Genome-Wide Reprogramming of Transcript Architecture by Temperature Specifies the Developmental States of the Human Pathogen Histoplasma
[['Environmental human pathogens have evolved the ability to survive in human hosts as well as diverse environmental reservoirs .', 'Thus a hallmark of environmental pathogens is their capacity to adapt to varied growth conditions such as differences in temperature , alterations in nutrient sources , as well as exposure to the host immune system .', 'The dimorphic human fungal pathogen , Histoplasma capsulatum ( Hc ) , is one such environmental pathogen that responds to an increase in temperature inside a mammalian host by dramatically altering its cellular morphology and gene expression programs to cause disease .', 'The environmental , infectious form of Hc grows in the soil as a saprophyte in a multicellular hyphal or “mold” form that produces vegetative spores called conidia [1 , 2] .', 'Infection occurs when humans inhale conidia or hyphal fragments .', 'Upon exposure to mammalian body temperature , Hc transitions into a unicellular , budding yeast ( Fig 1A ) that is capable of causing primary disease in immunocompetent individuals [3–6] .', 'The temperature-regulated differentiation of Hc between the hyphal and yeast forms can be recapitulated in the laboratory simply by switching the temperature from room temperature ( RT ) to 37°C , making Hc a unique organism for studying the regulation of gene expression during multicellular development , environmental signal transduction , and adaptation of a pathogen to a mammalian host .', 'Hc cells respond to temperature through a transcriptional regulatory circuit that controls cell morphology as well as the expression of 5–15% of the Hc transcriptome that is differentially regulated between yeast and hyphal cells [8–10] .', 'In addition , transcripts encoding many of the known Hc virulence factors are enriched in the yeast-phase of growth [11] , making yeast-enriched transcripts intriguing virulence factor candidates .', 'To this end , microarrays [8 , 9 , 12–14] and RNA-sequencing ( RNA-seq ) [10] have begun to elucidate the transcriptomes of yeast , hyphal , and conidial cell types to describe transcript expression patterns that specify these three major cell types important for the Hc pathogenic lifecycle .', 'Despite these efforts , our understanding of the Hc transcriptome and cell-morphology specific regulatory programs remains incomplete partly due to challenges inherent in deciphering intron-rich , densely populated eukaryotic fungal transcriptomes .', 'To date , Hc transcriptomics studies have been performed using two North and Central American Hc var .', 'capsulatum strains , G217B and G186AR ( see Fig 1B ) .', 'Yet , Hc is found globally ( North , South , and Central America , Southeast Asia , and Africa ) and phylogenetic analysis of Hc clinical isolates has revealed that Hc comprises at least 8 genetically and geographically distinct lineages that span 3–13 million years of evolutionary distance [7 , 15] ( Fig 1B ) .', 'In addition to its genetic diversity , Hc can be segregated into two varieties ( var . ) based on clinical outcomes: Hc var .', 'duboisii and Hc var .', 'capsulatum .', 'Hc var .', 'capsulatum is represented in all of the major Hc lineages and infection with this variety predominantly manifests as pulmonary and systemic disease [4] .', 'Hc var .', 'duboisii , conversely , is geographically restricted to Western and Central Africa and causes cutaneous and subcutaneous skin as well as bone lesions in a systemic infection that is distinguished in name from classical disease as African histoplasmosis [16 , 17] .', 'The virulence mechanisms and basic biology of the causative agent of African histoplasmosis , Hc var .', 'duboisii , as well as Hc var .', 'capsulatum strains from additional , more divergent lineages , remain uninvestigated .', 'In this work , we employ recent advances in next-generation sequencing and de novo transcriptome reconstruction methodologies to refine transcript architecture and define transcript expression programs across divergent isolates of Hc that cause classical ( G217B , G186AR , H143 ) or African ( H88 ) histoplasmosis .', 'Using comparative transcriptomics of two temperature-regulated states , we broaden our understanding of the basic biology and pathogenesis of Hc by defining