extfieldsofstudy: Biology Medicine

text:
Active VSG Expression Sites in Trypanosoma brucei Are Depleted of Nucleosomes

ABSTRACT African trypanosomes regulate transcription differently from other eukaryotes. Most of the trypanosome genome is constitutively transcribed by RNA polymerase II (Pol II) as large polycistronic transcription units while the genes encoding the major surface proteins are transcribed by RNA polymerase I (Pol I). In bloodstream form Trypanosoma brucei, the gene encoding the variant surface glycoprotein (VSG) coat is expressed in a monoallelic fashion from one of about 15 VSG bloodstream form expression sites (BESs). Little is known about the chromatin structure of the trypanosome genome, and the chromatin state of active versus silent VSG BESs remains controversial. Here, we determined histone H3 occupancy within the genome of T. brucei, focusing on active versus silent VSG BESs in the bloodstream form. We found that histone H3 was most enriched in the nontranscribed 50-bp and 177-bp repeats and relatively depleted in Pol I, II, and III transcription units, with particular depletion over promoter regions. Using two isogenic T. brucei lines containing marker genes in different VSG BESs, we determined that histone H3 is 11- to 40-fold depleted from active VSG BESs compared with silent VSG BESs. Quantitative PCR analysis of fractionated micrococcal nuclease-digested chromatin revealed that the active VSG BES is depleted of nucleosomes. Therefore, in contrast to earlier views, nucleosome positioning appears to be involved in the monoalleleic control of VSG BESs in T. brucei. This may provide a level of epigenetic regulation enabling bloodstream form trypanosomes to efficiently pass on the transcriptional state of active and silent BESs to daughter cells.

In eukaryotes nuclear DNA is packaged into linear arrays of nucleosomes, which provides one of the main determinants of accessibility to DNA binding proteins (37,60,68,82). Nucleosomes consist of ϳ146 bp of DNA wrapped around a histone octamer composed of two copies each of histones H2A, H2B, H3, and H4 (44). The ability to change how DNA is packaged within nucleosomes allows variation in the accessibility of different DNA binding sites and permits fine modulation of promoter activity (33). Recently, there have been major advances in our understanding of how chromatin structure impacts the regulation of RNA polymerase II (Pol II) transcription units. New developments (72) have enabled the determination of the global organization of nucleosomes in organisms including budding yeast, Drosophila, and humans (3,35,36,48,71,83). These studies have shown evidence for nucleosome depletion at transcriptionally active regulatory regions, with the level of nucleosome occupancy inversely proportional to the rate of transcription initiation at the promoter (3,36,83). However, nucleosome remodeling at promoters does not always appear to be simply a consequence of transcriptional activity but is also thought to mechanistically regulate transcription through modulating the access of trans-acting factors (74).
Our understanding of the role that chromatin structure plays in the regulation of RNA Pol I transcription has relatively lagged behind that of Pol II (reviewed in references 6, 21, and 49). This is in part due to the repetitive nature of the nearly identical ribosomal DNA (rDNA) transcription units, which in Saccharomyces cerevisiae (and presumably most eukaryotes) are the only transcription units transcribed by Pol I (54). Approximately half of the rDNA repeats are transcriptionally active at any time (11)(12)(13). Silencing of the inactive rDNA units is mediated by the nucleolar remodeling complex (NoRC) (69), which silences the inactive rRNA genes by changing nucleosome positioning (38,77). Despite the clear role that chromatin remodeling plays in transcriptional regulation of the rRNA, the precise nature of the chromatin present in active versus silent rRNA transcription units remains unclear (49). Studies using psoralen cross-linking (11) or more recent analyses combining this with chromatin endogenous cleavage (ChEC) (50) have suggested that transcriptionally active rDNA units are essentially devoid of nucleosomes. However, other recent studies have argued that active rDNA has a nucleosomal structure (30).
In African trypanosomes the majority of the trypanosome genome is constitutively transcribed as extensive Pol II transcription units. Very few regulatory regions have been defined, and only a few promoters have been well characterized. Trypanosomes appear to largely lack gene regulation at the level of transcription and, instead, rely on posttranscriptional control acting at the level of RNA processing and stability (10). Due to this very different way of regulating expression of their genes, it is unclear whether nucleosome positioning plays a role in trypanosomes similar to that of other eukaryotes.
Trypanosomes are further unconventional in that they use Pol I to transcribe not only the multicopy rDNA but also the genes encoding their major surface proteins: variant surface glycoprotein (VSG) in the bloodstream form or procyclin in the procyclic (insect mid-gut stage) form (22,32,64). This unique ability to use an unorthodox RNA polymerase to transcribe some of their protein-coding genes is presumably made possible by trans-splicing, which adds a capped 39-nucleotide spliced leader (SL) RNA to the 5Ј end of each mRNA, thereby making an uncapped Pol I-derived transcript available for translation (34,39,42,47). In order to provide the large amount of SL RNA necessary for trans-splicing, the trypanosome has up to 200 SL RNA transcription units which are highly transcribed by Pol II. The SL RNA promoter is the only Pol II promoter in trypanosomes which has been well characterized (14,23,70).
In bloodstream form T. brucei antigenic variation of a VSG coat is used to escape host antibodies. This is accomplished through monoallelic exclusion of VSG expression, whereby within a single cell only one of about 15 telomeric VSG blood stream form expression sites (BESs) is transcribed at a time by Pol I (7,9,58). The nature of the chromatin structure of VSG BESs has been a controversial issue. Earlier studies have argued that there are no detectable differences in nucleosomal organization between active and silent VSG BESs, although active VSG BESs are sensitive to digestion by endonucleases including single-strand-specific endonucleases (20,51,57). Studies using exogenous T7 RNA polymerase as a probe for chromatin accessibility also did not find evidence that active VSG BESs in bloodstream form T. brucei were more accessible for transcription than inactive ones (52). Recently, however, the role of chromatin in the downregulation of silent BESs is being reevaluated as chromatin remodeling proteins have been shown to be important for VSG BES control (15). First, the chromatin remodeling protein T. brucei ISWI (TbISWI) has been shown to be important for VSG BES silencing in both bloodstream and insect forms of T. brucei (28). In addition, downregulation of the histone modification protein DOT1B affects the kinetics of VSG BES switching, arguing that histone modification plays a role in monoallelic transcription (16).
The way in which African trypanosomes regulate transcription is unique compared with most eukaryotes, and little is known about the chromatin structure of the trypanosome genome. Here, we investigated nucleosome distribution within the genome of both bloodstream and insect forms of T. brucei. In general, we find that nucleosomes are enriched on the nontranscribed repeat arrays and are depleted from the promoter regions of a variety of different transcription units (including examples transcribed by Pol I,  II, and III) in both bloodstream and insect forms of T. brucei. We used two isogenic bloodstream form T. brucei lines containing marker genes in silent and active VSG BESs to investigate whether differences in chromatin structure exist between VSG BESs when they are either transcriptionally active or silent. Strikingly, we find that the active VSG BES in bloodstream form T. brucei is particularly depleted of nucleosomes compared with all other regions analyzed and shows a more open chromatin structure than silent VSG BESs. This difference may provide a level of epigenetic control whereby bloodstream form trypanosomes are able to efficiently pass on the transcriptional states of active and silent BESs to daughter cells.

