Key Cis-regulatory Elements Research Articles

Transcription factor networks disproportionately enrich for heritability of blood cell phenotypes.

Most phenotype-associated genetic variants map to non-coding regulatory regions of the human genome. Moreover, variants associated with blood cell phenotypes are enriched in regulatory regions active during hematopoiesis. To systematically explore the nature of these regions, we developed a highly efficient strategy, Perturb-multiome, that makes it possible to simultaneously profile both chromatin accessibility and gene expression in single cells with CRISPR-mediated perturbation of a range of master transcription factors (TFs). This approach allowed us to examine the connection between TFs, accessible regions, and gene expression across the genome throughout hematopoietic differentiation. We discovered that variants within the TF-sensitive accessible chromatin regions, while representing less than 0.3% of the genome, show a ~100-fold enrichment in heritability across certain blood cell phenotypes; this enrichment is strikingly higher than for other accessible chromatin regions. Our approach facilitates large-scale mechanistic understanding of phenotype-associated genetic variants by connecting key cis-regulatory elements and their target genes within gene regulatory networks.

Epigenomic identification of vernalization cis-regulatory elements in winter wheat

BackgroundWinter wheat undergoes vernalization, a process activated by prolonged exposure to low temperatures. During this phase, flowering signals are generated and transported to the apical meristems, stimulating the transition to the inflorescence meristem while inhibiting tiller bud elongation. Although some vernalization genes have been identified, the key cis-regulatory elements and precise mechanisms governing this process in wheat remain largely unknown.ResultsIn this study, we construct extensive epigenomic and transcriptomic profiling across multiple tissues—leaf, axillary bud, and shoot apex—during the vernalization of winter wheat. Epigenetic modifications play a crucial role in eliciting tissue-specific responses and sub-genome-divergent expressions during vernalization. Notably, we observe that H3K27me3 primarily regulates vernalization-induced genes and has limited influence on vernalization-repressed genes. The integration of these datasets enables the identification of 10,600 putative vernalization-related regulatory elements including distal accessible chromatin regions (ACRs) situated 30Kb upstream of VRN3, contributing to the construction of a comprehensive regulatory network. Furthermore, we discover that TaSPL7/15, integral components of the aging-related flowering pathway, interact with the VRN1 promoter and VRN3 distal regulatory elements. These interactions finely regulate their expressions, consequently impacting the vernalization process and flowering.ConclusionsOur study offers critical insights into wheat vernalization’s epigenomic dynamics and identifies the putative regulatory elements crucial for developing wheat germplasm with varied vernalization characteristics. It also establishes a vernalization-related transcriptional network, and uncovers that TaSPL7/15 from the aging pathway participates in vernalization by directly binding to the VRN1 promoter and VRN3 distal regulatory elements.

OsTBP2.1, a TATA-Binding Protein, Alters the Ratio of OsNRT2.3b to OsNRT2.3a and Improves Rice Grain Yield.

The OsNRT2.3a and OsNRT2.3b isoforms play important roles in the uptake and transport of nitrate during rice growth. However, it is unclear which cis-acting element controls the transcription of OsNRT2.3 into these specific isoforms. In this study, we used a yeast one-hybrid assay to obtain the TATA-box binding protein OsTBP2.1, which binds to the TATA-box of OsNRT2.3, and verified its important role through transient expression and RNA-seq. We found that the TATA-box of OsNRT2.3 mutants and binding protein OsTBP2.1 together increased the transcription ratio of OsNRT2.3b to OsNRT2.3a. The overexpression of OsTBP2.1 promoted nitrogen uptake and increased rice yield compared with the wild-type; however, the OsTBP2.1 T-DNA mutant lines exhibited the opposite trend. Detailed analyses demonstrated that the TATA-box was the key cis-regulatory element for OsNRT2.3 to be transcribed into OsNRT2.3a and OsNRT2.3b. Additionally, this key cis-regulatory element, together with the binding protein OsTBP2.1, promoted the development of rice and increased grain yield.

POU-M2 promotes juvenile hormone biosynthesis by directly activating the transcription of juvenile hormone synthetic enzyme genes in Bombyx mori.