core yeast ( parasitic , disease-causing form ) and hyphal ( infectious form ) transcript expression patterns that exhibit conserved regulatory patterns .', 'Systematic analysis of our improved Hc transcript architecture further revealed that a subset of the Hc transcriptome ( ~ 2% ) exhibited dramatically different leader sequence lengths ( often referred to as the 5’ untranslated region; UTR ) between these two Hc developmental cell states .', 'To begin to understand the biological significance of longer leader transcripts , we probed their ribosome occupancy using ribosome profiling .', 'Consequently , we defined several categories of genes , including those that are regulated at both the transcriptional and translational level in response to temperature-initiated developmental programs .', 'Phenotypic analysis revealed that this exquisite regulation is necessary for appropriate cell-type specification by temperature , the key signal for infectious and parasitic states .'], ['To compare yeast and hyphal transcript expression patterns across Hc lineages , we grew each Hc strain ( G217B , G186AR , H88 , H143 ) at 37°C for yeast-phase growth and at room temperature ( RT ) for hyphal phase growth ( Fig 1C ) .', 'Total RNA was isolated from biological duplicates of yeast or hyphal cultures and strand specific cDNA libraries were created from poly ( A ) -enriched RNA and sequenced using paired-end deep-sequencing .', 'The 4 Hc strains that we selected for transcriptomics analysis each have sequenced genomes; however , the Hc gene predictions vary among strains and often lack accurate models of 5’ leader and 3’ UTR regions .', 'Therefore we constructed de novo transcript calls leveraging our paired-end RNA-seq data with the aim of improving transcript models , generating high confidence ortholog mapping across Hc strains , and directly identifying poly ( A ) + transcribed regions .', 'To do so we combined yeast and hyphal sequencing reads for each Hc strain and used these pooled reads to generate per-strain reference transcriptomes across the 4 Hc lineages ( see Fig 2A; S1 Table; S1–S12 Data; Materials and Methods ) .', 'Hc RNA species without polyadenylated 3’ ends ( i . e . , potential small non-coding RNA ) were not enriched in our sequencing libraries and thus are likely not represented in these transcript models .', 'Our genome-guided transcriptome assembly pipeline yielded 12 , 175–12 , 889 transcripts for the 4 Hc strains with 9 , 580–9 , 844 of assembled transcripts predicted to be protein coding ( Table 1 ) .', 'This falls within the range of 9 , 229–11 , 329 protein coding genes predicted from Hc genomic sequences by ab initio gene prediction algorithms ( Table 1 ) .', 'These Hc transcript models do not take into account any alternate transcript isoforms that may exist within or between Hc cell types ( i . e . , transcript forms that arise due to intron retention ) ; however this is an interesting area for future refinement and investigation .', 'The assembled transcripts were overall larger in size than the predicted transcript set ( S1 Fig ) , which is mostly due to the improved annotation of 5’ leader and 3’ UTR transcript ends ( Fig 2B and 2C ) .', 'In addition to augmenting 5’ and 3’ transcript end annotations , we noticed many cases where assembled transcripts had more accurate intron boundaries than predicted transcripts resulting in improved annotation of coding sequence ( CDS ) regions ( S2 Fig ) .', 'In order to compare transcription across strains , groups of orthologous genes among the 4 Hc strains ( orthogroups ) were determined using two independent methods , Mercator [18] and InParanoid [19] , which gave similar numbers of per-strain ortholog assignments ( Table 1 ) .', 'For downstream analyses , we chose the Mercator method for determining Hc orthogroups since it incorporates both BLASTP [20] homology and genomic synteny for a more stringent determination of orthogroups .', 'Using Mercator we assembled 6 , 791 unique Hc orthogroups that were present in all four strains ( see Materials and Methods; Table 1 ) .', 'Genes not assigned to orthogroups by this method could include in-paralogs ( gene duplications that occurred after strain divergence ) , improperly assembled transcripts that led to inappropriate open reading frame ( ORF ) predictions , and genes truly unique to a given strain .', 'Of the 6 , 791 Hc orthogroups , 423 ( 6 . 