Beyond the transcript: chromatin implications in trans-splicing in Trypanosomatids

Beyond the transcript: chromatin implications in trans-splicing in Trypanosomatids | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Beyond the transcript: chromatin implications in trans-splicing in Trypanosomatids View ORCID Profile Romina Trinidad Zambrano Siri , View ORCID Profile Paula Beati , View ORCID Profile Lucas Inchausti , View ORCID Profile Pablo Smircich , View ORCID Profile Guillermo Daniel Alonso , View ORCID Profile Josefina Ocampo doi: https://doi.org/10.1101/2025.07.08.663533 Romina Trinidad Zambrano Siri 1 Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas , Vuelta de Obligado 2490, C1428ADN Buenos Aires, Argentina 4 Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires , Buenos Aires, Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Romina Trinidad Zambrano Siri Paula Beati 1 Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas , Vuelta de Obligado 2490, C1428ADN Buenos Aires, Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Paula Beati Lucas Inchausti 2 Laboratorio de Bioinformática, Departamento de Genómica, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE) , Montevideo, Uruguay 3 Sección Genómica Funcional, Facultad de Ciencias, Universidad de la República (UdelaR) , Montevideo, Uruguay Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lucas Inchausti Pablo Smircich 2 Laboratorio de Bioinformática, Departamento de Genómica, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE) , Montevideo, Uruguay 3 Sección Genómica Funcional, Facultad de Ciencias, Universidad de la República (UdelaR) , Montevideo, Uruguay Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pablo Smircich Guillermo Daniel Alonso 1 Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas , Vuelta de Obligado 2490, C1428ADN Buenos Aires, Argentina 4 Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires , Buenos Aires, Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Guillermo Daniel Alonso For correspondence: galonso{at}dna.uba.ar jocampo{at}ingebi-conicet.gov.ar Josefina Ocampo 1 Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas , Vuelta de Obligado 2490, C1428ADN Buenos Aires, Argentina Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Josefina Ocampo For correspondence: galonso{at}dna.uba.ar jocampo{at}ingebi-conicet.gov.ar Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Trypanosoma cruzi, Trypanosoma brucei and Leishmania major , usually known as TriTryps, are the causal agents of animal and human sickness, and are characterized by having complex life cycles, alternating between a mammalian host and an insect vector. Their genes are organized in long transcriptional units that give rise to polycistronic transcripts which maturate into mRNA by a process known as trans-splicing. Among those genes, an important subset is composed of multicopy genes, which play crucial roles in host invasion and immune evasion. Here, we predicted the most likely trans-splicing acceptor sites (TASs) for TriTryps and found that the average chromatin organization is very similar among them with a mild nucleosome depletion at the TASs, and the same layout is observed in most of the genome. A detailed examination of the nucleosome landscapes resulting from different levels of chromatin digestion in T. brucei shows that an MNase-sensitive complex is protecting the TASs, and it is at least partly composed of histones. Additionally, comparative analysis for single and multi-copy genes in T. cruzi revealed a differential chromatin structure at the TASs suggesting a novel mechanism to guarantee the fidelity of trans-splicing in trypanosomatids. Introduction The protozoan parasites Trypanosoma cruzi, Trypanosoma brucei and Leishmania major , usually known as TriTryps, are responsible for Chagas disease, Sleeping sickness, and Leishmaniasis respectively. They belong to the group of neglected tropical diseases and together affect more than 30 million people worldwide 1 . These parasites have complex life cycles alternating between an insect vector and a mammalian host. To cope with this alternation through different hostile environments, TriTryps need to adapt their gene expression to the specific requirements and challenges they face, including being able to achieve host invasion. Among the strategies orchestrated to achieve this aim, these parasites count on multi-copy genes encoding for specialized surface proteins involved in virulence or immune system evasion 2 – 6 . One peculiarity of TriTryps is that their genes are organized into directional gene clusters (DGCs) that encode polycistronic transcription units (PTUs) which are further processed into monocistronic units by a co-transcriptional process that involves polyadenylation and trans-splicing 7 . This maturation involves a cleavage event coupled to the addition of a Spliced Leader sequence (SL) at the 5’-end and a poly(A) tail at the 3’-end of each mRNA 8 . Although in TriTryps gene expression is mainly regulated post-transcriptionally, there is substantial