Research ArticlePLANT SCIENCES

Chromatin architectural proteins regulate flowering time by precluding gene looping

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Science Advances  11 Jun 2021:
Vol. 7, no. 24, eabg3097
DOI: 10.1126/sciadv.abg3097

Abstract

Chromatin structure is critical for gene expression and many other cellular processes. In Arabidopsis thaliana, the floral repressor FLC adopts a self-loop chromatin structure via bridging of its flanking regions. This local gene loop is necessary for active FLC expression. However, the molecular mechanism underlying the formation of this class of gene loops is unknown. Here, we report the characterization of a group of linker histone-like proteins, named the GH1-HMGA family in Arabidopsis, which act as chromatin architecture modulators. We demonstrate that these family members redundantly promote the floral transition through the repression of FLC. A genome-wide study revealed that this family preferentially binds to the 5′ and 3′ ends of gene bodies. The loss of this binding increases FLC expression by stabilizing the FLC 5′ to 3′ gene looping. Our study provides mechanistic insights into how a family of evolutionarily conserved proteins regulates the formation of local gene loops.

INTRODUCTION

Eukaryotic DNA is spatially and functionally organized with its associated proteins in the form of chromatin. Nucleosomes are the fundamental subunit of chromatin. Each nucleosome is a complex of ~146 base pairs (bp) of DNA wrapped around a histone octamer. Nucleosomes play an essential role in the formation of higher-order chromatin structures and orchestrate transcriptional regulation (13). Research into the role of nucleosome structures, histone modifications, and nucleosome-binding proteins is beginning to reveal sophisticated mechanisms by which the fate of gene expression is determined in response to developmental and environmental stimuli in eukaryotes (3, 4). In addition, there are growing evidences that chromosome structure plays a vital role in controlling gene expression, although there is limited understanding of the nuclear proteins that contribute to structural interactions among nucleosomes (57).

Nucleosomes are connected through a segment of linker DNA, which often associates with other proteins like linker histone proteins (H1 or H5). Linker histones are the most divergent class of histones, but they all contain an evolutionarily conserved N-terminal globular domain (GH1 domain), which binds to the nucleosome dyad and interacts with the linker DNA (8, 9). In addition to the GH1 domain, linker histones generally contain a positively charged C-terminal domain that can interact with DNA. It has been known that linker histones can function as architectural proteins that induce chromatin conformation changes through cooperative binding of both the GH1 domain and the C-terminal domain to its target (10, 11).

The high-mobility group (HMG) proteins are another set of chromatin architectural proteins. The HMG proteins were originally isolated via biochemical purification of chromatin proteins, and they are the most abundant nonhistone proteins (12, 13). They bind to DNA and nucleosomes and generally act as architectural elements that modulate multiple DNA-dependent processes, including replication and transcription (14, 15). Higher eukaryotes contain three classical families of HMG proteins based on their DNA-binding domains: HMGA, HMGB, and HMGN (16, 17). The HMGB family contains HMG-boxes, and the HMGN family contains nucleosome-binding domains (16, 17). The HMGA subfamily was grouped together because these proteins preferentially bind to the minor groove of AT-rich regions of DNA via several AT-hook motifs (16, 17). The AT-hook motif is a conserved DNA-binding motif commonly found in eukaryotes (18). HMGA proteins affect local chromatin structure in several ways, including bending, straightening, unwinding, and looping of substrate DNA (19), and they have been implicated in numerous DNA-based cellular processes.

In the flowering plant Arabidopsis thaliana, GH1 domain–containing proteins have been systematically annotated (20). A subgroup of plant GH1 domain–containing proteins has a C-terminal domain that has similarity to the mammalian HMGA proteins and thus was designated as the GH1-HMGA clade. Furthermore, similar arrangements of the GH1 domain and AT-hook motifs also exist in animals as well as in yeast, nematode, and insect species. However, plant GH1-HMGA proteins are restricted to angiosperms, implying that they are recently evolved in the plant lineage. Therefore, convergent evolution may have resulted in this group of H1 variants in diverse organisms. The biological function of plant GH1-HMGA proteins is not known, but they play fundamental roles in chromatin structure in other organisms (20).

In Arabidopsis, FLOWERING LOCUS C (FLC) has been an excellent model system to identify chromatin regulators, both protein and noncoding RNA components, and to unravel mechanistic details of epigenetic regulation (2123). In addition, FLC chromatin contains loops that influence transcriptional activity (22, 24, 25). Although the presence of topologically associated domains (TADs) in Arabidopsis is not as clear as in mammals, plants also use a three-dimensional spatial organization of the genome, including gene loops, as a means of architecturally regulating gene expression (2630). The formation of gene loops is prominent in Arabidopsis (27) and perhaps constitutes the basic unit of higher-order nucleosome structures that affect transcriptional activity. However, there is only a limited understanding of the mechanism underlying the formation of gene loops.

