Sc Genes Research Articles

Abstract 5823: Studying the role of microRNAs that target stem cell genes in the emergence of tumor heterogeneity in development of human colorectal cancers

Abstract We investigated miRNAs that are implicated to target genes that encode different stem cell markers (SC) during tumorigenesis of colorectal cancer (CRC), one of the leading causes of cancer deaths in the US. One reason behind the lack of efficacy in cancer drug therapeutics is due to the development of tumor heterogeneity. We hypothesize that different miRNAs target different SC genes, which leads to the emergence of multiple cancer SC (CSC) subpopulations and tumor heterogeneity. Flow cytometry helped evaluate the expression of multiple SC markers including ALDH1, CD166, LGR5, and LRIG1 in the HT29 CRC cell line. CSC sub-populations were isolated using fluorescence activated cell sorting (FACS) and the expression of miRNAs in each CSC subpopulation was measured using NanoString profiling. The differential expression of miRNAs between CSC subpopulations helped rank the different levels of miRNA upregulation in each of the CSC subpopulations. The mRNAs predicted to be targeted by the most significantly upregulated miRNAs were identified using bioinformatics analysis via NanoString and mRNAs were classified according to their predicted function by David functional annotation analysis. Altogether, our results indicate that multiple CSC subpopulations exist in the HT29 cell-line, and we identified unique miRNA signatures for each CSC subpopulation. Moreover, the mRNAs predicted to be targeted by the miRNAs in each CSC subpopulation were found to have different functional classifications. We conclude that multiple CSC subpopulations emerge in HT29 cells due to the expression of unique miRNAs that target different SC genes (and their co-expressed genes), resulting in the emergence of multiple CSC subpopulations and tumor heterogeneity. Citation Format: Victoria A. Stark, Caroline O. B. Facey, Lynn M. Opdenaker, Bruce M. Boman. Studying the role of microRNAs that target stem cell genes in the emergence of tumor heterogeneity in development of human colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5823.

Newly identified breast luminal progenitor and gestational stem cell populations likely give rise to HER2-overexpressing and basal-like breast cancers

Breast Cancer (BrC) is a common malignancy with genetically diverse subtypes. There is evidence that specific BrC subtypes originate from particular normal mammary cell populations. However, the cell populations that give rise to most BrC subtypes are unidentified. Several human breast scRNAseq datasets are available. In this research, we utilized a robust human scRNAseq dataset to identify population-specific marker genes and then identified the expression of these marker genes in specific BrC subtypes. In humans, several BrC subtypes, HER2-enriched, basal-like, and triple-negative (TN), are more common in women who have had children. This observation suggests that cell populations that originate during pregnancy give rise to these BrCs. The current human datasets have few normal parous samples, so we supplemented this research with mouse datasets, which contain mammary cells from various developmental stages. This research identified two novel normal breast cell populations that may be the origin of the basal-like and HER2-overexpressing subtypes, respectively. A stem cell-like population, SC, that expresses gestation-specific genes has similar gene expression patterns to basal-like BrCs. A novel luminal progenitor cell population and HER2-overexpressing BrCs are marked by S100A7, S100A8, and S100A9 expression. We bolstered our findings by examining SC gene expression in TN BrC scRNAseq datasets and S100A7-A9 gene expression in BrC cell lines. We discovered that several potential cancer stem cell populations highly express most of the SC genes in TN BrCs and confirmed S100A8 and A9 overexpression in a HER2-overexpressing BrC cell line. In summary, normal SC and the novel luminal progenitor cell population likely give rise to basal-like and HER2-overexpressing BrCs, respectively. Characterizing these normal cell populations may facilitate a better understanding of specific BrCs subtypes.

