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Published in final edited form as: Mol Cell. 2013 Sep 19;52(2):10.1016/j.molcel.2013.08.024. doi: 10.1016/j.molcel.2013.08.024

Chromatin remodelers fine-tune H3K36me-directed deacetylation of neighbor nucleosomes by Rpd3S

Chul-Hwan Lee 1, Jun Wu 1, Bing Li 1,*
PMCID: PMC3825818  NIHMSID: NIHMS518049  PMID: 24055344

Summary

Chromatin remodelers have been implicated in the regulation of histone modifying complexes. However the underlying mechanism remains poorly understood. The Rpd3S histone deacetylase complex is recruited by elongating RNA polymerase II to remove histone acetylation at coding regions in a manner that is dependent on methylation of lysine 36 on histone 3 (H3K36me), and Rpd3S prefers dinucleosomes. Here, we show that the binding of Rpd3S to dinucleosomes and its catalytic activity are sensitive to the length of nucleosomal linker in a nonlinear fashion. Intriguingly, we found that H3K36me on one nucleosome stimulates Rpd3S to deacetylate the neighboring nucleosomes when those two nucleosomes are within an optimal distance. Finally, we demonstrate that chromatin remodelers enhance Rpd3S activity by altering nucleosomal spacing, suggesting that chromatin remodelers prime chromatin configuration to fine-tune subsequent histone modification reactions. This mechanism is important for accurate temporal control of chromatin dynamics during the transcription elongation cycle.

Introduction

Chromatin dynamics is regulated by combined mechanisms of histone post-translational modifications (PTM), chromatin remodeling, histone variant incorporation, histone eviction and DNA modifications (Li et al., 2007a). To react to complex nuclear signals, most chromatin regulatory mechanisms are intricately connected. Chromatin remodeling factors are regulated by PTMs. Histone acetylation by SAGA facilitates Swi/Snf targeting and remodeling at the promoter regions (Hassan et al., 2001; Krebs et al., 1999); Likewise, RSC activity was enhanced by acetylation at coding regions (Carey et al., 2006); Histone H3K4 methylation helps to direct the remodeler NURF to the HOX gene loci (Wysocka et al., 2006). These phenomena are consistent with the signaling functions of histone modifications (Jenuwein and Allis, 2001). Although chromatin remodelers have been implicated in regulating histone modifications, the detailed molecular mechanisms have been poorly understood.

Eukaryotic genomes are packed into arrays of nucleosomes, which are mostly organized according to transcription units (Jiang and Pugh, 2009). Genome-wide nucleosome positioning is determined by the combination of multiple mechanisms, including intrinsic DNA sequence preferences, ATP-dependent chromatin remodelers, histone chaperones and RNA pol II transcription machinery (Struhl and Segal, 2013). DNA sequence properties contribute significantly at promoters, where nucleosome-free regions (NFR) are formed and the −1/+1 nucleosome boundary is established (Kaplan et al., 2009). Chromatin remodelers play major roles in establishing positioning at promoter proximal regions (Gkikopoulos et al., 2011; Zhang et al., 2011). Interestingly, host cells seem to be mostly responsible for nucleosome positioning of an artificially introduced exogenous genome, suggesting that transcription factors, remodelers and Pol II transcription machinery are the main determinants for the nucleosome pattern in vivo (Hughes et al., 2012). A survey of nucleosome and transcription profiles in a large number of yeast mutants reached a similar conclusion and further suggested that combined regulatory mechanisms offered more positional genome flexibility (van Bakel et al., 2013). Lastly, different remodelers act on specific regions and collectively produce net directionality in moving nucleosomes (Yen et al., 2012). Despite a comprehensive understanding of how nucleosomes are positioned and their driving forces, the functional consequences of nucleosome positioning remain elusive. Remodelers can mobilize nucleosomes at promoters to adjust the accessibility of DNA binding sites for transcription factors, which in turn influence the recruitment of co-activators/co-repressors (Li et al., 2007a). However, at coding regions, the situation is rather counter-intuitive. The protection/exposure model cannot be applied as easily because most cryptic transcription factor binding sites or cryptic promoters are not located according to transcription units. Why do cells employ a large number of ATP-dependent remodelers to position nucleosome at these regions?