Here, we report that members of the Arabidopsis GH1-HMGA family are chromatin architectural factors and redundantly promote floral transition through the repression of FLC expression. We demonstrated that GH1-HMGA proteins directly repress FLC by preventing the formation of the 5′ to 3′ gene loop, which facilitates FLC transcriptional activation.

RESULTS

Characterization of the roles of GH1-HMGA family in flowering in Arabidopsis

The mammalian HMGA family of proteins plays roles in various biological processes by influencing chromatin structure and transcription (19). Our phylogenetic analysis identified members of the GH1-HMGA clade in Arabidopsis as the closest homologs to mammalian HMGA proteins (Fig. 1A). Unlike the canonical mammalian HMGA, three members of Arabidopsis GH1-HMGA, including GH1-HMGA1 (HON4), GH1-HMGA2 (HON5), and GH1-HMGA3, contain a conserved GH1 domain at their N terminus in addition to four to six AT-hook motifs (20). A distant member, which we arbitrarily called GH1-HMGA4, has an N-terminal GH1 domain but contains C terminus without recognizable AT-hook, was also grouped with the GH1-HMGA cluster (Fig. 1A and fig. S1, A and B) (20). Both the GH1 domain and the AT-hook motifs can bind to nucleosomes, indicating that Arabidopsis GH1-HMGA proteins may function as architectural factors that influence chromatin structure (19).

Fig. 1 Characterization of the HMGA family of proteins in Arabidopsis.

(A) Phylogenetic tree of Arabidopsis GH1 domain–containing proteins. Human and mouse HMGA variants were used as outgroups. (B) Morphology of representative 5-week-old plants grown under long-day (LD) conditions at 22°C. Scale bar, 5 cm. (C) Flowering times of plants measured under LD. Error bars: ±SD (n ≥ 30); significantly distinct groups were determined by one-way ANOVA followed by Tukey post hoc test for multiple comparisons (letters indicate statistically distinct groups; P < 0.05). (D) Flowering time of plants grown in short-day (SD) condition at 22°C. Error bars: ±SD (n ≥ 15); significantly distinct groups were determined by one-way ANOVA followed by Tukey post hoc test for multiple comparisons (letters indicate statistically distinct groups; P < 0.05). (E) Differentially expressed genes (DEG) identified by RNA-seq of two biological replicates. Genes with more than 1.5-fold change were defined as DEGs; FDR < 0.05. (F) Enriched GO biological pathways of the up-regulated DEGs in hon45 mutant compared to Col-0. (G) qRT-PCR quantification of FLC mRNA level in 10-day-old seedlings grown under LD conditions at 22°C. Error bars: ±SD (n = 3).

Considering that the biological functions of plant GH1-HMGA proteins are mostly unknown, we isolated and analyzed corresponding T-DNA insertional loss-of-function mutants (fig. S2, A and B). We found that hon4, hon5, and gh1-hmga3 single-mutant plants show a slight but reproducible late-flowering phenotype (Fig. 1, B and C, and fig. S2C). Subsequent genetic analyses showed that hon4hon5 (hon45) double-mutant plants display a more pronounced late-flowering phenotype, and the late flowering was further enhanced by stepwise introgressions of gh1-hmga3 and gh1-hmga4 mutations; thus, the members of the GH1-HMGA gene family redundantly control the floral transition in Arabidopsis (Fig. 1, B and C, and fig. S2C). A delay in the flowering of mutants was also observed in short days, showing that the late flowering is not due to a compromised photoperiodic response in the mutants (Fig. 1D).

Transcriptome analysis of hon45 mutants

To explore the roles of the GH1-HMGA gene family in plant development, we performed RNA sequencing (RNA-seq) analysis using hon45 mutants. The transcriptome analysis identified 524 differentially expressed genes (DEGs) in hon45 mutants (Fig. 1E and dataset S1). Gene ontology (GO) analysis showed no significant enrichment of GO terms for down-regulated genes. On the other hand, several significant GO terms were identified for up-regulated genes, including biological pathways involved in photosynthesis, disease, and responses to environmental stimuli including light and temperature, implying that the GH1-HMGA family of proteins may function to repress these classes of genes (Fig. 1F). Next, we sought genes implicated in floral transition that may contribute to the late-flowering phenotype of the hon45 mutant. Notably, transcripts of the major floral repressor FLC increase significantly in the hon45 mutant, and this observation was further confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay (Fig. 1G and dataset S1). FLC mRNA levels in higher-order mutants display a positive correlation with their flowering times (Fig. 1, C and G), supporting the idea that the members of GH1-HMGA clade redundantly repress FLC to promote flowering.

