Chromosome Remodeling Research Articles

Identification of the Polo-like kinase substrate required for homologous synapsis in C. elegans.

The synaptonemal complex (SC) is a zipper-like protein structure that aligns homologous chromosome pairs and regulates recombination during meiosis. Despite its conserved appearance and function, how synapsis occurs between chromosome axes remains elusive. Here, we demonstrate that Polo-like kinases (PLKs) phosphorylate a single conserved residue in the disordered C-terminal tails of two paralogous SC subunits, SYP-5 and SYP-6, to establish an electrostatic interface between the SC central region and chromosome axes in C. elegans . While SYP-5/6 phosphorylation is dispensable for the ability of SC proteins to self-assemble, local phosphorylation by PLKs at the pairing center is crucial for SC elongation between homologous chromosome axes. Additionally, SYP-5/6 phosphorylation is essential for asymmetric SC disassembly and proper PLK-2 localization after crossover designation, which drives chromosome remodeling required for homolog separation during meiosis I. This work identifies a key regulatory mechanism by which localized PLK activity mediates the SC-axis interaction through phosphorylation of SYP-5/6, coupling synapsis initiation to homolog pairing.

Evolutionary accumulation of FKS1 mutations from clinical echinocandin-resistant Candida auris

Introduction: Drug resistance to echinocandins, first-line drugs used to treat Candida auris infection, is rapidly emerging. However, the accumulation of mutations in genes other than FKS1 (before an isolate develops to resistance via FKS1 mutations), remains poorly understood. Methods Four clinical cases and 29 isolates associated with the incremental process of echinocandin resistance were collected and analyzed using antifungal drug susceptibility testing and genome sequencing to assess the evolution of echinocandin resistance. Findings: Six echinocandin minimum inhibitory concentration (MIC)-elevated C. auris strains and seven resistant strains were isolated from the urinary system of patients receiving echinocandin treatment. Meanwhile, phylogenetic analyses illustrated that the echinocandin-resistant strains were closely related to other strains in the same patient. Genomic data revealed that the echinocandin-resistant strains had FKS1 mutations. Furthermore, three categories (ECN-S/E/R) of non-synonymous mutant SNP genes (such as RBR3, IFF6, MKC1, MPH1, RAD2, and MYO1) in C. auris appeared to be associated with the three-stage-evolutionary model of echinocandin resistance in C. glabrata: cell wall stress, drug adaptation, and genetic escape (FKS mutation). Interpretation: Echinocandin-resistant C. auris undergoes spatial and temporal phase changes closely related to echinocandin exposure, particularly in the urinary system. These findings suggest that FKS1 mutations mediate an evolutionary accumulation of echinocandin resistance followed by modulation of chromosome remodeling and DNA repair processes that ultimately lead to FKS1 hot spot mutations and the development of drug resistance. This study provides an in-depth exploration of the molecular pathways involved in the evolution of Candida auris echinocandin resistance.

MST1 interactomes profiling across cell death in esophageal squamous cell carcinoma

Abstract Objectives Resistance to apoptosis in esophageal squamous cell carcinoma (ESCC) constitutes a significant impediment to treatment efficacy. Exploring alternative cell death pathways and their regulatory factors beyond apoptosis is crucial for overcoming drug resistance and enhancing therapeutic outcomes in ESCC. Methods Mammalian Ste 20-like kinase 1 (MST1) is implicated in regulating various cell deaths, including apoptosis, autophagy, and pyroptosis. Employing enhanced ascorbate peroxidase 2 (APEX2) proximity labeling coupled with immunoprecipitation-mass spectrometry (IP-MS), we elucidated the interactomes of MST1 across these three cell death paradigms. Results Proteomic profiling unveiled the functional roles and subcellular localization of MST1 and its interacting proteins during normal proliferation and various cell death processes. Notably, MST1 exhibited an expanded interactome during cell death compared to normal proliferation and chromosome remodeling functions consistently. In apoptosis, there was a notable increase of mitosis-associated proteins such as INCENP, ANLN, KIF23, SHCBP1 and SUPT16H, which interacted with MST1, alongside decreased expression of the pre-apoptotic protein STK3. During autophagy, the bindings of DNA repair-related proteins CBX8 and m6A reader YTHDC1 to MST1 were enhanced. In pyroptosis, LRRFIP2 and FLII which can inhibit pyroptosis increasingly binding to MST1. Conclusions Our findings delineate potential mechanisms through which MST1 and its interactomes regulate cell death, paving the way for further investigation to validate and consolidate these observations.

