Comparative analysis of gene expression in tea plant (Camellia sinensis (L.) Kuntze) under low-temperature stress

Low-temperature stress is one of the main factors limiting the distribution and reducing the yield of many subtropical crops, including the tea crop. Efficient breeding to develop frost-tolerant cultivars requires a reliable set of genetic markers for identifying resistance donors, and that is why it is necessary to reveal the specific genetic response in frost-tolerant genotypes in comparison with frost- susceptible ones. In this work, we performed a comparative analysis of the expression of 18 tea genes (ICE1, CBF1, DHN1, DHN2, DHN3, NAC17, NAC26, NAC30, bHLH7, bHLH43, P5CS, WRKY2, LOX1, LOX6, LOX7, SnRK1.1, SnRK1.2, SnRK1.3) under cold and frost conditions in two tea genotypes, tolerant and susceptible. Low-temperature stress was induced by placing the potted plants in cold chambers and lowering the temperature to 0…+2 °С for 7 days (cold stress), followed by a decrease in temperature to –4…–6 °С for 5 days (frost stress). Relative electrical conductivity of leaf was measured in response to the stress treatments, and a significant difference in the frost tolerance of the two tea genotypes was confirmed. Cold exposure did not lead to a change in the electrical conductivity of leaf tissue. On the other hand, frost treatment resulted in increased REC in both genotypes and to a greater extent in the susceptible genotype. Increased expression of all the genes was shown during cold and frost. The genes that were strongly expressed in the tolerant tea genotype were revealed: ICE1, CBF1, DHN2, NAC17, NAC26, bHLH43, WRKY2, P5CS, LOX6, SnRK1.1, SnRK1.3. These genes can be proposed as markers for the selection of frost-tolerance donors in tea germplasm collections. Additionally, it was shown that the tolerant genotype is characterized by an earlier response to stress at the stage of cold acclimation. The study of the expression of the identified genes in different organs of tea plants and in different exposures to low temperature is relevant for further investigations

Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities

Background Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. Methods We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. Results We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. Conclusions Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia

Clinical and molecular characterization of early T-cell precursor leukemia: a high-risk subgroup in adult T-ALL with a high frequency of FLT3 mutations

A subgroup of pediatric acute T-lymphoblastic leukemia (T-ALL) was characterized by a gene expression profile comparable to that of early T-cell precursors (ETPs) with a highly unfavorable outcome. We have investigated clinical and molecular characteristics of the ETP-ALL subgroup in adult T-ALL. As ETP-ALL represents a subgroup of early T-ALL we particularly focused on this cohort and identified 178 adult patients enrolled in the German Acute Lymphoblastic Leukemia Multicenter studies (05/93–07/03). Of these, 32% (57/178) were classified as ETP-ALL based on their characteristic immunophenotype. The outcome of adults with ETP-ALL was poor with an overall survival of only 35% at 10 years, comparable to the inferior outcome of early T-ALL with 38%. The molecular characterization of adult ETP-ALL revealed distinct alterations with overexpression of stem cell-related genes (BAALC, IGFBP7, MN1, WT1). Interestingly, we found a low rate of NOTCH1 mutations and no FBXW7 mutations in adult ETP-ALL. In contrast, FLT3 mutations, rare in the overall cohort of T-ALL, were very frequent and nearly exclusively found in ETP-ALL characterized by a specific immunophenotype. These molecular characteristics provide biologic insights and implications with respect to innovative treatment strategies (for example, tyrosine kinase inhibitors) for this high-risk subgroup of adult ETP-ALL

Genes underlying cold acclimation in the tea plant (<i>Camellia sinensis</i> (L.) Kuntze)

The article reviews the latest studies showing the diversity of genetic mechanisms and gene families underlying the increased cold and frost tolerance of tea and other plant species. It has been shown that cell responses to chilling (0…+15°C) and freezing (&lt; 0°C) are not the same and gene expression under cold stress is genotype-specific. In recent decades, progress has been made in understanding the genetic mechanisms underlying the cold response of plants – ICE1 (inducer of CBF expression 1), CBF (C-repeat-binding factor), COR (cold-regulated genes) pathways and signaling have been discovered. The ICE, CBF and DHN gene groups play a key role in the cold acclimation of the tea plant. The accumulation of CBF transcripts occurs after 15 min of chilling induction, and longer cold stress leads to accumulation of CBF transcripts. It is shown that the transcripts of the CsDHN1, CsDHN2 and CsDHN3 genes accumulate at a higher level in resistant genotypes of tea in comparison with susceptible cultivars during freezing. CBF-independent pathways include genes involved in metabolism and transcription factors such as HSFC1, ZAT12, CZF1, PLD (phospholipase D), WRKY, HD-Zip, CsLEA, LOX, NAC, HSP, which are widely distributed in plants and are involved in the basic mechanisms of tea resistance to cold and frost. The most recent studies show an important role of miRNA in the mechanisms of response to chilling and freezing in tea. The data obtained on different plant species may correlate with the mechanisms of frost tolerance of tea and are the basis for future studies of the signaling pathways of response to cold in the tea plant. The results of the research emphasize the need to further explore the ways in which various genes regulate the tolerance of tea to cold stress to find the molecular markers of frost tolerance

Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities

Background: Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. Methods: We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. Results: We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. Conclusions: Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia.publishedVersionPeer reviewe

RBSP3 (HYA22) is a tumor suppressor gene implicated in major epithelial malignancies

Chromosome 3p21.3 region is frequently (>90%) deleted in lung and other major human carcinomas. We subdivided 3p21.3 into LUCA and AP20 subregions and discovered frequent homozygous deletions (10-18%) in both subregions. This finding strongly implies that they harbor multiple tumor suppressor genes involved in the origin and/or development of major epithelial cancers. In this study, we performed an initial analysis of RBSP3/HYA22, a candidate tumor suppressor genes located in the AP20 region. Two sequence splice variants of RBSP3/HYA22 (A and B) were identified, and we provide evidence for their tumor suppressor function. By sequence analysis RBSP3/HYA22 belongs to a gene family of small C-terminal domain phosphatases that may control the RNA polymerase II transcription machinery. Expression of the gene was drastically (>20-fold) decreased in 11 of 12 analyzed carcinoma cell lines and in three of eight tumor biopsies. We report missense and nonsense mutations in tumors where RBSP3/HYA22 was expressed, growth suppression with regulated transgenes in culture, suppression of tumor formation in severe combined immunodeficient mice, and dephosphorylation of ppRB by RBSP3/HYA22, presumably leading to a block of the cell cycle at the G(1)/S boundary