Functional characterization of HON4 and HON5

Our results indicated that members of the GH1-HMGA gene family positively promote floral transition through the repression of FLC. Accordingly, the hon45 mutant plants were transformed with HON4 or HON5 genomic sequences fused to the Myc epitope for molecular complementation. We found that the hon45 late-flowering phenotype could be complemented by either gHON4-Myc or gHON5-Myc (Fig. 2A and fig. S3A). A representative complemented line for each transgene was selected and further verified by detecting the Myc-fused proteins (Fig. 2A and fig. S3B). Correlated with the flowering phenotype, the elevated level of FLC mRNA expression in hon45 mutants was restored in the complementation lines to a comparable level to that in the wild type (Fig. 2C). In addition, the expression level of FT, a floral integrator downstream of FLC, is also recovered in the complementation lines (Fig. 2D). By analyzing transgenic plants in which β-glucuronidase (GUS) gene is fused in frame with HON4 or HON5 genomic copies, we found that HON4 and HON5 have similar expression pattern (Fig. 2E and fig. S3E); both HON4 and HON5 are expressed in the shoot apical meristem, supporting their redundant role in promoting floral meristem formation (Fig. 2E). We also detected strong GUS staining in root tissues as well as in the vasculature of cotyledons and expanded leaves (Fig. 2E and fig. S3E). Similar expression patterns of HON4 and HON5 were observed from green fluorescent protein (GFP)–tagged transgene lines with a strong signal at the shoot apex, and the GFP signal is also observed throughout cotyledons (fig. S3F). Consistent with their potential role as chromatin architectural proteins, HON4-GFP and HON5-GFP exclusively localize in the nucleus of plant cells (Fig. 2F).

Fig. 2 Functional characterization of HON4 and HON5.

(A) Representative 35-day-old plants grown under LD at 22°C showing the molecular complementation by Myc-tagged transgenes. Scale bar, 5 cm. (B) Flowering times of representative complementation lines grown under LD at 22°C. Error bars: ±SD (n ≥ 30). (C) Relative expression levels of FLC mRNA in 10-day-old seedlings grown under LD at 22°C. Bars indicate SD of three biological replicates. (D) Relative expression level of FT mRNA in 10-day-old seedlings grown under LD at 22°C condition. Error bars: ±SD (n = 3). (E) GUS staining of 7-day-old seedlings that carry a transgene to express HON4-GUS and HON5-GUS fusion proteins in hon45 mutant background. Scale bar, 2 mm. (F) HON4-GFP and HON5-GFP fusion proteins localize in the nucleus of root cells. Same subcellular localizations were observed from other tissues. Scale bar, 200 μm. DAPI, 4′,6-diamidino-2-phenylindole.

GH1-HMGA gene family acts through FLC to regulate flowering

Our gene expression analysis suggests that FLC may be a target of GH1-HMGA family of proteins in regulating flowering time (Figs. 1G and 2C). We addressed their genetic relationship by introducing the null flc-3 mutation (31) into hon45 and gh1-hmga quadruple (honq) mutants. Genetic assays revealed that the GH1-HMGA gene family promotes flowering mainly through FLC, as the flc-3 mutation could mostly reverse the late flowering of both hon45 and honq mutants back to that of the wild type (Fig. 3, A and B, and fig. S4A). FLC represses flowering by suppressing the transcription of floral integrator genes, including FT (32). In agreement with the flowering trait, the dramatic reduction of FT mRNA in honq mutant was also restored to the level similar to the wild type by flc-3 mutation (Fig. 3C), demonstrating that the members of the GH1-HMGA gene family modulate FT expression through FLC.

Fig. 3 Arabidopsis GH1-HMGA family genetically acts through FLC to regulate flowering.

(A) Introduction of flc-3 mutation rescues the late-flowering phenotype of honq. Scale bar, 5 cm. (B) Total leaf number of plants grown under LD at 22°C. Error bars: ±SD (n ≥ 30); two-tailed Student’s t test, ***P < 0.001. (C) Expression changes of FT show that FLC is required for the GH1-HMGA family proteins to promote the floral transition. (D) Genetic analysis of hon45 mutant with autonomous pathway mutants. Error bars: ±SD (n ≥ 20). (E) Changes in FLC expression during vernalization treatment.

To explore the tissues in which HON4 and HON5 regulate the FLC transcription, we crossed the transgenic line carrying GUS fused with the entire FLC genomic region (FLC-GUS) (33) into the hon45 mutant plant (fig. S4B). Consistent with previous reports (33, 34), we detected FLC-GUS signals throughout vascular tissues of young seedlings (fig. S4C). Compared to the wild-type Col-0 background, the overall patterns of FLC-GUS staining are not altered in hon45 mutants; however, a much higher level of FLC-GUS signal is detected in hon45 mutants (fig. S4C). Moreover, the FLC spatial expression patterns overlap with HON4 and HON5 expression domains (Fig. 2E and fig. S4C), implying that the GH1-HMGA family directly represses FLC transcription in the tissues where FLC is actively expressed.