Dual activities of a silencing information regulator complex in yeast transcriptional regulation and DNA-damage response.

The Saccharomyces cerevisiae silencing information regulator (SIR) complex contains up to four proteins, namely Sir1, Sir2, Sir3, and Sir4. While Sir2 encodes a NAD-dependent histone deacetylase, other SIR proteins mainly function as structural and scaffold components through physical interaction with various proteins. The SIR complex displays different conformation and composition, including Sir2 homotrimer, Sir1-4 heterotetramer, Sir2-4 heterotrimer, and their derivatives, which recycle and relocate to different chromosomal regions. Major activities of the SIR complex are transcriptional silencing through chromosomal remodeling and modulation of DNA double-strand-break repair pathways. These activities allow the SIR complex to be involved in mating-type maintenance and switching, telomere and subtelomere gene silencing, promotion of nonhomologous end joining, and inhibition of homologous recombination, as well as control of cell aging. This review explores the potential link between epigenetic regulation and DNA damage response conferred by the SIR complex under various conditions aiming at understanding its roles in balancing cell survival and genomic stability in response to internal and environmental stresses. As core activities of the SIR complex are highly conserved in eukaryotes from yeast to humans, knowledge obtained in the yeast may apply to mammalian Sirtuin homologs and related diseases.

The male pachynema-specific protein MAPS drives phase separation in vitro and regulates sex body formation and chromatin behaviors in vivo

Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male pachynema-specific protein (MAPS) in pachynema progression might be mediated by its liquid-liquid phase separation invitro and in cellulo. MAPS forms distinguishable liquid phases, and deletion or mutations of its N-terminal amino acids (aa) 2-9 disrupt its secondary structure and charge properties, impeding phase separation. Maps-/- pachytene spermatocytes exhibit defects in nucleus compartmentalization, including defects in forming sex bodies, altered nucleosome composition, and disordered chromatin accessibility. MapsΔ2-9/Δ2-9 male mice expressing MAPS protein lacking aa 2-9 phenocopy Maps-/- mice. Moreover, a frameshift mutation in C3orf62, the human counterpart of Maps, is correlated with nonobstructive azoospermia in a patient exhibiting pachynema arrest in spermatocyte development. Hence, the phase separation property of MAPS seems essential for pachynema progression in mouse and human spermatocytes.

The environmentally-regulated interplay between local three-dimensional chromatin organisation and transcription of proVWX in E. coli

Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS (histone-like nucleoid structuring protein)-regulated, osmoresponsive proVWX operon of Escherichia coli at different osmolarities and provide in vivo evidence for transcription regulation by NAP-mediated chromosome re-modelling in bacteria. By consolidating our in vivo investigations with earlier in vitro and in silico studies that provide mechanistic details of how H-NS re-models DNA in response to osmolarity, we report that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX. The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. Our results also reveal additional structural features associated with changes in proVWX transcript levels such as the decompaction of local chromatin upstream of the operon, highlighting that further complexity underlies the regulation of this model operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria.