evidence that chromatin punctuation by epigenetic factors exerts additional modulation 9 – 15 . In eukaryotic cells, chromatin arrangements are well orchestrated in the nucleus to regulate DNA exposure, so nucleosomes have been proposed as major determinants of DNA accessibility 16 . While in vitro , DNA sequence preferences determine nucleosome formation, i n vivo , the outcome will be the result of the interplay with ATP-dependent chromatin remodellingremodeling complexes, non-histone DNA binding proteins, histone variants, histones post-translational modifications, and transcription 17 – 19 . Regarding the influence of DNA sequences, regions rich in poly AT or poly GC are refractory to bend around the histone octamer 20 – 23 , while tracks of DNA with a 10 bp periodicity of AT dinucleotides facilitate the bending of the DNA around the histone core 24 , 25 . In TriTryps, it has been previously observed that dinucleotide repeats are non-uniformly distributed along the DGCs, suggesting that DNA sequence composition might influence genome compartmentalization and gene expression 26 . Additionally, it was described that the genome of T. cruzi is compartmentalized into a ‘core compartment’, with lower GC content mainly harboring conserved single-copy genes; and a ‘disruptive compartment,’ which exhibits high GC content and is mainly composed of multi-copy genes 27 . In general, nucleosome distribution on DNA sequences is organized into regular arrays where every nucleosome is spaced from the neighboring ones by a stretch of DNA called linker. In model organisms, nucleosomes are regularly spaced and phased over coding regions relative to the transcription start site of genes and present nucleosome depleted regions (NDRs) at promoters surrounded by well-positioned nucleosomes at +1 and -1 position 28 – 30 . The general bases of chromatin landscape are conserved from yeast to humans, but in more complex organisms this regular pattern is mainly associated with highly transcribed genes, while silenced genes usually do not have clear NDR or phased nucleosomes 31 . In trypanosomes, there are no canonical promoter regions. Instead, transcription is initiated from dispersed promoters and in general they coincide with divergent strand switch regions 32 , 33 . Consistent with an ongoing passage of RNA polymerase II, the nucleosome maps reported for TriTryps revealed poor nucleosome organization with no average spacing or phasing 33 – 36 . From our perspective, the most relevant observation made from the previous chromatin studies in TriTryps is that average nucleosome occupancy changes around the TASs, suggesting a potential role of chromatin in trans-splicing. However, a proper comparison of the nucleosome maps from the three organisms is missing. In this work, we performed a thorough comparison of MNase-seq data publicly available for the stages present in the insect vectors in TriTryps, generated by others and in our laboratory 34 – 37 . Consistent with the original works, from average nucleosome occupancy plots we observed a mild NDR at the TASs in T. cruzi , a shallower trough preceded by an MNase protected footprint in L. major , and an MNase protection at the TAS in T. brucei . Nevertheless, when analyzing comparable levels of digested chromatin, we unveiled that both, T. cruzi and T. brucei , present a nucleosome depletion at the trans-splicing acceptor sites (TASs). Additionally, we analyze different levels of MNase-digested samples for T. brucei and we demonstrate that an MNase-sensitive complex is protecting the TASs. This complex is at least partly composed of histones as shown by MNase-ChIP-seq data for histone H3 and is detected both in T. brucei and T. cruzi , suggesting a conserved protection of the TASs and sensitivity to MNase in trypanosomes. Moreover, comparative analysis for single and multi-copy genes in T. cruzi revealed that their TASs are differentially protected from MNase digestion. This observation unveils that the NDRs formation at the TASs in epimastigotes occurs more efficiently at genes that need to be expressed at that life-stage. Furthermore, by analyzing dinucleotide frequencies around TASs we observe different patterns for single and multi-copy genes, possibly implying that transcript maturation is additionally granted by the underlying DNA sequence composition in a stage-independent manner. Results There is a distinctive average nucleosome arrangement at the TASs in TriTryps Genome-wide nucleosome mapping by MNase-seq for the parasitic forms present in the insect vector has been performed for TriTryps: L. major 34 , T. brucei 37 and T. cruzi 35 , 36 . Some similarities and some differences from these original works could be inferred. However, a proper systematic comparison is still missing. Therefore, we performed a parallel analysis of the individual datasets following the same informatics workflow for all of them as we described before 36 . The list of raw data we used for every analysis is summarized in detail in Table S1. To make a fair comparison, it is important to contrast