MCB Accepts, published online ahead of print on 19 March 2007 Mol. Cell. Biol. doi:10.1128/MCB.02044-06 Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 D Genome-wide dynamics of SAPHIRE, an essential complex for gene activation and chromatin boundaries E T D o Matthew Gordon1, Derick Holt1, Anil Panigrahi1, Brian T. Wilhelm2, Hediye Erdjument- w P n Bromage3, Paul Tempst3, Jürg Bähler2, and Bradley R. Cairns1* lo a d 1Howard Hughes Medical Institute, DepartmEent of Oncological Sciences, Huntsman Cancer ed Institute, 2000 Circle of Hope, University of Utah, Salt Lake City, UT 84112. 2The Sanger fr o Institute, Wellcome Trust GenoCme Campus, Hinxton, Cambridge, CB10 1HH, UK. m 3Memorial Sloan-Kettering Cancer Center 1275 York Avenue, New York, NY 10021. h t t p : / C /m c b . a A s m . *Corresponding Author: o r g Bradley R. Cairns, Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope, Salt Lake / o City, UT 84112. n E-mail: [email protected] A p ril 4 , 2 0 1 9 b y g u Word Counts: e s Introduction/Results/Discussion – 6,041 t Materials and Methods – 989 Running Title: S. pombe SAPHIRE complex activates transcription 2 1 Abstract 2 Here we characterize a four-protein nucleosome-binding complex from S. pombe termed 3 SAPHIRE that includes two orthologs of human LSD1, a histone demethylase. SAPHIRE D 4 complex is essential for cell viability, whereas saphire mutants lacking key conserved catalytic E 5 residues are viable but thermosensitive, suggesting that SAPHIRE has both an important 6 enzymatic function and an essential non-enzymatic function. SAPHIRET is present in (or D o 7 adjacent to) particular heterochromatic loci, and also the transcription start site region of many w P n lo a 8 highly active Pol II genes. However, ribosomal protein genes are notably SAPHIRE deficient. d E e d 9 SAPHIRE promotes activation, as target genes are selectively attenuated in saphire mutants. fr o m C 10 Interestingly, saphire mutants display increased promoter H3K4 di-methylation, a modification h t t p : / 11 typically associated Cwith euchromatin. SAPHIRE localization is dynamic, as activated genes /m c b . 12 rapidly acquire SAPHIRE. Furthermore, saphire mutants dramatically shift a heterochromatin- a A s m . 13 euchromatin boundary in Chr1, suggesting a novel role in boundary regulation. o r g / o n A p r il 4 , 2 0 1 9 b y g u e s t 3 1 Introduction 2 Chromatin is a dynamic material that partitions chromosomes into functional domains 3 (telomeres, centromeres, rDNA arrays), and also partitions individual genes into segments, each D 4 with a distinct role in transcriptional regulation. All chromatin regions contain arrays of E 5 nucleosomes, which consist of 147 base pairs of DNA wrapped around an octameric disk of 6 histone proteins (24, 34). However, different chromatin regions have dTistinctive characteristics D o 7 including: 1) differences in chromatin composition, including histone variants, linker histones, w P n lo a 8 and associated non-histone chromatin architectural proteins, 2) structural diversity (nucleosome d E e d 9 positioning or compaction), and 3) variation in covalent modifications of the DNA and histones fr o m C 10 (34). h t t p : / 11 Covalent hisCtone modifications serve as docking sites for protein domains present in /m c b . 12 specific factors, which in turn endow the region with unique characteristics (46). Methylation of a A s m . 13 lysines in the unstructured termini (tails) of histones regulates a wide array of DNA-templated o r g / o 14 processes, including transcription (12, 47). Methylation of lysines in histone tails is mediated by n A p 15 two families of enzymes: the DOT family and the SET family (30, 40). These enzymes can ril 4 , 2 16 mono-, di- or tri-methylate lysines, and even the subtle difference between di- and tri- 0 1 9 17 methylation of the same lysine residue can be discriminated by recognition domains, and b y g u 18 therefore recruit distinct factors (21, 33, 52). e s t 19 Di-methylation of histone H3 lysine 4 (H3K4me2) is generally thought to make 20 chromatin permissive to transcription, whereas tri-methylation of the same residue (H3K4me3) is 21 associated only with transcriptionally active genes (38). Set1p, the protein which is responsible 22 for all H3K4 methylation in budding yeast (6, 19) bears several autoregulatory domains which 23 control whether the enzyme di- or tri-methylates (6, 39). This result argues that a balance 24 between these two very similar methyl marks is critical for homeostasis. Indeed, ChIP- 4 1 microarray experiments in budding yeast demonstrated that, on average, these two marks are not 2 identically distributed across genes. H3K4me3 is concentrated at the 5’ end of transcribed genes, 3 whereas H3K4me2 tends to peak in the middle of genes, but shifts closer to the 3’ end when D 4 genes are highly transcribed (23, 35). Although several factors that bind H3K4 methylated tails 5 have been identified, it is still unclear what role H3K4me2 and H3K4me3 play in tEranscription. 