ATF7IP2, a meiosis-specific partner of SETDB1, is required for proper chromosome remodeling and crossover formation during spermatogenesis

Meiotic crossovers are required for the faithful segregation of homologous chromosomes and to promote genetic diversity. However, it is unclear how crossover formation is regulated, especially on the XY chromosomes, which show a homolog only at the tiny pseudoautosomal region. Here, we show that ATF7IP2 is a meiosis-specific ortholog of ATF7IP and a partner of SETDB1. In the absence of ATF7IP2, autosomes show increased axis length and more crossovers; however, many XY chromosomes lose the obligatory crossover, although the overall XY axis length is also increased. Additionally, meiotic DNA double-strand break formation/repair may also be affected by altered histone modifications. Ultimately, spermatogenesis is blocked, and male mice are infertile. These findings suggest that ATF7IP2 constraints autosomal axis length and crossovers on autosomes; meanwhile, it also modulates XY chromosomes to establish meiotic sex chromosome inactivation for cell-cycle progression and to ensure XY crossover formation during spermatogenesis.

The composition dynamics of transposable elements in human blastocysts.

Transposable elements (TEs) are mobile DNA sequences that can replicate themselves and play significant roles in embryo development and chromosomal structure remodeling. In this study, we investigated the variation of TEs in blastocysts with different parental genetic backgrounds. We analyzed the proportions of 1137 TEs subfamilies from six classes at the DNA level using Bowtie2 and PopoolationTE2 in 196 blastocysts with abnormal parental chromosomal diseases. Our findings revealed that the parental karyotype was the dominant factor influencing TEs frequencies. Out of the 1116 subfamilies, different frequencies were observed in blastocysts with varying parental karyotypes. The development stage of blastocysts was the second most crucial factor influencing TEs proportions. A total of 614 subfamilies exhibited different proportions at distinct blastocyst stages. Notably, subfamily members belonging to the Alu family showed a high proportion at stage 6, while those from the LINE class exhibited a high proportion at stage 3 and a low proportion at stage 6. Moreover, the proportions of some TEs subfamilies also varied depending on blastocyst karyotype, inner cell mass status, and outer trophectoderm status. We found that 48 subfamilies displayed different proportions between balanced and unbalanced blastocysts. Additionally, 19 subfamilies demonstrated varying proportions among different inner cell mass scores, and 43 subfamilies exhibited different proportions among outer trophectoderm scores. This study suggests that the composition of TEs subfamilies may be influenced by various factors and undergoes dynamic modulation during embryo development.

Genomic profiles of chromosome remodeling pathway and their correlation with TMB, MSI, PD-L1, and immune microenvironment in biliary tract tumors.

e16181 Background: Immunotherapy combined with chemotherapy has become a promising first-line treatment for biliary tract tumor (BTC) patients. A previous clinical trial with a small sample size revealed that impaired functions of chromatin remodeling genes might be a potential predictive biomarker of combined immunotherapy for BTC. However, genomic profiles of chromosome remodeling pathway and their correlation with tumor mutation burden (TMB), microsatellite instability (MSI), programmed death ligand-1 (PD-L1) and immune microenvironment in Chinese BTC patients remain unknown. Methods: A total of 2031 BTC patients (cholangiocarcinoma [CCA], N=1476; gallbladder carcinoma [GBC], N=555) were included in this study, and their tumor tissue samples were subject to next generation sequencing using a 733-gene panel. Multiple immunofluorescence using for immune microenvironment related markers (including PD-1, PD-L1, CD8, CD68, CD163, CD3, CD4, CD20, CD56 and FoxP3) detection was conducted in 49 patients. Prognostic survival data were obtained from two clinical sequencing cohorts (CCA: ICGC-2017, N=417; GBC: MSK-2022, N=233) and were used to explore the association between chromatin remodeling genes mutation and prognosis. Results: In total, 57.9% (1175/2031) of BTC patients harbored alternations in genes involved in chromatin remodeling pathway and the top three alteration frequency somatic genes were ARID1A (19%), KMT2C (11%), ARID2/KMT2D/PBRM1 (8% each), respectively. The commonly alternation types were missense (48%), frameshift indel (25%) and nonsense (17%), respectively. The TMB level of altered group was significantly higher than wild group (median TMB, mutant-type vs. wild-type = 6.145 vs. 3.352 Muts/Mb, P<0.0001), but no significant difference was found in the PD-L1 expression level between the two groups. A total of 43 patients were identified as MSI-high, all of whom harbored alternations in genes involved in chromatin remodeling pathway. Compared with the wild group, the infiltration level of CD20+ B cells in tumor parenchyma was significantly higher in the altered group. Furthermore, there was no difference in overall survival (OS) between altered group and wild group (median OS, 18.99 vs. 20.97 months; HR=1.164 [95%CI: 0.9048-1.498], P=0.2360) in two clinical sequencing cohorts without immunotherapy. Conclusions: The results revealed the molecular basis of chromosome remodeling genes as the predictive biomarker of combined immunotherapy for BTC and exclude its effect on prognosis.