Juvenile hormone (JH) plays a key role in preventing larval precocious metamorphosis, maintaining larval state, controlling adult sexual development and promoting insect egg maturation. Genetic studies have shown that POU factor ventral veins lacking regulates JH synthesis to control the timing of insect metamorphosis. However, how POU factor regulates JH synthesis is largely unknown. Here, we found POU-M2 was highly expressed in corpora allata (CA) and specifically localized in the nucleus of CA. The overexpression of POU-M2 promoted the expression of JH synthase genes and kr-h1 and enhanced the activity of JH synthase genes promoter. Further, POU-M2 promoted the transcription of JH acid O-methyltransferase (JHAMT) by directly binding to the key cis-regulatory elements -207, -249 and -453 within the proximal regions of JHAMT promoter. Both the POU domain and homeodomain were vital for the activation of POU-M2 on JHAMT transcription. Our study reveals the mechanism by which POU-M2 regulates JHAMT transcription.

PeakVI: A deep generative model for single-cell chromatin accessibility analysis

SummarySingle-cell ATAC sequencing (scATAC-seq) is a powerful and increasingly popular technique to explore the regulatory landscape of heterogeneous cellular populations. However, the high noise levels, degree of sparsity, and scale of the generated data make its analysis challenging. Here, we present PeakVI, a probabilistic framework that leverages deep neural networks to analyze scATAC-seq data. PeakVI fits an informative latent space that preserves biological heterogeneity while correcting batch effects and accounting for technical effects, such as library size and region-specific biases. In addition, PeakVI provides a technique for identifying differential accessibility at a single-region resolution, which can be used for cell-type annotation as well as identification of key cis-regulatory elements. We use public datasets to demonstrate that PeakVI is scalable, stable, robust to low-quality data, and outperforms current analysis methods on a range of critical analysis tasks. PeakVI is publicly available and implemented in the scvi-tools framework.

Architecture and evolution of the cis-regulatory system of the echinoderm kirrelL gene.

The gene regulatory network (GRN) that underlies echinoderm skeletogenesis is a prominent model of GRN architecture and evolution. KirrelL is an essential downstream effector gene in this network and encodes an Ig-superfamily protein required for the fusion of skeletogenic cells and the formation of the skeleton. In this study, we dissected the transcriptional control region of the kirrelL gene of the purple sea urchin, Strongylocentrotus purpuratus. Using plasmid- and bacterial artificial chromosome-based transgenic reporter assays, we identified key cis-regulatory elements (CREs) and transcription factor inputs that regulate Sp-kirrelL, including direct, positive inputs from two key transcription factors in the skeletogenic GRN, Alx1 and Ets1. We next identified kirrelL cis-regulatory regions from seven other echinoderm species that together represent all classes within the phylum. By introducing these heterologous regulatory regions into developing sea urchin embryos we provide evidence of their remarkable conservation across ~500 million years of evolution. We dissected in detail the kirrelL regulatory region of the sea star, Patiria miniata, and demonstrated that it also receives direct inputs from Alx1 and Ets1. Our findings identify kirrelL as a component of the ancestral echinoderm skeletogenic GRN. They support the view that GRN subcircuits, including specific transcription factor-CRE interactions, can remain stable over vast periods of evolutionary history. Lastly, our analysis of kirrelL establishes direct linkages between a developmental GRN and an effector gene that controls a key morphogenetic cell behavior, cell-cell fusion, providing a paradigm for extending the explanatory power of GRNs.