2% ) were not in any of the previously predicted Hc transcript sets and exhibited a striking pattern of short CDS regions relative to overall transcript lengths ( S3 Fig ) .', 'Only a small percentage of these novel orthogroups had conserved Pfam-A domains ( 4% ) [21] or predicted secretion signals ( 9% as determined by Phobius [22 , 23] ) , and they exhibited lower transcript expression levels in comparison to the full orthogroup set ( S4 Fig ) .', 'In other eukaryotic species , poly ( A ) + long non-coding RNAs ( lncRNAs ) often have lower expression levels than protein-encoding transcripts [24] .', 'Thus , this set of novel orthogroups in Hc may include cases of poly ( A ) + non-coding RNA species with spurious ORF predictions as well as small proteins or peptides missed by ab initio genome prediction algorithms .', 'Overall , however , 90% of Hc orthogroups were predicted to encode at least one Pfam-A domain [21] indicating that the vast majority of Hc orthogroups determined from our RNA-seq transcriptome assemblies represent bona fide protein coding transcripts .', 'We used eXpress [25] to determine transcript expression levels across yeast and hyphal cell morphologies for the 4 Hc strains .', 'eXpress estimates of FPKM values ( fragments per kilobase of exon per million aligned fragments ) were well-correlated between biological replicates ( S2 Table ) ; therefore we took the mean of log2 FPKM values of biological replicates for downstream analyses of Hc yeast and hyphal expression patterns ( S13 Data ) .', 'To define a global picture of evolutionarily conserved programs of gene regulation across Hc lineages , we identified transcripts enriched in the yeast ( parasitic form ) or hyphal ( infectious form ) state from the 6 , 791 Hc orthogroups ( S5 Fig; S13–S15 Data ) .', 'Minor lineage-specific differences in gene expression were observed , though these were largely of unknown biological significance .', 'In terms of conserved patterns of gene expression , we found 139 yeast-phase enriched transcripts ( core yeast-phase transcripts: Y/H log2 ≥ 1 . 5 ) and 291 hyphal-phase enriched transcripts ( core hyphal-phase transcripts: Y/H log2 ≤ - 1 . 5 ) conserved in differential expression pattern among the 4 Hc strains ( S5B and S5C Fig; S13 Data ) .', 'Core yeast-phase enriched transcripts included two characterized virulence factors ( CBP1 , SOD3 ) [26 , 27] , siderophore biosynthesis and transport genes ( SID3 , SID4 , ABC1 ) important for yeast cell iron acquisition [28] , and 6 putative transcription factors ( CSR1 , CHA4 , MEA1 , RYP1 , RYP4 , XBP1 ) including RYP1 and RYP4 , which are master transcriptional regulators of Hc yeast-phase growth [8 , 9] .', 'We also identified 8 cell wall modifying enzymes ( AMY2 , CFP8 , CTS1 , DCW1 , ENG1 , GEL2 , OCH1 , SKN1 ) with transcripts upregulated in the yeast phase that are predicted to be involved in the biosynthesis or remodeling of cell wall polysaccharides such as β-glucan , chitin , and mannan; the upregulation of these transcripts in yeast may reflect differences inherent to yeast versus hyphal cell division and growth .', 'Core hyphal-phase enriched transcripts included 18 putative transcription factors ( see S13 Data ) , many of which are uncharacterized and are members of the fungal-specific Zn2C6 DNA binding domain family of transcriptional regulators ( Pfam Accession: PF00172 ) .', 'In addition , we noticed that many core hyphal-phase enriched transcripts encoded enzymes such as cytochrome p450s , polyphenol oxidases ( also known as tyrosinases ) , oxidoreductases , and peroxidases ( see S13 Data ) .', 'The hyphal-phase enrichment of these enzymes , which are often involved in nutrient acquisition or the production of toxins , melanin , and other secondary metabolites , is likely reflective of the saprophytic lifestyle of Hc hyphal cells .', 'Examining the relative abundances of yeast-enriched transcripts ( using per-state mRNA FPKMs ) indicated that some of the most abundant core yeast-enriched transcripts ( CBP1 , GH17/CFP4 , SOD3 , ENG1 ) encode proteins known to be secreted by yeast cells [29] ( S6 Fig ) .', 'Since bacterial and fungal pathogens often utilize secreted proteins as virulence effectors [30 , 31] , we were interested in identifying additional conserved yeast-phase enriched transcripts encoding proteins with secretion signals .', 'Prediction of secretion signal peptides for the conserved yeast-phase enriched ORFs ( using Phobius [22 , 23] ) revealed that secreted proteins were significantly more likely to be differentially expressed than non-secreted proteins ( p = 0 . 