Germ-cell-specific transcriptome analysis illuminates the chromatin and ubiquitin pathway in autism spectrum disorders.

Accumulating epidemiological studies have suggested a positive association between advanced paternal age at conception and the increased risk of neurodevelopmental outcomes such as autism spectrum disorder (ASD) in their children. Recent biological studies using human sperm have identified increased de novo mutations in aged fathers, and hyper- or hypomethylation has been identified in the sperm from aged rodents. Dysregulation of DNA methylation in sperm may explain the transgenerational effects on the pathogenesis of ASD. However, compared to these epigenetic changes in the sperm of aged males, the effects of inherited predisposition from germ cells are largely unknown. Here, we use single-cell transcriptome data sets from 13 cell lines, including 12 ASD-associated CNVs models and control, that are performed neural differentiation from mouse embryonic stem cells. This study performed comprehensive bioinformatic analyses such as gene ontology (GO), network, pathway, and upstream regulator analyses. Through these analyses, we identify several susceptible pathways, such as chromatin and ubiquitin, in addition to translational and oxidative phosphorylation. Our results suggest that dysregulation of epigenetic chromosome remodeling and ubiquitin-proteasome pathway in the germ cell is a possible modulator for subsequent differentiated cells, sperm, and egg, as a risk factor for the neurodevelopmental disorder.

Heat shock factor 5 establishes the male germ-line meiotic sex chromosome inactivation through regulation of Smarca4

Meiotic sex chromosome inactivation is an essential event in male germ cell development, which is directed by DNA damage response signaling independent of Xist RNA to silence the transcription activity of the sex chromosomes. However, the specific mechanism of establishment and maintenance of meiotic chromosome silencing is still unclear. Here we identify the HSF5 as a testicular specific protein and the expression of which was at the onset of meiosis pachytene stage to round sperm. When the function of the HSF5 was lost, meiosis sex chromosome remodeling and silencing fail, followed by activation of CHK2 checkpoint leads to germ cell apoptosis. Furthermore, we found that SMARCA4 in the linking the HSF5 to MSCI and uncover additional factors with meiotic sex chromosome remodeling. Together, our results demonstrate a requirement for HSF5 activity in spermatogenesis and suggest a role for the mammalian HSF5-SMARCA4 in programmed meiotic sex chromosome remodeling and silencing events that take place during meiosis.

Three-dimensional chromosome re-modelling: The integral mechanism of transcription regulation in bacteria.

Nucleoid-associated proteins (NAPs) are architectural proteins of the bacterial chromosome and transcription factors that dynamically organise the chromosome and regulate gene expression in response to physicochemical environmental signals. While the architectural and regulatory functions of NAPs have been verified independently, the coupling between these functions in vivo has not been conclusively proven. Here we describe a model NAP - histone-like nucleoid structuring protein (H-NS) - as a coupled sensor-effector that directly regulates gene expression by chromatin re-modelling in response to physicochemical environmental signals. We outline how H-NS-binding partners and post-translational modifications modulate the role of H-NS as a transcription factor by influencing its DNA structuring properties. We consolidate our ideas in models of how H-NS may regulate the expression of the proVWX and hlyCABD operons by chromatin re-modelling. The interplay between chromosome structure and gene expression may be a common - but, at present, under-appreciated - concept of transcription regulation in bacteria.