6 However, deletion of SET1 leads to defects in gene activation, highlighting the importance of T D 7 these marks (5). o w P n lo 8 Histone methylation is not restricted to transcriptional activation, as histone lysine a d E e d 9 methylation at alternative locations on the H3 tail promotes repression. First, methylation of f r o m C 10 H3K9 is present at sites of constitutive heterochromatin, and recruits repressive proteins such as h t t p : 11 HP1 to enforce the sCilent state of these domains (10). Second, H3K36 methylation in the body //m c b 12 and 3’ end of transcribed genes recruits a repressive complex that inhibits spurious transcription . a A s m 13 initiation from cryptic promoters inside the gene (7, 13-15, 29) .o r g / 14 Histone methylation is not a static mark in vivo, as shown by the discovery of histone o n A p 15 lysine demethylases (HDMs). The largest family of HDMs is the Jumonji (JMJ) domain histone r il 4 , 16 demethylases. To date, five JMJ HDMs have been characterized, with substrate specificities 2 0 1 9 17 directed toward H3K9, H3K36 or both (18, 49-51, 53). Thus, this class of HDMs appears to b y g 18 demethylate repressive methyl marks. Significantly, the active site chemistry of JMJ domains u e s t 19 allows for demethylation of mono-, di- or tri-methylated lysine residues. 20 However, only one protein has been isolated that can demethylate H3K4: Lsd1. LSD1 21 belongs to a class of proteins called nuclear amine oxidases (NAOs), which are defined by the 22 presence of an N-terminal SWIRM domain followed by an amine oxidase domain (43). The 23 SWIRM domain is found in many nuclear proteins, most of which are members of chromatin 24 associated multi-protein complexes (e.g. SAGA HAT complex, SWI/SNF remodeler complex) 5 1 (1). The amine oxidase domain is a flavin-adenine dinucleotide (FAD) dependent oxido- 2 reductase. Amine oxidases use the oxidative potential of FAD to break a carbon-nitrogen bond 3 that forms the substrate amine. This reaction can also be thought of as the dealkylation of an D 4 amine, as is the case in a histone demethylation reaction. Importantly, this class of enzymes 5 cannot oxidize quaternary amines; thus LSD1 can only demethylate mono- and di-mEethylated 6 H3K4 (3, 43) T D 7 Although most of the data regarding LSD1 point to a role in transcriptional repression, a o w P n lo 8 recent study observed that Lsd1 is recruited to the PSA gene during activation by the androgen a d E e d 9 receptor (AR) (20, 27, 43, 44). siRNA mediated knockdown of Lsd1 protein caused a drastic f r o m C 10 decrease in activation of AR-responsive reporter genes, suggesting that Lsd1 can be an activator. h t t p : 11 Knockdown studiesC also demonstrated that Lsd1 is required for demethylation of repressive //m c b 12 H3K9 methylation when PSA is activated. These studies suggested that Lsd1 demethylated . a A s m 13 H3K9 to promote activation. However, these results are also consistent with Lsd1 activity being .o r g / 14 required upstream of another H3K9 demethylase, rather than being an H3K9 demethylase itself. o n A p 15 Indeed, a JMJ domain protein (JHDM2A) with robust in vitro H3K9me2 demethylase activity r il 4 , 16 using purified components is also recruited to PSA upon activation by AR. siRNA studies 2 0 1 9 17 demonstrate that JHDM2A knockdown has profound defects in H3K9 demethylation at the PSA b y g 18 gene upon activation, whereas Lsd1 knockdown effects are much more modest (53). u e s t 19 Thus, many important questions remain to be answered about NAOs. For example, most 20 of the work done to date has focused on Lsd1 function at a limited number of gene targets (see 21 Discussion), and it remains to be determined whether the principles elucidated so far are general. 