000314 ) , suggesting that many secreted proteins could have phase-specific roles .', 'In addition , we noticed that there were many small ( ≤ 200 AAs ) putative secreted proteins ( Figs 3A and S6 ) that exhibited a conserved C-terminal , 6-cysteine spacing pattern reminiscent of some insect toxins [32] .', 'We examined existing hidden Markov models ( HMM ) of cysteine-rich protein domains to determine whether this Hc 6-cysteine motif belonged to any known protein families .', 'Through this analysis , we found that many Hc ORFs with the 6-cysteine motif had homology to a cystine knot ( or knottin ) gene family [34] .', 'Knottin domains are comprised of 3 interwoven disulfide bonds that form one of the smallest known stable globular domains [35] , making these proteins extremely resistant to chemical , heat , and proteolytic stresses [36] .', 'Knottins can be found in fungi , insects , plants , and animals [34] , and their 3-disulfide bond core can present constrained loop structures that mediate protein-protein interactions [37] .', 'Functionally , knottin proteins have been shown to act as pore formers , ion-channel inhibitors , as well as protease inhibitors [38–40] .', 'Phylogenetic analysis of proteins with identifiable knottin domains ( of the Fungi1 knottin family [34 , 41] ) in Hc as well as other related fungal species , revealed that this family appears to have undergone an expansion in the Ajellomycetaceae family of human fungal pathogens ( Hc , Paracoccidioides brasiliensis , Blastomyces dermatitidis ) ( Figs 3B and S7; S16–S18 Data ) .', 'Notably , knottins are absent from many Saccharomycetes fungi such as Saccharomyces cerevisiae and Candida albicans ( S7 Fig ) .', 'Hc transcripts encoding this knottin domain are predominantly yeast-phase enriched ( S8 Fig ) and are intriguing candidates for virulence effectors that could mediate host-pathogen interactions .', 'While examining our improved annotation of transcript boundaries in the 4 assembled Hc transcriptomes , we noticed that a subset of transcripts exhibited differences in the size of their leader regions between yeast and hyphal cell types .', 'For example , we observed that some transcripts in yeast cells displayed longer leader regions as compared to their cognate hyphal transcripts ( Fig 4 ) .', 'This was a phenomenon that we had previously seen for a handful of Hc yeast-enriched transcripts while examining transcript abundance and architecture in G217B yeast and hyphal cells using Northern blotting and 5’ RACE ( 5’ rapid amplification of cDNA ends ) [13] .', 'Given the high-resolution nature of RNA-seq data , we were able to identify leader regions that were differential in length between Hc morphologies genome-wide across all 4 strains .', 'To do so , we employed our existing transcript assembly pipeline to determine per-cell type ( yeast and hyphae ) transcript structures for each Hc strain ( see Materials and Methods; S19–S26 Data ) .', 'Globally , we found that most leader regions were similar in length between yeast and hyphae in all 4 Hc strains as expected ( S9 and S10 Figs ) .', 'However , systematic analysis of leader regions using per-cell type assembled transcript structures identified 187 transcripts with conserved , differentially sized leader regions between yeast and hyphal cells ( see Materials and Methods; S10 Fig; S13 Data ) .', 'The conservation of differential leader transcript structure in all 4 Hc lineages examined suggests that changes to 5’ transcript length are not a stochastic transcriptional outcome , but are evolutionarily conserved and likely regulated by the cell .', 'We found that the majority of transcripts that exhibited differential leader length are longer in the yeast phase compared to the hyphal phase of Hc growth ( 138/187 differential leaders; S10 Fig; S13 Data ) .', 'We did , however , find 49/187 conserved transcripts with longer leaders in hyphal cells ( S10 Fig; S13 Data ) , indicating that differential leader length is not an artifact of sample preparation or produced exclusively by one Hc cell type .', 'We examined the