samples that have achieved a similar level of MNase digestion. To corroborate this feature, we represented the length distribution of the sequenced DNA molecules for each sample into histograms (Fig. S1). The levels of digestion achieved for T. cruzi and T. brucei samples are in a good range for nucleosome core mapping, since most of the sequenced DNA molecules are ∼147 bp. In the case of L. major the samples are less digested; therefore, we considered this bias for result interpretations. In T. brucei , despite the histograms resemble those of T. cruzi , it is worth mentioning that the samples were gel-purified before library preparation. Therefore, we cannot rule out that the original samples could have been less digested and that only the nucleosome-size fraction was sequenced. One of the most interesting features that arose in the original articles is the fact that there is a distinctive average nucleosome arrangement at intergenic regions around the TASs in TriTryps that differs from the rest of the genome. Hence, to make a comparative analysis of average chromatin organization, we predicted the TASs for the three organisms using UTRme 38 . This program makes a prediction of the 5’ untranslated region (5’UTR) and as an approximation of the TAS, we used the 5’ end of the 5’UTR region predicted with the best score in each case ( Fig. 1a ). A list containing the genomic coordinates for the predicted TASs for each TriTryp is detailed in Table S2. Consistent with the original works, we observed that average chromatin organization only shows a mild change around the TASs with no regular nucleosome phasing in any TriTryp ( Fig. 1b and Fig. S2a, top panels). Remarkably, we corroborated the presence of a mild NDR around the TASs in T. cruzi , a shallower trough preceded by a small footprint of MNase protection in L. major and a protection centered at the TAS in T. brucei , as previously reported 34 – 37 . Download figure Open in new tab Figure 1. Chromatin organization around TASs in each TriTryp (A) Schematic representation of TASs prediction from 5’UTR regions obtained with UTRme (Radio et. al., 2017). (B) Average nucleosome occupancy (top panels), heatmaps for each region in a 1 kb window (boaom panels). The signals scored for DNA molecules in the nucleosomal-size range (120-180bp) are represented. (C) 2D occupancy plots showing nucleosome density relative to the TAS (boaom panels) for one representative data set of T. cruzi, T. brucei and L. major respectively. Red: High nucleosome density; blue: low nucleosome density. Given that average patterns can mask gene-to-gene variability, we also represented nucleosome occupancy into heatmaps for every individual region relative to the TAS in a 1 kb window. We could observe that the chromatin landscape is not just an average, but it is maintained in most of the detached regions represented for TriTryps ( Fig. 1b and Fig. S2a, lower panels). To determine the size of the sequenced molecules, that indirectly unveil the size of the molecules responsible for protecting the DNA from MNase digestion, and to know their location relative to the TAS, we represented the data into 2-dimensional plots (2D-plots) as previously described 36 , 39 ( Fig. 1c and Fig. S2b). Consistently with the length distribution histogram, most of the DNA molecules are ∼150 bp for T. cruzi and T. brucei , in a wider range for the latest, and a bit longer for L. major . Note that for L. major and T. brucei the samples were gel-purified before library preparation and that has implications on the range of the fragments detected in each case. Additionally, 2D-plots exposed that the DNA molecules protecting the TASs in T. brucei or the spliced-out fragment in L. major have the size of a nucleosome core particle, consistent with previous reports that describe the presence of a well-positioned nucleosome at those specific points 34 , 37 . However, given that the original samples were gel-purified, we cannot rule out that additional molecules could be involved in protecting DNA from digestion. In T. cruzi , those DNA protecting molecules around the TASs were not detected, but it is probably due to the extent of the MNase digestion. Overall, this analysis suggests the presence of some conserved MNase sensitive complex in or near the TASs, although some uniqueness might be involved in each TriTryp. An MNase sensitive complex occupies the TASs in T. brucei To explore whether the protection observed at the TASs in T. brucei is due to an MNase-sensitive complex sitting in or near that point, we analyzed samples exposed to different levels of digestion using different datasets publicly available (Fig. S3 and table S1). By representing average nucleosome density relative to the TAS we could observe that, for early digested timepoints, the TASs are covered by some protecting complex. As the digestion proceeds, the complex is less pronounced, reaching a minimum where a trough is observed (High digestion), consistent with the presence of an MNase sensitive complex ( Fig. 2a ). Moreover, heatmap and 2D-plot