Considering that the GH1-HMGA family proteins are previously unknown regulators of FLC transcription, we tested their genetic relationship with several other FLC regulators. Mutations in HON4 and HON5 show additive effects on the late-flowering phenotypes of autonomous pathway mutants, fca-9, fve-4, and fld-3 (Fig. 3D), implying that the GH1-HMGA family functions independently of FCA, FVE, and FLD in the repression of FLC. We also tested the hon45 mutant for its vernalization response by the introgression of hon45 mutants into the winter-annual FRI-Col genetic background (35). Additive effects were observed in terms of both flowering time and FLC expression with vernalization treatment, indicating that FRI and the GH1-HMGA family act in parallel to regulate FLC (Fig. 3E). hon45 mutants show a slow reduction of FLC and later flowering upon vernalization, although gradual repression of FLC by cold is still observed (Fig. 3E).

Trimethylation at histone H3 Lys27 (H3K27me3) and trimethylation at histone H3 Lys36 (H3K36me3) are two epigenetic markers that antagonize each other to fine-tune FLC expression (36). Although clear derepression of FLC in hon45 and higher-order mutants was observed, no apparent difference in either H2K27me3 or H3K36me3 at FLC is observed in hon45 mutants (fig. S4, D and E). Our above results collectively suggested that the GH1-HMGA family proteins may regulate FLC through a previously unknown mechanism.

GH1-HMGA family members bind to FLC chromatin

Next, we used the complemented lines harboring the Myc epitope to test whether HON4 and HON5 directly associate with FLC chromatin by chromatin immunoprecipitation (ChIP) followed by qPCR. We detected substantial enrichment of both HON4-Myc and HON5-Myc to the same region of ~600 bp upstream of the FLC transcription start site, corresponding to the canonical promoter of FLC (Fig. 4A), demonstrating that members of the GH1-HMGA family directly regulate FLC expression through physical association with FLC chromatin.

Fig. 4 Genome-wide study of HON5 occupancy.

(A) The upper part is a diagram of FLC gene structure with numbers marking the positions of PCR amplicons used for ChIP-qPCR. The lower panel is the ChIP-qPCR results performed across the FLC locus. Error bars: ±SD (n = 3). Primers used for qPCR are listed in table S1. (B) Genome-wide average profile of HON5-Myc ChIP-seq signals. (C) IGV browser track of HON5 binding at the FLC locus. Track in red color shows HON5-Myc ChIP signal; track in black color shows background from Col-0 immunoprecipitated with anti-Myc antibody. (D) Distribution of annotated HON5-Myc ChIP-seq peaks within defined genomic regions. (E) The two most significant DNA motifs associated with HON5 binding are AT-rich sequences. Those two motifs are enriched in the HON5 binding region at FLC, and the positions of corresponding motifs were highlighted in fig. S5.

To better understand the molecular function of the GH1-HMGA family, we identified HON5 targets at the genome-wide level by using ChIP sequencing (ChIP-seq) (table S2). More than 21,000 HON5 binding peaks were determined (dataset S2), including the one at the FLC promoter that is identical to the region detected by ChIP-qPCR (Fig. 4, A and C). HON5 shows distinct binding patterns that peak at 5′ and 3′ flanking regions of protein-coding genes (Fig. 4C). Most (~76%) of the HON5 binding sites are clustered within 3 kb upstream of the transcription start site (TSS) or 1 kb downstream of the transcription end site (TES), with relatively higher occupancy towards the 5′ end of genes (Fig. 4, B and D). However, few binding signals were observed across the gene body region. Therefore, genome-wide distribution patterns of HON5 indicate that it generally functions at the flanking regions of protein-coding genes (Fig. 4B).

Motif analysis of HON5 binding sites identified DNA motifs primarily composed of adenines and thymines, known as the binding motifs of AT-rich interaction domain (ARID)–containing family proteins (Fig. 4E) (37). Six such AT-rich motifs are clustered within 150 bp of HON5-enriched regions at the FLC promoter (Fig. 4, A and C, and fig. S5A), supporting their importance in mediating the binding of HON5 to FLC chromatin. Therefore, plant GH1-HMGA family proteins show similar functional property to mammalian HMGAs in terms of DNA-binding preference toward AT-rich regions of DNA (15, 19, 38). Given that GC- and AT-rich chromatin may differ in conformation and modification, we tested whether HON5 enrichment is associated with certain histone modifications. However, we did not find any genome-wide correlation among tested histone modifications, including H3K4me3, H3K27me3, and H3K36me3 (fig. S6). This is consistent with our finding that no obvious change in these modifications was observed at FLC in hon45 mutants (fig. S4, D and E). In addition, there is no correlation between HON5 occupancy and the level of gene expression or gene size (fig. S6), implying that the GH1-HMGA family proteins may function in a previously unknown manner.

The GH1-HMGA family proteins preclude FLC gene looping

The distinct patterns of HON5 occupancy revealed by ChIP-seq analysis prompted us to check whether it also binds to the 3′ end of FLC (Fig. 4B). A sharp HON5 binding signal was observed at the 3′ region of the FLC locus (Fig. 4C and fig. S5B), which corresponds to the promoter of antisense long noncoding RNA, COOLAIR (39, 40). Given that COOLAIR is known to be involved in the down-regulation of FLC transcription, we examined whether HON5 and its related members regulate FLC by altering COOLAIR transcription. However, there is no significant change in the level of both distal and proximal COOLAIR transcripts in hon45-FRI compared to the wild-type FRI-containing line (fig. S7, A and B).

