Abstract

Background

   Over the course of its intraerythrocytic developmental cycle (IDC), the
   malaria parasite Plasmodium falciparum tightly orchestrates the rise
   and fall of transcript levels for hundreds of genes. Considerable
   debate has focused on the relative importance of transcriptional versus
   post-transcriptional processes in the regulation of transcript levels.
   Enzymatically active forms of RNAPII in other organisms have been
   associated with phosphorylation on the serines at positions 2 and 5 of
   the heptad repeats within the C-terminal domain (CTD) of RNAPII. We
   reasoned that insight into the contribution of transcriptional
   mechanisms to gene expression in P. falciparum could be obtained by
   comparing the presence of enzymatically active forms of RNAPII at
   multiple genes with the abundance of their associated transcripts.

Results

   We exploited the phosphorylation state of the CTD to detect
   enzymatically active forms of RNAPII at most P. falciparum genes across
   the IDC. We raised highly specific monoclonal antibodies against three
   forms of the parasite CTD, namely unphosphorylated, Ser5-P and
   Ser2/5-P, and used these in ChIP-on-chip type experiments to map the
   genome-wide occupancy of RNAPII. Our data reveal that the IDC is
   divided into early and late phases of RNAPII occupancy evident from
   simple bi-phasic RNAPII binding profiles. By comparison to mRNA
   abundance, we identified sub-sets of genes with high occupancy by
   enzymatically active forms of RNAPII and relatively low transcript
   levels and vice versa. We further show that the presence of active and
   repressive histone modifications correlates with RNAPII occupancy over
   the IDC.

Conclusions

   The simple early/late occupancy by RNAPII cannot account for the
   complex dynamics of mRNA accumulation over the IDC, suggesting a major
   role for mechanisms acting downstream of RNAPII occupancy in the
   control of gene expression in this parasite.

Electronic supplementary material

   The online version of this article (doi:10.1186/1471-2164-15-959)
   contains supplementary material, which is available to authorized
   users.

Background

   The malaria parasite Plasmodium falciparum undergoes a 48 h life cycle
   from the moment of red blood cell invasion through to the production
   and release of mature progeny. In the course of this intraerythrocytic
   developmental cycle (IDC), the mRNA level for many genes rises and
   falls once at a point that correlates with the time its protein product
   is needed. Such results have led to the proposal of a “just in time”
   model of plasmodial gene expression in which mRNAs accumulate just as
   their products are required during the IDC [[41]1] Many factors affect
   mRNA levels, including transcriptional initiation, transcriptional
   elongation, mRNA processing, mRNA export, and mRNA stability. While
   control of Plasmodium gene expression at the level of transcription has
   been demonstrated [[42]2–[43]5], several recent studies provide
   evidence that post-transcriptional mechanisms must play a major role as
   well. For example, P. falciparum shows a widespread chromatin opening
   and histone H2A.Z recruitment in the intergenic regions throughout the
   IDC. Although this modification has been associated with actively
   transcribed chromatin in other species, in Plasmodium falciparum this
   histone variant is also recruited early to genes whose transcripts do
   not appear until much later [[44]6–[45]8]. A similar disconnect is seen
   with component of the basal transcriptional machinery, TBP and TFIIE,
   which are broadly recruited to Plasmodium genes regardless of
   corresponding transcript abundance [[46]9]. Moreover, nuclear run-ons
   correlated active transcription of some selected genes with the levels
   of their transcripts across the IDC. While some loci showed clear
   positive correlation between transcriptional activity and mRNA
   abundance, others revealed striking discrepancies strongly indicative
   of post-transcriptional regulation [[47]10] and consistent with similar
   discordances seen in humans [[48]11]. Additionally, transcript
   stability has been demonstrated to vary by an average of six fold
   between ring and schizont stages, and is correlated with a progressive
   loss of RNA degrading enzymes [[49]12]. Last, parasites deficient in
   the post-transcriptional regulator CAF1 display major shifts in peaks
   of mRNA accumulation [[50]13]. Such findings are consistent with major
   post-transcriptional control during the IDC.

