CLAMP promotes globally enhanced chromatin accessibility of the male X-chromosome

To examine whether CLAMP regulates chromatin accessibility, we identified nuclease-accessible regions genome-wide using a recently developed assay that is based on chromatin digestion in a MNase titration series followed by high-throughput sequencing [13] (Fig 1A). Using a range of concentrations of MNase enzyme provides richer information as compared to digestion with a single concentration [13–18]. After sequencing of digested fragments, the number of reads for any specific region of the genome is computed for each MNase titration point (four points arranged from highest to lowest concentration) and a linear regression line is fit to the data. The normalized slope of the line provides a metric called the MNase accessibility (MACC) score that represents how readily an individual region of the genome is digested with MNase. Regions of open chromatin are easily accessible to MNase at low enzyme concentrations (which results in high read counts) and become over-digested at higher MNase concentrations (low read counts). This produces a positive slope of the regression line, resulting in positive MACC scores (Fig 1A). In contrast, regions that are less accessible require increased amounts of MNase enzyme to produce high read counts for nucleosome-size fragments, resulting in negative MACC scores (Fig 1A). In this way, the MACC assay quantitatively profiles both open and closed regions of chromatin simultaneously, an advantage compared with other currently available methods for profiling chromatin accessibility [19].

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Fig 1. CLAMP establishes the global open chromatin environment on the male X-chromosome.

A) Chromatin is separated into four samples which are digested under differing concentrations of MNase. The digested samples are prepared for high-throughput sequencing and the number of reads obtained within a region of the genome is plotted for all MNase concentrations from highest to lowest. A linear regression line is fit to the data points, and the slope of the line derives the MNase accessibility (MACC) score. B) The overall distribution of accessibility scores measured by the MACC value is shown for the X-chromosome (blue) and autosomes (red) of Control (gfp), clamp, and msl2 RNAi treated male (S2) and female (Kc) cells. The male X-chromosome has overall higher MACC values than the autosomes. This accessibility is reduced after clamp RNAi. In females, MACC values for the X-chromosomes and autosomes are similar and are reduced slightly following clamp RNAi treatment. For all box and whisker plots, the 95% confidence interval is shown with a notch around the median line. C) For both the X-chromosomes and autosomes, the difference in MACC value (Δ MACC) between control and RNAi treatment for an individual 100bp bin was calculated to account for differences in chromosome number. In males, the change in MACC scores indicates a reduction in X-chromosome accessibility following clamp RNAi but not msl2 RNAi. D) The location of significant changes in accessibility (p-value < 0.01) was classified as either within a gene body (blue), at TSS or TTS (red), at enhancers (green), or in unannotated regions (purple). Details for region definitions are in the Methods. For all RNAi treatments, the largest proportion of accessibility changes is located within gene bodies. E) Average MACC values were plotted over a 20kb window centered on a CLAMP ChIP-seq peak [9] for both control RNAi (blue) and clamp RNAi (green) conditions. The lighter shading surrounding the darker average line represents the 95% confidence interval. On both male and female X-chromosomes there is a strong reduction in accessibility that extends approximates +/- 7kb from the peak center. A similar, but less pronounced effect is also seen on autosomes in males and females.

https://doi.org/10.1371/journal.pone.0186855.g001

To test our hypothesis that CLAMP promotes global opening of the chromatin on the male X-chromosome, we performed MNase-seq titrations in Drosophila male (S2) and female (Kc) tissue culture cells. S2 and Kc tissue culture cells are often compared to study Drosophila dosage compensation [20,21] because they provide a more uniform cell population than whole animals or tissues. Moreover, the MSL complex binding sites in cell culture are almost identical to those in the whole organism [21]. We reduced protein levels of CLAMP or the core MSL complex component, MSL2, in S2 cells using an RNAi strategy reported to be highly efficient in several previous studies [9,20,22]. Furthermore, we performed clamp RNAi but not msl2 RNAi in Kc cells because MSL complex is not present in these female cells. As a control, we used a gfp RNAi treatment that activates the RNAi pathway but does not target any gene in the Drosophila genome [9].

We first tested clamp RNAi efficiency using immuno-blotting and determined that no detectable CLAMP protein remains after clamp RNAi as we have previously reported (S1A Fig) [9]. To test efficiency of msl2 RNAi, we used qRT-PCR because MSL2 is an unstable protein (S1B Fig). In addition, we quantified residual MSL complex function by measuring abundance of roX2, which is transcriptionally activated and stabilized by MSL complex (S1C Fig). We determined that in addition to reduced msl2 transcript, little roX2 remained, consistent with strong MSL complex depletion (S1B and S1C Fig) [23].

After validating RNAi efficiency, we proceeded to investigate how chromatin accessibility changes in response to depletion of CLAMP or MSL complex. We first examined the overall distribution of MNase accessibility (MACC) scores within an experiment to compare cell types and conditions on a global level (S2A Fig). Additionally, we plotted MACC scores for biological replicates separately and determined that our replicates were in agreement with each other (S2B Fig). We next separated MACC values for the X-chromosome and autosomes in male (S2) and female (Kc) cells to investigate differences in accessibility between chromosomes (Fig 1B, S2C and S2D Fig). The 95% confidence intervals are shown with notches around the median lines in all box plots and therefore the lack of overlap between notches or reference lines indicates that differences between samples are statistically significant. The significance levels of MACC score differences were further evaluated by a Mann-Whitney test (see S1 Table for p-values). On average, we observed higher accessibility scores for the male X-chromosome compared to autosomes in our control RNAi condition, consistent with the hyper-transcription of this chromosome (Fig 1B and S2C Fig, control RNAi).