MATERIALS AND METHODS
Trypanosome strains and culturing. Bloodstream form and procyclic-form T. brucei subsp. brucei strain 427 was used for all experiments. Wild-type procyclic cells were grown at 27°C in SDM-79 medium containing 10% fetal bovine serum (8). Bloodstream form trypanosomes were grown at 37°C in HMI-9 medium containing 10% fetal bovine serum and 10% SerumPlus (SAFC Biosciences) (26). Two isogenic bloodstream form T. brucei cell lines containing a hygromycin resistance gene downstream of the promoter of the VSG221 BES and a neomycin resistance gene downstream of the promoter of the VSGVO2 BES were used (T. brucei HNI). In T. brucei HNI (221ϩ) the VSG221 BES was active, and the VSGVO2 BES was silent, while in T. brucei HNI (VO2ϩ) the VSGVO2 BES was active, and the VSG221 BES was silent (66).
ChIP. Chromatin immunoprecipitation (ChIP) was performed essentially as described previously (43). In brief, cultures of T. brucei were fixed in 1% formaldehyde for 20 min at room temperature. The reaction was stopped by the addition of glycine to a final concentration of 125 mM. Chromatin was sonicated to an average DNA size of about 200 to 500 bp using a BioRuptor (Wolf Laboratories) and clarified by centrifugation at 15,000 ϫ g for 5 min at 4°C. The sonicated extract was precleared using protein A-Sepharose CL-4B beads (GE Healthcare) and incubated for 16 h either with or without the relevant antihistone antibody. Chromatin from 7 ϫ 10 7 cell equivalents was used per immunoprecipitation (IP). Antibodies used for the ChIP experiments include antihistone H3 (ab1791; AbCam) at a concentration of 2 g per IP, anti-T. brucei histone H4 (kind gift of the George Cross laboratory) at a concentration of 10 g per IP (75), and anti-histone H2A (07-146; Upstate) at a final dilution of 1:58. The protein-DNA complexes were incubated with protein A-Sepharose CL-4B beads for 2 h and eluted from the beads after a wash in 1% SDS-0.1 M NaHCO 3 preheated to 65°C. The cross-links were reversed by adding NaCl to a final concentration of 325 mM and incubated at 65°C overnight. Following RNase A and proteinase K treatment, DNA was purified using a QIAquick PCR purification kit (Qiagen). Each ChIP experiment was performed two or three times, as indicated in the figure legends, and the results were analyzed by hybridization of slot blots or quantitative PCR (qPCR).
Slot blot analysis. ChIP material was analyzed on slot blots using radiolabeled probes. Twenty microliters of input chromatin (10% of total) and immunoprecipitated DNA was diluted in TE (Tris-EDTA) buffer (pH 8.0) and denatured by heating to 65°C for 30 min in a final concentration of 0.3 M NaOH. The denatured DNA were neutralized by the addition of ammonium acetate, pH 7.0, to a final concentration of 1.0 M and immediately transferred to Hybond XL membrane (GE Healthcare) using a Schleicher and Schuell Minifold II slot blot apparatus. Membranes were hybridized with a probe for the 50-bp repeats directly flanking VSG BESs (84) or the 177-bp repeats comprising the bulk of the T. brucei minichromosomes (81). The radioactive probes were labeled with [␣-32 P]dCTP using Ready To-Go DNA Labeling Beads and Illustra ProbeQuant G-50 Micro Columns according to the manufacturer's instructions (GE Healthcare). Membranes were hybridized with radiolabeled probes at 42°C overnight and washed to an end stringency of 0.3ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Blots were exposed to a PhosphorImager screen (Bio-Rad), and the percentage of DNA immunoprecipitated was determined using Quan-tityOne software (Bio-Rad).
Quantitative PCR. The ChIP material obtained was analyzed using qPCR. Input chromatin (10% of total) and immunoprecipitated material were analyzed using the primers listed in Table S1 in the supplemental material. All qPCR samples were amplified in triplicate in a 20-l total reaction volume containing 19 l of qPCR reaction mix (LightCycler480 SYBR green Master Mix; Roche), 0.5 M (each) forward and reverse primers, and 1 l of the DNA template. Reaction mixtures were amplified using a LightCycler480 Real-Time PCR System (Roche) according to the manufacturer's instructions. All PCR primers and conditions were optimized to produce a single amplicon of the correct size.
Micrococcal nuclease digestion of T. brucei chromatin. Digitonin was used to permeabilize bloodstream form T. brucei prior to micrococcal nuclease (MNase) treatment, as previously described (43,51). In brief, 3 ϫ VSG EXPRESSION SITE CHROMATIN STRUCTURE 137 10 5 cells l Ϫ1 . Six units of MNase (Worthington Biochemicals) was added to the cell suspension (two units per 1 ϫ 10 8 cells) and incubated for 5 min at 37°C. The reaction was stopped by the addition of EDTA and EGTA to a final concentration of 10 mM. Nuclei were pelleted at 10,000 ϫ g for 10 min at 4°C, and the supernatant was discarded. Chromatin was solubilized by resuspending the nuclear pellet in 200 l of RSB buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl 2 , 5 mM EDTA, 5 mM EGTA, 0.05% NP-40, 200 mM NaCl). Cell debris was removed by centrifuging at 10,000 ϫ g for 10 min at 4°C. A 15-l sample of the supernatant was removed as an input control, and the remaining supernatant was loaded immediately on a preprepared 5 to 30% sucrose gradient, as described below. Sucrose density gradient fractionation of multinucleosomes. MNase-digested DNA was separated into mono-, di-, tri-, and tetranucleosome fractions using 5 to 30% sucrose gradients, essentially as described previously (24). Discontinuous sucrose gradients were prepared containing 2 ml each of 5, 10, 15, 20, 25, and 30% sucrose solution in gradient buffer (10 mM Tris-HCl [pH 7.5], 0.25 mM EDTA, 100 mM NaCl, 0.1 mM PMSF, and 0.5 g/ml each of pepstatin A, leupeptin, and aprotinin) 24 h prior to use and stored at 4°C. The supernatant from the MNase treatment was carefully loaded on the 5 to 30% sucrose gradient and centrifuged at 40,000 rpm (Beckman SW41 Ti rotor) for 16 h at 4°C. Twenty-four 500-l fractions were isolated. Proteins were removed by treating fractions with proteinase K for 30 min at 37°C, followed by phenol-chloroform and chloroform extractions and ethanol precipitation. Aliquots from each of the MNase-digested DNA fractions were analyzed on a 2% agarose gel for the presence of mono-and multinucleosomes. Fractions containing di-/trinucleosomes (fractions 18 to 21) and tri-/tetranucleosomes (fractions 22 and 23) were pooled for analysis by qPCR using the primer pairs indicated in the figures, sequences of which can be found in Table S1 in the supplemental material. The data were plotted as the fold difference between T. brucei HNI (221ϩ) and T. brucei HNI (VO2ϩ). Error bars in the figures reflect standard deviations of the average signal obtained from three independent experiments.