The Set2-Rpd3S pathway is important for maintaining chromatin integrity and suppressing cryptic transcription at coding regions (Li et al., 2007a). The Rpd3S histone deacetylase complex is recruited by elongating RNA pol II to remove histone acetylation in a histone H3K36me-dependent manner (Carrozza et al., 2005; Drouin et al., 2010; Govind et al., 2010; Joshi and Struhl, 2005; Keogh et al., 2005). Recently, Set2-mediated K36me was also shown to suppresses histone exchange (Venkatesh et al., 2012), in part by recruiting chromatin remodeler Isw1. com to transcribed genes (Smolle et al., 2012). K36me3 and K36me2 are both responsible for directing the function of Rpd3S (Chu et al., 2007; Li et al., 2009; Youdell et al., 2008). The level of K36me3 is proportional to the transcription frequency of underlying genes (Pokholok et al., 2005) while K36me2 displays a uniform level at transcribed regions (Rao et al., 2005). Therefore, a fraction of nucleosomes should not contain any K36me2 or K36me3 at less frequently transcribed regions, where the Set2-Rpd3S pathway is more critical (Li et al., 2007b). How can Rpd3S act efficiently at those nucleosomes that lack essential signals? Given that Rpd3S binds to two nucleosomes at a time (Huh et al., 2012), we postulated that methylated nucleosome may direct Rpd3S function at unmethylated neighboring nucleosomes. We began our investigation by examining if the linker length of dinucleosomes affects Rpd3S function. We showed that the binding of Rpd3S to dinucleosomes and its catalytic activity are sensitive to the length of linker DNA in a non-linear fashion. Intriguingly, we found that H3K36me on one nucleosome stimulates Rpd3S to deacetylate the neighboring nucleosomes when they are close. Finally, we demonstrated that chromatin remodelers enhance Rpd3S activity by altering nucleosomal spacing, suggesting that one of critical functions of chromatin remodelers is to prime chromatin configuration for subsequent histone modification reactions. We will discuss the importance of this mechanism in accurate temporal control of chromatin dynamics during the transcription elongation cycle.

Results

The binding of Rpd3S to dinucleosomes is sensitive to the length of linker DNA in a non-linear fashion

We previously showed that Rpd3S nucleosome binding and histone deacetylation activity were minimally affected by nucleosome positioning when mononucleosomes were used as substrates (Huh et al., 2012). Because Rpd3S prefers dinucleosomes (Huh et al., 2012), we decided to test the influence of nucleosome spacing on Rpd3S activity. To this end, we designed a series of chromatin templates that only differed by the length of their linker DNA (Figure 1A). We found that Rpd3S senses linker difference in a non-linear fashion and displays a modest preference for 30–40 bp linkers (Figure 1C, D). This binding pattern is independent of H3K36me, since unmodified dinucleosomes display a similar trend (Supp. Figure 1A, B). Even with the longest linker that we used (90 bp), the binding of Rpd3S is still much stronger on dinucleosomes than mononucleosomes, suggesting that Rpd3S simultaneously contacts two nucleosomes (Huh et al., 2012). Despite their overall weaker binding, dinucleosomes with longer linkers seem to be more conducive to the binding of the second Rpd3S complex. Moreover, changing the rotational orientations of two nucleosomes by increasing the length of the linker DNA by 5 bp (DNA ends rotating 180°) results in minimal influence on Rpd3S binding (Supp. Figure 1C), which is consistent with the symmetric nature of nucleosomes and the flexibility of histone tails.

Figure 1. The binding of Rpd3S to dinucleosomes is sensitive to the length of linker DNA in a non-linear fashion.

Figure 1

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(A) A schematic illustration of dinucleosome templates. All nucleosomes are identical except for the length of linker DNA and their positions were confirmed in (B). (B) Nucleosome positioning of reconstituted dinucleosomal templates was confirmed by Bgl1 restriction digestion. As DNA probes were 32P-labeled at the 5′ ends, only the left-side (601-L) nucleosomes and residual undigested dinucleosomes can still be visualized upon digestion. Their migration patterns indicated the correct translational positions of 601-L nucleosomes and the dinucleosomes. (C) The binding of Rpd3S to dinucleosomes with different linker DNA were measured by EMSA assays. (D) Quantification of the results from (C). Data are represented as mean +/− SEM.