22 Because most of the data point to a repressive function for NAOs, it is particularly important to 23 determine if the activation function seen at PSA is broadly applicable and dependent upon H3K9 24 demethylation. Importantly, as Lsd1 is present in larger complexes, it is important to determine 6 1 the catalytic and non-catalytic roles of this class of proteins, and the contributions of associated 2 proteins to Lsd1 function. Finally, the only biological function so far attributed to NAOs is 3 transcriptional regulation and, since chromatin modifications impact many aspects of D 4 chromosome biology, it is imperative to explore other possible roles for NAOs. 5 Here, we report the genome-wide dynamics of SAPHIRE, a novel NAO comEplex from S. 6 pombe. Our results demonstrate that SAPHIRE promotes gene activation, most likely through T D 7 enzymatic and non-enzymatic means. SAPHIRE occupancy is dynamic and shifts to newly o w P n lo 8 activated genes during heat shock. Furthermore, mutant analysis reveals that defects in a d E e d 9 transcription of SAPHIRE targets are associated with increased H3K4me2 of the affected genes. f r o m C 10 Finally, we identify a new role for NAOs in maintaining the boundary between euchromatin and h t t p : 11 heterochromatin at aC telomere. //m c b . a A s m . o r g / o n A p r il 4 , 2 0 1 9 b y g u e s t 7 1 Results 2 Purification of the SAPHIRE complex 3 We recognized two proteins in S. pombe that contained three domains of interest: a D 4 SWIRM domain, an FAD-dependent amine oxidase domain, and an HMG(B) domain. As the E 5 SWIRM and HMG(B) domains are closely associated with chromatin function, we initially 6 pursued these proteins as potential new chromatin enzymes that could bTe studied in a genetically D o 7 tractable organism. The amine oxidase domain of these proteins contains the key catalytic w P n lo a 8 residues present in known amine dealkylases, suggesting that these S. pombe proteins have the d E e d 9 capacity for amine demethylation. The two S. pombe paralogs are highly similar to each other fr o m C 10 (BLAST E-value 2X10-84) and share the same domain order. The human protein Lsd1 is highly h t t p : / 11 similar (BLAST E-vCalue 4X10-14), and shares with the S. pombe proteins key catalytic residues, /m c b . 12 the SWIRM domain, and the same domain order. a A s m . 13 To characterize the biochemical properties of these S. pombe NAO paralogs and to o r g / o 14 identify associated proteins, we purified each to homogeneity. To facilitate their purification, we n A p 15 integrated a DNA sequence into the 3’ end of each gene (separately, at the chromosomal locus) ril 4 , 2 16 that encoded an in-frame fusion of the tandem affinity purification (TAP) epitope. Integrations 0 1 9 17 were performed separately in diploids, with sporulation and dissection generating tagged haploid b y g u 18 progeny that grew as well as their untagged siblings, demonstrating that the tags do not impair e s t 19 function. Thus, the two strains used for purification contained one tagged NAO and one 20 untagged NAO. Purification of either NAO to homogeneity yielded the same four proteins, as 21 identified by mass spectrometry: the tagged NAO, the untagged NAO, and two additional 22 proteins of 60 and 50 kDa (Figure 1A). Mass spectrometric analysis also showed that the 23 additional bands in these lanes are proteolytic products of these four proteins, thus both 24 purifications yielded the same four-protein complex. We call the complex SAPHIRE: a 8 1 SWIRM-Amine oxidase and PHD protein complex involved in regulating gene expression 2 (shown later), with members designated by their apparent molecular weight: Saf140p, Saf110p, 3 Saf60p, Saf50p. The systematic names are: saf140+, SPAC23E2.02; saf110+, SPBC146.09c; D 4 saf60+, SPAC30D11.08c, saf50+, SPCC4G3.07c. Thus, the two NAO paralogs are both present 5 in the isolated complex. Interestingly, the additional 60 kDa and 50 kDa proteins aEre themselves 6 paralogs (BLAST E-value 7X10-44), and both contain a single PHD domain (Figure 1B). Gel T D 7 filtration of the entire complex provided an apparent mass of ~350kDa, suggesting a simple o w P n lo 8 tetramer (data not shown). a d E e d 9 f r o m C 10 SAPHIRE Binds Nucleosomes h t t p : 11 As SAPHIRCE contains multiple domains for chromatin association, we used //m c b 12 electrophoretic mobility shift assays (EMSA) to assess interaction with mononucleosomes. We . a A s m 13 find that SAPHIRE binds to mononucleosomes with Kd of about 8nM (Figure 1C and data not .o r g / 14 shown). Specificity for this interaction was confirmed with a polyclonal antibody raised against o n A p 15 Saf110p, which supershifted the nucleosome-SAPHIRE complex (Figure 1C, lane 3). r il 4 , 16 To date, the only Lsd1-related protein that has demonstrated histone lysine demethylation 2 0 1 9 17 activity in vitro is Lsd1 itself. Extensive biochemical testing of both native and recombinant b y g 18 SAPHIRE has not revealed appreciable activity. This is consistent with the work of others on u e s t 19 these S. pombe orthologs who have not observed activity in vitro (31). Therefore, we focused on 20 the biological and genomic impact of removing the conserved catalytic residue of these enzymes, 21 as described below. 