Relative to the control RNAi, msl2 RNAi had no effect on the average chromatin accessibility scores of the X-chromosome or autosomes in agreement with a recent study [7] (Fig 1B and S2C Fig, msl2 RNAi). In contrast, clamp RNAi reduced accessibility scores of the male X-chromosome to levels below that of autosomes (Fig 1B and S2C Fig, clamp RNAi). In females, clamp RNAi had no average effect on autosomes or the X-chromosomes relative to control RNAi (Fig 1B and S2D Fig). We further performed a direct pair-wise comparison of the accessibility scores within 100-bp bins between the control and clamp or msl2 depleted samples. This approach showed that clamp RNAi treatment resulted in significantly more genomic locations (bins) with decreased accessibility than an increased accessibility on the male X-chromosome compared with the autosomes (Fig 1C and S2E Fig). Therefore, CLAMP and not MSL complex promotes the globally enhanced accessibility of chromatin on the male X-chromosome compared with autosomes.

Next, we performed additional analyses that further validated our observation that clamp RNAi specifically decreases chromatin accessibility of the male X-chromosome. First, to rule out a possibility of global differences in MACC values between samples, we determined that the average MACC profiles around a random set of genomic locations are similar for different RNAi conditions (S5B Fig). Second, we determined that clamp RNAi treatment decreases accessibility of the male X-chromosome when MACC values were median-shifted to zero for each sample independently (S2F Fig). Third, to check if the presence of a single male X-chromosome in male cells compared to two female X-chromosomes and two copies of each autosome can be a source of artifacts in the MACC analysis, we recomputed MACC values counting each X-linked read twice to compensate for the single X-chromosome in males (S2G Fig).

Even though global shifts in chromatin accessibility were only observed on the male X-chromosome after clamp RNAi (Fig 1B), many changes in chromatin accessibility occur throughout the genome after clamp and msl2 RNAi treatments. To further characterize these changes in accessibility, we categorized genomic loci with significant (p-value < 0.01) changes in accessibility based on their location: within gene bodies, at transcription start or termination sites (TSS/TTS), at enhancers, or in unannotated regions (see Methods for details on region definitions). We found that the majority of accessibility changes for both clamp and msl2 RNAi treatments occur within gene bodies (>65% for both treatments), consistent with the most frequent genomic location of CLAMP and MSL complex binding sites (Fig 1D). To account for differences in size of each genomic feature investigated, we also normalized counts of loci with accessibility changes by the percentage of the genome covered by each feature. Following clamp RNAi treatment in females, there is a roughly equal distribution of accessibility changes amongst the different genomic features (S3A Fig). In males, clamp RNAi results in an over-representation of changes at enhancers, indicating that CLAMP may have a role in regulating chromatin organization at these sites (S3A Fig). We conclude that CLAMP alters chromatin accessibility at a diverse set of genomic loci.

CLAMP and MSL complex both mediate chromatin accessibility changes at CES

Diverse locus-specific changes are often masked when investigating global effects in a genome- or chromosome-wide manner. Therefore, we next investigated how accessibility changes locally at specific sites in response to clamp RNAi. We first generated average profiles to examine MACC scores around previously defined CLAMP ChIP-seq peaks [9] (Fig 1E, the average MACC value is shown with a thick line and 95% confidence intervals are shown as lighter shading around it). Targeting clamp by RNAi has a long distance effect on chromatin accessibility that extends approximately +/-7kb from the peak center. This influence on chromatin accessibility over long distances occurs independent of MSL complex because we observe similar effects on male autosomes and in females. Additionally, CLAMP functions more strongly at regions surrounding CLAMP peaks on the X-chromosome compared with autosomes, which is consistent with the non-sex-specific enrichment of CLAMP on this chromosome [9]. Interestingly, in females, the long-distance effect does not mirror the effect directly at the CLAMP peak. While CLAMP enhances chromatin accessibility beyond peak centers, it modestly represses chromatin accessibility at its peak centers, suggesting that CLAMP differentially regulates chromatin depending on the distance from the peak center. It is possible that CLAMP acts with different cofactors to modulate its function at peak centers compared with its longer range effects on chromatin accessibility [24,25].

To test whether changes in accessibility correlate with CLAMP occupancy levels, we further integrated our previously generated CLAMP ChIP-seq data [9] with the MACC data. We found that in general, regions with the greatest CLAMP occupancy tended to have higher accessibility values (S3B Fig). This property is not unique to CLAMP because the correlation between factor occupancy level and degree of chromatin accessibility has been previously demonstrated in Drosophila embryos [26]. However, we observed this trend in both males and females and found it was not specific to the X-chromosome (S3B Fig). Overall, the correlation between increased CLAMP occupancy and increased MACC scores on all chromosomes in both males and females suggests that this relationship is present genome-wide.

Due to their high levels of enrichment for CLAMP and MSL complex, we examined chromatin accessibility changes at CES in response to clamp and msl2 RNAi treatments. Using MACC scores from 264 CES [9], we plotted the average MACC value +/- 500bp around the CES center (dark lines in all plots in Fig 2). We also calculated the 95% confidence intervals for all profiles, which are represented by the lighter shading surrounding the central average lines. We compared the effects of CLAMP on chromatin accessibility at CES to a randomized control data set (S3C Fig) to determine the specificity of the observed effects. In male cells treated with control RNAi, chromatin was most accessible at CES centers compared to neighboring regions and this accessibility was reduced after either clamp or msl2 RNAi (Fig 2A and 2B, see S1 Table for p-values). There was a focused reduction in accessibility directly at CES centers following msl2 RNAi treatment, suggesting a specific role for MSL complex in promoting chromatin accessibility at CES centers (Fig 2B). In contrast, we observed decreases in accessibility after clamp RNAi in male cells at and beyond CES centers, consistent with the ability of CLAMP to promote chromatin accessibility changes over long distances from its site of occupancy (Fig 1E).