H3K36me facilitates Rpd3S deacetylation of neighboring nucleosomes at proper distances

We next sought to determine if the histone deacetylation activity of Rpd3S is sensitive to linker length. We previously showed that using homogenously acetylated nucleosomes, Rpd3S activity mostly correlates with its affinity: dinucleosomes display stronger activity than mononucleosomes while methylated nucleosomes are better than unmodified ones (Huh et al., 2012). To evaluate the overall HDAC activity of Rpd3S and test if H3K36me on one nucleosome influences the neighboring unmodified nucleosome in the same assay, a new strategy was designed. Hetero-dinucleosomes were generated by ligating two mononucleosomes via a unique non-palidromic restriction site so that different patterns of PTM can be separately placed on each nucleosome (Figure 2A). Here, only the right side nucleosome (601-R or Right) contains 3H-labeled acetylated lysines (AcK) for measuring the deacetylase activity, whereas the left side nucleosome (601-L or Left) carries either unmodified or methylated H3K36 as signals. Three representative linker lengths (15 bp, 30 bp, and 70 bp) were chosen, and the positioning of the resulting dinucleosomes was confirmed by restriction digestion (Figure 2B). When overall activities of similarly modified dinucleosomes were compared, Rpd3S activity correlated with its affinity to nucleosomes (Supp. Figure 2A–C). More importantly, when we compared the activities of dinucleosomes with the same linker but different PTM patterns (Figure 2C–E), a clear neighboring effect was observed. For dinucleosomes with shorter linkers (Figure 2C, D; green lines vs. red lines), H3K36me on 601-L significantly stimulated Rpd3S to deacetylate adjacent unmethylated 601-R. To our surprise, the neighboring effect was lost when the linker was extended to 70 bp (Figure 2E), although the affinity of Rpd3S to this template as measured by EMSA clearly indicated that Rpd3S still contacts both nucleosomes (Figure 1C and 2H). Furthermore, H3K36me does not stimulate Rpd3S HDAC activity in trans (Supp. Figure 3). These data collectively suggest that H3K36me can broadly recruit Rpd3S to the dinucleosome templates (Figure 2F–H); however, its stimulatory effects on Rpd3S catalytic activity are restricted to close neighbors. As a control, we showed that when H3K36me and acetylation were placed on the same nucleosome (601-R) within the 70 bp dinucleosomes, H3K36me can effectively elevate Rpd3S activities (Figure 2E; blue vs. red), indicating that the orientation of Rpd3S binding to dinucleosomes is important for its enzymatic outcome.

Figure 2. Histone H3K36 methylation regulates Rpd3S HDAC activity toward neighboring nucleosomes within proper linker distance.

Figure 2

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(A) Preparation of hetero-dinucleosome templates. Gel-purified mononucleosomes (designated as Right or 601-R) containing either unmodified (white-filled) or K36me3 (black-filled) histone H3, were labeled with 3H acetyl-CoA using Ada2-TAP (H3 acetylation) and NuA4 (H4 acetylation). 601-R nucleosomes were ligated at a Bgl1 site to 601-L nucleosomes that carry various lengths of linker DNA. The resulting dinucleosomes were gel-purified and directly applied to HDAC assays in (C–E) or end-labeled using 32P-γATP for EMSA assays in (F–H). (B) Confirmation of proper nucleosome positioning of the hetero-dinucleosomes. The restriction analyses were performed similarly to that described in Figure 1B, except that in this case both ends of the DNA probes were labeled. Therefore, both digested mononucleosomes can be seen. Lane 19–26 contain the mono-nucleosomes before they were ligated to form dinucleosomes, and serve as marks for the correct migration pattern of digested dinucleosomes. (C–E)Histone deacetylase assays showing that the HDAC activity of Rpd3S toward neighboring nucleosomes depends on the length of linker DNA. Data are represented as mean +/− SEM. (F–H) The binding affinity of Rpd3S to each hetero-dinucleosome was measured using gel mobility shift assays. Data are represented as mean +/− SEM.