Two competing chromatin loops have been identified at the FLC locus (22, 24). A gene loop between 5′ and 3′ of FLC flanking regions is known to be necessary for the active FLC transcription (22, 24, 25, 28). However, the regulatory factors involved in the formation of the FLC gene loop are not known. Because HON5 binds to the same regions where the 5′ to 3′ FLC gene loop forms, we investigated whether the GH1-HMGA gene family plays a role in the formation of the FLC gene loop. By using chromosome conformation capture (3C) followed by quantitative PCR (22, 25, 41), we found that the frequency of the 5′ to 3′ gene looping at FLC markedly increased (more than fourfold) in the honq mutant compared to the wild-type Col-0 (Fig. 5A). Moreover, the enhanced FLC gene looping in the honq mutant is restored to near the wild-type level in the complemented line (Fig. 5A and fig. S8, A and B). Similarly, we observed that the FLC gene looping significantly increased in honq-FRI compared to the wild-type FRI-Col (Fig. 5B). The frequency of FLC gene looping is more robust in honq-Col compared to FRI-Col (Fig. 5C), despite the fact that the level of FLC transcription in FRI-Col is four times higher than that in honq mutants in the Col-0 background (fig. S8B). Therefore, the GH1-HMGA family proteins contribute to the repression of FLC by preventing the 5′ to 3′ gene looping at FLC, independent of the FRI complex.

Fig. 5 GH1-HMGA family of proteins precludes FLC gene loop formation.

(A) The upper panel shows the relative locations at FLC in 3C-qPCR experiments. Dpn II restriction sites are indicated with vertical red lines. The lower panel is the quantitative relative interacting frequency of FLC 5′ and 3′ regions determined by 3C-qPCR. Error bars: ±SD (n = 2 × 2; biological replicates × technical replicates). (B and C) Quantitative relative interacting frequency of FLC 5′ and 3′ regions determined by 3C-qPCR. Error bars: ±SD (n = 2 × 2; biological replicates × technical replicates). Primers used for 3C are listed in table S1. (D) Detection of transcriptional initiation form of RNA Pol II levels at the FLC promoter region. The numbers on the X-axis correspond to the positions of PCR amplicons shown in Fig. 4A. Error bars: ±SD (n = 2 × 2; biological replicates × technical replicates). (E) A model to depict the role of the GH1-HMGA family proteins in precluding the FLC gene looping. The left part shows that FLC self-looping is stabilized by unknown factors, which promote FLC transcription. The right part shows that members of the GH1-HMGA family, including HON5, bind to the FLC promoter and region downstream of the terminator to prevent gene loop formation by antagonizing with the unknown factors and RNA Pol II, which, in turn, suppress FLC transcription.

It has been proposed that the FLC 5′ to 3′ looping may create a favorable condition for transcription by facilitating the recycling of RNA polymerase II (RNA Pol II) at FLC (24, 25). This prompted us to examine the level of transcription-initiation form of RNA Pol II, Ser5-phosphorylated Pol II (Ser5-P Pol II) (42) at the FLC locus. Consistent with the change in the level of FLC gene looping and transcription, we found that the level of Ser5-P Pol II at FLC increases in hon45 mutant compared to the wild type, and this accumulation is restored in the complementation line (Fig. 5D). Moreover, we detected a relatively higher level of Ser5-P Pol II at the region corresponding to the HON4 and HON5 binding sites (Fig. 4A and 5D). Therefore, our results demonstrated that the binding of GH1-HMGA family proteins to FLC flanking regions disrupts the formation of gene loop and thus alters local chromatin structures necessary for effective FLC transcription (Fig. 5E).

DISCUSSION

Here, we characterized the GH1-HMGA gene family for their roles in floral transition in Arabidopsis. We showed that the late flowering observed in higher-order mutants is due to the elevated level of the floral repressor FLC. By a classical definition (43, 44), the GH1-HMGA gene family belongs to the autonomous pathway genes, which regulate FLC. hon45 and higher-order mutants, among the members of the family, still retain the photoperiod response, and their late-flowering phenotypes are suppressed by flc mutation, which is a classical definition of autonomous pathway mutants (Fig. 1, C and D) (43, 44). Moreover, additive effects of GH1-HMGA family mutations were observed in all tested mutant backgrounds (Fig. 3, D to F), suggesting that this group of proteins regulates FLC through an unknown molecular mechanism.