Molecular and Chromosomal Evolution of DsTc1_5 and DsPogo_8 Elements (Tc1-Mariner) in Species of the Dichotomius Genus (Coleoptera: Scarabaeidae): Contributions to the Speciation Process

In Dichotomius genus, transposable elements (TE) have been related to chromosome remodeling, genomic evolution, and, possibly, to the speciation process. The objective of this study was to verify the interpopulational and interspecific conservation/variation of Tc1-Mariner elements (possibly autonomous) in Dichotomius species, aiming to identify possible contributions in the speciation process of this group. The analysis was performed on four species of Dichotomius, belonging to the Selenocopris subgenus. We verified the presence of the DsPogo_8 and DsTc1_5 elements by PCR and sequencing. We also isolated and sequenced the 28S and 16S rRNA genes aiming at the phylogenetic reconstruction of the analyzed species. Chromosomal mapping of TEs DsTc1_5 and DsPogo_8 was performed by fluorescent in situ hybridization. The results revealed the presence of the elements in the different species analyzed, except for DsTc1_5 in D. (S.) geminatus. These results suggest a vertical inheritance, with the presence of these elements in the common ancestor of these species. In the analyzed species, the nucleotide similarity of DsTc1_5 was higher than that of the 28S and 16S rRNA genes, suggesting the occurrence of horizontal transfer. The phylogenetic tree indicated that the absence of DsTc1_5 in D. (S.) geminatus is related to stochastic loss of this TE. Chromosomal mapping revealed dispersed signals, with predominance in euchromatic regions and wide variation in the chromosomal localization pattern of DsTc1_5 and DsPogo_8, both interpopulational and interspecific. This variation indicates that DsTc1_5 and DsPogo_8 may have contributed to prezygotic and postzygotic isolation, thus contributing to the speciation of these species.

Chromatin landscape, DSB levels, and cKU-70/80 contribute to patterning of meiotic DSB processing along chromosomes in C. elegans.

Programmed DNA double-strand break (DSB) formation is essential for achieving accurate chromosome segregation during meiosis. DSB repair timing and template choice are tightly regulated. However, little is known about how DSB distribution and the choice of repair pathway are regulated along the length of chromosomes, which has direct effects on the recombination landscape and chromosome remodeling at late prophase I. Here, we use the spatiotemporal resolution of meiosis in the Caenorhabditis elegans germline along with genetic approaches to study distribution of DSB processing and its regulation. High-resolution imaging of computationally straightened chromosomes immunostained for the RAD-51 recombinase marking DSB repair sites reveals that the pattern of RAD-51 foci throughout pachytene resembles crossover distribution in wild type. Specifically, RAD-51 foci occur primarily along the gene-poor distal thirds of the chromosomes in both early and late pachytene, and on both the X and the autosomes. However, this biased off-center distribution can be abrogated by the formation of excess DSBs. Reduced condensin function, but not an increase in total physical axial length, results in a homogeneous distribution of RAD-51 foci, whereas regulation of H3K9 methylation is required for the enrichment of RAD-51 at off-center positions. Finally, the DSB recognition heterodimer cKU-70/80, but not the non-homologous end-joining canonical ligase LIG-4, contributes to the enriched off-center distribution of RAD-51 foci. Taken together, our data supports a model by which regulation of the chromatin landscape, DSB levels, and DSB detection by cKU-70/80 collaborate to promote DSB processing by homologous recombination at off-center regions of the chromosomes in C. elegans.

Emerging Roles of Epigenetic Regulators in Maintaining Hematopoietic Stem Cell Homeostasis.

Hematopoietic stem cells (HSCs) are adult stem cells with the ability of self-renewal and multilineage differentiation into functional blood cells, thus playing important roles in the homeostasis of hematopoiesis and the immune response. Continuous self-renewal of HSCs offers fresh supplies for the HSC pool, which differentiate into all kinds of mature blood cells, supporting the normal functioning of the entire blood system. Nevertheless, dysregulation of the homeostasis of hematopoiesis is often the cause of many blood diseases. Excessive self-renewal of HSCs leads to hematopoietic malignancies (e.g., leukemia), while deficiency in HSC regeneration results in pancytopenia (e.g., anemia). The regulation of hematopoietic homeostasis is finely tuned, and the rapid development of high-throughput sequencing technologies has greatly boosted research in this field. In this chapter, we will summarize the recent understanding of epigenetic regulators including DNA methylation, histone modification, chromosome remodeling, noncoding RNAs, and RNA modification that are involved in hematopoietic homeostasis, which provides fundamental basis for the development of therapeutic strategies against hematopoietic diseases.