   During transcription, many steps of mRNA synthesis and processing are
   integrated through the C-terminal domain (CTD) of the largest subunit
   of RNAPII (RPB1). A hallmark of RPB1 in most eukaryotes is the presence
   of a repeating heptapeptide motif in the CTD [[51]14]. In most
   eukaryotes, the heptad repeat has the consensus sequence YSPTSPS and is
   present in many copies ranging from 52 in humans to 26 in Saccharomyces
   cerevisiae. Moreover, the number and sequence of repeats is highly
   invariant within and even between species. By contrast, the CTD of
   Plasmodium spp. differs by the inclusion of a lysine at position 7 of
   the heptad repeat (YSPTSPK), contains fewer repeats and shows much
   greater variability in repeat number between and within Plasmodium
   species [[52]15, [53]16].

   Among other modifications, the serine residues at positions 2 and 5
   (and 7 in many organisms), can be phosphorylated and intensive effort
   has gone into trying to understand the role of these modifications in
   gene expression. Much attention has focused on the functional
   consequences of an unphosphorylated CTD, mono-phosphorylation at
   position 5 (Ser5-P), and di-phosphorylation at positions 2 and 5
   (Ser2/5-P). A long-held model proposes that the enzymatic activity of
   RNAPII is determined by the phosphorylation status of the CTD. In this
   model, RNAPII bearing a hypophosphorylated CTD is enzymatically inert,
   while Ser5-P is required for RNAPII to initiate transcription, and
   Ser2/5-P then confers the elongating and highly processive activity of
   RNAPII [[54]14]. However, a revised model suggests that the
   phosphorylation status of the CTD may simply be a correlative marker of
   RNAPII activity [[55]17]. Thus, while the exact functions of
   phosphorylation events at the CTD are a matter of debate, there is a
   strong consensus that the presence of Ser5-P and especially Ser2/5-P
   are marks of transcriptionally active polymerase.

   We have exploited the phosphorylation state of the RNAPII CTD to assess
   the engagement of most P. falciparum genes with the transcriptional
   machinery across the IDC. This allowed us to assess the extent to which
   RNAPII occupancy correlates with the mRNA accumulation. Our data
   indicate that P. falciparum genes are divided into two classes
   depending on whether peak RNAPII binding occurs early or late during
   the IDC. When comparing RNAPII occupancy to mRNA abundance, we
   identified subsets of genes with high occupancy and relatively low RNA
   levels and vice versa. We find that genes at which RNAPII is detected
   early (8 to 32 hours post-infection (hpi)) are shorter in length, have
   more introns and shorter first exons when compared to genes at which
   RNAPII is detected late (40-48 hpi). Furthermore, we find distinct
   RNAPII occupancy patterns for the early and late genes along the gene
   length. Comparison of RNAPII occupancy with various histone
   modifications identifies chromatin changes that precede, accompany and
   follow RNAPII recruitment. Together our results show that occupancy by
   presumed active forms of RNAPII cannot account for the highly
   orchestrated pattern of mRNA accumulation and loss over the IDC. We
   suggest that post-initiation mechanisms such as transcriptional
   elongation and mRNA stability may play predominant roles in determining
   the dynamics of mRNA abundance during infection of red blood cells.

Results

Production of monoclonal antibodies specific for three forms of the CTD of
the large subunit of RNAPII

   Steps in the generation and use of monoclonal antibodies in this study
   are summarized in Figure [56]1A. To examine the genome-wide
   distribution of RNAPII, we raised highly specific monoclonal antibodies
   against three forms of the P. falciparum RPB1 CTD: unmodified CTD,
   Ser5-P-bearing CTD, and CTD having the Ser2/5-P double phosphorylation.
   The phosphorylated forms of RPB1 have been associated with RNAPII
   molecules that are functionally engaged with bound genes.
   Unphosphorylated heptad repeats may be associated with inert forms of
   the polymerase, but could also be present as one or more copies within
   a CTD that also contains phosphorylated motifs. The specificity of the
   antibodies was confirmed in several ways. First, the antibodies showed
   high specificity in ELISA by serial dilution (data not shown) and,
   second, by competition with the immunizing peptide in a peptide
   competition assay (Figure [57]1B-D). These results establish that each
   antibody specifically recognizes its cognate peptide and not the other
   two. Third, the appropriate sized band for P. falciparum RBP1 is also
   observed on Western blot of nuclear lysates following
   immunoprecipitation using each of the three monoclonal antibodies
   (Figure [58]1E). A well-characterized commercially available antibody
   (8WG16) that recognizes unphosphorylated and Ser5-P CTD [[59]18]
   likewise detects a similarly sized band corresponding to RPB1 in a
   Western blot of cell lysate. Although all antibodies (including 8WG16)
   detect additional bands, stringent washing leaves a unique band
   corresponding to the expected molecular weight of RPB1 as the major
   species detected by the αSer2/5-P antibody (indicated by W* in
   Figure [60]1E). Fourth, IP followed by mass spectrometry revealed that
   both the αSer5-P and αSer2/5-P antibodies precipitated RPB1 as well as
   two additional subunits of RNAPII, namely RPB2 and RPB3, with high
   confidence. No RNAPII subunits were precipitated by a control IgG
   (Additional file [61]1: Table S1). Together, these results show that
   these antibodies are specific for the CTD of RPB1 in P. falciparum and
   are suitable for use in the chromatin immunoprecipitation assay (ChIP).