Our analysis revealed the Arabidopsis GH1-HMGA family members are the closest homologs to mammalian HMGA proteins (Fig. 1A). However, the Arabidopsis GH1-HMGA family proteins are unique in that they contain the GH1 domain, which is the signature motif of H1 linker histone proteins (20). A recent study showed that mammalian HMGA proteins display widespread bindings with only a preference to AT-rich regions (45). Our ChIP-seq analysis also showed that HON5 has pervasive genome-wide occupancy with over 21,000 peaks (dataset S2), and GH1-HMGA proteins preferentially bind to AT-rich regions as well (Fig. 4E and fig. 5, A and B). A total of 5611 genes have at least one nearby HON5 binding signal, and 108 genes with the HON5 peak are differentially expressed in hon45 mutants (Fig. 1E and dataset S3). Relatively minor changes in transcriptome were also reported in mouse embryonic stem cells (45), suggesting that only a limited number of loci are sensitive to the loss of this class of chromatin architectural proteins. A previous study reported that hon4 mutants exhibited multiple growth defects, including short roots, small and sharp leaves, short inflorescences, and total sterility (46). However, we did not observe any developmental abnormality in single mutants or in any higher-order mutants (Fig. 1B), except for the late flowering due to the derepression of FLC.

Although the GH1-HMGA family of proteins shares some similarities with known HMGA and H1-linker proteins, genome-wide occupancy patterns of the GH1-HMGA family of proteins are unique. Their occupancies peak at both 5′ and 3′ end of protein-coding genes (Fig. 4B), and the depletion of the GH1-HMGA family of proteins resulted in the enhanced formation of a gene loop at the FLC locus. In Arabidopsis, gene loops have been systematically identified, and the packing of its genome is predicted to adopt units of gene bodies (27, 28). In a previous study, 1792 genes were found to contain self-loops between the 5′ and 3′ portion of their transcribed region (27, 28). It should also be noted that the formation of a gene loop could be inducible in response to stimuli and also be expected to be tissue specific (22, 24, 2729, 47). Therefore, the number of genes with self-looping is likely to be underestimated. Besides the FLC locus, whether GH1-HMGA family proteins control gene loop formation at other loci remains to be determined.

One of the FLC transcriptional activators, the FRI complex, has been shown to be necessary for FLC 5′ to 3′ gene loop formation (24). Our data revealed that the FRI complex is not required for GH1-HMGA family proteins to govern gene looping at FLC (Fig. 5, A to C). Although all examined H3K4me3, H3K27me3, and H3K36me3 histone modifications unlikely contribute to the regulatory role of GH1-HMGA on FLC gene looping (fig. S4, D and E, and fig. S6), a recent study showed that the RNA Pol II complex plays an active role in gene loop formation (48). Our ChIP-qPCR data show that the enrichment of transcription-initiation form of RNA Pol II at FLC promoter occurs in a HON4- and HON5-dependent manner (Fig. 5D). The binding of GH1-HMGA family proteins to FLC promoter and region downstream of the terminator appears to create chromatin structures that adversely affect the recycling of RNA Pol II, and thus prevents 5′ to 3′ gene looping (Fig. 5E).

Although the presence of gene loop has been reported in many species (6, 22, 27, 49), the regulators that affect the formation of gene loop is not well understood and may be divergent among species (49, 50). Our work identified the GH1-HMGA family proteins as regulators of the formation of gene loop at FLC. Further characterization of this group of proteins will shed light on the molecular mechanisms underlying gene loop formation and their function in various biological processes.

MATERIALS AND METHODS

Plant materials and growth conditions

The hon4 (SALK_071403), hon5 (SALK_116292), gh1-hgma3 (SALK_078336), and gh1-hmga4 (CS824818) mutants in the Columbia (Col-0) background were obtained from Arabidopsis Biological Resource Center (ABRC). Mutants were crossed with the FRI-Col to generate lines in the FRI background. Primers for transfer DNA (T-DNA) insertion genotyping are listed in table S1. Sterilized seeds were sown on agar plates, stratified at 4°C for 3 days, and then moved to the growth chamber with long-day conditions (16 hours light, 8 hours dark) at 22°C for 7 days. After that, plants were transplanted to soil and transferred to either long-day or short-day (8 hours light, 16 hours dark) growth chambers for further assay. Flowering time was measured by counting the total number of leaves (rosette and cauline leaves) at the bolting stage. For the vernalization treatment, seeds were germinated on agar plates for 10 days and vernalized at 4°C under short-day conditions. After the vernalization treatment, plants were transplanted to soil and transferred to growth chambers (22°C) under long-day conditions for flowering time test or harvested for RNA isolation.

Transgenic plants

Genomic sequences of HON4 and HON5 were amplified by PCR and cloned into pENTR and then transferred into pGWB16, pGWB203, and pGWB604 binary vectors using Gateway System (Invitrogen). Sequences were confirmed by Sanger sequencing and used for complementation of the mutant lines. All binary vectors were transformed into Agrobacterium tumefaciens GV3101 strain. Plants of hon4hon5 double mutant were transformed with a flower dip method. Homozygous transgenic plants harboring single T-DNA insertion were selected on antibiotic plates. Primers used for gene cloning were listed in table S1.