representation have enlighten that, at an early digestion point (Low digestion) the complex that protects the TASs is only accessible in part of the genome and it has a footprint that is heterogeneous in size; but at intermediate digestion timepoint (Intermediate digestion 1) it is homogenously observed in the whole genome and mostly nucleosome-size ( Fig. 2b and Fig. S3b). This analysis shows how important it is to check the level of digestion reached by a given sample when comparing them, since different regions of the genome are not equally accessible. Therefore, if we compare similar levels of digested sample for T. brucei ( Fig. 2 , intermediate 2) and T. cruzi ( Fig. 1b ), we can observe a comparable chromatin organization with NDRs formation at the TASs. This observation suggests a conserved pattern of chromatin landscape with a potential role in mRNA maturation among TriTryps. Download figure Open in new tab Figure 2. Differential sensitivity to MNase at the TASs in T. brucei . ( A ) Average nucleosome occupancy and (B) heatmaps showing nucleosome density relative to the TAS for each region in a 1 kb window for procyclic forms of T. brucei exposed to different levels of MNase digestion. Red: High nucleosome density; blue: low nucleosome density. The signals scored for every DNA molecule sequenced (0-500bp) are represented. Red: High nucleosome density; blue: low nucleosome density. The MNase sensitive complexes protecting the TASs in T. brucei and T. cruzi are at least partly composed of histones Despite most of the time DNA protection to MNase digestion is mediated by nucleosomes, on occasions other non-histone binding complexes could be involved 40 , 41 . To understand the nature of the MNase protection at TASs observed in T. brucei , we analyzed data obtained by MNase-ChIP-seq of histone H3 publicly available 33 , 42 (Table S1). By analyzing average occupancy of histone H3 relative to the TAS, we could observe that the MNase protection previously observed at the reference point in T. brucei disappeared. Instead, we detected a mild trough as reported 33 ( Fig. 3a , middle panel and Fig. S4a, left panel). Download figure Open in new tab Figure 3. TASs resistance to MNase digestion is partly mediated by a histone component. Average H3 occupancy (top panels) and heatmaps (boaom panels) relative to the TAS for one representative experiment of MNase-ChIP-seq of histone H3 (A) T. brucei and (B) T. cruzi . Red: High nucleosome density; blue: low nucleosome density. As we discussed before, the protection of the TASs is tightly connected to the extent of the sample digestion. To be sure we had not missed any partial histone protection of the TAS due to a differential digestion, we analyzed the average signal of histone H3 in the sequenced molecules not only for those fragments belonging to the nucleosome-size range (120-180bp, middle panel), but also for dinucleosomes-size (180-300bp, left panel) or subnucleosome-size (50-120bp, right panel) ranges. We could observe that only when sorting fragments smaller than a nucleosome we could detect a partial protection of the TASs mediated by histones ( Fig. 3a ). Although we cannot prove that the TASs are entirely protected by histones in T. brucei , we show that the MNase-sensitive complex sitting at the TASs is at least partly composed of histones. To investigate if the TASs protection only occurs in T. brucei or it could be extended to other trypanosomatids, we performed the same analysis using MNase ChIP-seq data for histone H3 from T. cruzi 43 , using datasets with comparable levels of MNase digestion (Fig. S4b). We could observe that the same average chromatin organization was displayed around the TASs in T. cruzi , not only in the average representation but also in every region relative to the TAS, as illustrated in heatmaps ( Fig. 3b and Fig. S4a, right panel). This observation suggests that chromatin protection and sensitivity to MNase digestion at the TASs are similar for both parasites. The TASs of single and multi-copy genes are differentially protected by nucleosomes It was previously described that the genomes of trypanosomatids are compartmentalized into core regions holding mainly single-copy genes and species-specific disruptive regions that encode multigene families 27 . Particularly in T. cruzi , these two subsets of genes differ not only in their genomic distribution, but they also present different chromatin organization and gene expression levels. On one hand, single-copy genes display a more open chromatin, higher levels of gene expression and faster transcription rates compared to multi-copy genes 44 – 46 . To unveil if there was any difference at their TASs, we analyzed their average chromatin organization and we observed that the TASs are differentially protected from MNase digestion. Remarkably, single-copy genes harbor more accessible TASs with a mild NDR, while multi-copy genes show TASs fully occupied by nucleosomes ( Fig. 4a and S5a left panel). These chromatin arrangements are consistent with the higher levels of gene expression observed