Landscape and dynamics of accessible chromatin during pigmentation process in green, white and purple sea cucumber Apostichopus japonicus

Cis-regulatory sequences, such as enhancers, control development and physiology by regulating gene expression. The identification and functional characterization of these distal regulatory elements remains challenging, especially in non-model aquatic organisms like sea cucumbers. Apostichopus japonicus, an important echinoderm species, exhibits diverse body colour. Pigmentation is an important stage of growth and development in this species, but the regulatory mechanism remains poorly understood, especially the extent to which epigenetic changes regulate pigmentation. Herein, we globally profiled chromatin accessibility using the assay for transposase-accessible chromatin sequencing technique during pigmentation in green, white and purple sea cucumbers. We have got 27, 817 peaks in total and discovered 1427 peaks showing differentially accessed DNA sites across the genome, which were categorized into four distinct clusters via unsupervised hierarchical clustering. Among three colour morphs, white sea cucumber exhibited the most extensive changes and the greatest differences from the other two colour morphs. Global increases in chromatin accessibility occur in the late pigmentation stage suggesting that active function in the late pigmentation stage drives body colour formation. The screened key cis-regulatory elements KIT, RPS6KB2 and IRF8 may contributed to the albinism, slow growth and poor disease resistance of white sea cucumber. In conclusion, our study provides a framework for understanding the accessible chromatin dynamics of body colour diversity during echinoderm pigmentation process and a new avenue to unravel the transcriptional regulatory mechanisms that facilitate the occurrence of relevant molecular events.

Knockdown of 60S ribosomal protein L14-2 reveals their potential regulatory roles to enhance drought and salt tolerance in cotton

BackgroundCotton is a valuable economic crop and the main significant source of natural fiber for textile industries globally. The effects of drought and salt stress pose a challenge to strong fiber and large-scale production due to the ever-changing climatic conditions. However, plants have evolved a number of survival strategies, among them is the induction of various stress-responsive genes such as the ribosomal protein large (RPL) gene. The RPL gene families encode critical proteins, which alleviate the effects of drought and salt stress in plants. In this study, comprehensive and functional analysis of the cotton RPL genes was carried out under drought and salt stresses.ResultsBased on the genome-wide evaluation, 26, 8, and 5 proteins containing the RPL14B domain were identified in Gossypium hirsutum, G. raimondii, and G. arboreum, respectively. Furthermore, through bioinformatics analysis, key cis-regulatory elements related to RPL14B genes were discovered. The Myb binding sites (MBS), abscisic acid-responsive element (ABRE), CAAT-box, TATA box, TGACG-motif, and CGTCA-motif responsive to methyl jasmonate, as well as the TCA-motif responsive to salicylic acid, were identified. Expression analysis revealed a key gene, Gh_D01G0234 (RPL14B), with significantly higher induction levels was further evaluated through a reverse genetic approach. The knockdown of Gh_D01G0234 (RPL14B) significantly affected the performance of cotton seedlings under drought/salt stress conditions, as evidenced by a substantial reduction in various morphological and physiological traits. Moreover, the level of the antioxidant enzyme was significantly reduced in VIGS-plants, while oxidant enzyme levels increased significantly, as demonstrated by the higher malondialdehyde concentration level.ConclusionThe results revealed the potential role of the RPL14B gene in promoting the induction of antioxidant enzymes, which are key in oxidizing the various oxidants. The key pathways need to be investigated and even as we exploit these genes in the developing of more stress-resilient cotton germplasms.

Insights into the dynamics of cyclic diguanosine monophosphate I riboswitch using molecular dynamics simulation

Riboswitches are key cis-regulatory elements, present at 5' UTRs of several prokaryotic mRNAs, involved in gene expression regulation by binding selectively to specific ligands followed by conformational changes. However, understanding of ligand-free riboswitch, conformational changes between the ligand-free and ligand-bound riboswitch and binding mechanism of ligand to the aptamer of riboswitch is limited. In the present paper we describe the structural and dynamical properties of ligand-free c-di-GMP I riboswitch aptamer and its possible binding mechanism with c-di-GMP by means of all-atom molecular dynamics (MD) simulations. Various analyses such as principal component analysis, cross correlation dynamics analysis, network analysis and trajectory analyses were carried out. The ligand-free structure shows stable conformation with folded P2, P3 and an unwind P1 helix with open binding pocket at helix-join-helix junction while the ligand-bound structure showed closed binding pocket structure compared to the ligand-free structure. The junction residues significantly showed anti-correlations with P1 helix and weak correlated motions with each other in the open conformation of the ligand-free aptamer of riboswitch. The networks analysis of binding pocket residues suggested interaction among the identified key residues of the binding pocket (G20, A47, C92) and P1 helix illustrating the role of P1 helix in binding of ligand. The structural insights, on c-di-GMP I riboswitch, presented in this paper can be useful for the development of therapeutics against V. cholerae. The understanding of the c-di-GMP I riboswitch at dynamic molecular level provides a potential solution for riboswitch drug design.