Figure 1.

   Figure 1
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   Validation of monoclonal antibodies against RNA polymerase II. (A)
   Schematic of the work flow. P. falciparum samples were harvested every
   8 h across the 48 h life-cycle at time points (TP) 1 through TP6. The
   same culture was used for triplicate ChIP-on-chip and transcriptome
   analyses. (B–D) Peptide competition assays. Monoclonal antibodies were
   raised against peptides corresponding to the unmodified heptad repeat
   (anti-CTD), the heptad repeat monophosphorylated at position 5
   (αSer5-P) and diphosphorylated at positions 2 and 5 (αSer2/5-P). Each
   monoclonal antibody was pre-incubated with each of the three
   immunogenic peptides derived from the RNAPII CTD. Reactivity against
   the immunizing peptide was then tested by ELISA. The concentration of
   competing peptide in μg/ml is given to the right of each graph. (E)
   Parasite nuclear protein lysate from an asynchronous culture was
   immunoprecipitated (IP) with each of the three monoclonal antibodies
   raised in this study or a non-specific IgG control. The precipitates
   were separated by 6% SDS-PAGE and Western blot (W) performed with the
   indicated antibodies. The appropriately sized band for P. falciparum
   RPB1 (279 KDa) was observed with all three antibodies (arrowhead). Two
   panels are shown for antibody α-Ser2/5-P and represent the same Western
   blot following a moderate and more extensive (W*) series of washes.
   Note that the only significant specific band is of the correct
   molecular mass for RPB1. No specific bands are detected by the α-CTD
   and α-Ser2/5 antibodies following IP with control IgG. The commercial
   anti-CTD antibody 8WG16 detects a band of the correct molecular mass
   for P. falciparum RPB1 along with several faster running species. The
   positions of the marker bands in KDa are given to the right of the
   panels they describe.

RNA polymerase II shows a bi-phasic binding distribution across the P.
falciparumIDC

   We next used these P. falciparum anti-RNAPII antibodies in ChIP-on-chip
   analysis to map the position and enzymatic status of RNAPII across the
   entire P. falciparum genome over the 48 h of the IDC at 8 h intervals.
   The commercial anti-CTD antibody, 8WG16, was also used in parallel. We
   used custom-made microarrays spotted with 70-mer oligonucleotides
   covering both coding and intergenic regions [[63]19, [64]20].