Phylogenetic tree analysis

Protein sequences were obtained from The Arabidopsis Information Resource (TAIR) and UniProt databases. MEGA 7 software was used to construct phylogenetic trees with maximum-likelihood estimation and 1000 bootstrap. The tree was rooted using human and mouse HMGA proteins as an outgroup for both GH1-HMGA and histone H1 in Arabidopsis.

RNA expression analysis

Total RNA was extracted from whole seedlings 10 days after germination unless otherwise specified using TRIzol (Invitrogen). Extracted RNA was treated with deoxyribonuclease (DNase) I (Promega) for 30 min at 37°C to remove genomic DNA. Purified RNA was quantified on NanoDrop (Thermo Fisher Scientific), and 1 μg of RNA was used for first-strand complementary DNA (cDNA) synthesis using oligo(dT) primers. Synthesized cDNA products were diluted threefold with water and then used for real-time qRT-PCR analyses with Maxima SYBR Green Master Mix (Thermo Fisher Scientific) on the ViiA 7 Real-Time PCR System (Life Technologies). Relative gene expressions were determined by normalizing to the levels of PP2A. Primer sequences for qRT-PCR are listed in table S1.

Transcriptomic analysis

Whole seedlings grown on half-strength Murashige and Skoog (MS) medium under short-day conditions were collected at zeitgeber time 6. Total RNAs were extracted using TRIzol (Invitrogen) and treated with DNase I (Promega) to eliminate traces of genomic DNA. Sequencing libraries were prepared with 500 ng of total RNA following NEBNext Poly(A) mRNA Magnetic Isolation Module [New England Biolabs (NEB) #E7420]. Libraries were assessed on a bioanalyzer (Agilent High Sensitivity DNA Assay) and sequenced on an Illumina NextSeq 500 platform. RNA-seq clean reads were aligned to TAIR10 genome release using HISAT2 with default parameters. Gene expression was quantified as counts per million reads mapped (CPM). DEGs were determined with edgeR over two biological replicates. Genes with more than 1.5-fold change relative to Col-0 and false discovery rate (FDR) < 0.05 were considered as DEGs. GO term enrichment was performed over the sets of DEGs with the online tools (http://geneontology.org).

Chromatin immunoprecipitation

About 2 g of 10-day-old seedlings was harvested and cross-linked in 1% formaldehyde solution under a vacuum for 25 min. Cross-linking was stopped by adding 0.125 M glycine and vacuumed for 5 min. Cross-linked seedlings were rinsed in 10 mM Hepes buffer three times and dried with paper towels. Samples were ground into fine powder in liquid nitrogen. ChIP assays were performed following the Abcam ChIP protocol (https://abcam.com/protocols) with minor adjustments. Immunoprecipitations were performed by using c-Myc antibody (9E10, Santa Cruz Biotechnology) combined with protein G magnetic beads (Thermo Fisher Scientific). Input DNA and immunoprecipitated DNA samples were purified by PCR purification kits from Qiagen. Eluted DNA samples were used for either ChIP-qPCR or ChIP-seq.

ChIP-seq analysis

ChIP assays were conducted by using both Col-0 and HON5-Myc transgenic plants. One immunoprecipitated DNA sample from Col-0 (Col-0-IP) and immunoprecipitated DNA from two replicates of HON5-Myc (HON5-IP) as well as pooled input DNA (HON5-Input) were selected for sequencing. ChIP-seq libraries were prepared using the NEBNext ChIP-seq Library Prep Kit and sequenced on an Illumina NextSeq 500 platform. Sequencing reads were mapped to the Arabidopsis reference genome (TAIR10) with Bowtie2. Mapped reads were normalized using deepTools and visualized using IGV. As both Col-0-IP and HON5-Input track show background signals, we selected Col-0-IP as the control for the following assays. To check the enrichment of HON5 relative to gene start and end positions, we calculated the scores of HON5 per gene using deepTools. Each gene was defined as the interval from TSS to TES plus 3 kb upstream and 1 kb downstream. In total, 33,602 such regions annotated from TAIR10 were analyzed. All the regions were then scaled and stacked, and the average score was plotted to show the relative enrichment of HON5 over genes. Genome-wide HON5 peak distribution was analyzed by categorizing the Arabidopsis genome into nonoverlapping elements including TSS, TES, 5′ untranslated region (UTR), 3′UTR, Exon, Intron, Intergenic, and TSS & TES. TSS & TES is where two genes are closely located, and thus, the TSS of one gene overlapped with the TES of another gene. The percentage of HON5 peaks that fell into each category was calculated and shown in the pie chart. Motif analysis was carried out by extracting the ±300-bp sequences surrounding HON5 peak summits and submitting these regions to MEME (multiple expectation maximizations for motif elicitation)-ChIP motif discovery module against DNA affinity purification sequencing (DAP-seq) datasets (51). The estimated statistical significance (E value) and sequence logo was generated for each motif.