Genome-wide characterization, expression profiling, and post-transcriptional study of GASA gene family

The GASAs are a family of plant cysteine-rich small peptides widely distributed in the plant kingdom and involved in the plant development and stress response. In the current study, GASA gene family was widely compared in dicots and monocots based on their structure and expression. In addition, the post transcription/translation modifications of GASA proteins were predicted to more understanding the regulatory mechanisms. The phylogenetic analysis revealed that GASA genes of studied monocot plants were more similar than dicot plants. Also, the leucine-rich repeat domain, motif 4, was identified in the C terminal of studied GASA proteins that may affect the function of GASA proteins. Furthermore, most studied-GASA proteins were unstable based on instability index values. Besides, our result revealed that rice-GASA genes are more GC rich than studied dicots (Arabidopsis and tomato). The GASA genes showed differential expression in response to biotic and abiotic stress, and hormone treatments and also key cis-regulatory elements were observed in their promoter site, indicating that GASA genes as an important gene family have diverse roles and functions. Also, the strong interactions among Dof, BBR-BPC, ERF, and MYB transcription factors family and GASA genes were predicted in Arabidopsis, tomato, and rice. The results of the present study can be useful in further studies related to the function of GASA genes.

Function analysis and stress-mediated cis-element identification in the promoter region of VqMYB15

ABSTRACT The transcription factor MYB15 plays an important role in grape basal immunity, and its promoter can be used as a potential target in resistance breeding. However, the regulatory mechanisms of cis-elements in its promoter region under a variety of stresses remain unclear. In this study, we identified some putative cis-regulatory elements present upstream of MYB15 in Vitis quinquangularis Shanyang (pVqMYB15_SY) and subsequently characterized the function of these elements using reporter assays. Our results showed that TCA-elements 1 and 2, ABRE, MYC and 3-AF1 binding site 1 are key cis-regulatory elements in pVqMYB15_SY and play important roles in plant bio/abiotic stress resistance.

NET-CAGE characterizes the dynamics and topology of human transcribed cis-regulatory elements.

Promoters and enhancers are key cis-regulatory elements, but how they operate to generate cell type-specific transcriptomes is not fully understood. We developed a simple and robust method, native elongating transcript-cap analysis of gene expression (NET-CAGE), to sensitively detect 5' ends of nascent RNAs in diverse cells and tissues, including unstable transcripts such as enhancer-derived RNAs. We studied RNA synthesis and degradation at the transcription start site level, characterizing the impact of differential promoter usage on transcript stability. We quantified transcription from cis-regulatory elements without the influence of RNA turnover, and show that enhancer-promoter pairs are generally activated simultaneously on stimulation. By integrating NET-CAGE data with chromatin interaction maps, we show that cis-regulatory elements are topologically connected according to their cell type specificity. We identified new enhancers with high sensitivity, and delineated primary locations of transcription within super-enhancers. Our NET-CAGE dataset derived from human and mouse cells expands the FANTOM5 atlas of transcribed enhancers, with broad applicability to biomedical research.

Tissue Regeneration Enhancer Elements: A Way to Unlock Endogenous Healing Power.

Regenerative capacity is widespread throughout almost all animal phyla. However, the distribution pattern remains incompletely understood. Various examples show that very closely related species display different regenerative capacities. Why and how have diverse regenerative capacities evolved across species? One prevailing thought in the field of regeneration is that most regeneration-associated factors are evolutionarily conserved, suggesting the existence of an innate tissue regeneration ability in all species. However, its regulation is differentially controlled in distinct species, resulting in heterogeneous regenerative capabilities. In this review, we discuss regeneration-associated enhancers, the key cis-regulatory elements controlling gene expression, their underlying molecular mechanisms, and their influence on regenerative capacity. Understanding the regulatory mechanisms of regeneration enhancers can provide fundamental insights into tissue regeneration and further help us develop therapeutic strategies to unlock latent healing powers in humans. Developmental Dynamics 248:34-42, 2019. © 2018 Wiley Periodicals, Inc.