To address Rpd3S binding orientation and its primary deacetylating targets, we asked if the preferred function of Rpd3S is to recognize H3K36me nucleosomes and then deacetylate neighboring nucleosomes or to deacetylate H3K36me nucleosomes directly. To this end, we created dinucleosomes that are equally modified except that different mononucleosomes are labeled with 3H-AcK for detection in HDAC assays (Figure 3A). Sequential acetylation was carried out first using radioactive or cold acetyl CoA, then cold acetyl CoA. Equal acetylation on all mononucleosomes was confirmed prior to ligation (Figure 3B). Two types of dinucleosomes were generated: one containing H3K36me within the 3H-AcK nucleosomes (the end product on the left in Figure 3A); and the other one carrying H3K36me and 3H-AcK on separate nucleosomes (the end product on the right in Figure 3A). As expected, we found that 30 bp dinucleosomes displayed the highest overall activity (the total of black and grey bars in Figure 3D). Since the black bars were higher than the grey bars in all three cases (Figure 3D), this result suggested that Rpd3S by default prefers H3K36 methylated nucleosomes within dinucleosome templates. Interestingly, given an optimal linker (30 bp), Rpd3S is capable of deacetylating both nucleosomes similarly (Figure 3D middle). When the two nucleosomes are too close (15 bp), Rpd3S showed less activity towards the K36me neighbor nucleosomes. However, when two nucleosomes are far apart, Rpd3S only deacetylates methylated nucleosomes but not unmethylated neighbors (Figure 3D). The low activity (with 70 bp linker length; grey bar) was resulted from H3K36me-independent binding of Rpd3S to dinucleosomes. In summary, these data suggest that Rpd3S primarily deacetylates K36me nucleosomes, and its activity towards neighbors depends on linker length (Figure 3E). For dinucleosomes separated by a long linker, although Rpd3S is flexible enough to bind to two nucleosomes simultaneously, it may be difficult for its catalytic subunit Rpd3 to reach the neighboring nucleosomes for deacetylation (Figure 3E).

Figure 3. Deacetylation preference of Rpd3S on dinucleosome substrates.

Figure 3

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(A) The strategy to generate hetero-dinucleosome templates in which each nucleosome contains an equal amount of acetyl-groups. During the first round of the acetylation reaction, 3H-labeled acetyl-CoA or cold acetyl-CoA were used for either 601-L nucleosomes or 601-R nucleosomes. Subsequently, additional acetylation was carried out for all mononucleosomes using cold acetyl-coA to reach saturated acetylation levels on all nucleosomes. After ligation and gel-purification, the resulting two types of dinucleosomes are identical in total acetylation status except for 3H-labeling depending on the reaction. (B) The acetylation levels of each mononucleosome after two rounds of acetylation were monitored by western blotting using anti acetylated H3 or acetylated H4 antibodies. An antibody against histone H4 was used to demonstrate the loading consistency. (C) The final hetero-dinucleosomes prepared above were resolved on a 3.5% acrylamide gel, and the dinucleosome concentrations were quantified based on the amount of DNA. (D) Quantification of HDAC results based on three independent experiments. Data are represented as mean +/− SEM. (E) A model for different mechanisms of action of Rpd3S on dinucleosomes containing different linker DNA. The thicknesses of the arrows reflect the strength of HDAC activity. 30bp (mid) is near optimal distance in that Rpd3S can efficiently deacetylate both methylated nucleosome and neighboring unmethylated nucleosome; 15bp (left) represents the situation where two nucleosomes are too close and Rpd3S can function on neighboring nucleosomes but with less efficiency; At 70bp (right) distance, Rpd3S can no longer deacetylate neighboring nucleosomes.

Chromatin remodelers fine-tune the Rpd3S HDAC activity by altering nucleosomal spacing

Many chromatin remodeling complexes have been shown to regulate nucleosome spacing at transcribed genes where Rpd3S functions (Struhl and Segal, 2013). Since Rpd3S prefers a certain linker length, we speculate that chromatin remodelers may fine-tune Rpd3S activity by adjusting the spacing between nucleosomes. To test this hypothesis, we utilized a dinucleosome template (70 bp) with which H3K36me fails to facilitate Rpd3S deacetylation of neighbors (Figure 2). Our rationale is that if mobilization of nucleosomes by chromatin remodelers activates the neighboring effect, we would be able to detect increased HDAC activity (Figure 4A). Multiple ATP-dependent remodeling complexes that are functionally relevant in vivo (as discussed later) were chosen. As shown in Figure 4B, we detected a significantly increased HDAC activity of Rpd3S after incubating dinucleosomes with chromatin remodelers in the presence of ATP. However, when similar experiments were performed with mononucleosomes, these remodelers did not induce any evident changes (Figure 4C), suggesting that remodeling per-se is not sufficient to mediate the observed stimulation. This result also ruled out the possibility that momentarily increased accessibility of histones during the remodeling process gave rise to higher Rpd3S activity.