Histone H3 is enriched in nontranscribed repeat sequences compared with regions highly transcribed by Pol II or Pol III.
Most of the trypanosome genome is transcribed as extensive polycistronic transcription units extending for many tens of kilobases (10,75), with relatively few regions remaining nontranscribed. Notable exceptions which are kept transcriptionally silent include the 50-bp and 177-bp repeat arrays. The 50-bp repeats form the upstream flanks of all known VSG BESs and typically extend for 40 to 50 kb (66,73,84). The 177-bp repeat arrays comprise the bulk of the transcriptionally silent T. brucei minichromosomes (81). This highly abundant class of about 100 small chromosomes (each ranging between 50 to 150 kb) makes up about 10% of the T. brucei nuclear genome (81).
To determine the distribution of histone H3 over different types of genomic regions in T. brucei, we first looked at histone H3 occupancy over the nontranscribed 50-bp and 177-bp repeat arrays. Histones are extremely well conserved within eukaryotes, and an antibody against the C terminus of human histone H3 specifically recognizes T. brucei histone H3 (46). Chromatin immunoprecipitation (ChIP) using formaldehyde cross-linked T. brucei chromatin was performed with the antihistone H3 antibody or no antibody as a negative control. Input chromatin (10% of total) and immunoprecipitated material were analyzed by slot blot using radiolabeled probes for either the 50-bp repeats or the 177-bp repeats (Fig. 1A). The signal on the blots was quantitated, and values obtained from the no-antibody control were subtracted from the values obtained using the anti-histone H3 antibody. In both bloodstream form and procyclic-form T. brucei, about 8% of the input DNA was immunoprecipitated with anti-histone H3 antibody, showing that histone H3 is very abundant in these nontranscribed regions ( Fig. 1D).
We next looked at the distribution of histone H3 on actively transcribed regions of the T. brucei genome. As with the majority of genes in T. brucei, the genes encoding the RNA polymerase I large subunit (Pol I) and ␥-tubulin are present in large polycistronic transcription units constitutively transcribed by Pol II in both bloodstream form and procyclic form T. brucei. Quantitative PCR (qPCR) was used to determine the relative abundance of these two genes in the material immunoprecipitated with the anti-histone H3 antibody. The primers used for the qPCR experiments are shown in Table S1 in the supplemental material. In both life cycle stages, about 5 to 6% of the input DNA was immunoprecipitated (Fig. 1D), indicating that these Pol II-transcribed genes contain less histone H3 and are in a more open chromatin conformation than the silent repeats.
In trypanosomes, maturation of the precursor mRNAs requires the addition of a 39-nucleotide spliced leader (SL) RNA onto the 5Ј end of every mRNA (39). In order to provide the large amount of SL RNA necessary for trans-splicing, the trypanosome has up to 200 SL RNA transcription units which are transcribed by RNA Pol II (19,70). The repetitive SL genes are highly transcribed regions in the T. brucei genome, and are the only Pol II-transcribed genes with a well-characterized promoter (14,23,70). Here, we used ChIP followed by qPCR to determine the distribution of histone H3 in the genomic region containing the SL RNA transcription unit repeats. Figure 1B depicts a portion of a genomic region containing 10 to 11 tandem SL RNA transcription units, as well as a retrotransposable element called the SL-associated conserved sequence (SLACS), which has integrated exclusively in the SL RNA genes (1,55). Using ChIP-qPCR, we found that the SLACS elements contain amounts of histone H3 similar to those of the genes encoding the Pol I large subunit or ␥-tubulin, which are also transcribed in both life cycle stages by Pol II (Fig. 1D). However, approximately 3-fold less histone H3 was found on the SL gene promoters, and about 2-fold less histone H3 was present on the SL RNA genes themselves or the SL RNA spacer region (Fig. 1D). This indicates that the SL RNA transcription units are more transcriptionally open than those areas containing SLACSs.
We next determined the distribution of histone H3 across the 5S rDNA transcription units transcribed by RNA Pol III. 5S rRNA is an integral component of the large subunit of ribosomes, and a schematic depicting part of a region of eight tandem 5S rRNA transcription units is shown in Fig. 1C. The histone H3 distribution across the 5S rDNA region was slightly lower than that found within the Pol II-transcribed polycistronic transcription units and was constant between both T. brucei life cycle stages (Fig. 1D).
Histone H3 is particularly depleted around the promoters of the Pol I-transcribed rDNA transcription units. It has been shown in a variety of eukaryotes that the ribosomal DNA transcription units are transcribed at a very high rate by Pol I. In T. brucei there are about 11 to 12 rDNA transcription units arranged over six to seven chromosomes (4). In order to determine the distribution of histone H3 across these highly transcribed rDNA transcription units, we designed qPCR primers spanning the length of an rDNA transcription unit as 138 STANNE AND RUDENKO EUKARYOT. CELL on February 11, 2021 by guest http://ec.asm.org/ well as in the nontranscribed rDNA spacer (80) ( Fig. 2A). We performed ChIP-qPCR in both bloodstream form and procyclic-form T. brucei and found that the rDNA promoter region contains approximately 3-fold less histone H3 (approximately 1 to 2% input precipitated) than the nontranscribed rDNA spacer (5 to 6% of the input was immunoprecipitated) (Fig.  2B). This is compatible with an open chromatin structure that is depleted of nucleosomes immediately around the transcriptionally highly active Pol I promoters. Additionally, a gradient of histone H3 distribution was found along the rDNA transcription unit, with levels that were lowest immediately downstream of the promoter and gradually increasing up to those observed in the spacer region (Fig. 2B). Histone H3 is depleted from the procyclin gene promoters in both bloodstream form and procyclic form T. brucei. In most eukaryotes RNA Pol I exclusively transcribes rDNA transcription units. However, T. brucei is unique in its ability to use Pol I to transcribe the major cell surface proteins of both procyclic form and bloodstream form trypanosomes (22,32,64). The surface coat of procyclic form trypanosomes is composed of members of the procyclin family of glycoproteins including the EP and GPEET procyclin variants (62,63), of which one EP procyclin transcription unit is depicted in Fig. 3A. Using ChIP-qPCR analysis, we found that histone H3 is more than 3-fold depleted on the procyclin EP promoter in both bloodstream and procyclic T. brucei (less than 1.5% ChIP material immunoprecipitated) compared with regions upstream of the EP procyclin transcription unit (Fig. 3B). The EP procyclin genes contain more histone H3 than the promoter region (about 3% of input immunoprecipitated) (Fig. 3B); however, this is 2-fold less than that observed in genes present in the Pol II-transcribed polycistronic arrays, indicating a more open chromatin structure (Fig. 1D).
Analysis of these two Pol I transcription units (rDNA and procyclin) is complicated by the fact that the genes within them are present in multiple, nearly identical copies. In a number of other eukaryotes, it has been shown that about half of the rDNA transcription units are transcriptionally active (11). In T. brucei this is also likely to be the case, although this issue has not yet been resolved. Multiple procyclin transcription units are also present, and within a single cell multiple units can be simultaneously transcriptionally active (reviewed in reference 62). In order to better study the chromatin structure of a single Pol I transcription unit, we investigated the chromatin structure of VSG BESs since in bloodstream form T. brucei a single VSG BES is transcribed by Pol I in a monoallelic fashion (9,22). Active VSG expression sites are dramatically depleted of histones compared with silent VSG expression sites. VSG BESs are extensive telomeric transcription units typically varying be-tween 40 to 60 kb and containing a variety of expression siteassociated genes (ESAGs) in addition to the telomeric VSG gene (Fig. 4A) (25). Although different members of individual ESAG gene families are polymorphic, they remain highly similar to each other in sequence (25), which greatly complicates the analysis of BES transcriptional states. Here, we use two isogenic bloodstream form T. brucei cell lines containing different single-copy marker genes in both the active and silent VSG BES (Fig. 4B) to investigate the distribution of histone H3 along the length of VSG BESs as well as between silent and active BESs. T. brucei HNI (221ϩ) has an active VSG221 BES with a hygromycin resistance gene inserted behind the promoter and a neomycin resistance gene inserted behind the promoter of the silent VS-GVO2 BES. In T. brucei HNI (VO2ϩ) the VSG221 BES is silent, and the VSGVO2 BES is active.
Using ChIP-qPCR, we first used these cell lines to investigate the distribution of histone H3 along the length of VSG BESs. In T. brucei 427 there are approximately 15 BESs (25), all of which are silent except one. Due to the high degree of sequence similarity between BESs, most of the primer pairs used for amplifying genes within the BES would be expected to recognize most if not all BESs (BES Fig. 4C) (25). The distribution of histone H3 over BESs was found to be particularly low over the "core" BES promoter (29) (Fig. 4C, qPCR primer pair c) in isogenic T. brucei strains expressing either the VSG221 or VSGVO2 BES (Fig. 4C, red or blue bars, respectively). Levels of histone H3 over the BES core promoter were about 2-fold lower than in the region upstream of the BES promoter (primer pair a) and about 3-fold lower than levels in ESAG6/7 (primer pair e). The low level of histone H3 present at the BES core promoter is consistent with data from Figueiredo and Cross (17)  attenuates relatively rapidly downstream of the BES promoter in all except for the active BES (2,65,79).
We next investigated the distribution of histone H3 between silent and active BESs by performing ChIP-qPCR analysis on the single-copy marker genes present in our two isogenic T. brucei cell lines (Fig. 4C). We found that there is a striking 11to 40-fold difference in distribution of histone H3 on active versus silent BESs. Less than 0.5% of input was immunoprecipitated from the single-copy genes present in the actively transcribed VSG221 BES (Fig. 4C, primer pairs i to k) and VSGVO2 BES (Fig. 4C, primer pairs l and m) compared to over 4% when the same genes were silent. This indicates that the active VSG BES is in a far more open chromatin conformation than the silent BESs. As expected, in the isogenic T. brucei strain where the VSG221 BES has been silenced and the VSGVO2 BES activated, the distribution of histone H3 over the two BESs is reversed (Fig. 4C).
To determine whether other histones exhibit a similar distribution pattern between silent and active BESs, we performed additional ChIP experiments using antibodies against histone H4 and histone H2A. qPCR analysis was performed using a subset of the BES primer pairs and ␥-tubulin as a control. As observed with the histone H3 ChIP experiments, histone H4 was found to be significantly depleted (5-to 10-fold) on the active BES compared with the silent BES (Fig. 5A) while histone H2A was 2-to 4-fold depleted (Fig. 5B). Taken together, these data strongly suggest that the active BES in bloodstream form T. brucei is depleted of nucleosomes. In insect form T. brucei, all BESs are downregulated as VSG is not necessary in this life cycle stage. BES transcriptional silencing appears to be more "leaky" in procyclic form than in bloodstream form T. brucei, and significant levels of transcription extending from most, if not all, BES promoters has been reported (2,65,79). Using a subset of the BES qPCR primer pairs, we next investigated the distribution of histone H3 along BESs in procyclic-form T. brucei. Less histone H3 was present on the BES core promoter than the rest of the BES, compatible with the transcriptional activity observed in this life cycle stage (Fig. 6).
Active VSG expression sites are depleted of nucleosomes. To ensure that the results obtained from the ChIP experiments were not an artifact of cross-linking, we investigated BES chromatin structure using an alternative experimental approach. In order to confirm that the relative depletion of histones over the active BES observed in the ChIP experiments indeed reflected reduced nucleosome occupancy, we examined the relative sensitivity of the active and silent BES to digestion by micrococcal nuclease (MNase). Rather than using the traditional MNase experimental procedure requiring large numbers of cells and Southern blotting, we used an approach which involves fractionating MNase-digested chromatin with sucrose gradients, followed by qPCR. This newly developed approach has been used to establish nucleosome depletion over mammalian promoters and provides a more sensitive and quantitative way of investigating chromatin structure than older Southern blottingbased methods (18,40). Genomic areas which are depleted for nucleosomes are in a more open chromatin structure and are therefore hypersensitive to digestion by MNase. Using this reasoning, less qPCR product would be expected to be amplified from oligonucleosomal DNA template in regions of the genome that are in a relatively more open chromatin state than in those genomic regions with a more closed chromatin structure.
Chromatin from permeabilized T. brucei HNI (221ϩ) and HNI (VO2ϩ) cell lines was partially digested with MNase to yield oligonucleosomes. This preparation was then separated on 5 to 30% sucrose gradients, and DNA was isolated from the collected fractions (Fig. 7A). Fractions enriched in di-and trinucleosomes were pooled to form nucleosomal fraction I (Fig. 7A, NI), as were fractions enriched in tri-and tetranucleosomes (Fig. 7A, NII). These two pools of oligonucleosomal DNA were then subjected to qPCR using a subset of the primer pairs used in the ChIP experiments ( Fig. 7B and C) and plotted as the fold difference in the amount of DNA amplified between the two isogenic T. brucei lines. For the housekeeping genes (Pol I, ␥-tubulin, and ␣-tubulin) and two regions common to all BESs (BES promoter and ESAG6/7), equivalent  Fig. 4B. ChIP experiments were performed using an antibody against histone H4 or no antibody as a negative control. Quantitative PCR was used to analyze two regions common to all BESs: the BES core promoter (primer c in Fig. 4A) and ESAG7/6 (ESAG6 and primer e in Fig. 4A). Additionally, the five single-copy sequences, shown schematically in Fig. 4B, specific for either the VSG221 or VSGVO2 BES were analyzed: i, hygromycin; j, pseudogene; k, VSG221; l, neomycin; and m, VSGVO2. ChIP data from the VSG221-expressing cell line are indicated with red bars, and data from the VSGVO2-expressing line are shown with blue bars. As a control, histone H4 presence in the ␥-tubulin locus (␥Tub) was also analyzed. Signals are expressed as a percentage of input immunoprecipitated, with the results shown being the average of three independent experiments; the standard deviations are indicated with error bars. (B) ChIP was performed as described for panel A, except that an anti-histone H2A antibody was used. Signals from T. brucei cell lines containing an active VSG221 (red bars) or an active VSGVO2 (blue bars) gene are expressed as a percentage of input immunoprecipitated and are the average signal obtained from two independent experiments. The primers used for qPCR analysis are as indicated in panel A.
FIG. 6. Histone H3 distribution over VSG expression sites in procyclic form T. brucei. ChIP was performed in procyclic form T. brucei using either an anti-histone H3 antibody or no antibody. Quantitative PCR was used to analyze three regions common to all BESs with the primers indicated in Fig. 4A: the BES core promoter (ESProm, primer c in Fig. 4A), the area immediately downstream of the BES promoter (ESProm Dn; primer d), and ESAG7/6 (ESAG6; primer e). Additionally, the single-copy VSG221 gene (primer k in Fig. 4B) was amplified. The results are expressed as the percentage of input immunoprecipitated after the signal obtained from the no-antibody control was subtracted. Three independent experiments were performed, and the standard deviations are indicated with error bars.