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Fig 2. CLAMP and MSL complex promote accessibility at CES.

A) MACC values at Chromatin Entry Sites (CES) in male (S2) cells are greatest at the peak center (control RNAi, blue). RNAi treatment of clamp reduces accessibility at the CES peak and +/- 500 bp beyond the peak (green). For all panels, the lighter color indicates 95% confidence intervals, while the darker line represents the average MACC value. B.) A reduction in MSL complex following msl2 RNAi only reduces accessibility directly at the CES peak (purple). C) The average MACC scores around CES in female Kc cells indicates a reduction in MACC values distal to the CES center, extending +/- 500bp beyond the peak. D) MACC scores at the three subgroups of CES indicate clamp RNAi (green) results in a decrease in accessibility in CES Groups A and B, but not in Group C. E) RNAi targeting msl2 (purple) results in a loss of accessibility at Group A CES only at the CES peak. There is a small reduction in accessibility in Group B sites and no effect of msl2 RNAi treatment at Group C sites. F) MACC scores from Kc cells are plotted for the three subgroups of CES. In females, clamp RNAi (green) results in a decrease in MACC values around CES in Group A, and to a lesser extent Group B. CES in Group C exhibit an increase in MACC values following clamp RNAi.

https://doi.org/10.1371/journal.pone.0186855.g002

In female cells, clamp RNAi reduced chromatin accessibility outside of CES but not at CES centers, suggesting an MSL-independent function for CLAMP in promoting chromatin accessibility beyond the CES center (Fig 2A and 2C). The effect of clamp RNAi at CES in female cells is similar to what we observed on average for all CLAMP peaks (Fig 1E). Our data suggest that while MSL complex functions to promote chromatin accessibility at CES centers, CLAMP functions independently of MSL complex to promote accessibility outside of CES peak centers.

Next, we investigated the effect of clamp and msl2 RNAi at previously established subclasses of CES that differ with respect to the inter-dependent binding relationship between CLAMP and MSL complex [9]. CES were previously categorized into three subgroups based on the dependence of CLAMP occupancy on MSL complex [9]: 1) At Group A CES (176 sites), CLAMP occupancy is fully dependent on the presence of MSL complex; 2) At Group B CES (43 sites), CLAMP occupancy is partially dependent on MSL complex; 3) At Group C CES (45 sites), CLAMP occupancy is independent of MSL complex. To determine differences in accessibility between the three subclasses of CES, we examined how either clamp or msl2 RNAi affects average CES chromatin accessibility for each group.

We found that both clamp and msl2 RNAi reduced chromatin accessibility at Group A CES most strongly followed by Group B CES (Fig 2D and 2E, Groups A and B, see S1 Table for p-values). While msl2 RNAi reduced accessibility directly at the CES center, clamp RNAi reduced accessibility extending at least 500-bp beyond the center for both Group A and Group B CES. This effect is consistent with our earlier observation that CLAMP promotes chromatin accessibility over long distances (Fig 1E). Furthermore, the enhanced ability of CLAMP and MSL complex to promote chromatin accessibility at Group A and B CES is consistent with our previous hypothesis that establishing chromatin accessibility at CES requires the coordinated recruitment of CLAMP and MSL complex [9]. In contrast to Group A and B CES, chromatin accessibility at Group C CES was largely unaffected by either clamp or msl2 RNAi (Fig 2D and 2E, Group C). Therefore, in the absence of MSL complex, CLAMP does not affect chromatin accessibility at Group C sites, possibly due to redundancy with another similar factor. We conclude that while both CLAMP and MSL complex promote chromatin accessibility at the majority of CES in male cells, there is a small subset of CES (Group C) that regulate their accessibility via CLAMP- and MSL-independent mechanisms.

In female cells, clamp RNAi affected chromatin accessibility at CES relative to control RNAi treatment, but the changes in accessibility differed from those observed in male cells. At the CES that largely require MSL complex for enhanced CLAMP occupancy (Group A CES), clamp RNAi reduced chromatin accessibility outside of the CES peak but not at the peak (Fig 2F, Group A). This suggests that in males MSL complex and CLAMP function together at Group A CES to promote chromatin accessibility, but in females CLAMP functions in an MSL-independent manner outside of CES centers. In contrast to other classes of CES, clamp RNAi enhanced chromatin accessibility at Group C CES in females (Fig 2F, Group C), similar to the average effect observed at all CLAMP peaks in females (Fig 1E). It is likely that CLAMP interacts with different co-factors in males and females, such as MSL complex, to differentially regulate chromatin accessibility at the same locus. Consistent with this hypothesis, we previously reported that CLAMP can have opposing roles in gene regulation at the same locus in males versus females [10]. Overall, we conclude that in males, MSL complex regulates chromatin accessibility precisely at CES centers, whereas CLAMP has a broader role in regulating accessibility beyond the centers of CES that is independent of MSL complex.