   Following hybridization and analysis of the microarrays, we selected
   all those probes exhibiting a significantly oscillating profile with p
   <0.05 and ≥1.5 fold change across the life cycle. This cut-off resulted
   in the retention of a subset of probes for each of the three
   antibodies: 2,936 probes with the αCTD (unphosphorylated) antibody;
   4,462 probes with the αSer5-P antibody; and 4,255 probes with the
   αSer2/5-P antibody. We addressed the temporal order within the dataset
   by creating a phaseogram of RNAPII occupancy. The genome-wide profiles
   reveal a striking periodicity involving the majority of genes
   (Figure [65]2A). Most genes can be divided in two groups: those that
   bind RNAPII early during the IDC and those that bind RNAPII late,
   regardless of the phosphorylation state of the CTD. This division into
   early versus late is most dramatic for the Ser2/5-P form of RNAPII
   which is the elongating form of the polymerase. The data show a
   striking switch between T4 (32 hpi) and T5 (40 hpi) such that the
   Ser2/5-P form binds maximally to one large fraction of genes early in
   the IDC until T4 (1,600 genes), but then switches to a largely mutually
   exclusive set of genes late in infection during T5 and T6 (1,359). Only
   11% of genes are present in both categories due to differential signals
   from probes within the same gene. A similar observation is made for
   data sets obtained with the other two antibodies against the unmodified
   CTD and the Ser5-P; however, the switch between early and late binding
   profiles for these two forms occurs earlier between T3 (24 hpi) and T4
   (32 hpi). Differential occupancy of the different forms of RNAPII
   argues against a bias inherent to the experimental procedure or the
   microarrays themselves. Importantly, ChIP-on-chip with the commercial
   8WG16 antibody likewise revealed a segregation of early vs late signals
   for the majority of probes in a pattern resembling those obtained with
   the α-CTD and α-Ser5-P antibodies (Additional file [66]2: Figure S1A).
   This is consistent with the demonstration that 8WG16 recognizes the
   unmodified and Ser5-P forms of the heptad repeat [[67]18]. Hierarchical
   clustering of the datasets also highlights the distinct distribution of
   RNAPII in just two prominent groups, one dominated by loci where RNAPII
   binds early in the IDC and another dominated by loci where the binding
   is late (Additional file [68]2: Figure S1A and Additional file [69]3:
   Figure S2). The timings of the switches are identical to those revealed
   in the phaseograms.

Figure 2.

   Figure 2
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   ChIP-on-chip reveals two classes of genes distinguished by recruitment
   of RNAPII either early or late in the IDC. (A) Three phaseograms
   (blue-yellow scale) representing temporal occupancy patterns for the
   three forms of RNAPII detected in this study: unphosphorylated CTD,
   Ser5-P and Ser2/5-P. The data were filtered to include only those loci
   where RNAPII shows an oscillating profile with p <0.05 and a fold
   change ≥1.5 across the life cycle. The yellow-blue color scale
   represents the mean-centered log transformed ratios of ChIP/input
   values across the IDC (T1 to T6). Two prominent probe clusters binding
   the Ser2/5-P form of RNAPII early vs late in the IDC are marked with
   orange rectangles. The phaseogram on the far right (green-red scale)
   depicts the phase of peak gene expression for the P. falciparum
   transcriptome across the IDC. (B-C) ChIP-qPCR confirms the presence of
   two classes of genes distinguished by enrichment in RNAPII binding
   either early or late in the IDC. Using immunoprecipitated DNA,
   quantitative real time PCR was carried out on representative genes
   chosen from the early (B) and late (C) categories to validate the ChIP
   occupancy profiles of Ser2/5-P RNAPII across the IDC. Primers for real
   time PCR were designed to amplify 200–300 bp regions around the
   respective probe on the microarray. The x-axis gives time points T1 to
   T6 and the y-axis shows the fold change. Solid lines represent real
   time PCR profiles, whereas dashed lines give the microarray
   (ChIP-on-chip) profile at the same probe region. The fine dotted line
   represents the mRNA levels across the six time points determined by
   hybridization of cDNA to the microarrays. Error bars show standard
   deviation of triplicate determinations. See also Additional files
   [71]2, [72]3 and [73]4: Figures S1, S2 and S3.

   To confirm results obtained by microarray, we performed ChiP-qPCR at
   each of the six time points for six genes that bind the elongating
   (Ser2/5-P) form of RNAPII early in the IDC and four that bind late.
   Although qPCR reveals a greater dynamic range than the microarray
   analysis, the results show that all early genes acquire peak RNAPII
   recruitment early in the IDC at either time point 3 or 4 (24 and 32
   hpi), while late genes do not display maximal RNAPII binding until time
   point 6 (48 hpi) (Figure [74]2B,C). These findings strongly validate
   the results of ChIP-on-chip and confirm the presence of two gene
   classes distinguished by early or late enrichment in RNAPII binding
   over the course of the IDC.