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STANNE AND RUDENKO EUKARYOT. CELL on February 11, 2021 by guest http://ec.asm.org/ levels of DNA were amplified from the NI and NII fractions using both T. brucei HNI (221ϩ) and HNI (VO2ϩ), indicating that the chromatin structure within these regions does not differ between these two cell lines.
Using primers for the five single-copy genes (Fig. 4B), we found that DNA from silent BESs was about 3-fold more enriched in the di-/trinucleosomal fraction than DNA from the active BES (Fig. 7B, NI), indicating that the active BES is indeed more sensitive to digestion by MNase. Comparable results were obtained using the tri-/tetranucleosomal fraction (Fig. 7C, NII). This result is similar to that observed in the ChIP experiments using anti-histone antibodies and confirms that the active BES is depleted of nucleosomes. As a control, qPCR analysis was performed on "naked" DNA that was FIG. 7. The active VSG expression site in T. brucei is relatively depleted of nucleosomes. (A) Chromatin from the T. brucei HNI (221ϩ) or HNI (VO2ϩ) cell lines was partially digested by MNase and fractionated through sucrose gradients to obtain mono-and oligonucleosomes. DNA was extracted from the input material as well as fractions 9 to 24 (Frac. nr.) and electrophoresed on a 2% agarose gel stained with ethidium bromide. Size markers are indicated on the left, and the locations of mono-, di-, tri-, and tetranucleosomes are indicated on the right. Oligonucleosomal fractions comprised primarily of di-and trinucleosomes were pooled to serve as the template DNA in subsequent qPCR analysis (NI). In addition, fractions containing primarily tri-and tetranucleosomes were pooled (NII). Three independent experiments were performed, and a representative gel is shown. (B) qPCR was used to amplify oligonucleosomal DNA from the NI pool of fractions containing primarily di-and trinucleosomes. These data are plotted as the fold difference in amount of DNA amplified (indicating nucleosome occupancy) between the two isogenic T. brucei lines expressing either VSG221 or VSGVO2. Red bars indicate the values obtained from the VSG221-expressing cells divided by the values obtained from the VSGVO2-expressing cell line (221/VO2). Blue bars represent the reciprocal calculation (VO2/221). Three housekeeping genes were monitored as a control and include ␥-tubulin (␥tub), ␣-tubulin (␣tub), and the large subunit of RNA Pol I. Two primer pairs common to all BESs were also used and include the BES core promoter (ESPro) and ESAG7/6 (represented in Fig. 4A as primers c and e, respectively). The five single-copy sequences specific for either the VSG221 or VSGVO2 BES were analyzed (shown schematically in Fig. 4B as primers i to m). The results presented are the average from three independent experiments, with the standard deviations indicated with error bars. (C) qPCR was used to amplify DNA from the NII pool containing primarily tri-and tetranucleosomes, as described in panel B. Average values are shown from three independent experiments, with standard deviations indicated with error bars.  Fig. S1A in the supplemental material). As expected, no relative enrichment between the different BESs was found, providing further support that nucleosome positioning is responsible for the differences observed between silent and active expression sites (see Fig. S1B in the supplemental material).