Recently, a new subgroup of pioneer CES called PionX sites was identified at which MSL complex is hypothesized to directly contact DNA [27]. Out of 43 PionX sites located on the X-chromosome, 23 are Group A CES. We found that at PionX sites, there was a modest decrease in accessibility that was stronger after clamp RNAi than msl2 RNAi (S3B Fig, see S1 Table for p-value). These results suggest that CLAMP promotes an open chromatin environment at PionX sites that may allow MSL complex to directly contact DNA.

CLAMP promotes changes in chromatin accessibility over X-linked gene bodies

To define how CLAMP and MSL complex contribute to chromatin accessibility within and surrounding genes, we rank-ordered all annotated genes by CLAMP ChIP-seq occupancy levels and generated heat maps of MACC scores across gene bodies (S4A Fig). We found that regions with higher CLAMP occupancy exhibited enhanced chromatin accessibility at TSS and TTS under control RNAi conditions in both males and females (S4A Fig, Control).

To better define changes in accessibility between the control and RNAi treatments, we plotted the difference in accessibility between RNAi conditions (RNAi—control). These heat maps were rank-ordered based on the magnitude of accessibility change over the gene body following either clamp or msl2 RNAi treatment (Fig 3A). We also determined the percentage of genes that either increase (RNAi—control > 0) or decrease (RNAi—control < 0) in accessibility (numbers shown next to the corresponding heat maps). We found only modest changes in chromatin accessibility on the X-chromosome and autosomes after msl2 RNAi in males (Fig 3A and S4A Fig, msl2 RNAi). In contrast, clamp RNAi resulted in a broad loss of chromatin accessibility over the majority (86%) of male X-linked genes (Fig 3A and S4A Fig, clamp RNAi), while male autosomes and female chromosomes exhibited nearly equal percentages of genes with increased vs. decreased accessibility (Fig 3A and S4A Fig).

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Fig 3. CLAMP directly affects accessibility of X-linked gene bodies in both males and females.

A) The difference in MACC value (RNAi treatment—control) over gene bodies of all annotated genes was ranked by the magnitude of decrease (top) to increase (bottom) in accessibility. RNAi of msl2 shows only a modest change in accessibility, whereas clamp RNAi results in accessibility changes in both males and females. Shown to the right of each heat map is the percent of genes that decrease (MACC<0, blue line) or increase (MACC>0, red line) in accessibility following the indicated RNAi treatment. B) Average MACC profiles across gene bodies are shown for male (S2) and female (Kc) cells separated by X-chromosomes and autosomes. X-linked gene bodies in both males and females have reduced accessibility after clamp RNAi treatment (green). The dark line represents the average MACC value, while 95% confidence intervals are represented by the lighter shading. C) The difference in MACC scores (Δ MACC) between control and RNAi over gene bodies was calculated. Plotted is the distribution of difference values for all chromosomes together (all), or separately for the X-chromosome and autosomes. Plotted is the median difference in MACC scores with the 95% confidence interval indicated by a notch around the median line.

https://doi.org/10.1371/journal.pone.0186855.g003

Next, we analyzed the average effect of clamp RNAi over gene bodies after separating genes based on the enrichment of CLAMP within their TSS region. We determined groups by ranking CLAMP ChIP-seq enrichment from highest to lowest and dividing genes into two groups, “high” and “low” (see Methods). For genes highly enriched for CLAMP, clamp RNAi reduced chromatin accessibility over gene bodies on the X-chromosome more strongly than autosomes in both males and females (Fig 3B). This effect was specific to genes that are highly enriched for CLAMP because lowly enriched genes exhibited little difference in accessibility over gene bodies between the control and clamp RNAi treatments (S4B Fig). Taken together these results show that regions with a high enrichment of CLAMP have stronger changes in accessibility over gene bodies after clamp RNAi than those that are lowly enriched for CLAMP.

Consistent with the enrichment of CLAMP occupancy over X-linked gene bodies in both males and females [11], female cells exhibited an average X-specific decrease in accessibility over gene bodies that is similar to males (Fig 3B). Unlike male cells where 86% of X-linked genes decrease in accessibility following clamp RNAi, females have only a modestly larger percentage of genes with a decrease in accessibility compared to autosomes (Fig 3A, compare 53% to 46%). We asked how female cells could have a strong average X-specific reduction in accessibility after clamp RNAi when markedly fewer genes decrease in accessibility compared to males (compare 53% in females to 86% in males. To address this, we calculated the change in MACC scores along gene bodies between the control and RNAi treatments and plotted the distribution of these differences for all chromosomes combined, and the X-chromosome and autosomes separately (Fig 3C, see S1 Table for p-values). We found that genes on the female X-chromosome exhibited a stronger magnitude of decrease in chromatin accessibility over gene bodies compared to female autosomes. Therefore, the X-specific decrease seen on the average gene profiles in females (Fig 3B) is likely driven by a subset of X-linked genes (56%) that have a strong decrease in accessibility. The observation that CLAMP binds and alters chromatin accessibility more strongly on X-linked gene bodies than autosomal gene bodies in both males and females [9,11], suggests an MSL-independent function for CLAMP in promoting an open chromatin environment at a subset of X-linked gene bodies.