sizes of a subset of longer leader transcripts in yeast and hyphal cells via Northern blotting , which confirmed the differential transcripts sizes between Hc cell morphologies ( S11 Fig ) .', 'Northern blot analysis of longer leader transcripts also highlighted that some of these transcripts represent a population of transcripts of variable sizes ( S11 Fig ) .', 'This is analogous to emerging observations of complex transcript structure in S . cerevisiae where variations in 5’ and 3’ transcript boundaries give rise to many different transcript isoforms for a given gene [42 , 43] .', 'Differential sizes of transcript leader regions between cell states or in cells encountering changing environmental conditions is a recently observed genome-wide phenomenon in eukaryotes [44–48] .', 'While the biological function of differential leader regions is often unclear , changes in leader length have been proposed to influence the translational outcome of protein coding ORFs either by introducing regions of RNA that can serve to regulate translation ( e . g . , upstream ORFs; uORFS ) [44] or by introducing an alternate start codon that encodes a new protein variant [42] .', 'Thus we hypothesized that differential leader transcripts in Hc may be translationally regulated between Hc developmental states .', 'To investigate this possibility we adapted ribosome profiling to Hc .', 'Ribosome profiling is based on deep-sequencing of ribosome-protected RNA fragments or “footprints” and has been well-established as a method to probe translational regulation in eukaryotic cells [44 , 49–51] .', 'Analogous to ribosome profiling developed in the model yeast organism S . cerevisiae [49] , we digested total RNA from yeast or hyphal G217B Hc cells with RNase I and then separated and fractionated the RNA with a sucrose density gradient to collect Hc 80S monosomes ( Fig 5A and 5B ) .', 'Polysome profiles of undigested RNA from yeast and hyphal vegetative cells were noticeably different ( Fig 5A and 5B ) , most likely illustrating the distinct modes of growth for these Hc cell types .', 'Hc yeast grow and divide as unicellular budding yeast cells while Hc hyphal cells are connected by septa to form filamentous , branched hyphae that grow as highly polarized multicellular structures .', 'In Ascomycete fungi , hyphal cells exhibit specialization and often possess differences in growth rate , cell size , or access to nutrients [52] , leading to a heterogeneous mix of cell states per hyphae .', 'We collected biological replicate samples of yeast and hyphae monosomes as well as poly ( A ) -enriched RNA from the same samples for ribosome profiling and RNA-seq , respectively .', 'Ribosome footprint density and mRNA abundance measurements ( calculated as the number of ribosome-protected or mRNA fragments per kilobase of coding sequence per million mapped reads; FPKM ) were reproducible between yeast and hyphal biological replicates ( S12 Fig ) .', 'With ribosome profiling data we can also determine the translational efficiency ( TE ) of transcripts ( calculated as the ratio of ribosome footprint density to mRNA abundance over a coding region ) , which is a measure of the extent to which a transcript is translationally regulated .', 'Importantly , measurements of TE in Hc were reproducible between biological replicates ( Fig 5C and 5D; S27 Data ) and TE measurements for genes expressed in yeast or hyphal cells spanned similar orders of magnitude ( S13A Fig ) .', 'Additionally , ribosome footprint FPKMs for transcripts involved in basic cellular functions thought to be important for growth or maintenance of both cells types ( ACT1 , GAPDH , TEF1 , CDC2 , TUB1 , and TUB2 ) were overall remarkably similar between yeast and hyphae ( S13B Fig; S27 Data ) .', 'However , we want to stress that the ribosome profiling data sets provide relative measures of translational efficiencies and not absolute measures of translation .', 'Together these data indicate that ribosome profiling is a robust method to probe genome-wide ribosome occupancy as well as the translational regulation of transcripts in the developmentally distinct morphologies of Hc .', 'Transcriptome-wide ribosome occupancy measurements did not correlate perfectly with mRNA abundance in Hc yeast or hyphae ( Fig 6A and 6B ) .', 'This suggested that expression of a subset of Hc genes is controlled at