22 9 1 SAPHIRE Is Essential for Viability 2 To determine the importance of SAPHIRE, we constructed gene deletion mutations of 3 each subunit in diploids. We then sporulated the heterozygous diploids and examined the D 4 phenotypes of haploid progeny. We find that saf140+, saf60+, and saf50+ are essential genes, as 5 viability segregated 2:2 in four-spore tetrads (data not shown). Somewhat surprisinEgly, 6 saf110+/saf110∆ diploids produced four viable spores, with the saf110∆ progeny always T D o 7 displaying extremely slow growth (Figure 2A). As 2:2 segregation of slow growth was observed w P n lo 8 in a large number of tetrads, and was always linked to saf110∆::kanMX, the phenotype was a d E e d 9 likely not a result of background mutation. Importantly, normal growth could be restored by f r o m C 10 transformation with a plasmid bearing only saf110+, confirming the null phenotype (data not h t t p : / 11 shown). In additionC to slow growth, saf110∆ null strains are temperature sensitive (Figure 2A). /m c b 12 To test whether saf110+ and saf140+ are partially redundant, we overexpressed saf110+ or .a A s m . 13 saf140+ (using high-copy plasmids) in heterozygous diploids (saf110+/saf110∆ or o r g / o 14 saf140+/saf140∆) and tested whether we could acquire haploid segregants, where the high copy n A p r 15 plasmid conferred growth ability to the reciprocal null. We find that a high copy saf110+ il 4 , 2 16 plasmid will not suppress the inviability of a saf140∆ strain, nor will a high copy saf140+ 0 1 9 b 17 plasmid suppress the extremely slow growth of a saf110∆ strain. These experiments suggest y g u e 18 that the Saf140 and Saf110 proteins make largely unique contributions to SAPHIRE. s t 19 20 SAPHIRE NAO Mutants Lacking a Conserved Catalytic Residue Are Viable 21 Saf140p, Saf110p, and Lsd1 share a high degree of similarity to FAD-dependent amine 22 oxidases such as monoamine oxidase (MAO, which dealkylates neurotransmitters) and 23 polyamine oxidase (PAO, which dealkylates spermine and spermidine). Although these 10 1 enzymes have different substrates, they all dealkylate an amine, using flavin reduction to reduce 2 the amine to an imine, followed by imine hydrolysis to generate two products; in the case of 3 Lsd1, the products derived from monomethyl-lysine are formaldehyde and lysine. The catalytic D 4 residue that has been the most extensively studied is a specific lysine. In all crystal structures 5 examined, this lysine positions the lone water molecule in the active site into a bridEging position 6 with the flavin N5 atom of the FAD cofactor (2, 4, 9, 45). This lysine is absolutely conserved in T D 7 all Lsd1 orthologues, all PAOs, all MAOs, and is the only residue in the catalytic pocket that is o w P n lo 8 absolutely conserved in the distantly-related sarcosine oxidases. Crystal structures and/or a d E e d 9 alignments identify the corresponding catalytic lysine as K661 in Lsd1, K300 in maize PAO, f r o m C 10 K862 in Saf140p, and K604 in Saf110p (Figure 2B). In all cases tested, including LSD1 and h t t p : 11 maize PAO, mutatioCn of this residue alone produced no detectable activity in the resultant //m c b 12 protein. . a A s m 13 We determined the importance of this lysine on SAPHIRE function by replacing it with .o r g / 14 alanine. As Saf140p and Saf110p have a lysine adjacent to this position, we also replaced o n A p 15 K861/K603 with alanine, resulting in the double KK861-862AA and KK603-604AA mutations r il 4 , 16 (KK→AA), to further ensure an impact of these substitutions on catalytic activity. Strains 2 0 1 17 bearing saf140KK→AA and saf110KK→AA were constructed by replacing one of the two wild-type 9 b y g 18 alleles with a KK→AA mutant allele at the endogenous genomic locus in a diploid, sporulating, u e s t 19 and then isolating haploid KK→AA progeny. Separately, saf140KK→AA or saf110KK→AA mutants 20 were viable, with the saf110KK→AA mutant displaying a moderate Ts- phenotype. Interestingly 21 the saf140KK→AA saf110KK→AA double mutant was also viable, though displaying a pronounced 22 temperature sensitivity (Figure 2C). These phenotypes could not be attributed to a significant 23 reduction in SAPHIRE stability, as SAPHIRE abundance and integrity was similar in the double 24 mutant at the permissive and non-permissive temperatures (Figure 2D), though we have not