Figure 4. Chromatin remodeling factors fine-tune the Rpd3S HDAC activity on dinucleosomes by altering nucleosomal spacing.

Figure 4

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(A) The experimental design. (B) Rpd3S HDAC activity on dinucleosomes is enhanced by indicated chromatin remodeling complexesin an ATP-dependent manner. Data are represented as mean +/− SEM. (C) Chromatin remodeling per-se does not increase Rpd3S activity. Similar experiments were performed as in (B) except that mono-nucleosomes with indicated modification pattern were used as substrates. Data are represented as mean +/− SEM. (D–F) ISWI family of ATPases mediates nucleosome sliding on dinucleosome templates. (D) Chromatin remodeling assay using dinucleosome substrates containing a 70 bp linker DNA. The reactions were directly loaded on a 3.5% native PAGE gel to resolve dinucleosome populations that are positioned differently. (E) Predicted positioning changes of each nucleosome caused by Isw1-TAP and ACF-mediated dinucleosome sliding. Restriction sites Not1 and Bgl1, which are just outside each nucleosome, are used to monitor nucleosome movement. (F) Restriction site protection assays for dinucleosome sliding suggest that both nucleosomes moved toward the center. Following the remodeling assay described above, the reaction mixtures were subjected to restriction digestion with Not1 or Bgl1. The resulting digested and undigested nucleosomes were resolved on a 4% PAGE gel.

To directly measure ISWI-mediated chromatin remodeling activity on dinucleosomes, we performed EMSA-based sliding assays. As shown in Figure 4D, the migration patterns of nucleosomes after reactions indicated that remodeled populations remained at the dinucleosome state (Figure 4D). We performed restriction enzyme mapping to examine the final states of dinucleosomes remodeled by ISWI. As illustrated in Figure 4E, the Not I and Bgl I cutting sites are just outside of the nucleosome boundary, thus any movement of the nucleosome toward the center would block the access of enzymes to their targets. The digestion patterns after remodeling reactions suggest that both nucleosomes move toward each other (Figure 4F). Therefore, we conclude that chromatin remodelers facilitate Rpd3S deacetylation by mobilizing nucleosome arrays to the optimal spacing.

Discussion

Nucleosome spacing matters

In this study, we demonstrated that optimal spacing of nucleosome arrays is not only important for the overall binding and deacetylase activity of Rpd3S, but also controls the ability of Rpd3S to function at nucleosomes adjacent to its K36me targets. Indeed, the sensitivity to linker length seems to be a common property for other chromatin regulators. For instance, Heterochromatin protein 1 (HP1) preferentially binds to arrays with short DNA linkers (Canzio et al., 2011); the PRC2 histone methyltransferase is more active on dense nucleosomal arrays because the neighboring histone H3 can allosterically stimulate EZH2 catalytic activity (Yuan et al., 2012). For all three examples above, chromatin binding complexes form a bridge between nucleosomes. HP1 is a relatively small protein that needs to oligomerize to mediate the connection (Canzio et al., 2011), whereas Rpd3S and PRC2 exist in multi-subunit complexes and can achieve this as monomeric forms (Ciferri et al., 2012; Huh et al., 2012). The structures of both complexes appear to be very flexible (Ciferri et al., 2012) (unpublished results, Ruan, Li and Asturias), giving them the potential to accommodate different chromatin configurations. In contrast to general perceptions, linker DNA seems to be a rather rigid structure. DNA twistability and bendability have been measured by various methods with some minor discrepancies (Savelyev et al., 2011). However, the general consensus is that the binding persistence length of dsDNA ranges from 90 bp to 150 bp, meaning that DNA shorter than this length would be almost straight and require great force to bend or twist significantly (Geggier and Vologodskii, 2010; You et al., 2012). Therefore, the physiological length of linker DNA is typically shorter than 90 bp, resulting in inflexibility of the linker. The concept of a rigid DNA linker is highly consistent with the length dependence of various complexes that we described above and that have been reported by others (Canzio et al., 2011; Yuan et al., 2012). Therefore, it is the chromatin regulators that must adapt to different chromatin conformations to achieve optimal binding (Figure 3E). Interestingly, two repressive factors (PRC2 and HP1) appear to prefer shorter linkers and compact arrays; whereas Rpd3S functions at actively transcribed genes and favors a longer spacer length, which may be conducive for a dynamic process during multiple rounds of transcription.