Relationship between transcriptional activity and mRNA abundance

   To characterize the relationship between the genome-wide binding of
   distinct forms of RNAPII and mRNA abundance, we calculated the
   Pearson’s Correlation Coefficient (PCC or r) between the dynamic RNAPII
   occupancy (p <0.05 and 1.5 fold change) and the expression levels of
   corresponding mRNAs. We established two subsets (Figure [75]3). In one
   subset, increased RNAPII occupancy (yellow) was positively correlated
   with high mRNA levels (red). In the other subset, the correlation was
   negative, meaning that there was either high mRNA expression without
   detectable RNAPII binding, or that we observed RNAPII binding without
   corresponding mRNA expression. With respect to the Ser2/5-P isoform of
   RNAPII, 1,740 probes were highly positively correlated with gene
   expression (right panel), while 1,200 probes were strongly negatively
   correlated (left panel) (Figure [76]3C). Similar observations were also
   made with the unmodified CTD and Ser5-P isoforms of RNAPII
   (Figure [77]3A,B) as well as with the 8WG16 commercial antibody
   (Additional file [78]2: Figure S1B). Approximately a third of the
   probes showed no significant correlation. The above results thus reveal
   four subsets of genes with respect to RNAPII occupancy and mRNA
   abundance: genes which bind RNAPII early and are either positively or
   negatively correlated with mRNA levels (early/positive and
   early/negative), and genes which bind RNAPII late (late/positive and
   late/negative).

Figure 3.

   Figure 3
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   Relationship between RNAPII occupancy and mRNA abundance. Correlation
   between the genome-wide detection of unmodified CTD (panel A), Ser5-P
   (panel B) and Ser2/5-P (panel C) forms of RNAPII and mRNA levels was
   calculated using Pearson’s correlation coefficient (PCC) between the
   mean-centered profile of all probes with p <0.05 and the corresponding
   gene transcripts. The data have been presented as bar graphs with bins
   of PCC ranging from −1 to +1 on the x-axis plotted against the number
   of probes falling into each bin on the y-axis. The phaseograms in the
   right-hand panels represent probes which are highly positively
   correlated with gene expression (r ≥0.4), and in the left-hand panels
   display probes showing negative correlation with gene expression
   (r ≤ -0.4). One-third of the probes do not show any correlation. The
   order of the probes represented in the phaseograms for RNAPII binding
   is the same as those used in the mRNA phaseogram. On the basis of this
   analysis, we identify four subsets of genes with respect to the
   correlation between RNAPII binding and mRNA abundance. These are
   early/positive (RNAPII binds early in the IDC and is positively
   correlated with mRNA levels), early/negative and late/positive and
   late/negative.

   We assessed the genomic characteristics of RNAPII-Ser2/5-P-associated
   early and late genes. We mapped the genes that showed either early or
   late binding of RNAPII Ser2/5-P onto the 14 P. falciparum chromosomes.
   All chromosomes were seen to bear potential clusters (Additional file
   [80]4: Figure S3); however, closer examination revealed that genes
   within such clusters do not share a common expression profile, arguing
   against any functional organization and consistent with a previous
   study suggesting that P. falciparum lacks chromosomal domains with
   common expression profiles [[81]1]. We mapped several genomic
   descriptors known to associate with gene expression regulation in human
   [[82]21], including gene length, GC content, number of introns and
   intron length. We found that genes binding RNAPII Ser2/5-P at early
   times (T1 to T4) are short with lower GC content by comparison to late
   genes (T5 and T6) which are significantly longer (Additional file
   [83]5: Figure S4A, B). In addition, the early genes had a greater
   number of introns when compared to late genes (Additional file [84]5:
   Figure S4C; see below). The presence of introns has been linked to
   higher transcriptional efficiency [[85]22]. We assessed the length of
   the first exon and found that late genes have longer first exons when
   compared to early genes (Additional file [86]5: Figure S4D). Thus,
   early-transcribed genes are shorter in length, have more introns and
   shorter first exons. Short gene length and short first exons are
   properties of highly expressed genes in human cells [[87]23, [88]24].

Differential distribution of RNAPII along the transcription unit
distinguishes genes binding RNAPII early vs late in the IDC