CRISPR/Cpf1-mediated mutagenesis and gene deletion in industrial filamentous fungi Aspergillus oryzae and Aspergillus sojae

In industrial applications such as fermentation and heterologous protein production, various Aspergillus oryzae and A. sojae strains are used. Although genetic engineering techniques have been developed for these filamentous fungi, applying such classical techniques to many strains is difficult. Therefore, the establishment of innovative technologies applicable to various industrial strains is required. We previously developed a genome editing technology using the CRISPR/Cas9 system for the efficient genetic engineering of A.oryzae; however, this system is limited by its protospacer adjacent motif sequence. In A.sojae, no genetic engineering using genome editing has been developed. In this study, we aimed to develop a genome editing technology using the Cpf1 nuclease for the genetic engineering of A.oryzae and A.sojae. AMA1-based genome editing vectors bearing codon-optimized cpf1 expression cassettes were constructed, and guide RNA expression cassettes were inserted into the Cpf1 genome editing vectors. Using the resultant plasmids, we performed mutagenesis of the AowA and sC genes in A.oryzae and the AswA gene in A.sojae. We deleted these genes by co-introducing the Cpf1 genome editing plasmid and the donor plasmid. Our study demonstrates that the CRISPR/Cpf1 system can be used as an efficient alternative to the CRISPR/Cas9 system to genetically engineer A.oryzae and as a new approach for efficient genetic engineering of A.sojae.

Abstract 2360: Identifying differentially expressed miRNAs in CRC stem cell subpopulations

Abstract Our research goal is to discover the molecular mechanisms behind tumor heterogeneity in CRC. We previously showed that CRCs contain multiple subpopulations of cancer stem cells (CSCs) which may explain the occurrence tumor heterogeneity and resistance to treatment. To determine how CSC sub-populations might arise, we are studying miRNA expression in CRC SCs. MicroRNAs are known to regulate SC phenotype and are found to be dysregulated in many cancers. Hypothesis: Tumor heterogeneity results from existence of multiple CSC subpopulations that are regulated by distinct miRNAs. Accordingly, we are using bioinformatics and miRNA profiling to identify miRNAs that target SC genes in CRCs. Indeed, our miRNA expression profiling of normal and malignant ALDH+ human colonic SCs showed that miRNA92a targets the SC gene LRIG1 and upregulation of miRNA92a leads to decreased LRIG1 expression. We also discovered that miRNA23b targets the SC gene LGR5 and miRNA23b is upregulated in ALDH+ CSCs. We have identified several other candidate miRNAs that are predicted to target CD166, ALDH1A1, BMI1, LRG5, and LRIG1 SC genes. We are currently in the process of validating whether these miRNAs contribute to emergence of specific CSC sub-populations in CRCs. Thus, identifying miRNAs that regulate CSC subpopulations should provide new strategies to modulate CSC composition in order to sensitize tumors to treatments. Citation Format: Victoria A. Stark, Vignesh Viswanathan, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Identifying differentially expressed miRNAs in CRC stem cell subpopulations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2360.

Phylogenomics and biogeography of Wisteria: Implications on plastome evolution among inverted repeat‐lacking clade (IRLC) legumes

AbstractThe genus Wisteria (Fabaceae) is disjunctly distributed in eastern Asian and eastern North American temperate deciduous forests, and it is widely cultivated around the world as spectacular garden plants. It is a member of inverted repeat‐lacking clade (IRLC). The IRLC Species are characterized by the loss of an IR region in their plastomes, which has long been of great interest. In this research, we report whole plastome sequences from all four Wisteria species and a Wisteriopsis japonica, combining these with existing data to explore phylogenetic relationships and biogeography of Wisteria, as well as plastome evolution of IRLC species. Phylogenetic analyses recognized a clade containing Glycyrrhiza–Wisteriopsis–Wisteria as sister to the remaining genera of IRLC. North American Wisteria frutescens and the three Asian species formed reciprocal clades, and Wisteria brachybotrys was sister to Wisteria floribunda and Wisteria sinensis. Wisteria may have originated in Japan near the boundary of the Oligocene and Miocene. The disappearance of Bering Land Bridge in the late Miocene might lead to the Eastern Asian–Eastern North American disjunction of Wisteria. Allopatric speciation of Wisteria between the Japanese archipelago and the Asian continent in the Quaternary increased the species richness of eastern Asia in comparison with eastern North America. Synonymous substitution rates (dS) of protein‐coding genes in the IRLC species were around 2‐fold (SC genes) or 11‐fold (IR genes) higher than those of non‐IRLC species. For both SC and IR genes, herbaceous legumes have around 3‐fold higher dS than woody ones. Both loss of one IR region and herbaceous habit elevated substitution rates of the plastomes.