CLAMP promotes chromatin accessibility at transcription start and termination sites genome-wide

In addition to gene bodies, CLAMP is highly enriched at TSS [9], where we observed CLAMP-mediated changes in accessibility at genes with the greatest CLAMP occupancy (S4A Fig). To investigate changes in accessibility around TSS in greater detail, we plotted average MACC values around TSS for genes with either a high or low enrichment of CLAMP defined as described as above (Fig 4). Following clamp RNAi, we observed a reduction in accessibility upstream of the TSS, approximately within the nucleosome-depleted region and the -1 nucleosome position, for genes where CLAMP is highly enriched independent of whether the genes are located on the X-chromosome or autosomes in males or females (Fig 4A). This change in accessibility after clamp RNAi was not observed at TSS with low CLAMP enrichment (S5A Fig). Further, these changes are specific to TSS because a randomized TSS control data set does not produce the same result (S5B Fig). We additionally observed some increase in accessibility immediately downstream of TSS in female cells after clamp RNAi treatment, which does not extend beyond 400 bp into the gene body (Fig 4A).

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Fig 4. CLAMP promotes nucleosome positioning at 5’ends genome-wide.

A) Average MACC profiles around transcription start sites (TSS) are shown for male (S2) and female (Kc) cells separated by X-chromosomes and autosomes. TSS at genes with highly bound CLAMP in both males and females are reduced in accessibility after clamp RNAi treatment (green) compared to control (blue). The dark line represents the average MACC value, while 95% confidence intervals are represented by the lighter colors. B) Average MNase-seq read frequency was calculated for the four MNase experiments and plotted +/- 1 Kb flanking annotated TSS. Upon clamp RNAi (green), there is a decrease in read frequency upstream of the TSS with a concurrent increase in occupancy within gene bodies.

https://doi.org/10.1371/journal.pone.0186855.g004

To further investigate this shift in the position of chromatin accessibility after clamp RNAi treatment, we generated average read frequency profiles around genes displaying the shift pattern. We selected 150 bp sized sequence fragments for our analysis under the assumption that these are enriched for mononucleosome-associated DNA. While these profiles largely represent nucleosome occupancy, MNase-seq data contain up to several percent of fragments associated with other chromatin-bound factors [13]. Importantly, such non-nucleosomal DNA fragments often originate from the regulatory loci including nucleosome-depleted regions at the TSS of active genes. Our results indicate that after clamp RNAi, there is a loss of precise positioning of occupancy peaks, specifically those that flank nucleosome-depleted region upstream of the TSS on both the X-chromosome and autosomes in both male and female cells (Fig 4B, S5C and S5D Fig). Furthermore, we observed the largest changes in read frequency using the lowest 1.5 Unit MNase concentration, indicating that the position of accessible nucleosomes and other chromatin-associated factors associated with accessible chromatin regions are most dependent on CLAMP (S5C and S5D Fig). Therefore, CLAMP functions to promote the specific localization of chromatin accessibility upstream of the TSS throughout the genome in both male and female cells.

In addition to TSS, we also investigated chromatin changes over TTS because CES are biased towards the 3’ ends of genes [12]. We observed CLAMP-mediated changes in accessibility at TTS for genes with the greatest CLAMP occupancy (S4A Fig). Similar to TSS and gene bodies, TTS exhibited a reduction in accessibility following clamp RNAi on the male X-chromosome (S5E Fig), which we also observed on the male autosomes, as well as all female chromosomes (S5E Fig). We conclude that like TSS, CLAMP promotes accessibility at TTS genome-wide.

CLAMP differentially regulates transcription on the male X-chromosome compared with autosomes

To analyze the relationship between transcriptional and chromatin changes upon CLAMP depletion, we used the Start-seq technique [28]. The Start-seq technique specifically measures nascent RNAs produced by engaged RNA Polymerase II (Pol II) near transcription start sites, or Start-RNAs. Start-seq is a more precise measure of promoter Pol II activity than mRNA-seq, which also measures elongation, mRNA export, and stability. Using our Start-seq data, we redefined TSS based on the location where Pol II is engaged to generate a new subgroup of TSS called observed TSS (obsTSS) (S2 obsTSS: 9,740; Kc obsTSS: 10,067; TSS in FBv5.57: 13,903) [28]. We found that after clamp RNAi, approximately 10% of obsTSS were significantly changed (p<0.05) in their Start-RNA abundance in male cells, while approximately 7% were significantly changed in female cells (S2: 1,007 out of 9740: 10.3%; Kc: 680 out of 10,067: 6.8%).

We next determined the chromosomal distribution of significantly (p<0.05) changed Start-RNAs after clamp RNAi. We found that the significantly changed Start-RNAs were evenly distributed amongst all chromosomes in females, and modestly biased towards the X-chromosome in male cells, suggesting a role for CLAMP in transcriptional regulation on all chromosomes (Fig 5A and S5F Fig). For each chromosome, we then further examined the genes that displayed significantly increased (hatching) or decreased (no hatching) Start-RNA levels after clamp RNAi (Fig 5B and S5G Fig).

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Fig 5. CLAMP functions to regulate transcription genome wide through regulating nucleosome positioning.