the level of translation and thus could reflect cell-type specific translational regulation between Hc cell morphologies .', 'To investigate this , we first examined ribosome footprint density measurements to identify transcripts with different levels of translation between yeast and hyphal cells .', 'Through this analysis we found subsets of transcripts that were predominantly translated only in one cell type ( yeast = 200 transcripts and hyphae = 490 transcripts; Fig 6C; S27 Data ) and reasoned that these transcripts encoded proteins with biological functions that are specific to a given cell type .', 'Since protein abundance in a cell is influenced by both transcript levels as well as the rate at which transcripts are translated , we also examined the translational efficiency of transcripts in yeast and hyphal cells .', 'Clustering of yeast and hyphal TE values for transcripts that exhibited translational regulation highlighted at least 6 categories of translational regulation between yeast and hyphae ( Fig 6D; S28–S29 Data ) .', 'Most noticeably , we identified a large class of genes with low translational efficiency in both yeast and hyphae ( Fig 6D- Category B ) ; not surprisingly many transcripts in this category encode proteins that are not expected to serve a function in Hc vegetative yeast or hyphae under the growth conditions used in these experiments .', 'For example , genes predicted by homology to be involved in mating/meiosis ( HOP1 , MEI2 , STE2 , STE6 ) , light sensing during circadian rhythm ( WHC2 ) , as well as conidial ( asexual spore ) development and biology ( CATA , RDS1 , CON132 ) showed low levels of translational efficiency in both yeast and hyphal cells .', 'We also found many transcripts with robust translational efficiency in both cell types ( Fig 6D- Category E ) that included genes involved in routine cellular functions such as fatty acid biosynthesis ( ACC1 , FOX2 , FAA1 ) , cytoskeletal organization ( ACT1 , BUD6 , ARP1 , SLA1 ) , and nuclear and mitochondrial transport ( KAP95 , TOM20 , KAP108 , TIM16 ) .', 'Categories where we observed translational efficiency differences between yeast and hyphae ( Fig 6D- Category A , C , D , F ) included some previously identified yeast and hyphal-enriched transcripts , indicating that a subset of cell-type enriched transcripts are regulated at both the transcriptional and translational level by the temperature-dependent developmental program .', 'Having established ribosome profiling as a method to measure ribosome occupancy and translational efficiency in Hc yeast and hyphae genome-wide , we next examined the translational outcomes of differential leader transcripts .', 'Comparing yeast and hyphal TE values for the set of longer leader transcripts revealed that many exhibited altered translational regulation compared to their cognate shorter transcript isoforms ( Figs 7 and S14; S30–S33 Data ) .', 'Correlation of ribosome footprint densities on differential leader regions with changes in TE values ( by examining a 2 or 1 . 5 fold change in CDS TE ) between short and long transcript forms resulted in classification of leaders into broad regulatory patterns ( Figs 8 and S15 ) .', 'The most striking pattern we observed was differential leader ribosome density that correlated with a decrease in TE , reminiscent of uORF mechanisms of translational repression ( Fig 8- Category a ) .', 'We also observed longer leaders with no ribosome density and strong repression of downstream CDS translation ( Fig 8- Category d ) via unknown mechanisms .', 'In addition , we found examples of robust leader ribosome occupancy that resulted in no change to the TE , which may represent cases of N-terminal protein extensions and uORFs that don’t influence translation of the CDS ( Fig 8- Category b ) .', 'Together these data suggest that as many as half of the observed longer leaders may be involved in translational repression of the associated CDS .', 'The features distinguishing such regulatory leaders remain to be elucidated experimentally .', 'While examining longer leader transcripts that exhibited strong repression of CDS translation ( Fig 8- Category a , d ) , we noticed that many of these transcripts had longer leaders in the yeast phase and were additionally regulated at the level of transcription between yeast and hyphae ( Fig 7A ) .', 'Specifically , a