Transcriptome sequencing revealed the inhibitory mechanism of ketoconazole on clinical Microsporum canis.

BackgroundMicrosporum canis is a zoonotic disease that can cause dermatophytosis in animals and humans.ObjectivesIn clinical practice, ketoconazole (KTZ) and other imidazole drugs are commonly used to treat M. canis infection, but its molecular mechanism is not completely understood. The antifungal mechanism of KTZ needs to be studied in detail.MethodsIn this study, one strain of fungi was isolated from a canine suffering with clinical dermatosis and confirmed as M. canis by morphological observation and sequencing analysis. The clinically isolated M. canis was treated with KTZ and transcriptome sequencing was performed to identify differentially expressed genes in M. canis exposed to KTZ compared with those unexposed thereto.ResultsAt half-inhibitory concentration (½MIC), compared with the control group, 453 genes were significantly up-regulated and 326 genes were significantly down-regulated (p < 0.05). Quantitative reverse transcription polymerase chain reaction analysis verified the transcriptome results of RNA sequencing. Gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the 3 pathways of RNA polymerase, steroid biosynthesis, and ribosome biogenesis in eukaryotes are closely related to the antifungal mechanism of KTZ.ConclusionsThe results indicated that KTZ may change cell membrane permeability, destroy the cell wall, and inhibit mitosis and transcriptional regulation through CYP51, SQL, ERG6, ATM, ABCB1, SC, KER33, RPA1, and RNP genes in the 3 pathways. This study provides a new theoretical basis for the effective control of M. canis infection and the effect of KTZ on fungi.

Corneal nonmyelinating Schwann cells illuminated by single-cell transcriptomics and visualized by protein biomarkers.

The cornea is the most innervated tissue in the human body. Myelinated axons upon inserting into the peripheral corneal stroma lose their myelin sheaths and continue into the central cornea wrapped by only nonmyelinating corneal Schwann cells (nm‐cSCs). This anatomical organization is believed to be important for central vision. Here we employed single‐cell RNA sequencing (scRNA‐seq), microscopy, and transgenics to characterize these nm‐cSCs of the central cornea. Using principal component analysis, uniform manifold approximation and projection, and unsupervised hierarchal cell clustering of scRNA‐seq data derived from central corneal cells of male rabbits, we successfully identified several clusters representing different corneal cell types, including a unique cell cluster representing nm‐cSCs. To confirm protein expression of cSC genes, we performed cross‐species validation, employing corneal whole‐mount immunostaining with confocal microscopy in mouse corneas. The expression of several representative proteins of nm‐cSCs were validated. As the proteolipid protein 1 (PLP1) gene was also expressed in nm‐cSCs, we explored the Plp1‐eGFP transgenic reporter mouse line to visualize cSCs. Specific and efficient eGFP expression was observed in cSCs in adult mice of different ages. Of several putative cornea‐specific SC genes identified, Dickkopf‐related protein 1 was shown to be present in nm‐cSCs. Taken together, our findings, for the first time, identify important insights and tools toward the study nm‐cSCs in isolated tissue and adult animals. We expect that our results will advance the future study of nm‐cSCs in applications of nerve repair, and provide a resource for the study of corneal sensory function.

Multiple signatures of a disease in potential biomarker space: Getting the signatures consensus and identification of novel biomarkers.