for single-copy genes compared to multi-copy genes (Fig. S5b). This observation suggests that, despite transcription being mainly regulated post-transcriptionally in trypanosomes, chromatin organization might represent another layer of modulation to guarantee the appropriate maturation of the transcripts. A similar trend for compartmentalized chromatin organization is observed in T. brucei (Fig. S5b). Download figure Open in new tab Figure 4. Chromatin organization at TASs in single and multi-copy gene families. (A) Average histone H3 occupancy (top panel) and heatmaps (boaom panels) in a 1 kb window relative to the TAS for single (green) and multi-copy genes (blue). Red: High histone H3 density; blue: low histone H3 density. (B) Average dinucleotide frequency for AA/TT/AT/TA (purple), CG/CC/GC/CG (orange) and other possible combinations (light-blue) in a 100bp window relative to the TAS for single and multi-copy genes. Given that DNA sequence is among the major determinants for nucleosome positioning 17 and that it was previously described that in T. cruzi the genome compartments present different GC content 27 , we wonder whether the difference in nucleosome organization between single and multi-copy genes can be influenced by their DNA sequence. Hence, we analyzed the average frequency of AT and CG containing dinucleotides relative to the TAS for these two groups of genes in a 100bp window. We observed that while for single-copy genes DNA is enriched in AT containing dinucleotides particularly upstream of the TASs, for multi-copy genes, there is some oscillation between AT and GC containing dinucleotides ( Fig 4b ). Thus, while the long stretches of AT dinucleotides observed in single-copy genes are less likely to assemble into nucleosomes, stretches with a periodic alternation between AT and CG dinucleotides present in multi-copy genes might be more favorable for wrapping around the histone core. The different composition possibly implies that the appropriate transcript maturation might be additionally granted by the underlying DNA sequence in a stage-independent manner. Discussion Despite gene expression in trypanosomatids is mainly regulated post-transcriptionally, it was shown that the genome of the trypanosomes is organized into chromatin-folding domains underlying that chromatin and DNA accessibility to some extent control gene expression 13 , 47 . Here, by analyzing MNase-seq data from the parasitic forms present in the insect vector, we made a contrasting study of genome-wide chromatin organization in TriTryps. To do so, we performed a thorough and systematic analysis of the available datasets following the same informatics pipeline for one representative strain of each organism: T. cruzi CL Brener, T. brucei 427 and L. major Friedlin. Consistent with previous observations, they have in common a poorly organized chromatin with nucleosomes that are not strikingly positioned or phased, being the most remarkable characteristic the presence of a peculiar change in nucleosome arrangements around the TASs ( Fig. 1b and Fig. S2a). The earliest genome-wide nucleosome mapping by MNase-seq performed in TriTryps was done in L. major , where the presence of a well-positioned nucleosome at the spliced-out region followed by a shallowed trough at the TASs was reported 34 . Afterwards, in T. brucei , a similar study revealed a mild nucleosome depletion upstream of the first gene of the DGCs, coincident with divergent strand switch regions but described the presence of a well-positioned nucleosome at the TASs for internal genes 37 . Almost in parallel, MNase-ChIP-seq for histone H3 was performed in T. brucei showing a nucleosome depletion upstream of every gene of the DGCs 33 . Later on in T. cruzi , a nucleosome depletion was observed upstream of every gene 35 ; and our group described that these NDRs co-localized with the predicted TASs 36 . In this work, we corroborated the original observations and brought to light that the most distinctive feature of average chromatin patterns shared by TriTryps is a peculiar change in average chromatin organization around TASs. Moreover, the change in the chromatin landscape observed at that point is not only an average but is observed for every TAS along their genomes ( Fig. 1b and Fig. S2a). By analyzing the extent of sample digestion, in L. major and T. brucei we exposed that these samples were less digested than those for T. cruzi , suggesting that the MNase protecting complex at the spliced-out region or the TASs could have been destroyed during the more extensive digestion experienced by T. cruzi samples ( Fig.1c , Fig. S1 and S2b). Despite the sizes of the sequenced DNA molecules look similar between T. cruzi and T. brucei , those from the latter were gel-purified before library preparation. This is possibly the reason why the MNase protecting complexes were more preserved in T. brucei . To expose the MNase-sensitive nature of the TASs protecting complex, we analyzed MNase-seq datasets available for T. brucei in which the samples achieved different levels of