To check the correlation between HON5 enrichment and histone modifications of its neighboring gene (52), we extracted the pairs of HON5 peak and its closest gene using BEDtools. In total, 21,164 HON5-gene unique pairs were obtained (median distance of 223 bp; mean distance of 526 bp). In cases where the downstream and upstream genes showed the same distance to the HON5 peak, we assigned two pairs to include both genes. The level of HON5 was calculated by averaging the coverage within each peak. The levels of H3K27me3, H3K36me3, and log-transformed transcription were calculated by averaging the coverage within each gene. Unlike those modifications that spread across the gene body, H3K4me3 is largely concentrated in 5′ end regardless of gene length; therefore, we calculated H3K4me3 levels by extracting the maximum coverage value within each gene. Pairwise correlation analysis was carried out using R stats package. The corresponding Pearson correlation coefficients were calculated and displayed together with the scatter plot and linear trendline for each pair.

Chromosome conformation capture

3C assays were conducted as previously described with minor modifications (41). Nuclei were isolated from 1% formaldehyde cross-linked 10-day-old Arabidopsis seedlings and treated with 0.3% SDS at 65°C for 40 min followed by 30 min at 37°C. SDS was sequestered with 1% Triton X-100 for 60 min at 37°C. Chromatin was digested overnight by 400 U of Dpn II restriction enzyme (NEB) at 37°C. Restriction enzymes were inactivated by the addition of 1.6% SDS and incubated at 65°C for 20 min, and then 2% Triton X-100 was added to sequester SDS. Ligations were performed for 5 hours at 16°C using 200 U of T4 DNA ligase (Invitrogen) followed by 2 hours at room temperature. Reverse cross-linking was performed at 65°C for 6 hours. After proteinase K (NEB) treatment, ligated DNA was purified by phenol/chloroform/isoamyl-alcohol (25:24:1) extraction and ethanol precipitation. Quantitative PCR was performed to calculate the relative interaction frequencies between the two regions. An FLC region without Dpn II digestion was amplified as a loading control to normalize the DNA concentrations of different samples. The primer efficiencies were corrected using a control template that contains equal amounts of all possible ligation products from a Dpn I–digested plasmid harboring 11 kb of assayed FLC genomic region. Primers used for 3C-qPCR are listed in table S1.

Histochemical GUS staining

Plant materials were submerged in X-Gluc solution [0.5 mg/ml; 0.1 M monosodium phosphate (pH 7.0), 10 mM EDTA, 0.1% Triton X-100, 0.5 mM potassium ferrocyanide, and 0.5 mM potassium ferricyanide], vacuumed for 5 min, and kept at 37°C. Subsequent materials were decolorized in 70% ethanol and imaged with a stereomicroscope.

Statistical analysis

Two-tailed Student’s t test and one-way analysis of variance (ANOVA) were conducted using Excel.

Accession numbers

Arabidopsis Genome Initiative gene identifiers are as follows: FLC (AT5G10140), FT (AT1G65480), HON4 (AT3G18035), HON5 (AT1G48620), GH1-HMGA3 (AT1G14900), GH1-HMGA4 (AT5G08780), PP2A (AT1G13320), ACT2 (AT3G18780), ACT7 (AT5G09810), H1.1 (AT1G06760), H1.2 (AT2G30620), H1.3 (AT2G18050), GH1-Myb1 (AT1G49950), GH1-Myb2 (AT5G67580), GH1-Myb3 (AT3G49850), GH1-Myb4 (AT1G17520), GH1-Myb5 (AT1G72740), GH1-Myb6 (AT1G54230), GH1-Myb7 (AT1G54240), and GH1-Myb8 (AT1G54260).

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/7/24/eabg3097/DC1

https://creativecommons.org/licenses/by-nc/4.0/

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

REFERENCES AND NOTES

Acknowledgments: We acknowledge the Texas Advanced Computing Center (TACC; http://tacc.utexas.edu) at The University of Texas at Austin for providing high-performance computing resources that have contributed to the research results reported within this paper. We also wish to thank R. Amasino for comments on the manuscript. Funding: This work was supported by NIH R01GM100108 and NSF IOS 1656764 to S.S. Author contributions: B.Z., Y.X., J.K., and S.S. conceived and implemented the method and performed the experiments and data analysis. B.Z. and S.S. drafted the manuscript. S.S. advised on the design and implementation and interpretation of results and edited the manuscript. All authors read and approved the final manuscript. Competing interests: The authors declare that they no competing interests. Data and materials availability: The data supporting the findings of this study are available within the paper and in the Supplementary Materials. A reporting summary for this article is available in the Supplementary Materials. ChIP-seq and RNA-seq data, including raw reads and FPKM expression tables, were deposited in the NCBI Gene Expression Omnibus (GEO) database under accession number GSE163850. Specific materials generated during this study are available upon request.

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