Epigenetic response of imprinted domains during carcinogenesis

BackgroundImprinted domains have been identified as targets for aberrant DNA methylation during carcinogenesis, but it remains unclear when these epigenetic alterations occur and how they contribute to tumor progression. Epigenetic instability at key cis-regulatory elements within imprinted domains can concomitantly activate proto-oncogenes and turn off tumor suppressor genes. Thus, to further characterize the epigenetic response of imprinted domains during carcinogenesis, we compared the stability of DNA methylation at a variety of cis-regulatory elements within imprinted domains in two fundamentally different mouse tumors, benign and malignant, induced by the KrasG12D mutation.ResultsWe report that imprinted domains remain stable in benign processes but are highly susceptible to epigenetic alterations in infiltrative lesions. The preservation of DNA methylation within imprinted domains in benign tumors throughout their duration suggests that imprinted genes are not involved with the initiation of carcinogenesis or the growth of tumors. However, the frequent detection of DNA methylation changes at imprinting control regions in infiltrative lesions suggest that imprinted genes are associated with tumor cells gaining the ability to defy tissue boundaries.ConclusionOverall, this study demonstrates that imprinted domains are targeted for DNA hypermethylation when benign tumor cells transition to malignant. Thus, monitoring DNA methylation within imprinted domains may be useful in evaluating the progression of neoplasms.

Transcriptomic analysis of rice aleurone cells identified a novel abscisic acid response element.

Seeds serve as a great model to study plant responses to drought stress, which is largely mediated by abscisic acid (ABA). The ABA responsive element (ABRE) is a key cis-regulatory element in ABA signalling. However, its consensus sequence (ACGTG(G/T)C) is present in the promoters of only about 40% of ABA-induced genes in rice aleurone cells, suggesting other ABREs may exist. To identify novel ABREs, RNA sequencing was performed on aleurone cells of rice seeds treated with 20μM ABA. Gibbs sampling was used to identify enriched elements, and particle bombardment-mediated transient expression studies were performed to verify the function. Gene ontology analysis was performed to predict the roles of genes containing the novel ABREs. This study revealed 2443 ABA-inducible genes and a novel ABRE, designated as ABREN, which was experimentally verified to mediate ABA signalling in rice aleurone cells. Many of the ABREN-containing genes are predicted to be involved in stress responses and transcription. Analysis of other species suggests that the ABREN may be monocot specific. This study also revealed interesting expression patterns of genes involved in ABA metabolism and signalling. Collectively, this study advanced our understanding of diverse cis-regulatory sequences and the transcriptomes underlying ABA responses in rice aleurone cells.

Genome-Wide Identification and Analysis of Genes, Conserved between japonica and indica Rice Cultivars, that Respond to Low-Temperature Stress at the Vegetative Growth Stage.

Cold stress is very detrimental to crop production. However, only a few genes in rice have been identified with known functions related to cold tolerance. To meet this agronomic challenge more effectively, researchers must take global approaches to select useful candidate genes and find the major regulatory factors. We used five Gene expression omnibus series data series of Affymetrix array data, produced with cold stress-treated samples from the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), and identified 502 cold-inducible genes common to both japonica and indica rice cultivars. From them, we confirmed that the expression of two randomly chosen genes was increased by cold stress in planta. In addition, overexpression of OsWRKY71 enhanced cold tolerance in ‘Dongjin,’ the tested japonica cultivar. Comparisons between japonica and indica rice, based on calculations of plant survival rates and chlorophyll fluorescence, confirmed that the japonica rice was more cold-tolerant. Gene Ontology enrichment analysis indicate that the ‘L-phenylalanine catabolic process,’ within the Biological Process category, was the most highly overrepresented under cold-stress conditions, implying its significance in that response in rice. MapMan analysis classified ‘Major Metabolic’ processes and ‘Regulatory Gene Modules’ as two other major determinants of the cold-stress response and suggested several key cis-regulatory elements. Based on these results, we proposed a model that includes a pathway for cold stress-responsive signaling. Results from our functional analysis of the main signal transduction and transcription regulation factors identified in that pathway will provide insight into novel regulatory metabolism(s), as well as a foundation by which we can develop crop plants with enhanced cold tolerance.