Remodelers prime chromatin structure for subsequent modifications

Here, using a defined biochemical system, we demonstrated that chromatin remodelers can fine-tune the structure of nucleosomal arrays and thus control the activity of downstream chromatin regulators (Figure 4). This mechanism appears to be shared by other chromatin modification enzymes as well. Chromatin remodeler ACF regulates nucleosomal spacing and is required for PRC2-mediated repression (Fyodorov et al., 2004). The chromosome binding of histone acetyltransferase complex ATAC and histone H4-K12 acetylation are compromised in ISWI mutants, suggesting that NURF is required for ATAC to access the chromatin (Carre et al., 2008). Histone deacetylase complex SHREC contains an ATPase subunit Mit1, which is also required for full functionality of SHREC (Sugiyama et al., 2007).

The neighboring effect enables Rpd3S to work efficiently at regions where not all nucleosomes are methylated

Rpd3S can efficiently deacetylate unmethylated nucleosomes that are close to its K36me targets. This feature is a crucial for Rpd3S, considering its essential function at less frequently transcribed genes (Li et al., 2007b) where not all nucleosomes are methylated at H3K36. Because of its sensitivity to nucleosome spacing, our model predicts that if nucleosome spacing is compromised upon deletion of the Isw1 remodeler, Rpd3S would not function properly. This should in turn lead to increased acetylation levels at coding regions. Indeed, a recent genome-wide study demonstrated that deletion of ISW1 increases histone acetylation levels at transcribed genes (Smolle et al., 2012). Based on these results, we propose a model to interpret how Rpd3S and chromatin remodelers coordinate in order to maintain the integrity of transcribed chromatin. Upon the passage of RNA pol II, nucleosomes need to be re-assembled with the help of histone chaperones that are associated with elongating Pol II (Venkatesh et al., 2012). The Isw1 remodeler prevents incorporation of newly synthesized histones, which are hyper-acetylated, and instead favors recycling locally displaced histones (Smolle et al., 2012). Upon the completion of the nucleosome, Isw1 and other spacing factors slide the nucleosome to a proper position. Then, Rpd3S removes acetylation marks and stabilizes the nucleosome by reducing its fluidity. In one scenario, deacetylation locks a nucleosome into a fixed position, and the preference of Rpd3S serves as a part of the ruler for nucleosome linker length. Alternatively, deacetylated nucleosomes can be further mobilized by other remodeling factors to their eventual position. In the latter case, the preferred distance for Rpd3S (30–40 bp) may not necessarily be equal to the steady-state nucleosomal linker length.

Experimental Procedures

All chromatin templates, plasmids and primers are listed in the Supplemental Tables 1, 2 and 3 respectively. Procedures for protein purification, plasmid construction, nucleosome template preparation, EMSA assays, the nucleosome-based HDAC assay and the remodeling-coupled HDAC assay are provided in the supplemental experimental procedures.

Supplementary Material

01

Highlights.

  • Rpd3S senses linker length of dinucleosomes in a non-linear fashion.

  • H3K36me facilitates Rpd3S to deacetylate close proximity neighboring nucleosomes.

  • Chromatin remodelers fine-tune nucleosome spacing to control Rpd3S HDAC activity.

Acknowledgments

We thank Dr. Kadonaga for providing baculovirus constructs for ACF purification and Dr. Harrod for editorial assistance. BL is a W.A. “Tex” Moncrief, Jr. Scholar in Medical Research and is supported by grants from the National Institutes of Health (R01GM090077), the Welch Foundation (I-1713), the March of Dimes Foundation, and the American Heart Association.

Footnotes

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