subset of yeast-phase longer leader transcripts were transcriptionally and translationally repressed in yeast cells , yet were robustly transcribed and translated in hyphae ( Fig 7A- red box ) .', 'To validate whether these ribosomal profiling data corresponded to levels of protein accumulation , we expressed GFP under the control of the MS95 longer leader transcript promoter and leader region ( MS95p ) in Hc cells and examined GFP protein production at 37°C and RT .', 'MS95 has a longer leader transcript form in yeast cells that exhibits lower translational efficiency compared to its shorter hyphal form ( S16A Fig ) .', 'Assessment of GFP protein levels by Western blot in Hc cells expressing the MS95p – GFP construct and grown at 37°C or RT recapitulated the differential protein expression pattern we inferred from ribosome profiling ( S16B and S16C Fig ) .', 'Namely , the MS95 upstream region restricts expression at 37°C , but allows robust transcription and translation at RT .', 'Why Hc cells tightly restrict expression of Ms95 at 37°C is unclear as the biological function of Ms95 , a homolog of the DNA damage and heat-stress responsive protein Ddr48 in S . cerevisiae , remains unknown .', 'Much precedence exists for cells to employ translational regulation to achieve tight spatial or temporal control of protein expression during developmental processes [53] .', 'In this vein , we noticed that some longer leader transcripts have been implicated in governing Hc cell fate decisions .', 'Both RYP2 and RYP3 , master regulators of yeast cell morphology , have longer leader regions in hyphal cells .', 'RYP2 has ribosome density in its differential leader region and lower TE of the RYP2 CDS in hyphae versus yeast ( S17A Fig ) .', 'The role of the RYP3 hyphal longer leader region is unclear as there is only a slight reduction in TE of the RYP3 CDS in hyphae versus yeast ( S17B Fig ) .', 'Ryp2 and Ryp3 both associate with DNA to regulate transcription of yeast-phase enriched transcripts at 37°C; furthermore , disruption of RYP2 or RYP3 in Hc cells results in inappropriate hyphal growth of Hc at 37°C [9 , 54] .', 'Thus , tight restriction of Ryp levels during cellular development may be a mechanism used by Hc to control appropriate morphology in response to temperature .', 'We mined Hc longer leader transcripts for additional candidates that could be involved in regulating cell fate decisions by identifying longer leader transcripts with transcription factor domains or homology to developmental regulators in other fungal species .', 'This approach identified 14 putative developmental regulators with longer leader regions in Hc yeast or hyphal cells ( S3 Table ) .', 'One putative Hc developmental regulator we identified was WET1 , which we named based on its homology to the Aspergillus nidulans regulator of conidial ( asexual spore ) development , WetA [13 , 55] .', 'WET1 has a longer leader sequence in Hc yeast cells and its CDS region is translationally repressed in yeast while the shorter hyphal transcript form has robust ribosome CDS occupancy in hyphae ( Fig 9A ) .', 'To explore the idea that regulators of Hc developmental programs include longer leader transcripts that are tightly regulated between yeast and hyphal cell types , we placed the CDS of WET1 under the control of a heterologous Hc promoter , ACT1p ( Fig 9B ) .', 'Importantly ACT1 is not differentially translationally or transcriptionally regulated between Hc yeast and hyphae .', 'Ectopic expression of this ACT1p – WET1 construct in Hc yeast cells resulted in inappropriate hyphal growth of Hc at 37°C ( as compared to vector control cells; Fig 9C and 9D ) , while expression of the ACT1p – WET1 construct had no discernible effect on Hc hyphal morphology at RT ( as compared to vector control cells; Fig 9D ) .', 'These data indicate that restricting Wet1 expression in yeast-phase cells is critical for maintenance of the yeast cell developmental program and that Hc longer leader transcripts are interesting candidates for Hc developmental regulators .'], ['Here we present improved transcript models for multiple clinical Histoplasma isolates as well as the first look at the translational landscape of this medically relevant human fungal pathogen .', 'Through this work we find that Hc alters transcript leader length between its morphologic cell types for a subset of