicated deleted domain mutants of set1 contain an N-terminal FLAG (3×) epitope. set1FH3K4me- denotes an H3K4me mutant due to the presence of a FLAG (3×) epitope at the C-terminus of set1. (C) Tf2 repression requires intact RRM2 and SET domains. (D–F) Set1 represses heterochromatic loci dependent and independent of H3K4me. Expression of Tf2 ORF and heterochromatic loci was analyzed by qRT-PCR. Fold changes relative to wildtype were normalized by act1 expression. (s.d., error bars; n = 3). Pericentromeric repeat dg (cen), silent mating type cenH (mat), subtelomeric prl70 (subtel).

Mentions: The protein architecture of fission yeast Set1 is highly conserved [22], [24], containing two putative RNA-recognition motifs (RRMs) termed RRM1 and RRM2 near the N-terminus [28], [29], an nSET domain responsible for interaction with certain COMPASS subunits [11], [30], a catalytic SET domain and a short post-SET (pSET) domain near its C-terminus [28] (Figure 1A). Previous studies from budding yeast have shown that H3K4me is affected by the loss of various domains of Set1 [11], [28], [29], [31]. We examined the status of H3K4me in S. pombe mutant strains lacking individual domains of set1. Loss of RRM1 abolished H3K4me3, substantially diminished H3K4me2 [22], and slightly decreased H3K4me1 compared to wildtype (Figures 1B and S1). An RRM2 deletion resulted in no appreciable decrease in H3K4me levels. Cells expressing Set1 with a deleted nSET, SET or pSET domain displayed a complete loss of H3K4me. S. cerevisiae cells expressing Set1 with a C-terminal TAP epitope showed reduced H3K4me levels [32] while an affinity-purified S. pombe equivalent Set1-TAP protein retained in vitro H3K4me activity [24]. We found that H3K4me was completely abolished in cells containing a FLAG (3×) epitope attached to the C-terminus of Set1 (set1FH3K4me-), likely a result of the epitope interfering with the interaction of the SET or pSET domain with the H3K4 substrate [33], [34].


Multifaceted genome control by Set1 Dependent and Independent of H3K4 methylation and the Set1C/COMPASS complex.

Mikheyeva IV, Grady PJ, Tamburini FB, Lorenz DR, Cam HP - PLoS Genet. (2014)

Set1 represses different classes of repetitive elements via distinct functional domains.(A) Schematic of S. pombe Set1 protein domain architecture. (B) H3K4 methylation (H3K4me) in set1 domain mutants. Mono (H3K4me1), di (H3K4me2), and tri (H3K4me3) methylation of H3K4 was analyzed from histone extracts of indicated set1 mutant strains by western blotting. Full-length FLAG-set1 and the indicated deleted domain mutants of set1 contain an N-terminal FLAG (3×) epitope. set1FH3K4me- denotes an H3K4me  mutant due to the presence of a FLAG (3×) epitope at the C-terminus of set1. (C) Tf2 repression requires intact RRM2 and SET domains. (D–F) Set1 represses heterochromatic loci dependent and independent of H3K4me. Expression of Tf2 ORF and heterochromatic loci was analyzed by qRT-PCR. Fold changes relative to wildtype were normalized by act1 expression. (s.d., error bars; n = 3). Pericentromeric repeat dg (cen), silent mating type cenH (mat), subtelomeric prl70 (subtel).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214589&req=5