BackgroundThe lack of consensus among reported gene signature subsets (GSSs) in multi-gene biomarker discovery studies is often a concern for researchers and clinicians. Subsequently, it discourages larger scale prospective studies, prevents the translation of such knowledge into a practical clinical setting and ultimately hinders the progress of the field of biomarker-based disease classification, prognosis and prediction.MethodsWe define all "gene identificators" (gIDs) as constituents of the entire potential disease biomarker space. For each gID in a GSS of interest ("tested GSS"/tGSS), our method counts the empirical frequency of gID co-occurrences/overlaps in other reference GSSs (rGSSs) and compares it with the expected frequency generated via implementation of a randomized sampling procedure. Comparison of the empirical frequency distribution (EFD) with the expected background frequency distribution (BFD) allows dichotomization of statistically novel (SN) and common (SC) gIDs within the tGSS.ResultsWe identify SN or SC biomarkers for tGSSs obtained from previous studies of high-grade serous ovarian cancer (HG-SOC) and breast cancer (BC). For each tGSS, the EFD of gID co-occurrences/overlaps with other rGSSs is characterized by scale and context-dependent Pareto-like frequency distribution function. Our results indicate that while independently there is little overlap between our tGSS with individual rGSSs, comparison of the EFD with BFD suggests that beyond a confidence threshold, tested gIDs become more common in rGSSs than expected. This validates the use of our tGSS as individual or combined prognostic factors. Our method identifies SN and SC genes of a 36-gene prognostic signature that stratify HG-SOC patients into subgroups with low, intermediate or high-risk of the disease outcome. Using 70 BC rGSSs, the method also predicted SN and SC BC prognostic genes from the tested obesity and IGF1 pathway GSSs.ConclusionsOur method provides a strategy that identify/predict within a tGSS of interest, gID subsets that are either SN or SC when compared to other rGSSs. Practically, our results suggest that there is a stronger association of the IGF1 signature genes with the 70 BC rGSSs, than for the obesity-associated signature. Furthermore, both SC and SN genes, in both signatures could be considered as perspective prognostic biomarkers of BCs that stratify the patients onto low or high risks of cancer development.

The identification and expression of achaete-scute genes in the branchiopod crustacean Triops longicaudatus

The achaete-scute ( ac/ sc) genes are a highly conserved family of transcription factors that play important roles in the development of neural cells in both vertebrates and invertebrates. As such, the study of arthropod ac/ sc gene expression during neurogenesis has become a model system for investigating the evolution of neural patterning. To date, ac/ sc gene expression has been investigated in insects, chelicerates, and myriapods. Here we present the identification of two ac/ sc genes from the branchiopod crustacean Triops longicaudatus. Triops longicaudatus achaete-scute homologs 1 and 2 ( Tl-ASH1 and Tl-ASH2) exhibit dynamic and distinct expression profiles during Triops neurogenesis. Tl-ASH1 expression initiates in nearly all cells of the neurogenic region and subsequently in clusters of cells evenly spaced along the length of the developing limbs. In contrast, Tl-ASH2 initiates expression after Tl-ASH1. In the CNS, only a subset of Tl-ASH1 cells appears to express Tl-ASH2. Similarly, in the PNS individual Tl-ASH2 positive cells appear to arise from the clusters of Tl-ASH1 expressing cells. Shortly after activating Tl-ASH2 expression, these cells enlarge and divide. The expression dynamics of ac/ sc genes in Triops parallel those observed in insects and contrasts with those found in chelicerates and myriapods.

RLR1 (THO2), required for expressing lacZ fusions in yeast, is conserved from yeast to humans and is a suppressor of SIN4

We isolated a mutation (rlr1-1; required for lacZ RNA) in the Saccharomyces cerevisiae (Sc) RLR1 gene as a suppressor of sin4, a component of the Mediator subcomplex of the RNA polymerase II holoenzyme and a determinant of chromatin structure. RLR1 encodes a deduced protein found also in fission yeast, nematode worms, and humans. The presence of these orthologs suggests that Rlr1 family members comprise a class of putative KEKE motif-containing proteins, characteristic of certain chaperones as well as regulators and subunits of the mammalian 20S proteasome. A role for RLR1 (THO2) in transcription appears to occur at a step subsequent to transcription initiation (see also Piruat, J.I. and Aguilera, A., 1998. EMBO J. 17, 4859-4872); Sc genes fused to the reporter gene lacZ were expressed at a very low level, while the corresponding native chromosomal genes were expressed at approximately normal levels in rlr1 mutants. Our studies show that rlr1 mutations cause a wide range of growth defects in addition to their novel affect on lacZ.

Cis-regulation of achaete and scute: shared enhancer-like elements drive their coexpression in proneural clusters of the imaginal discs.