Targeting regulation of VEGF by BPTF in non-small cell lung cancer and its potential clinical significance

PurposeVEGF facilitates tumor angiogenesis, and bevacizumab targeting VEGF is used in anti-tumor therapy. It is meaningful to clarify the upstream regulatory mechanism of VEGF. BPTF is important in chromosomal remodeling, and promotes the progression of tumors. However, its role in promoting tumor angiogenesis by targeting VEGF has not been fully reported. This study aims to elucidate the expression regulation of VEGF by BPTF and its clinical significance in NSCLC.Methods1. BPTF siRNA and shRNA plasmids were used to reduce the expression of BPTF by transfection in vivo and in vitro. BPTF, VEGF and CD144 expressions were examined by immunofluorescence and Western Blot. 2. The expressions of BPTF, VEGF, CD144 and CD31 were detected in lung adenocarcinoma samples by immunofluorescence, Western blot and immunohistochemical staining. 3. 26 lung adenocarcinoma patients treated by bevacizumab were divided into 2 groups according to the treatment efficacy. BPTF and VEGF expressions were analyzed.Results1. BPTF knockdown inhibited the expression of VEGF and CD144 in vivo and in vitro. 2. Compared with para-cancer tissues, BPTF, VEGF, CD144 and CD31 were highly expressed in lung adenocarcinoma. 3. In 75 lung adenocarcinoma specimens, BPTF and VEGF overexpression was correlated with lymph node metastasis and clinical stage. The 5-year survival rate of patients with BPTF and VEGF low expression was higher, and BPTF expression was positively correlated with VEGF expression. 4. Among 26 patients treated with bevacizumab, the patients with BPTF overexpression are more sensitive to the treatment.ConclusionsBPTF positively regulates VEGF expression and its high expression predicts a better efficacy of bevacizumab treatment in NSCLC.

Transcriptome and Metabolome Analyses Reveal Complex Molecular Mechanisms Involved in the Salt Tolerance of Rice Induced by Exogenous Allantoin.

Allantoin is crucial for plant growth and development as well as adaptations to abiotic stresses, but the underlying molecular mechanisms remain unclear. In this study, we comprehensively analyzed the physiological indices, transcriptomes, and metabolomes of rice seedlings following salt, allantoin, and salt + allantoin treatments. The results revealed that exogenous allantoin positively affects the salt tolerance by increasing the contents of endogenous allantoin with antioxidant activities, increasing the reactive oxygen species (ROS)–scavenging capacity, and maintaining sodium and potassium homeostasis. The transcriptome analysis detected the upregulated expression genes involved in ion transport and redox regulation as well as the downregulated expression of many salt-induced genes related to transcription and post-transcriptional regulation, carbohydrate metabolism, chromosome remodeling, and cell wall organization after the exogenous allantoin treatment of salt-stressed rice seedlings. Thus, allantoin may mitigate the adverse effects of salt stress on plant growth and development. Furthermore, a global metabolite analysis detected the accumulation of metabolites with antioxidant activities and intermediate products of the allantoin biosynthetic pathway in response to exogenous allantoin, implying allantoin enhances rice salt tolerance by inducing ROS scavenging cascades. These results have clarified the transcript-level and metabolic processes underlying the allantoin-mediated salt tolerance of rice.

Chromosome remodelling by SMC/Condensin in B. subtilis is regulated by monomeric Soj/ParA during growth and sporulation

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving adenosine triphosphate (ATP)-dependent dimerization and DNA binding, facilitating chromosome segregation in bacteria. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. Here, we show that a major redistribution of SMC complexes drives axial filament formation in a process regulated by ParA/Soj. Furthermore, and unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyze ATP. These results reveal additional roles for ParA/Soj proteins in the regulation of SMC dynamics in bacteria and yet further complexity in the web of interactions involving chromosome replication, segregation and organization, controlled by ParAB and SMC.