pgen-1004740-g001: Set1 represses different classes of repetitive elements via distinct functional domains.(A) Schematic of S. pombe Set1 protein domain architecture. (B) H3K4 methylation (H3K4me) in set1 domain mutants. Mono (H3K4me1), di (H3K4me2), and tri (H3K4me3) methylation of H3K4 was analyzed from histone extracts of indicated set1 mutant strains by western blotting. Full-length FLAG-set1 and the indicated deleted domain mutants of set1 contain an N-terminal FLAG (3×) epitope. set1FH3K4me- denotes an H3K4me mutant due to the presence of a FLAG (3×) epitope at the C-terminus of set1. (C) Tf2 repression requires intact RRM2 and SET domains. (D–F) Set1 represses heterochromatic loci dependent and independent of H3K4me. Expression of Tf2 ORF and heterochromatic loci was analyzed by qRT-PCR. Fold changes relative to wildtype were normalized by act1 expression. (s.d., error bars; n = 3). Pericentromeric repeat dg (cen), silent mating type cenH (mat), subtelomeric prl70 (subtel).
Mentions: The protein architecture of fission yeast Set1 is highly conserved [22], [24], containing two putative RNA-recognition motifs (RRMs) termed RRM1 and RRM2 near the N-terminus [28], [29], an nSET domain responsible for interaction with certain COMPASS subunits [11], [30], a catalytic SET domain and a short post-SET (pSET) domain near its C-terminus [28] (Figure 1A). Previous studies from budding yeast have shown that H3K4me is affected by the loss of various domains of Set1 [11], [28], [29], [31]. We examined the status of H3K4me in S. pombe mutant strains lacking individual domains of set1. Loss of RRM1 abolished H3K4me3, substantially diminished H3K4me2 [22], and slightly decreased H3K4me1 compared to wildtype (Figures 1B and S1). An RRM2 deletion resulted in no appreciable decrease in H3K4me levels. Cells expressing Set1 with a deleted nSET, SET or pSET domain displayed a complete loss of H3K4me. S. cerevisiae cells expressing Set1 with a C-terminal TAP epitope showed reduced H3K4me levels [32] while an affinity-purified S. pombe equivalent Set1-TAP protein retained in vitro H3K4me activity [24]. We found that H3K4me was completely abolished in cells containing a FLAG (3×) epitope attached to the C-terminus of Set1 (set1FH3K4me-), likely a result of the epitope interfering with the interaction of the SET or pSET domain with the H3K4 substrate [33], [34].

Bottom Line: Here, we show that the fission yeast Schizosaccharomyces pombe Set1 utilizes distinct domain modules to regulate disparate classes of repetitive elements associated with euchromatin and heterochromatin via H3K4me-dependent and -independent pathways.Intriguingly, we uncover a genome organization role for Set1C and H3K4me in mediating the clustering of Tf2s into Tf bodies by antagonizing the acetyltransferase Mst1-mediated H3K4 acetylation.Our study provides unexpected insights into the regulatory intricacies of a highly conserved chromatin-modifying complex with diverse roles in genome control.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Boston College, Chestnut Hill, Massachusetts, United States of America.

ABSTRACT
Histone modifiers are critical regulators of chromatin-based processes in eukaryotes. The histone methyltransferase Set1, a component of the Set1C/COMPASS complex, catalyzes the methylation at lysine 4 of histone H3 (H3K4me), a hallmark of euchromatin. Here, we show that the fission yeast Schizosaccharomyces pombe Set1 utilizes distinct domain modules to regulate disparate classes of repetitive elements associated with euchromatin and heterochromatin via H3K4me-dependent and -independent pathways. Set1 employs its RNA-binding RRM2 and catalytic SET domains to repress Tf2 retrotransposons and pericentromeric repeats while relying on its H3K4me function to maintain transcriptional repression at the silent mating type (mat) locus and subtelomeric regions. These repressive functions of Set1 correlate with the requirement of Set1C components to maintain repression at the mat locus and subtelomeres while dispensing Set1C in repressing Tf2s and pericentromeric repeats. We show that the contributions of several Set1C subunits to the states of H3K4me diverge considerably from those of Saccharomyces cerevisiae orthologs. Moreover, unlike S. cerevisiae, the regulation of Set1 protein level is not coupled to the status of H3K4me or histone H2B ubiquitination by the HULC complex. Intriguingly, we uncover a genome organization role for Set1C and H3K4me in mediating the clustering of Tf2s into Tf bodies by antagonizing the acetyltransferase Mst1-mediated H3K4 acetylation. Our study provides unexpected insights into the regulatory intricacies of a highly conserved chromatin-modifying complex with diverse roles in genome control.

Show MeSH