The pattern of bristles and other sensory organs on the adult cuticle of Drosophila is prefigured in the imaginal discs by the pattern of expression of the proneural achaete (ac) and scute (sc) genes, two members of the ac-sc complex (AS-C). These genes are simultaneously expressed by groups of cells (the proneural clusters) located at constant positions in discs. Their products (transcription factors of the basic-helix-loop-helix family) allow cells to become sensory organ mother cells (SMCs), a fate normally realized by only one or a few cells per cluster. Here we show that the highly complex pattern of proneural clusters is constructed piecemeal, by the action on ac and sc of site-specific, enhancer-like elements distributed along most of the AS-C (approximately 90 kb). Fragments of AS-C DNA containing these enhancers drive reporter lacZ genes in only one or a few proneural clusters. This expression is independent of the ac and sc endogenous genes, indicating that the enhancers respond to local combinations of factors (prepattern). We show further that the cross-activation between ac and sc, discovered by means of transgenes containing either ac or sc promoter fragments linked to lacZ and thought to explain the almost identical patterns of ac and sc expression, does not occur detectably between the endogenous ac and sc genes in most proneural clusters. Our data indicate that coexpression is accomplished by activation of both ac and sc by the same set of position-specific enhancers.

Cloning of the Candida glabrata TRP1 and HIS3 genes, and construction of their disruptant strains by sequential integrative transformation

The Candida glabrata (Cg) TRP1 and HIS3 genes have been isolated by complementation of the Saccharomyces cerevisiae (Sc) trpl and his3 mutants, respectively. Cg TRP1 encodes a polypeptide of 217 amino acids (aa), whose aa sequence is 58% identical to that of Sc TRP1. Cg HIS3 encodes a polypeptide of 210 aa, whose aa sequence is 73% identical to that of the Sc HIS3. Both Cg TRP1 and HIS3 were disrupted by sequential integrative transformation where the Sc URA3 was used as a selection marker for transformation. The resulting auxotrophic strain of his3 − and trpl − was used to examine the ability of the Sc genes to complement the Cg mutations; Sc HIS3 and TRP1 complemented the Cg his3 − and trpl − mutations, respectively.

The function of the proneural genes achaete and scute in the spatio-temporal patterning of the adult labellar bristles of Drosophila melanogaster.

The sensory precursors for labellar taste bristles develop from the labial disc in three distinct temporal waves occurring at 0 h, 8 h and 14 h of pupal development. In each temporal wave, transcripts for the achaete (ac) and scute (sc) genes are expressed in overlapping patterns in cells of the disc epithelium prior to the appearance of sensory mother cells (SMCs). No bristles form in mutant flies in which the ac and sc genes are absent. When the sc gene alone is deleted, a set of seven bristles fail to form. Pulses of ubiquitous sc + expression during pupal development, in a strain mutant for both ac and sc, can result in flies with all the labellar bristles at their correct positions. sc + pulses at times corresponding to the initiation of each of the waves of SMC specification in the disc was sufficient to restore bristle pattern. Bristles were not induced at ectopic positions and times as a result of the ubiquitous expression of sc +. These results suggest that the proneural genes ac and sc do not themselves set the pattern of the labellar bristles. Instead, they are required for the elaboration of the pattern set by other gene products. We also show that the formation and positioning of the later waves of bristles can take place even in the absence of bristles normally specified earlier.

Sensory mother cells are selected from among mitotically quiescent cluster of cells in the wing disc of Drosophila

ABSTRACT The large sensory bristles on the head and thorax of Drosophila are arranged in a precise pattern. Each bristle is formed by the progeny of a sensory mother cell (SMC) which is generated in the imaginal wing disc. The proneural genes ac and sc play an important role in the correct positioning of these SMCs by conferring to reproducibly located clusters of cells the competence to become SMCs. Indirect evidence suggested that the mitotic state of a cell could also play an important role in SMC positioning. In order to analyze the relation between the formation of SMC and the mitotic activity in the developing wing disc, we used BrdU immunolabeling to monitor the mitotic activity in an enhancertrap transformant line, A101, where the SMCs can be visualized. Our results indicate that SMCs arise from clusters of mitotically quiescent cells (MQC), and that the cell that becomes a SMC was itself arrested at the G2 stage of the cell cycle. The emergence of MQCs follows a precise temporal and spatial pattern which is not affected by the absence of the ac and/or sc genes. We propose that the reproducible emergence of MQCs could be an important factor in the formation of SMCs.