Comparative Cytogenetic and Sequence Analysis of U Small Nuclear RNA Genes in Three Ancistrus Species (Siluriformes: Loricariidae).

Ancistrus presents a wide karyotypic diversity, resulting from numeric and structural chromosomal rearrangements. It has been proposed that some genome-specific regions containing repetitive units could organize prone-to-break DNA sites in Loricariidae, triggering chromosomal rearrangements such as Robertsonian fusions (Rb fusions), centric fissions, translocations, and inversions. The tandemly repeats of the small nuclear RNAs (snRNAs) gene families are considered good cytogenetic markers for understanding chromosomal remodeling events among closely related species, but these snRNAs have been scarcely analyzed in Ancistrus. This study presented the nucleotide sequencing and comparative in situ location of U snRNA sequences from Ancistrus aguaboensis, Ancistrus cf. multispinis, and Ancistrus sp. (2n = 50, 52, and 50, respectively), aiming to provide information about snRNA clusters in the genome and chromosome evolution in Ancistrus. U snRNA nucleotide sequences of Ancistrus presented identity to orthologous copies and folded their secondary structures correctly. In situ localization and karyotyping of the three Ancistrus species revealed clustered copies of U2 and U5 snRNA gene families to a single chromosome site, one chromosome pair bearing U1 snRNA sequence, and one main locus of U4 snRNA sequence, besides scattered signals along the chromosomes. Previous studies related the participation of the rRNA gene families in centric fusion events, contributing to chromosome rearrangements and karyotype plasticity present in Loricariidae. In this study, homeologies in U snRNA loci chromosomal locations were detected, indicating the occurrence of conserved sites of these gene families in these three Ancistrus species with 2n = 50 or 52 chromosomes.

Sorbicillinoids hyperproduction without affecting the cellulosic enzyme production in Trichoderma reesei JNTR5

BackgroundMicrobial production of bioactive secondary metabolites is challenging as most of the encoding genes are silent; and even if they are activated, the biosynthetic pathways are usually complex. Sorbicillinoids with multifunctional bioactivities are examples of these problems, which if solved can result in a more sustainable, simple supply of these important compounds to the pharmaceutical industry. As an excellent producer of cellulosic enzymes, Trichoderma reesei can secrete various sorbicillinoids.ResultsHere, we obtained a T. reesei mutant strain JNTR5 from the random mutation during overexpression of gene Tr69957 in T. reesei RUT-C30. JNTR5 exhibited a significant constitutive increase in sorbicillinoids production without affecting the cellulosic enzyme production. Confocal laser scanning microscope (CLSM) results indicated that sorbicillinoids were distributed in both mycelium and spores of JNTR5 with blue and green fluorescence. Compared with RUT-C30, JNTR5 displayed different cell morphology, reduced growth rate, and increased sporulation, but a similar biomass accumulation. Furthermore, transcriptome analysis revealed that all genes belonging to the sorbicillinoid gene cluster were upregulated, while most cellulase-encoding genes were downregulated. The cell wall integrity of JNTR5 was damaged, which might benefit the cellulase secretion and contribute to the almost unchanged cellulase and hemicellulase activity given that the damaged cell wall can enhance the secretion of the enzymes.ConclusionsFor the first time, we constructed a sorbicillinoids hyperproduction T. reesei platform with comparable cellulosic enzymes production. This outperformance of JNTR5, which is strain-specific, is proposed to be attributed to the overexpression of gene Tr69957, causing the chromosome remodeling and subsequently changing the cell morphology, structure, and the global gene expression as shown by phenotype and the transcriptome analysis of JNTR5. Overall, JNTR5 shows great potential for industrial microbial production of sorbicillinoids from cellulose and serves as an excellent model for investigating the distribution and secretion of yellow pigments in T. reesei.