Transcriptional regulation of the IL-7Rα gene by dexamethasone and IL-7 in primary human CD8 T cells.

Interleukin-7 is essential for the development and maintenance of T cells, and the expression of the IL-7 receptor is tightly regulated at every stage of the T cell's lifespan. In mature CD8 T cells, IL-7 plays important roles in cell survival, peripheral homeostasis, and cytolytic function. The IL-7 receptor alpha-chain (CD127) is expressed at high levels on naïve and memory cells, but it is rapidly downregulated upon IL-7 stimulation. In this study, we illustrate the dynamicity of the CD127 promoter and show that it possesses positive as well as negative regulatory sites involved in upregulating and downregulating CD127 expression, respectively. We cloned the CD127 gene promoter and identified key cis-regulatory elements required for CD127 expression in mature resting primary CD8 T cells. The core promoter necessary for efficient basal transcription is contained within the first 262bp upstream of the TATA box. Additional positive regulatory elements are located between -1200 and -2406bp, conferring a further 2- to 4-fold enhancement in gene expression. While transcription of the CD127 gene is increased directly through a glucocorticoid response element located between -2255 and -2269bp upstream of the TATA box, we identified a suppressive region that lies upstream of 1760bp from the TATA box, which is likely involved in the IL-7-mediated suppression of CD127 transcription. Finally, we illustrated IL-7 does not bias alternative splicing of CD127 transcripts in primary human CD8 T cells.

Molecular cloning and promoter analysis of squalene synthase and squalene epoxidase genes from Betula platyphylla.

Betula platyphylla is a rich repository of pharmacologically active secondary metabolites known as birch triterpenoids (TBP). Here, we cloned the squalene synthase (SS) and squalene epoxidase genetic (SE) sequences from B. platyphylla that encode the key enzymes that are involved in triterpenoid biosynthesis and analyzed the conserved domains and phylogenetics of their corresponding proteins. The full-length sequence of BpSS is 1588bp with a poly-A tail, which contained an open reading frame (ORF) of 1241bp that encoded a protein of 413 amino acids. Additionally, the BpSE full-length sequence of 2040bp with a poly-A tail was also obtained, which contained an ORF of 1581bp encoding a protein of 526 amino acids. Their organ-specific expression patterns in 4-week-old tissue culture seedlings of B. platyphylla were detected by real-time PCR and showed that they were all highly expressed in leaves, as compared to stem and root tissues. Additionaly, both BpSS and BpSE were enhanced following stimulation with ethephon and MeJA. The expression of BpSS was enhanced by ABA, whereas BpSE was not. The SA treatment did not affect the BpSS and BpSE transcripts notably. Using a genome walking approach, promoter sequences of 965 and 1193bp, respectively, for BpSS and BpSE were isolated, and they revealed several key cis-regulatory elements known to be involved in the response to phytohormone and abiotic plant stress. We also found that the BpSS protein is localized in the cytoplasm. Opening reading frames of BpSS and BpSE were ligated into yeast expression plasmid pYES2 under control of GAL1 promoter and introduced into the yeast INVScl1 strain. The transformants were cultured for 12h, the squalene content of galactose-induced BpSS expression yeast cells was 13.2 times of control (empty vector control yeast cells) by high-performance liquid chromatography (HPLC) test method. And, the squalene epoxidase activity of induced BpSE expression yeast cell was about 11.8 times of control. These indicated that we cloned birch BpSS and BpSE that were indeed involved in the synthesis of triteropenoids. This is the first report wherein SS and SE from B. platyphylla were cloned and may be of significant interest to understand the regulatory role of SS and SE in the triterpenoids biosynthesis of B. platyphylla. This is the first report wherein SS and SE from B. platyphylla were cloned and may be of significant interest to understand the regulatory role of SS and SE in the biosynthesis of birch triterpenoids.