DISCUSSION
Here, we examined the distribution of nucleosomes throughout the genome of T. brucei using histone H3 as a marker. We find that histone H3 is enriched at the extensive nontranscribed 50-bp and 177-bp simple sequence repeats (8 to 10% of input precipitated) in both bloodstream form and procyclicform T. brucei, compatible with the idea that these nontranscribed regions are present in a more closed chromatin state. In contrast, histone H3 is relatively depleted in the Pol II transcription units (␥-tubulin or Pol I large-subunit genes with 5 to 6% of input precipitated) or Pol III-transcribed 5S rRNA transcription units (approximately 4% input precipitated), which is compatible with the regions being present in a relatively open chromatin state. Histone H3 is particularly depleted in the spliced leader (SL) arrays which are highly transcribed by Pol II, with only 2% of the input precipitated. These results are in agreement with what has been found in yeast, where histone occupancy has in general been shown to be inversely correlated with rates of transcription (3,35).
Within the SL RNA genomic loci we observed differences in chromatin structure and found about 3-fold more histone H3 present within characteristic SLACS retroposons (about 6% input precipitated) than within the SL transcription units themselves (about 2% input precipitated). This is in agreement with the observed reduced density of Pol II over the SLACS elements, indicating that transcription through the SLACS open reading frames (ORFs) is relatively inefficient compared with the SL RNA (55). Recently, similar observations have been reported for other trypanosomatids. In Trypanosoma cruzi relatively few nucleosomes are detected at the SL promoter (61), and in Leishmania tarentolae the SL RNA promoters and transcribed SL RNA genes are nucleosome free (27).
In the Pol I-transcribed rDNA we find that histone H3 occupancy is greatly reduced in the area immediately downstream of the rDNA promoter (approximately 1% input precipitated) compared with the nontranscribed rDNA spacer (5 to 6% input precipitated). Therefore, the promoter regions of T. brucei transcription units transcribed by all three RNA polymerases (Pol I, II, and III) appear to be particularly depleted of histone H3. This relative depletion of histone H3 at promoter regions is in agreement with previous studies in yeast which provide evidence for nucleosome depletion at active regulatory regions (3,35,83). Within the rDNA in T. brucei, we find evidence for a gradient of histone H3 distribution extending along the length of the rDNA transcription unit, ranging from relatively little histone H3 in the neighborhood of the rDNA promoter, up to higher levels of histone H3 at the end of the transcription unit and approaching levels found in the nontranscribed spacer. A gradient of histone H3 distribution over the rDNA transcription unit has also been shown in S. cerevisiae (30) although these data appear to show relatively less histone H3 in the nontranscribed spacer region than we found using T. brucei.
Procyclin is another Pol I transcription unit in T. brucei which encodes the major surface protein in insect form trypanosomes. We found approximately 3-fold less histone H3 on the procyclin EP promoter in both bloodstream form and procyclic form T. brucei in comparison with sequences upstream of these transcription units. These data are compatible with previous studies which found significant transcriptional activity from the procyclin promoter even in bloodstream form T. brucei (56,78), where the elongation of transcription appears to be developmentally controlled (78).
However, a complicating factor in the analysis of rDNA Pol I transcription units has been the repetitive nature of the nearly identical genes contained within them. This complication applies to the analysis of rDNA as well as procyclin transcription in T. brucei. Bloodstream form T. brucei is unique, however, in transcribing a single VSG BES by Pol I using strict monoallelic exclusion (7,9,53). We have been able to investigate the chromatin structure of different single-copy sequences within VSG BESs using isogenic T. brucei strains with two different VSG BESs in either "on" or "off" states. Here, we show that there is a striking depletion of histone H3 along the entire length of an active VSG BES, with an 11-to 40-fold difference between silent and active sites. Using antibodies against histone H4 and H2A in additional ChIP experiments, we identified a similar trend, albeit less pronounced, with 5-to 10-fold and 2-to 4-fold depletion, respectively, compared with the same BES in its silent state. The fact that VSG BESs are monoallelically expressed and contain, or can be modified to contain, single-copy sequences greatly facilitates the analysis of the chromatin architecture of these Pol I transcription units. Our results showing the dramatic depletion of histones within the active VSG BES are consistent with those from Figueiredo and Cross (17) and are in agreement with studies which argue that actively transcribed rRNA genes are largely devoid of histones (12,50).
We find more striking differences in the distribution of histone H3 than in histone H2A distribution in active versus silent VSG BESs. It is important to note, however, that the antihistone antibodies used here differ in how efficiently they are able to immunoprecipitate their target. Using the Pol II-transcribed ␥-tubulin genes as a control, the anti-histone H3 antibody immunoprecipitated ϳ5% of the input (Fig. 1D), and anti-histone H4 immunoprecipitated ϳ2% of the input (Fig.  5A) while anti-histone H2A immunoprecipitated only ϳ0.5% of the input (Fig. 5B). The observed variations in abundance of these histones could therefore be a consequence of different antibody affinities. However, in addition these results could be explained by nucleosome dynamics. Within the nucleosome, DNA is first wrapped around a core histone H3/H4 tetramer before two histone H2A/H2B dimers are added (44). Consistent with this, H2A/H2B dimers have been shown to be more dynamic in chromatin, and there is a relatively rapid exchange of histone H2B compared with the histone H3/H4 tetramers, which are more tightly and stably associated in chromatin (31). Our results showing relatively less enrichment of histone H2A in silent BESs is compatible with a scenario whereby histone H2A is less stably associated with the nucleosomes present in silent BESs than histone H3.
Histone H3 was found to be depleted from the multicopy VSG BES promoters in both bloodstream and insect form T. brucei, indicating a more open chromatin conformation. In bloodstream form T. brucei this depletion is about 2-fold greater than in the procyclic form T. brucei, with ϳ2% rather than 4% of the input immunoprecipitated. These results agree with the previous observation that a prominent nuclease-hypersensitive site is present within the core promoter of both active and inactive VSG BESs in bloodstream form T. brucei (51). Furthermore, it agrees with the extensive evidence that there is significant nonprocessive transcription from multiple "inactive" VSG BESs in both bloodstream and insect form T. brucei (2,65,79), which could explain the open chromatin structure in this region that we observe.
An inherent problem with ChIP experiments is the fact that formaldehyde cross-linking can potentially inhibit antibody access to more interior targets. In order to ensure that we were not observing an artifact related to this, we used an additional experimental approach that did not require cross-linking to investigate VSG BES chromatin structure. Micrococcal nuclease digestion followed by nucleosome fractionation provides a powerful tool to investigate chromatin structure in a quantitative fashion using limited material. Using this approach, we confirm our histone ChIP results and show that the active VSG BESs are in a more open chromatin structure than the silent VSG BESs. This observation that active VSG BESs are highly sensitive to digestion by micrococcal nuclease is in agreement with results obtained using Southern blot analysis of MNasedigested chromatin (17). These results in some ways contradict the findings from previous studies, which concluded that no difference exists in the nucleosome structure between active and silent VSG BESs.
Why has this difference in nucleosomal structure between the two different VSG BES activation states been missed in the past? Early studies comparing the nucleosomal structure of active versus silent BESs have argued that there is no detectably altered nucleosomal organization in active versus silent VSG BESs (20). However, this study did not compare the same VSG BES sequence (in either an active or silent state) with a single probe to allow effective comparison. In our study, using isogenic T. brucei strains, we have been able to compare the same set of single-copy sequences present within an BES in either a transcriptionally active or silent state, allowing us to directly compare differences. A later study concentrated on investigating the chromatin structure immediately around the BES core promoter in both an active and silent state in bloodstream form T. brucei and found a prominent DNase I-hypersensitive site in both cases (51). Our data are in agreement with the observation that most, if not all, VSG BES promoters are present in an open chromatin state (51). However, using two different methods, we find that the active VSG BES is depleted of nucleosomes along its entire length. An additional study using exogenous T7 RNA polymerase to probe for chromatin structure in T. brucei found that the silent VSG BESs in insect form but not bloodstream form T. brucei were refractory to transcription by T7 RNA polymerase (52). The authors argued from these data that chromatin remodeling plays a role in VSG BES silencing specifically in insect form T. brucei but did not find evidence for this being the case in bloodstream form T. brucei. However, it is unclear exactly which aspects of eu-karyotic chromatin can impede transcription by T7 RNA polymerase.
We do not know if the relative depletion of nucleosomes on the active VSG BES is a direct consequence of transcriptional activity or if this open chromatin structure is uncoupled from transcription itself. It has been shown in other experimental systems that nucleosome loss is not necessarily a consequence of transcription. For example, after the induction of a heat shock, initial loss of nucleosomes from the Drosophila heat shock locus occurs extremely rapidly and before the first RNA polymerases reach the corresponding region of the gene (59). This initial rapid loss of nucleosomes after heat shock was shown to be independent of transcription and to occur over a larger region than a single transcription unit (59). In these experiments, a second and later wave of nucleosome loss was dependent on transcription. Although we have found that the active VSG BES is significantly depleted of nucleosomes, we do not know the order of events and whether nucleosome depletion precedes VSG BES activation.
In summary, we show that highly transcribed loci within the T. brucei genome have reduced nucleosomes, with the active VSG BES showing the lowest nucleosomal occupancy of all regions investigated. The observed depletion of nucleosomes within the active VSG BES indicates that chromatin remodeling may play a critical role in the monoallelic exclusion necessary for VSG BES control in bloodstream T. brucei. The transcriptional states of active and repressed BESs are efficiently inherited in bloodstream form trypanosomes, suggesting the presence of epigenetic marks. The lack of nucleosomes on the active BES may be one of the factors involved in marking and propagating the epigenetic state of the BES from one generation to the next. Future studies will be necessary to allow us to determine exactly how this chromatin remodeling proceeds during VSG BES switching.