A) Of the total number of transcripts aligned to the genome in males, approximately 12% (X-chromosome, blue) and 10% (autosomes, red) are significantly (p<0.05) changed following clamp RNAi treatment. The percentage of significantly changed transcripts in females is roughly equivalent for the X-chromosome and autosomes and is ~7%. B) The percent of significantly changed transcripts (p<0.05) that decrease in abundance after clamp RNAi (Positive regulation, un-hatched) or increase in abundance after clamp RNAi (Negative regulation, hatched) is plotted. CLAMP functions predominantly to promote transcription on the male X-chromosome, while on the male autosomes, it functions more frequently as a negative regulator. In females, CLAMP functions positively and negatively on all chromosomes at approximately equal frequencies. C) Average read frequency from all MNase titrations was plotted +/- 1 kb centered around obsTSS on the X and autosomes for genes with a CLAMP peak within +/- 200 bp of the obsTSS. There is a decrease in nucleosome positioning upstream of the TSS and an increase downstream of TSS on all chromosomes in both males (left) and females (right). D and E) X-chromosome and autosome obsTSS were categorized into quartiles based on the change in transcript abundance following clamp RNAi as measured by Start-seq. The two graphs on the left are for males while the graphs on the right are for females. Shown are average MNase-seq read frequency profiles for the quartiles with the largest decrease in transcription after clamp RNAi (D) (positively regulated) and largest increase in transcription after clamp RNAi (E) (negatively regulated).

https://doi.org/10.1371/journal.pone.0186855.g005

The male X-chromosome had the greatest percentage of transcripts positively regulated by CLAMP (62%), which was expected given the role of CLAMP in male X-chromosome dosage compensation which increases transcript levels (Fig 5B and S5G Fig). While transcription levels and chromatin accessibility tend to be highly correlated, we cannot fully determine whether the observed transcriptional changes following clamp RNAi are due to changes in accessibility or vice versa. However, in the case of the male X-chromosome, it is likely that changes in chromatin accessibility are separable from the transcriptional changes associated with MSL-dependent dosage compensation even if they are not separable from other transcription changes for the following reason: While MSL complex mediates the transcriptional changes associated with dosage compensation [20,22], it does not promote the same chromosome-wide changes in chromatin accessibility that are facilitated by CLAMP (Fig 1B and 1C) [7]. Furthermore, Start-seq measures transcription initiation and early elongation, which is less regulated by MSL complex than elongation over gene bodies [22,29]. Therefore, the CLAMP-mediated transcriptional changes observed are more likely to be linked to changes in chromatin accessibility than to a loss of dosage compensation.

In contrast to the male X-chromosome, CLAMP negatively regulates approximately 65% of genes on all autosomal arms with an even stronger effect on the heterochromatic 4^th chromosome (Fig 5B and S5G Fig). In females, CLAMP functions at approximately equal frequencies as both a positive and negative regulator of transcription on all chromosomes (Fig 5B and S5G Fig). Overall, CLAMP functions to promote gene activation more frequently on the male X-chromosome compared to male autosomes (Fig 5A and 5B).

To define how CLAMP-regulated transcriptional changes correlate with its role in chromatin accessibility, we compared average MNase-seq read frequencies over X-linked and autosomal genes that were either positively or negatively regulated by CLAMP as we did previously for TSS (Figs 4B and 5C). We analyzed genes that are highly bound by CLAMP as determined by ChIP-seq analysis and regulated by CLAMP as determined by Start-seq analysis (Fig 5) to define effects that are most likely to be directly mediated by CLAMP, presumably in combination with different co-factors within different genomic contexts. Genes that are positively regulated by CLAMP exhibited strong positioning over 5’ ends and gene bodies independently of genomic location (Fig 5D, S6A and S6B Fig). This pattern is similar to the pattern observed along the male X-chromosome (Fig 4B). In contrast, genes negatively regulated by CLAMP exhibited reduced nucleosome positioning after clamp RNAi treatment (Fig 5E, S6A and S6B Fig). For these genes, changes occur specifically over the nucleosome-depleted region at promoters and not along gene bodies.

To examine whether changes in MNase-seq read frequency after clamp RNAi differ when genes are expressed at different levels, we generated similar read frequency profiles for quartiles of genes ranked from lowest to highest expression level based on Start-seq read abundance in the control RNAi condition. Genes were further separated into additional classes based on whether they were located on the X-chromosome or autosome and if they were positively or negatively regulated by CLAMP (S7 and S8 Figs). In general, genes within the lowest three quartiles of gene expression exhibit similar changes in occupancy profiles after clamp RNAi. In contrast, the chromatin structural changes after clamp RNAi at genes in the top quartile are the strongest. We conclude that CLAMP regulates the chromatin organization of the most highly expressed genes more strongly than lowly expressed genes, with similar effects on the X-chromosome and autosomes. Overall, genes positively regulated by CLAMP exhibit changes in chromatin accessibility more similar to the average profile for genes on the male X-chromosome compared with those negatively regulated by CLAMP. In addition, we observed the same chromatin accessibility changes at CLAMP-regulated genes in both male and female cells, suggesting that the mechanisms by which CLAMP regulates chromatin and transcription at promoters are shared between the sexes.

CLAMP promotes NURF recruitment to CES but not promoters in males

Thus far, our analysis does not specifically distinguish between changes in chromatin accessibility that result from changes in nucleosome occupancy or occupancy of other factors that interact with chromatin. To measure changes in nucleosome occupancy, we performed Chromatin Immunoprecipitation followed by qRT-PCR (ChIP qRT-PCR) for the core Histone 3 (H3). We first measured the effect of clamp RNAi on H3 enrichment within the nucleosome-depleted region of four promoters differentially regulated by CLAMP at the level of chromatin accessibility and transcription (two positively regulated and two negatively regulated) (S9A and S9B Fig). We observed no statistically significant difference in H3 enrichment in either males or females between the control and clamp RNAi treatment at any of the tested promoters (Fig 6A, S2 Table). This suggests that regulation of accessibility at TSS by CLAMP does not involve changes in nucleosome occupancy. We therefore conclude that the mechanism by which CLAMP regulates accessibility within the upstream TSS region occurs independent of histone occupancy. Instead, accessibility may be regulated by the recruitment of other factors known to influence chromatin accessibility at TSS such as pausing factors like the Negative Elongation Factor (NELF) complex or even the Pol II complex itself [30–32].