its transcriptome .', 'Transcript leader length is a biologically regulated outcome of transcription in Hc as our work uncovers many examples of leader extensions ( 187 transcripts ) that are evolutionarily conserved across Hc phylogenetic lineages .', 'Ribosome and mRNA density measurements of longer leader transcripts reveal a class of genes that are under tight translational and transcriptional regulation .', 'Further examination of this group of transcriptionally and translationally regulated genes indicated that some are involved in controlling Hc morphology and that their strict regulation may be necessary for appropriate developmental decisions .', 'Our ribosome profiling experiments suggested mechanisms of translational regulation for longer leader transcript CDS regions .', 'Namely , we observed cases where ribosome density on longer leader regions correlated with reduced translational efficiency of the downstream CDS , suggesting that this class of transcripts may be translationally repressed via well-studied mechanisms of uORF-mediated translational control [56] .', 'Alternatively , we observed cases of translationally repressed CDS regions of longer leader transcripts with no evidence of ribosome occupancy on the leader , indicating that translational repression may be regulated via uORF-independent mechanisms .', 'Future work will be needed to examine mechanisms of translational repression for this class of transcripts such as changes to mRNA secondary structure of the leader that inhibit ribosome scanning or cell-type specific factors that regulate ribosome function .', 'For example , leader sequences could specify subcellular localization of mRNAs , which could inhibit ( or facilitate ) translation .', 'Lastly , we identified longer leader transcripts that exhibited ribosome footprint density in the leader region directly upstream of the predicted CDS with little effect on translational efficiency .', 'We suspect that some of these longer leader transcripts could serve to encode alternate protein variants and thus represent cases where leader extensions increase proteome diversity .', 'We must al
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however , mechanisms of post-transcriptional gene regulation remain poorly studied .', 'The human fungal pathogen Histoplasma capsulatum ( Hc ) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular ( hyphae ) or unicellular ( yeast ) developmental states that function in infectivity or pathogenesis , respectively .', 'Here we used recent advances in next-generation sequencing to uncover a novel re-programming of transcript length between Hc developmental cell types .', 'We found that ~2% percent of Hc transcripts exhibit 5’ leader sequences that differ markedly in length between morphogenetic states .', 'Ribosome density and mRNA abundance measurements of differential leader transcripts revealed nuanced transcriptional and translational regulation .', 'One such class of regulated longer leader transcripts exhibited tight transcriptional and translational repression .', 'Further examination of these dually repressed genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature .']
['Eukaryotic cells alter their developmental programs in response to environmental signals .', 'Histoplasma capsulatum ( Hc ) , a ubiquitous fungal pathogen of humans , establishes unique transcriptional programs to specify growth in either a multicellular hyphal form or unicellular yeast form in response to temperature .', 'Since hyphae and yeast are specialized to function in infectivity or pathogenesis , respectively , Hc provides a clinically relevant system in which to query eukaryotic regulatory processes .', 'Here we used next-generation sequencing approaches to annotate the transcriptomes of four distinct Hc strains in response to temperature .', 'We found that a fraction of Hc transcripts have differential transcript architecture in hyphae and yeast , exhibiting 5’ leader sequences that differ markedly in length between morphogenetic states .', 'To begin to understand the effect of these differential leader sequences on expression , we performed the first ribosome density and mRNA abundance measurements in Hc , thereby uncovering transcriptional and translational control that contribute to cell-type regulation .']
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