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Angiotensin II signaling increases activity of the renal Na-Cl cotransporter through a WNK4-SPAK-dependent pathway

Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and high serum K+ levels (hyperkalemia). WNK4 has distinct functional states that regulate the balance between renal salt reabsorption and K+ secretion by modulating the activities of renal transporters and channels, including the Na-Cl cotransporter NCC and the K+ channel ROMK. WNK4's functions could enable differential responses to intravascular volume depletion (hypovolemia) and hyperkalemia. Because hypovolemia is uniquely associated with high angiotensin II (AngII) levels, AngII signaling might modulate WNK4 activity. We show that AngII signaling in Xenopus oocytes increases NCC activity by abrogating WNK4's inhibition of NCC but does not alter WNK4's inhibition of ROMK. This effect requires AngII, its receptor AT1R, and WNK4, and is prevented by the AT1R inhibitor losartan. NCC activity is also increased by WNK4 harboring mutations found in PHAII, and this activity cannot be further augmented by AngII signaling, consistent with PHAII mutations providing constitutive activation of the signaling pathway between AT1R and NCC. AngII's effect on NCC is also dependent on the kinase SPAK because dominant-negative SPAK or elimination of the SPAK binding motif in NCC prevent activation of NCC by AngII signaling. These effects extend to mammalian cells. AngII increases phosphorylation of specific sites on SPAK and NCC that are necessary for activation of each in mpkDCT cells. These findings place WNK4 in the signaling pathway between AngII and NCC, and provide a mechanism by which hypovolemia maximizes renal salt reabsoprtion without concomitantly increasing K+ secretion.

Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and high serum K ؉ levels (hyperkalemia). WNK4 has distinct functional states that regulate the balance between renal salt reabsorption and K ؉ secretion by modulating the activities of renal transporters and channels, including the Na-Cl cotransporter NCC and the K ؉ channel ROMK. WNK4's functions could enable differential responses to intravascular volume depletion (hypovolemia) and hyperkalemia. Because hypovolemia is uniquely associated with high angiotensin II (AngII) levels, AngII signaling might modulate WNK4 activity. We show that AngII signaling in Xenopus oocytes increases NCC activity by abrogating WNK4's inhibition of NCC but does not alter WNK4's inhibition of ROMK. This effect requires AngII, its receptor AT1R, and WNK4, and is prevented by the AT1R inhibitor losartan. NCC activity is also increased by WNK4 harboring mutations found in PHAII, and this activity cannot be further augmented by AngII signaling, consistent with PHAII mutations providing constitutive activation of the signaling pathway between AT1R and NCC. AngII's effect on NCC is also dependent on the kinase SPAK because dominant-negative SPAK or elimination of the SPAK binding motif in NCC prevent activation of NCC by AngII signaling. These effects extend to mammalian cells. AngII increases phosphorylation of specific sites on SPAK and NCC that are necessary for activation of each in mpkDCT cells. These findings place WNK4 in the signaling pathway between AngII and NCC, and provide a mechanism by which hypovolemia maximizes renal salt reabsoprtion without concomitantly increasing K ؉ secretion.
angiotensin II receptor ͉ hypertension ͉ distal convoluted tubule ͉ salt reabsorption ͉ thiazide A ldosterone is released from the adrenal glomerulosa in 2 different physiologic conditions: intravascular volume depletion and hyperkalemia. In the former, aldosterone promotes maximal renal Na-Cl reabsorption to preserve and restore intravascular volume, whereas in the latter renal K ϩ secretion is maximized. Classical explanations for these alternative responses have focused on acute changes in solute delivery to the distal nephron. For example, in volume depletion there is enhanced proximal reabsorption of Na ϩ , which reduces the distal electrogenic reabsorption of Na ϩ via the epithelial sodium channel (ENaC) that is required to establish the electrical gradient necessary for K ϩ secretion.
The rare autosomal dominant disease pseudohypoaldosteronism type II (PHAII) suggests there must be additional components that regulate the balance between renal salt reabsorption and potassium secretion. Patients with PHAII have chloridedependent hypertension and hyperkalemia despite otherwise normal renal function and normal aldosterone secretion, sug-gesting that they constitutively reabsorb Na-Cl at the expense of impaired K ϩ secretion. Missense mutations in the serinethreonine kinase WNK4 have been shown to cause PHAII (1). Subsequent studies in Xenopus oocytes (2-7), mammalian cells (8) and monolayers (9,10) have demonstrated that WNK4 regulates the balance between renal NaCl reabsorption and K ϩ secretion; this is achieved by orchestrating the activities of the Na-Cl cotransporter NCC, the K ϩ channel ROMK, the Na ϩ channel ENaC, and the paracellular Cl Ϫ flux pathway (11,12). Wild-type WNK4 inhibits NCC in Xenopus oocytes (2)(3)(4), and in mammalian cells [Cos-7 and polarized M1 cells (8)], however, missense mutations that cause PHAII abrogate this inhibition, increasing NCC activity; these effects are mediated at least in part by altering trafficking of NCC to the plasma membrane. Similarly, PHAII mutations allow increased activity of ENaC (6) and selectively increase paracellular Cl Ϫ conductance (9), effects that all promote maximal renal NaCl reabsorption. Conversely, although wild-type WNK4 also inhibits the K ϩ channel ROMK (the major mediator of distal renal K ϩ secretion), PHAIImutant WNK shows enhanced, not diminished, inhibition of ROMK (5). This latter effect prevents renal K ϩ secretion and promotes hyperkalemia. Thus, the kidneys of patients with PHAII behave as although a regulatory switch is stuck in a state that results in constitutive reabsorption of Na-Cl and inhibition of K ϩ secretion, accounting for the hypertension and hyperkaemia in affected patients.
These in vitro effects of WNK4 are duplicated in mouse models: mice harboring a single additional genomic copy of the wild-type WNK4 locus introduced as a BAC transgene show reduced expression of NCC, lower blood pressure, and predisposition to hypokalemia, whereas BAC transgenes harboring a PHAII mutation induce hypertension and striking hyperkalemia; both traits are reversed by NCC deficiency (13). These findings were confirmed by analysis of a mouse with knockin of a PHAII mutation (14).
These effects demonstrated that WNK4 has at last 2 distinct biochemical states and raised the possibility that WNK4 might  have a third state that could support K ϩ secretion without maximizing Na-Cl reabsorption. Phosphorylation of WNK4 in its C terminus by the aldosterone-induced kinase SGK (15) induces such a state; this phosphorylation alleviates inhibition of ENaC and ROMK, which would maximize K ϩ secretion. Thus, the different states of WNK4 can modulate distal renal activity from a basal state to one that either maximizes salt reabsorption or maximizes K ϩ secretion.
The different states of WNK4 correspond to the desired alternative responses to intravascular volume depletion and hyperkalemia. This suggests that the PHAII mutations mimic a natural state resulting from volume depletion, and beg the question of what the upstream regulatory signal might be. Signaling of the peptide hormone angiotensin II (AngII) through its G protein coupled receptor AT1R, is an attractive candidate, because AngII levels are markedly elevated by activation of the renin angiotensin system in response to volume depletion, but are not increased by hyperkalemia.
Here, by reconstitution experiments in Xenopus oocytes, we show that AngII signaling acts through WNK4 and the Ste20type kinase SPAK to increase NCC activity. This effect can be substituted in full by PHAII-mutant WNK4. We further show that AngII signaling increases phosphorylation at key regulatory sites on both SPAK and NCC in mammalian cells. Conversely, we show that AngII signaling does not reverse WNK4's inhibition of ROMK. These findings place WNK4-SPAK in the signaling pathway between AngII and NCC, and reveal a key role for this pathway in the renal response to intravascular volume depletion.