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Fig 6. CLAMP promotes recruitment of NURF to CES, but not promoters.

A) Chromatin Immunoprecipitation following by qRT-PCR was performed for Histone H3 at promoters of genes that are either positively (cg1116 and prosap) or negatively (cg1815 and nemo) regulated by CLAMP as measured by Start-seq. For all graphs, plotted is the average log₂ fold enrichment value normalized to cg15570, a gene with no CLAMP enrichment with +/- 1 standard error of the mean (S.E.M). At all promoters tested there is no effect of clamp RNAi on H3 occupancy. B) H3 occupancy was measured by ChIP at five CES from the three subclasses. The CES with the greatest synergy between CLAMP and MSL complex (Group A) exhibit the strongest increase in H3 occupancy following clamp RNAi. The different in enrichment at CES5C2 was determined to be significant (* = p-value <0.05) by using a student’s T-test to compare differences between the clamp and control RNAi conditions. C) Enrichment of the NURF complex component NURF301 was measured at the same promoters as H3 occupancy. No significant differences were detected between the control and clamp RNAi treatments with the exception of cg1815 in females (* = p-value <0.05). D) The effect of clamp RNAi on NURF occupancy was measured different subgroups of CES. There are observed reductions in NURF occupancy in males at CES within all groups with the exception of Group C CES.

https://doi.org/10.1371/journal.pone.0186855.g006

We also measured H3 occupancy at CES to test whether changes in accessibility at these sites after clamp RNAi result from changes in nucleosome occupancy. We selected CES that exhibit CLAMP-dependent decreases in chromatin accessibility (S9B and S9C Fig). In contrast to candidate promoters, we found that clamp RNAi increases histone occupancy at the two-tested Group A CES in males, where CES5C2 was determined to exhibit the most significant change (p-value <0.05) (Fig 6B and S2 Table). Thus, CLAMP can regulate chromatin accessibility either by depleting histones or affecting interactions of non-histone factors with chromatin.

To define a possible mechanism by which CLAMP decreases histone occupancy at CES, we measured how CLAMP regulates recruitment of the Nucleosome Remodeling Factor (NURF) chromatin-remodeling complex. Several lines of evidence suggest a possible functional link between CLAMP and NURF. First, NURF has been previously implicated in regulating the chromatin organization of the male X-chromosome [33]. Second, CLAMP and the NURF301 chromatin remodeler both repress the expression of the male-specific non-coding RNA on the X (roX) RNAs in females [10,34]. Third, independent proteomic analyses of the MSL complex and dREAM chromatin-modifying complex identified both CLAMP and NURF301 as interacting factors [35,36]. Fourth, the well-studied transcription factor, GAGA Factor (GAF) shares a similar GA-rich binding motif with CLAMP and is known to promote the recruitment of the NURF complex [37–41]. Therefore, we investigated whether CLAMP recruits NURF to regulate the chromatin accessibility of the X-chromosome.

To test whether CLAMP promotes NURF recruitment to CES, we examined the same CES that we profiled for H3 occupancy and performed ChIP-qRT-PCR using an antibody specific for the NURF301 protein after control or clamp RNAi treatment. Co-occupancy of CLAMP and NURF301 was confirmed at each of these sites using previously published CLAMP ChIP-seq and NURF301 ChIP-chip data from the modENCODE project (S9 Fig) [42]. At all CES tested, we observed that CLAMP promotes NURF301 recruitment in males but not in females (Fig 6D and S2 Table), suggesting a potential mechanism for mediating changes in chromatin accessibility of the male X-chromosome. Because the ability of CLAMP to recruit NURF is specific to MSL-dependent Group A and B CES in males, it is possible that cooperation with MSL complex is important for this process. In contrast, clamp RNAi does not alter NURF occupancy at most promoters tested in males or females (Fig 6C) suggesting that mechanisms by which CLAMP alters chromatin accessibility at CES and TSS are different, comprising two distinct context-specific roles for the same protein. Overall, our data suggest that an X-chromosome enriched ubiquitous transcription factor that can act over long distances (CLAMP) and a sex-specific protein complex that acts locally (MSL complex) function together to generate a highly specialized chromatin domain that coordinates gene activation on the male X-chromosome.

Cell culture conditions

Drosophila S2 and Kc cells were maintained at 25°C in Gibco Schnieder’s Drosophila media (ThermoFisher Scientific) supplemented with 10% heat-inactivated Fetal Bovine Serum and 1.4X Antibiotic-Antimycotic (ThermoFisher Scientific). Cells were passaged every 2–3 days to maintain appropriate cell density.

RNAi treatment of Drosophila cells

Generation of dsRNA targeting gfp (control), clamp, and msl2 for RNAi has been previously validated and described [9,20,57]. In this study, the templates for clamp and msl2 dsRNA were generated using PCR amplification from bacterial artificial chromosomes (BAC) available from the BACPAC resources program (clamp = Ch322 20C06, msl2 = Ch321 59O03) [58]. PCR products were used as templates to generate dsRNA with the T7 Megascript kit (Ambion, Inc.), followed by purification with the Qiagen RNeasy kit (Qiagen).