digestion. We could observe that the TASs protection was indeed closely related to the size-range of the sequenced DNA molecules ( Fig. 2 and Fig. S3). Deciphering the nature of the molecule/s that could be bound at those sites of the genome that are less sensitive to MNase, is one of the most relevant questions and a topic of ongoing research in the chromatin field. In yeast, there are a couple of examples where NDRs colocalize with the presence of non-histone complexes at gene promoters and at tDNA genes transcribed by RNA polymerase III 40 , 41 . To expose the presence of histones in the MNase-sensitive complex protecting the TASs in TriTryps, we analyzed MNase-ChIP-seq data for histone H3 from T. brucei and T. cruzi from similar levels of digested samples with an optimal range of digestion for nucleosome mapping as previously described 48 . We observed that, when analyzing nucleosome-size (120-180bp) DNA molecules or longer fragments (180-300bp), the TASs of either T. cruzi or T. brucei are mostly nucleosome-depleted. However, when representing fragments smaller than a nucleosome-size (50-120bp) some histone protection is unmasked ( Fig. 3 and Fig. S4). This observation suggests that the MNase sensitive complex sitting at the TASs is at least partly composed of histones. Unfortunately, there is no similar data available for L. major ; hence, whether the MNase protecting complex detected at the spliced-out region in L. major contains histones remains an open question. What contributes to NDRs formation in different organisms, is a subject of active investigation, but in general, NDRs represent accessible regions that are typically coincidental with regulatory regions. In model organisms NDRs are related to promoters, enhancers, origins of replication and tRNA genes. Regarding how those NDRs are formed and maintained, there are several models that involve the concerned activity of transcription factors, histone variants, chromatin remodelling complexes, the transcription initiation machinery and the potential presence of physical barriers 49 , 50 . Different ATP-dependent chromatin remodeling complexes work in a coordinated manner to keep the NDRs clear, help to position the +1 nucleosome and to organize and space nucleosomes on gene bodies 51 – 54 . In Trypanosomes, there are only a few DNA binding proteins; so, it is hard to think about a possible candidate for this role. Instead, this interaction could be bridged by other molecules, such as R-loops. Consistent with our hypothesis, in T. brucei R-loop enrichment was detected at intergenic regions coincident with lower histone density and, among R-loop interacting proteins some putative trans-splicing factors have been detected 55 , 56 . Based on this observation, we propose that in TriTryps the NDRs are formed at the TASs to guarantee the proper maturation of the transcripts when needed. Whether the NDRs favor the appropriate assembly of the trans-splicing machinery or the other way around, is still an open question. To test the feasibility of this hypothesis, we analyzed separately two subsets of genes: single-copy genes, which usually encode housekeeping functions for the stage of the parasite present in the insect vector; and, multi-copy genes, required for infection and immune system evasion 57 . We uncovered that most of the genes, belonging to the single-copy gene subset, show mild NDRs at the TASs, while multi-copy genes present TASs normally obstructed by nucleosomes ( Fig. 4a and S5a, left panel). This observation is consistent with previous reports that multi-copy genes are associated with higher nucleosome occupancy, lower levels of expression and transcription rates, as opposed to single-copy genes, which display a more open chromatin, higher expression levels and transcription rates 44 – 46 . Unfortunately, we could not obtain a full TAS list for multi-copy genes for T. brucei and L. major due to the characteristic of the transcriptomic data used to make UTR predictions and the repetitive nature of their genomes. However, there is a hint that in T. brucei nucleosomes display a similar average organization at these two groups suggesting it is a common feature in trypanosomatids (Fig S5b). This is supported by previous observations that in T. brucei and T. cruzi , the expression of multi-copy genes is also modulated by special isolation in the nucleoplasm 44 . Finally, we show that these two subsets of genes differ in the dinucleotide content, where single-copy genes differentiate by having a striking asymmetry in the percentage of AT-containing dinucleotides upstream of the TAS ( Fig 4b ). The fact that single-copy genes are enriched in poly AT tracks upstream of the TASs -more refractory to bend into nucleosomes-, while multi-copy genes bear a more periodical alternance between AT and GC dinucleotides -easier to bend around the histone octamer-, suggests that DNA sequence might be acting as a spare security gate. DNA sequence might contribute to keep the TASs more accessible at single-copy genes that require to be expressed most of the