Results
AngII Abrogates WNK4's Inhibition of NCC. As described in refs. 16 and 17, injection of NCC cRNA into Xenopus laevis oocytes resulted in a marked increase in thiazide-sensitive 22 Na ϩ uptake that is inhibited by coexpression of wild-type (WT) WNK4 (Fig.  1A). Although Xenopus oocytes do not endogenously express the AngII receptor AT1R, injection of AT1R cRNA induces expression and functional receptor signaling, and administration of exogenous AngII results in increased intracellular IP 3 and calcium (18). In the absence of WNK4, expression of AT1R with NCC in the presence or absence of AngII had no effect on NCC activity ( Fig. 1 A). Addition of WT-WNK4 resulted in AngIIregulated NCC activity: WT-WNK4 inhibited NCC activity in the absence of AngII, but this inhibition was completely eliminated by the addition of AngII (P Ͻ 0.001) ( Fig. 1 A). Timecourse experiments revealed that, in the presence of WNK4 and AT1R, AngII increased the activity of NCC within 1 to 2 minutes, and its maximal effect was achieved by Ϸ15 min (Fig.   1B and Fig. S1 A). The ability of AngII to increase NCC activity was completely blocked by losartan, a specific antagonist of AT1R (19), demonstrating that AngII's effect is receptordependent ( Fig. 1C and Fig. S1B). Together, these data show that AngII signaling increases NCC activity by elimination of WNK4's inhibitory activity.
Insensitivity of PHAII-Mutant WNK4 to Ang II Signaling. In vivo and in vitro studies have shown that WNK4 harboring missense mutations found in patients with PHAII mutations no longer inhibits NCC activity (2,13), an effect similar to the observed loss of WT-WNK4's inhibitory activity after activation of AT1R. This suggests that PHAII mutations might be functionally equivalent to the physiologic effect of AngII signaling on WT-WNK4. If this were true, we would expect AngII signaling to have no further stimulatory effect on NCC activity in the presence of PHAII-mutant WNK4. As previously shown, PHAII mutations WNK4-Q562E and WNK4-D561A both abrogate inhibition of NCC activity (2). AngII signaling in the presence of either WNK4-Q562E or WNK4-D561A had no further stimulatory effect on NCC activity ( Fig. 2A) (P ϭ 0.24 and P ϭ 0.58, respectively). This result is consistent with PHAII mutant WNK4 constitutively supplying the physiologic effect of activated AT1R in the absence of AT1R signaling.
Ang II's Stimulation of NCC Specifically Requires WNK4. The kinase domains of other WNK kinases are highly homologous to WNK4 and have been studied in Xenopus oocytes. Expression of WNK1 with NCC has no effect on NCC activity, whereas WNK3 is a powerful activator of NCC (3,20). To determine whether AngII signaling can regulate NCC activity in the presence of other WNKs, we measured the effect of activation of AT1R on NCC activity in oocytes expressing either WNK1 or WNK3. AngII signaling had no significant effect on NCC activity in the presence of WNK1 (Fig. 2B). Similarly, AngII signaling had no effect on NCC activity in the presence of WNK3 (Fig. 2C)  WNK4 also inhibits the potassium channel ROMK and unlike the loss of inhibition of NCC with PHAII mutations, inhibition of ROMK is augmented by PHAII mutations (5). We tested whether AngII signaling is able to modulate the effect of WNK4 on ROMK. In contrast to the loss of WNK4's inhibition of NCC, Fig. 1. AngII relieves WNK4's inhibition of NCC. Xenopus oocytes were injected with water or indicated cRNAs, incubated, and thiazide-sensitive Na ϩ influx was measured in the presence or absence of AngII and losartan as described in Methods. (A) The results of 8 different experiments are shown. The results demonstrate that WNK4 inhibits NCC activity, that AT1R in the absence of AngII has no effect, but in the presence of AngII, WNK4's inhibition of NCC is abrogated. (B) Time dependence of AngII effect. Groups of oocytes were exposed to AngII for 1, 5, 15, and 90 min. Asterisks denote Na ϩ influx that is significantly different from uptake observed in the absence of AngII. AngII's effect is rapid, reaching maximal values in Ϸ15 min. (C) Losartan prevents AngII's effect on NCC. Oocytes were exposed to losartan at 1 M for 15 min before addition of AngII. Losartan completely blocks the effect of AngII to increase NCC activity.
we found that AngII signaling did not relieve inhibition of ROMK by WNK4 (Fig. 3A). A mutation that mimics phosphorylation of WNK4 at a serum glucocorticoid kinase site (SGK site; WNK4-S1169D) has been shown to release ROMK and ENaC from WNK4 inhibition (15). If this regulatory site is used to help distinguish response to hyperkalemia and volume depletion, we might expect this modification not to alleviate inhibition of NCC. Similar to wild-type WNK4, WNK4-S1169D retained the ability to inhibit NCC. Importantly, AngII retains the ability to alleviate WNK4's inhibition of NCC in the presence of the S1169D mutation (Fig. 3B).

The Stimulatory Effect of AngII on NCC in the Presence of WNK4 Is
SPAK-Dependent. Recent data has shown that WNK1 and WNK4 lie upstream of the Ste20-related serine-threonine prolinealanine rich kinase (SPAK) and oxidative stress response 1 kinase (OSR1) to regulate the bumetanide-sensitive Na ϩ :K ϩ :2Cl Ϫ cotransporter NKCC1, a close relative of NCC in the SLC12A gene family (21)(22)(23)(24)(25). WNK3 also lies upstream of SPAK to activate the renal specific Na ϩ :K ϩ :2Cl Ϫ cotransporter NKCC2 (26). Moreover, SPAK is a key kinase for the activation of NCC by intracellular chloride depletion (27,28). These observations suggest that the pathway from AT1R to NCC may include SPAK.
Because oocytes express endogenous SPAK, we analyzed the effect of SPAK harboring the K104R mutation, which is catalytically inactive and functions as a dominant-negative inhibitor of the endogenous SPAK (7,26). SPAK-K104R prevented AngII's activation of NCC in the presence of WNK4 (Fig. 4A). These data provide evidence that the stimulatory effect of AngII on NCC in the presence of WNK4 is SPAK-dependent. As a control, wild-type SPAK cRNA was added to the injection mixture containing ATR1 and WNK4. In this condition, we observed a significant activation of NCC in the presence of AngII (Fig. 4A).
SPAK/OSR1-possess a Conserved C-Terminal (CCT) domain, which is capable of interacting with RFx(V/I) motifs present in WNK isoforms and substrates such as NCC (29). One such motif is present in the carboxyl terminal domain of WNK4 ( 994 VGRFQVT) and the amino terminal domain of NCC ( 15 CS-GRFTIS). To test whether either of these motifs are necessary for AngII's stimulatory effect on NCC in the presence of WNK4, we mutated each (WNK4-F997A; NCC-R18A). Elimination of the SPAK-binding motif in WNK4 did not affect the activation of NCC by activated AT1R (Fig. S2), suggesting that a physical interaction between WNK4 and SPAK via this motif is not required. In contrast, elimination of the SPAK-binding motif in NCC had 2 prominent effects: The basal activity of NCC was virtually eliminated and activation of NCC by AngII in the presence of WNK4 was abrogated (Fig. 4B). These results suggest that the path from AT1R to NCC requires both WNK4 and SPAK. Interestingly, however, NCC-R18A could still be activated by WNK3, suggesting that WNK3's activation of NCC uses an alternative pathway that is not SPAK-dependent (Fig. 4C).

AngII Induces Phosphorylation of SPAK and NCC in Mammalian Cells.
In oocytes (27) and in HEK-293 cells, NCC is activated by intracellular chloride depletion. This activation is accompanied by, and dependent on, phosphorylation of T53, T58, and S71 in rat NCC and homologous threonines T55 and T60 in human NCC (27,28). In HEK-293 cells or in mpkDCT cells NCC activation is also accompanied by increased phosphorylation of SPAK in the activating T233 and S373 sites (25,28). We analyzed the effect of AngII in mpkDCT cells on the phosphorylation of SPAK and NCC at these important regulatory residues using previously-characterized antibodies specific for phosphorylation at these sites (28). Phosphorylation at SPAK T233 and S373 was increased by both low chloride hypotonic stress and AngII, and  22 Na ϩ uptake was measured in oocytes injected with NCC and AT1R alone or together with wild-type WNK4 or WNK4 harboring either the Q562E or D561A PHAII mutation in the presence or absence of AngII. Unlike WT-WNK4, PHAII-WNK4s show no significant inhibition of NCC activity, and AngII signaling imparts no significant further increase in NCC activity. (B and C) AngII signaling does not alter NCC activity in the presence of WNK1 or WNK3. Thiazide-sensitive 22 Na ϩ uptake was measured as in panel A except that WNK1 or WNK3 has been substituted for WNK4. WNK1 has no effect on NCC in the presence or absence of AngII; WNK3 markedly increases NCC activity as previously described, however, AngII signaling does not modulate this effect. * , significantly different from the uptake observed in NCCϩAT1R group in the absence of AngII. Fig. 3. Effects of AngII on ROMK and effects of WNK41169D on NCC. (A) No effect of AngII signaling on ROMK activity. Oocytes were injected with ROMK cRNAϩAT1R with or without WNK4 cRNA. ROMK activity was assessed in the absence (white bars) or presence of AngII for 5 min (gray bars) or 10 min (black bars). WNK4 significantly inhibited ROMK activity, and this effect was not altered by AngII signaling. Asterisks denote significant differences from ROMKϩAT1R in the absence of WNK4 and AngII. (B) Effects of WNK4 S1169D on NCC activity. Oocytes were injected with cRNAs encoding NCCϩAT1R with and without WNK4-S1169D cRNA and 22 Na ϩ uptake was assessed. Values were normalized to uptake observed in NCCϩAT1R in the absence of AngII. WNK4-S1169D significantly inhibits NCC (white bar) and this inhibition is reversed by AngII signaling (black bar). * , significantly different from uptake observed in NCCϩAT1R group in the absence of AngII; ** , significantly different from the uptake observed in NCCϩAT1RϩWNK4-S1169D in the absence of AngII. this latter increase was inhibited by losartan (Fig. 5A). Similar effects were seen on NCC at T55 (Fig. 5B). These results extend the effects of AngII signaling on NCC from oocytes to mammalian cells.