RNAi was performed in T75 tissue culture flasks. A total of 1.2x10⁷ S2 or Kc cells suspended in 6mLs of Gibco Schenieder’s Drosophila media without FBS were added to a T75 tissue culture flask containing 135μg gfp, clamp, or msl2 dsRNA in 3mL ultra pure water. The cells were serum starved at room temperature for 45 minutes before adding 6mLs of Gibco Schneider’s Drosophila media supplemented with 10% FBS to the flask containing the dsRNA and cells. Cells were incubated for 6 days and upon collection efficiency of the RNAi treatment was determined before performing the desired experiment.

Validation of RNAi efficiency

MNase-titration and sequencing

Preparation of two biological replicates of Drosophila S2 and Kc cells for RNAi treatment with gfp, clamp, or msl2 was performed in T75 flasks. MNase titration was performed after 6 days of RNAi treatment as previously described [13].

Start-sequencing sample preparation and analysis

Start-sequencing was performed from three biological replicates of S2 and Kc cells that were treated for 6 days with either gfp (control) or clamp dsRNA. RNAi for Start-seq was performed as described above for MNase-seq with the exception that T150 flasks were used instead of T75. This resulted in a doubling of all reagents to perform the RNAi treatment. Total RNA was extracted from nuclei using Trizol reagent (Invitrogen) and transcription start site RNAs were isolated and prepared for sequencing as previously described [28]. Analysis for Start-seq was performed as previously described [28].

Chromatin immunoprecipitation of NURF301 and H3

Chromatin was prepared from gfp (control) and clamp dsRNA treated cells prepared in T150 flasks after 6 days of incubation. To prepare for chromatin immunoprecipitaton, cells were first scraped from the bottom of the flask to resuspend adhered cells. Formaldehyde (37% stock) was added to a final concentration of 1% and the cells were crosslinked for 10 minutes by shaking at room temperature at 110 rpm. The crosslinking reaction was quenched with the addition of 2.5M glycine to a final concentration of 125mM. Fixed cells were transferred to a 15mL falcon tube and placed on ice.

Following crosslinking and quenching, the media containing formaldehyde and glycine was removed after pelleting cells by centrifugation at 4°C for 5 minutes at 1,500xg. The cells were washed briefly by resuspending in 15mL of PBS-EDTA (1X PBS, 0.5mM EDTA pH 8.0, 0.2mM PMSF). Cells were pelleted as before and the supernatant removed. Next, the cells were resuspended in 6mL of ChIP wash A (10mM HEPES pH 7.6, 10mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 0.25% Triton X-100, 1X PI, 0.2mM PMSF, filter sterilized) and rotated for 10 minutes at 4°C. After rotation, the cells were pelleted once again and the supernatant removed. The cell pellet was then resuspended in 6mL ChIP wash B (10mM HEPES pH 7.6, 100mM NaCl, 1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 0.01% Triton X-100, 1X PI, 0.2mM PMSF, filter sterilized). The cells were rotated for 5 minutes at 4°C. A small aliquot was removed to determine total cell quantity using Trypan Blue (ThermoFisher Scientific). After washing the cells in ChIP wash B, the cells were pelleted flash frozen in liquid nitrogen for storage at -80°C.

Using the total cell count, cell pellets were resuspended in an appropriate amount of lysis buffer (0.1% SDS, 50mM Tris-HCL pH 8.0, 10mM EDTA pH 8.0) to obtain a cell concentration of 1x10⁸ cells/mL. The cells were lysed for 10 minutes by rotating at 4°C before aliquoting equal volumes (between 100-300uL) into Protein LoBind Safelock tubes (Eppendorf). The cell lysates were then sonicated for 3 cycles totaling 5 minutes each on high setting using a water bath sonicator (Bioruptor, Diagenode) programmed to pulse 30 sec on, 30 sec off. After sonication, replicate samples were pooled and centrifuged at 4°C for 10 minutes at a speed of 13,000 rpm to pellet the insoluble chromatin. Solubilized chromatin was transferred to a 15mL falcon tube, to which 9 volumes of ChIP dilution buffer was added (0.01% SDS, 16.7mM Tris-HCL pH8.0, 1.2mM EDTA pH 8.0, 1.1% Triton X-100, 167mM NaCl). Diluted chromatin was filtered on a PolyPrep chromatography column (BioRad) before aliquoting 1mL into Protein LoBind safelock tubes. Additionally, 400uL was removed for Input DNA. Chromatin aliquots were flash frozen for storage at -80°C until after Input was checked for chromatin size distribution.

Quantitative Real-Time PCR for analysis of enrichment

To determine enrichment of NURF301 or H3 to chromatin entry sites and promoters, three independent biological ChIP replicates were performed. Targets were amplified from 2uL of Input (1%) and immunoprecipitated DNA using validated primers at a concentration of 200nM. Primer sequences for qRT-PCR are in S3 Table. Two technical replicates for each sample were amplified using SYBR Green on an Applied Biosystems StepOnePlus^™ Real-Time PCR System. The obtained relative abundance values were then averaged and used for calculation of enrichment.

Internal normalization was performed using primers located within the cg15570 control gene, which is a genomic locus unbound by CLAMP as determined by ChIP-seq. Internally normalized values were then normalized to the 1% Input sample to determine log₂-fold enrichment of the immunoprecipitated sample compared to Input. Subsequently, the enrichment values from the three biological replicates were averaged and standard error of the mean was calculated.

Datasets

CLAMP ChIP-seq data and MSL CES coordinates were taken from GSE39271. To determine overlap between CLAMP and NURF301, browser tracks were taken from GSE20829 (S2 cells) and GSE32845 (Kc cells). Data from this study is deposited to NCBI GEO with GEO number GSE99894.

Data analysis