An SCF-FBXW7 Ubiquitin Ligase Mediated Feedback Loop Facilitates GATA Factor Switching and Reinforces Commitment to Terminal Erythroid Maturation

The GATA family transcription factors GATA2 and GATA1 play reciprocal roles during terminal erythroid and megakaryocytic maturation. GATA2 is expressed early in hematopoiesis and is required for early progenitor cell proliferation and survival. It must be down regulated in order for terminal maturation to occur. In contrast, GATA1 increases in expression during erythropoiesis and megakaryopoiesis, and is required for terminal maturation of these lineages. During this process, GATA1 displaces GATA2 at a large number of chromatin loci, typically leading to repression of early progenitor genes and activation of terminal maturation genes. This “GATA factor switch” is facilitated by the considerably shorter half-life of GATA2 (~30 min) compared to GATA1 (>4-6 hours). GATA2’s short half-life is mediated by ubiquitin-proteosomal degradation mechanisms. Yet the ubiquitin complex(es) responsible for GATA2-specific clearance has not been identified. In this study, we show that the Skip1-Cullin-Fbox (SCF) substrate recognition factor Fbxw7 is involved in GATA2 ubiquitin-mediated degradation. Proteomic and co-immunoprecipitation experiments show physical association of Fbxw7 with GATA2, but not GATA1. CRISPR/Cas9 deletion of Fbxw7 in G1ER4 cells results in elevated GATA2 steady-state protein levels, a prolonged GATA2 half-life, delayed GATA2 clearance upon estradiol induction, and impaired erythroid terminal maturation. Importantly, Fbxw7 mRNA and protein levels normally increase during terminal erythroid maturation and this occurs in a GATA1 dependent manner. We identified a key cis-regulatory element upstream of the Fbxw7 gene that is occupied by GATA2 during early erythropoiesis, but becomes bound by GATA1/TAL1 during late erythroid maturation in induced G1-ER4 cells and human CD34+ in vitro differentiated erythroblasts. This is accompanied by the acquisition of active enhancer histone marks at this site. Deletion of this regulatory element in G1ER4 cells blocks the GATA1 dependent increase in Fbxw7 mRNA transcript levels during erythroid cell maturation. Lastly, we identified a family with congenital hypoplastic anemia (lacking mutations in all known Diamond Blackfan Anemia genes) who harbor a germline Fbxw7missense mutation. Collectively, these findings identify GATA2 as a novel Fbxw7 substrate and uncover an ubiquitin-mediated post-transcriptional GATA factor feedback loop that reinforces GATA factor switching and commitment to terminal erythroid maturation. DisclosuresCantor:Amgen: Membership on an entity’s Board of Directors or advisory committees.

Isolation, Characterization and Promoter Analysis of Cell Wall Invertase Gene SoCIN1 from Sugarcane (Saccharum spp.)

Cell wall invertase (CIN) plays an important role in carbohydrate partitioning and regulation of sink-source interaction. In this study, a full length cDNA encoding CIN gene was cloned by RT-PCR and RACE-PCR from sugarcane. The open reading frame of 1731 bp encodes a protein of 577 amino acids with a predicted molecular mass of 141.04 kDa and theoretical PI of 4.67. A size of 4,537 bp DNA encoding SoCIN1 was isolated, which contains seven exons and six introns. Using genome walking approach, a promoter sequence of 974 bp of SoCIN1 was isolated, and prediction with PlantCARE and PLACE software revealed that it has several key cis-regulatory elements known to be involved in various biotic and abiotic plant stresses. Relative expression in specific tissues was done using qRT-PCR, and the results demonstrated that SoCIN1 expression level was higher in the immature leaves and maturing leaves than the matured leaves and internodes at elongation stage and processing maturing stage. The conditions of 15 % PEG and 6 °C could have a similar function for inducing the expression of SoCIN gene in leaves, while 15 % PEG, 100 mM NaCl and 6 °C could induce the expression of SoCIN gene in roots.