Discussion
Prior work in Xenopus oocytes has demonstrated that WNK4 regulates NCC and ROMK. Wild-type WNK4 reduces the activity of both, whereas PHAII mutations have opposite effects on NCC and ROMK, increasing activity of the former, while reducing function of the latter. These findings have provided an explanation for the observed chloride-dependent hypertension and hyperkalemia seen in humans and mice with PHAII-mutations.
These observations together have suggested that WNK4 might be a mediator of AngII signaling, normally contributing to the differential renal response to volume depletion and hyperkalemia. In this model, the PHAII mutations mimic constitutive AngII signaling in renal epithelium. A key part of this model is that AngII signaling should increase NCC activity. Consistent with this proposition, recent work has demonstrated that increased or decreased AngII levels in vivo, respectively increase or decrease expression of NCC at the plasma membrane in the DCT (30).
In the present study, we were able to reconstitute activation of NCC by AngII in the Xenopus oocyte. This reconstitution required exogenous AT1R, NCC and WNK4, and endogenous SPAK. Because all of these gene products are expressed in the native DCT, and because of the demonstrated effects of both AngII and WNK4 function on NCC in vivo, this signaling pathway is highly likely to be relevant in the native DCT. Our findings demonstrate that AngII signaling increases NCC activity through a pathway that requires WNK4 and SPAK. These observations suggest that AngII switches WNK4 from a state that inhibits NCC, to one that allows or promotes NCC activation; the observation that AngII induces no further increase in NCC activity when PHAII-WNK4 is expressed is consistent with PHAII-WNK4 mimicking the state induced by AngII.
Our findings also indicate that the effect of AngII on NCC requires modulation of SPAK activity. It is clear that AngII signaling has similar downstream effects to increase the phosphorylation of SPAK at threonine 233 and serine 373 and NCC at threonine 55 in both oocytes and mammalian cells (Fig. 5 A  and B) (31).
These findings collectively support the physiologic model of AngII and WNK4 activity in the DCT outlined in Fig. 6. In the setting of normal or expanded intravascular volume (Fig. 6A), in which the renin-angiotensin system is suppressed, WNK4 inhibits NCC by reducing the amount of NCC present in plasma membrane (2,8,30). In the setting of intravascular volume depletion (Fig. 4B), in which the renin-angiotensin system is activated, AngII signaling alleviates WNK4's inhibition of NCC, resulting in increased NCC activity. This is likely mediated through SPAK and the effect on NCC is likely increased trafficking to the plasma membrane (30). WNK1 has also been shown to be capable of activating SPAK in vitro (32); whether WNK1 fails to activate NCC in oocytes because a factor necessary for its activation is missing or because an inhibitory component is present is unknown. The model depicts AT1R in the basolateral membrane, however, the detection of renin and angiotensin-converting enzyme within the distal nephron (33,34) and the demonstration of an apical AT1R in the collecting Fig. 4. SPAK is required for AngII induced increase of NCC activity. (A) 22 Na ϩ uptake in oocytes injected with combinations of constructs that include wild-type SPAK or the dominant-negative SPAK-K104R (SPAK-KR). SPAK-KR prevents AngII signaling from increasing NCC activity, whereas wild-type SPAK supports a robust increase in NCC activity with AngII signaling. (B) Elimination of the SPAK binding site on NCC drastically reduces NCC activity. NCC-R18A (NCC-R18) mutates the unique SPAK binding site on NCC. Oocytes expressing this mutant NCC show markedly reduced activity that cannot be restored by AngII, suggesting a requirement for SPAK binding. (C) WNK3 can activate NCC-R18A. Oocytes were injected with NCC-R18A cRNA alone or together with WNK3 cRNA. Assessment of Na ϩ uptake indicates that WNK3 is still capable of activating NCC, showing that WNK3 stimulation of NCC activity does not require the unique SPAK binding site of NCC. The asterisk denotes difference from NCC-R18A control without WNK3. duct (35) open the possibility that AngII in the distal tubular fluid may influence distal tubule transport.
Thus, PHAII-mutant WNK4 has many features of constitutive AngII signaling in the DCT, resulting in unrestrained salt reabsorption by NCC and hypertension. Consistent with the proposal that AngII signaling promotes salt reabsorption without K ϩ secretion, AngII had no effect on WNK4's inhibition of ROMK (Fig. 3A) and WNK4 harboring mutations that mimic SGK phosphorylation maintained inhibition of NCC but was still capable of responding to AngII (Fig. 3B).
The molecular mechanism by which AngII modulates WNK4's regulation of NCC remains to be elucidated. AT1R is a typical heptahelical G protein-coupled receptor that, on AngII binding, elicits multiple cellular responses, predominantly via coupling to Gq/11 proteins (36). Gq/11-mediated inositol phosphate/Ca 2ϩ signaling is the primary transduction mechanism initiated by AngII in its major physiological target tissues, including DCT cells (37,38). PHAII mutations in WNK4 cluster in a negatively charged segment that bears some similarity to EF hand domains, raising the possibility that increased intracellular Ca 2ϩ concentrations could be a direct signal to change WNK4 activity by binding to this segment. There is presently, however, no direct evidence that this is the case. Further studies will be required to determine the molecular mechanism by which AngII signals to WNK4, and how this signal alters the downstream effects of WNK4.

Methods
Clones and Mutagenesis. The cDNAs used in this study are described in refs. 2, 7, 17, and 20, except for the AT1R that was obtained for Origine. The full length cDNA was sequenced and sublconed in to the pgh19 vector. Site-directed mutations (QuikChange; Stratagene) were performed to substitute phenylalanine 997 for alanine in WNK4 and arginine 18 for alanine in NCC. DNA sequencing was used to confirm all mutations. All primers were custom made (Sigma).
Assessment of the Na-Cl Cotransporter and ROMK Function. rNCC activity was assessed by functional expression in Xenopus laevis oocytes as described in refs. 17, 20, 39, and 40. Oocytes were injected with water or 10 ng cRNA per oocyte of NCC and different combinations of other cRNAs expressing AT1R and wild-type or mutant WNK4, WNK1, WNK3, and SPAK, as indicated in each experiment. Three to 4 days after injection, NCC activity was assessed by measuring tracer 22 Na ϩ uptake in the presence or absence of metolazone following a protocol that includes a 30-min preincubation in Cl Ϫ free solution and a 60-min incubation in uptake solution. Unless otherwise indicated, AngII stimulation was performed by including 100 pM AngII (Sigma) in the preincubation solution for 15 min before measurement of uptake. Inhibition of AT1R was performed with Losartan (1 M), which was added during the 30-min preincubation period. Mean Na ϩ uptake in the presence of matolazone was substracted from values obtained in its absence to yield the thaizidesensitive Na ϩ uptake attributable to NCC activity. ROMK activity was assessed as Ba 2ϩ -sensitive whole-cell K ϩ currents measured by a 2-electrode voltage clamp as described in refs. 5 and 15. Oocytes were injected with ROMK and AT1R cRNA with or without WNK4 cRNA and incubated for 2-3 days. Reported K ϩ currents refer to Ba 2ϩ -sensitive currents at ϩ 40 mV.
Phosphorylation at Regulatory Sites of NCC and SPAK. Immunoblots for NCC were performed using rabbit polyclonal antibody Chemicon AB3553 as described in ref. 28. In brief, 20 g of mpkDCT protein extract was fractionated on 3-8% SDS Page gels and transferred to a nitrocellulose membrane. After blocking with TBS-T 5% milk, blots were incubated overnight in the presence of the primary antibody, washed in TBS-T, and then incubated with the secondary Ab and washed again. Phosphorylation at specific sites on NCC and SPAK was measured by blotting with antibodies specific for phosphorylation at NCC T55 and SPAK T233 or S373 as described in ref. 28.
Data Analysis. All results presented are based on a minimum of 3 different experiments with at least 10 oocytes per group in each experiment. Statistical significance is defined as 2-tailed, with P Ͻ 0.05 considered significant, and the results are presented as mean Ϯ SEM. The significance of the differences between groups was tested by 1-way ANOVA with multiple comparisons using Bonferroni's correction. In hypovolemia, AngII levels and AT1R signaling are increased, WNK4 inhibition of SPAK/NCC is prevented, and NCC activity increases via increased trafficking of the phosphorylated cotransporter to the plasma membrane. (C) PHAII-WNK4 alleviates WNK4 inhibition of SPAK, leading to SPAK phosphorylation and increased delivery of NCC to the plasma membrane.