   According to one model of CTD function, phosphorylation of Ser5
   residues predominates near the promoter, whereas toward the end of the
   gene RNAPII is extensively phosphorylated on Ser2 residues [[89]25,
   [90]26]. To explore the phosphorylation status of the CTD at different
   positions along the transcription unit, we examined the distribution of
   the three forms of RNAPII along the gene for each of the four classes,
   namely early and late binders that positively correlated with the mRNA
   levels (Figure [91]4A, right panels) and early and late binders that
   negatively correlated with mRNA levels (Figure [92]4A, left panels). We
   observed very distinct profiles for genes that show a positive
   correlation between mRNA levels and peak RNAPII binding either early or
   late in the IDC. Promoter regions in P. falciparum are predicted to lie
   between 300 and 1650 bp upstream of the ATG [[93]27]. RNAPII
   accumulates in the promoter region of early binders, whereas for late
   binders RNAPII is enriched in the gene body (Figure [94]4A, right
   panel). Likewise, the Ser2/5-P form of RNAPII was concentrated within
   upstream presumptive promoter regions (–1,000 to –500) of genes
   expressed early and negatively correlating with mRNA levels
   (Figure [95]4A, left panel). On the other hand, the unmodified (CTD)
   and Ser5-P forms did not obviously follow this distribution. A
   goodness-of-fit test assigns a significant p value (<0.01) to the
   difference in RNAPII distribution (all three forms) between early and
   late binders (Figure [96]4B,C) suggesting that the transcriptional
   process in early vs late phases may be distinguished by mechanistic
   differences. However, it is possible that this has more to do with the
   transcription of shorter vs longer genes or the coupling of the
   splicing machinery to the CTD, since “late” genes are longer and have
   fewer introns, as noted above.

Figure 4.

   Figure 4
   [97]Open in a new tab

   Distribution of RNAPII along the gene length and enriched pathways. (A)
   The plots represent the average distribution along the gene of probes
   (with p <0.05) associated with each form of RNAPII. The start codon is
   taken as “0”. Data are organized into bins ranging from -500 bp
   upstream of the ATG to +4000 bp downstream and plotted against the
   percentage of total probes falling into each bin. The position of a
   probe along the x axis is the average of the positions of all probes
   within a given bin. While all genes are aligned at the ATG (“0”), they
   terminate at a wide range of positions downstream with an average at
   +2.5 kb. The plots on the right show the distribution of early/positive
   and late/positive loci (r >0.4), while those on the left plot the
   distribution of early/negative and late/negative loci (r <0.4). (B-C)
   The plots represent the average distribution of the genetic loci
   associated with each form of RNAPII as a function of gene length.
   Position distributions were plotted as histograms for each gene group
   with intervals of 500 bp from -2 kb to +5 kb. Histograms were plotted
   to display the binding positions in 500 bp intervals from -2 kb to 5 kb
   flanking the translational start codon at 0. A goodness-of-fit test was
   performed within each bin to calculate the probability of that region
   being equally bound by RNAPII for different gene groups. Regions with
   binding bias by group were assigned p <0.05 using the chi-square test.
   Data were assessed in this manner for (B) early/positive and
   late/positive and (C) early/negative and late/negative subsets. (D)
   Functionally enriched pathways (p <0.05) for each of the four gene
   groups. Colors refer to the three distinct databases that were used for
   pathway enrichment analysis.

   To determine whether the observed differential recruitment of RNAPII at
   early vs. late times of infection also reflects functional gene
   classes, we mapped early and late genes to known pathways and
   determined which pathways are over-represented in each group by using
   three pathway databases, namely Gene Ontology (GO), Kyoto Encyclopedia
   of Genes and Genomes (KEGG) and Malaria Parasite Metabolic Pathway
   (MPMP) (Figure [98]4D). We found that early genes were significantly
   enriched at loci associated with growth (ribosome structure and
   maturation), metabolism (starch and sucrose metabolism, purine and
   pyrimidine metabolism), and transcription and splicing. As expected,
   late genes were significantly enriched in proteins involved in
   host-parasite interactions and pathogenesis (merozoite invasion and
   motility, merozoite surface proteins) and cytoskeleton organization and
   biogenesis.

Relationship between RNAPII occupancy and chromatin modifications

   Our group and others have shown that chromatin influences gene
   expression in P. falciparum [[99]6, [100]19, [101]28–[102]34]. To
   examine the relationship between RNAPII occupancy and chromatin
   modification, we calculated the PCC (r) between dynamic RNAPII
   occupancy of unmodified CTD, Ser5-P and Ser2/5-P and 13 histone
   modifications determined in our previous study [[103]19]. We identified
   5 histone marks including H3K9ac, H3K56ac, H4ac4, H4K12ac and H4K20me1
   which showed clear positive correlation with Ser2/5-P RNAPII binding at
   high statistical confidence (Figure [104]5). All these histone
   modifications, in addition to H3K4me3, associate with transcriptionally
   active protein coding genes and are described as active marks [[105]30,
   [106]31, [107]33], and references therein.