time; while it might facilitate nucleosome formation to prevent the unnecessary expression of multi-copy genes, only meant to fulfill a very specific function. Alternatively, trans-acting factors might be required to generate or keep the NDRs but their interaction with DNA or R-loops at the TAS of single-copy genes might be more efficient. This differential chromatin organization resembles what is observed in more complex organisms where the presence of NDRs is associated with highly transcribed genes; while silenced genes usually have occluded promotores 31 . Given that in Trypanosomes transcription initiation is almost a constitutive process, we propose that the implications of chromatin are associated to modulate the maturation of the polycistronic transcripts into mature monocistronic units in a co-transcriptional manner. Therefore, the chromatin landscape might be modulating the speed of RNA pol II, forcing a pause near TASs and contributing to guarantee an appropriate maturation of the transcripts as previously observed for cis-splicing 58 . Future studies focused on mapping the binding of trans-splicing factors along the genomes and studying the dynamics of RNA polymerase II in TriTryps, will contribute to answering the unsolved maaers. Materials and Methods Data collection Informatics analysis were performed using publicly available data from Gene Expression Omnibus 59 as detailed in Table S1. MNase-seq and MNase-ChIP-seq data available for the parasite stadium detected in the insect vector for T. cruzi CL Brener strain, T. brucei 427 strain and L. major Friedlin strain were used for chromatin studies 34,37,42,43,60,61 . Statistics analysis, genome alignment and reference genomes used The sequence quality metrics were assessed using FastQC v0.11.9 ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). During this step, over represented sequences were detected and trimmed out using Cutadapt tool v3.5 ( https://cutadapt.readthedocs.io/en/stable/ ) 62 when required. Paired-end reads were aligned using Bowtie2 v2.4.4 ( https://bowtie-bio.sourceforge.net/bowtie2/index.shtml ) 63 against version TriTryp46 of the respective genomes retrieved from TriTrypDB 64 . For T. cruzi CL Brener, we built a genome combining the Esmeraldo-like haplotype, the non Esmeraldo-like haplotype and the extra regions not assigned to any haplotype as described before 60 . For T. brucei 427 the corresponding genome was used, compatible with a software applied in further steps. For L. major Friedlin its corresponding genome was used 65 . TAS prediction The most likely trans-splicing acceptor site (TAS) for each member of TriTryp was predicted as described before 60 . Briefly, the 5′untranslated regions (5’UTR) were predicted with UTRme 38 . Given the lack of transcriptomic data for CL Brener epimastigotes, the predictions were based on available RNA-seq data for the Y strain 66 using TriTryp46 Esmeraldo-like genome as a reference. Similarly, because of lacking transcriptomic data for 427 strain, T. brucei predictions were based on EATRO 1125 data 67 using TriTryp46 T. brucei 427. In the case of L. major Friedlin, predictions were based on available data for this strain 68 using TriTryp46 L. major Friedlin genome. As an approximation of the trans-splicing acceptor site, the 5’ end of the 5’UTR region was used ( Fig. 1a ). The list of the genomic coordinates for the predictions for each TriTryp is provided in Table S2. Length distribution histograms, BigWig files, average occupancy plots, 2D-plots and heatmaps Length distribution heatmaps, BigWig files, average occupancy plots and 2D-plots were generated from BAM files as described before 39 , 60 . Briefly, Bigwig files containing information of nucleosome occupancy (MNase-seq), histone H3 signal (from MNase-ChIP-seq) were generated by counting the number of times that a base pair was occupied. The signals were normalized by summing all the sequences covering a nucleotide and dividing that number by the average number of detected sequences per base pair across the genome. For MNase-seq and H3-IP the analysis was performed for all the sequenced fragments (50-500bp) or restricted to those fragments that belong to the dinucleosome-range (180-300bp) nucleosome-size range (120-180bp), or subnucleosome-range (50-120bp), as detailed in the figure legends. Average occupancy plot, 2D-plots and heatmaps were built using the TASs predictions with best score, obtained for each parasite, as reference point. In the case of T. cruzi only the Esmeraldo-like haplotype was represented but the alignments of the fastq files were made to the whole genome as explained above. For 2D-plots, the data was represented relative to the TAS in the x axis, while the size of the analyzed DNA fragments was represented in the y axis. A source code easy to adapt to any TriTryp is available at ( https://github.com/paulati/nucleosome ). To build heatmaps for the disaggregated regions around TAS, computeMatrix and plotHeatmap functions from deepTools version 3.5.1 ( https://test-argparse-readoc.readthedocs.io/en/latest/ ) were used 69 . BED6 files containing the genomic coordinates for the TASs predictions with best score were used as regions (detailed in Table S2) and, BigWig files generated for each analyzed dataset were used as score files. Average plots and heatmaps were represented either for all the genes with a predicted TASs or sorted for single and multi-copy genes of T. cruzi when stated. To generate the list of single-and multi-copy genes, gene IDs corresponding to multigene families (trans-sialidase and trans-sialidase-like, MASP, Mucins, GP63, RHS and DGF) were obtained by text searches using the current genome annotation on the “description” field. Single and low copy number genes were defined as those that did not belong to the latter gene list. For simplicity “single-copy” will be used to refer to these genes throughout the manuscript. The list is detailed in Table S3. Dinucelotide frecuency calculation The region surrounding every predicted TAS in a 100 bp window was annotated in a BED format where the start site was the position located 100 bp upstream of the TAS, and the end site was the position located 100 bp downstream of the TAS as depicted in Fig. 4b . The sequences of these regions, named TAS regions , were then saved in a FASTA format and used for further analysis. The periodicity of the AA/TT/AT/TA, GG/CC/GC/CG or other possible combinations of dinucleotides in the TAS region was analyzed and the average frequency of these dinucleotides was represented. The occurrence of undetermined dinucleotides “NN”, based on the genome annotations, was considered. Author Contributions J. O. and G. D. A. conceived the study. J.O. mentored the project. R.T.Z.S. performed data analysis and contributed to figure design. P. B. performed data analysis and developed the adapted bioinformatic tools. J. O. collaborated in data analysis. made the figures and wrote the first version of the manuscript. L.L. collaborated in data analysis. P.S. made UTR predictions. J. O. and R.T.Z.S. carried out data interpretation. All authors reviewed, edited, and approved the final version of the manuscript. Additional Information Supplementary Materials The following are available online. Competing interests The authors declare no competing interests. Acknowledgments We are grateful to Dr. David Clark for valuable discussions and to Santiago Carena for reading our manuscript. “This research was funded by the Agencia Nacional de Promoción Científica y Tecnológica”, PICT 2020-00473; and Concejo Nacional de Investigaciones científicas y técnicas (CONICET): Proyectos de Investigación Bianual (PIBBA) 2020-28720210100100CO; Proyectos de Investigación Plurianuales (PIP) 2021-2023-03073 and Joint Canada-Israel Health Research Program, IDRC-Project 109929”. J.O. and G.D.A are members of the Research Career of CONICET. R.T.Z.S. is Ph.D. fellow supported by ANPCYT and her PhD thesis is carried out at Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Funder Information Declared Proyectos de Investigación Científica y Tecnológica (PICT) , 2020-00473 Proyectos de Investigación Plurianuales (PIP) , 2021-2023-03073 Proyectos de Investigación Bianual (PIBBA) , 2020-28720210100100CO Joint Canada- Israel Health Research Program,IDRC , 109929 Footnotes romizambranos{at}gmail.com (R.T.Z.S.); pbeati{at}dna.uba.ar (P.B.); psmircich{at}fcien.edu.uy (P.S.); linchausti{at}fcien.edu.uy (L.I). References 1. ↵ Engels , D. & Zhou , X.-N. Neglected tropical diseases: an effective global response to local poverty-related disease priorities . Infect. Dis. poverty 9 , 10 ( 2020 ). OpenUrl PubMed 2. ↵ Pech-Canul , Á. de la C. , Monteón , V. & Solís-Oviedo , R.-L. A Brief View of the Surface Membrane Proteins from Trypanosoma cruzi . J. Parasitol. Res . 2017 , 1 – 13 ( 2017 ). OpenUrl 3. Freitas , L. M. et al. 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Share Beyond the transcript: chromatin implications in trans-splicing in Trypanosomatids Romina Trinidad Zambrano Siri , Paula Beati , Lucas Inchausti , Pablo Smircich , Guillermo Daniel Alonso , Josefina Ocampo bioRxiv 2025.07.08.663533; doi: https://doi.org/10.1101/2025.07.08.663533 Share This Article: Copy Citation Tools Beyond the transcript: chromatin implications in trans-splicing in Trypanosomatids Romina Trinidad Zambrano Siri , Paula Beati , Lucas Inchausti , Pablo Smircich , Guillermo Daniel Alonso , Josefina Ocampo bioRxiv 2025.07.08.663533; doi: https://doi.org/10.1101/2025.07.08.663533 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Genomics Subject Areas All Articles Animal Behavior and Cognition (7618) Biochemistry (17635) Bioengineering (13859) Bioinformatics (41846) Biophysics (21401) Cancer Biology (18534) Cell Biology (25423) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24286) Genetics (15582) Genomics (22463) Immunology (17700) Microbiology (40298) Molecular Biology (17141) Neuroscience (88429) Paleontology (666) Pathology (2825) Pharmacology and Toxicology (4813) Physiology (7633) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4284) Systems Biology (9808) Zoology (2267)