Searching for synaptonemal complex proteins and their genes.

As an alternative to the production and use of monoclonal antisynaptonemal complex (SC) antibodies to isolate SC genes, we have explored the use of polyclonal anti-SC antibodies to identify SC genes from a cDNA expression library. The method proved relatively simple, reliable, and fast and has yielded two SC genes. A homologue of one of these genes from a different species has previously been isolated in another laboratory.

Competence to develop sensory organs is temporally and spatially regulated in Drosophila epidermal primordia.

The Drosophila adult cuticle displays a stereotyped pattern of sensory organs (SOs). Its deployment requires the expression of the achaete (ac) and scute (sc) genes. Their products confer to cells of epidermal primordia (imaginal discs and histoblasts) the ability to become SO precursors (SOPs). In imaginal discs, ac and sc expression is spatially restricted to cell clusters within which one or a few cells become SOP(s). With the help of ubiquitous sc expression provided at different developmental times by a heat shock-sc (HSSC) chimeric gene, we have analyzed the response of epidermal primordia to the proneural action of the sc product, and have tested whether the patterned distribution of ac/sc products is necessary to position SOs correctly within the epidermis. Each primordium responds to HSSC expression by developing SOs only during a characteristic developmental period. In the absence of the endogenous ac and sc genes, most SOs induced by HSSC are of the correct type and are located in wild type positions. These results indicate that the capacity of primordia to respond to sc is temporally and spatially regulated, that specification of the type of SO does not depend on ac/sc, and that SO positioning utilizes topological information independent of the spatially restricted distribution of ac/sc products.

A near terminal pericentric inversion leads to nitrogen metabolite derepression in Aspergillus nidulans.

The mutation xprD-1, previously shown to be an allele of the areA gene and to lead to nitrogen metabolite derepression in Aspergillus nidulans, is shown to be associated with a near terminal pericentric inversion in linkage group III. The left arm break-point is between the adI and sC genes, and the right arm break-point is between the ornC and areA genes but just centromere proximal to areA. In crosses of xprD-1 strains to inversion-free strains one class of duplication-deficiency progeny is recovered. These progeny have two copies of the distal portion of the left arm beginning just before sC but lack a copy of areA and the region distal to it on the right arm. The viability of these duplication-deficiency progeny indicates that no indispensable gene can lie distal to areA, suggesting proximity of areA to the telomere. The inversion might increase expression of areA which, given the positive acting nature of this regulatory gene, would result in nitrogen metabolite derepression. If increased expression be the result of fusion to (or creation of) a more efficient promoter and/or ribosome binding sequence, areA must be transcribed towards the right arm telomere.

Genetic and accumulation studies in sulfite-requiring mutants of Aspergillus nidulans.

Using complementation tests and genetic mapping, mutants of two new genes, sE (sulfite less) and sF (thiosulfateless) were identified among 145 nitroso-guanidine-induced mutants of the sulfur metabolism in Aspergillus nidulans. Most of the other mutants (134) turned out to be alleles of the four established 'sulfite' genes, sA - sD. The new genes were found to be unlinked to any other s-gene; sE was mapped in linkage group VIII distal to cha and sF in VII. Mapping of several mutants allelic to one or the other of the closely linked sA or sC genes showed that these loci are two cistrons 1.1 cMo units apart.Results from studies using radioactive sulfate as a substrate for the different mutants showed35S-accumulation profiles by electrophoresis consistent with the assignment of the five sulfite genes to the following steps: sB(3), sulfate transport; sC(12), sulfurylation of ATP to produce APS; sD(50 or 205), phosphorylation of APS to PAPS; and sA(6) and sE(15), PAPS reduction to sulfite. Although the last two genes appear to control the same step, their35S-accumulation profiles are distinct. In addition, it was found that mutants of all previously established genes, including sA, are epistatic to sE(15) when single and double mutants are tested for resistance on selenate medium.